@article {pmid37230046, year = {2023}, author = {Li, W and Wang, Z and Jiang, Z and Yan, Y and Yao, X and Pan, Z and Chen, L and Wang, F and Wang, M and Qin, Z}, title = {MiR-3960 inhibits bladder cancer progression via targeting of DEXI.}, journal = {Biochemical and biophysical research communications}, volume = {668}, number = {}, pages = {8-18}, doi = {10.1016/j.bbrc.2023.05.055}, pmid = {37230046}, issn = {1090-2104}, abstract = {PURPOSE: MicroRNAs (miRNAs) are dominant cargo in exosomes and act as master regulators of cell function, inhibiting mRNA translation and affecting gene silencing. Some aspects of tissue-specific miRNA transport in bladder cancer (BC) and its role in cancer progression are not fully understood.<br><br>MATERIALS AND METHODS: A microarray was used to identify miRNAs in mouse bladder carcinoma cell line MB49 exosomes. Real-time reverse transcription polymerase chain reaction was used to examine the expression of miRNAs in BC and healthy donor serum. Western blotting and immunohistochemical staining were used to examine the expression of dexamethasone-induced protein (DEXI) in patients with BC. CRISPR-Cas 9 was used to knock out Dexi in MB49, and flow cytometry was performed to test cell proliferation ability and apoptosis under chemotherapy. Human BC organoid culture, miR-3960 transfection, and 293T-exosome-loaded miR-3960 delivery were used to analyze the effect of miR-3960 on BC progression.<br><br>RESULTS: The results showed that miR-3960 levels in BC tissue were positively correlated with patient survival time. Dexi was a major target of miR-3960. Dexi knockout inhibited MB49&#xa0;cell proliferation and promoted cisplatin- and gemcitabine-induced apoptosis. Transfection of miR-3960 mimic inhibited DEXI expression and organoid growth. In parallel, 293T-exosome-loaded miR-3960 delivery and Dexi knockout significantly inhibited subcutaneous growth of MB49&#xa0;cells in&#xa0;vivo.<br><br>CONCLUSION: Our results demonstrate the potential role of miR-3960-mediated inhibition of DEXI as a therapeutic strategy against BC.}, }
@article {pmid37228830, year = {2023}, author = {Schmidt, M and Kircheis, W and Simons, A and Potthast, M and Stein, B}, title = {A diachronic perspective on citation latency in Wikipedia articles on CRISPR/Cas-9: an exploratory case study.}, journal = {Scientometrics}, volume = {128}, number = {6}, pages = {3649-3673}, pmid = {37228830}, issn = {0138-9130}, abstract = {This paper analyzes Wikipedia's representation of the Nobel Prize winning CRISPR/Cas9 technology, a method for gene editing. We propose and evaluate different heuristics to match publications from several publication corpora against Wikipedia's central article on CRISPR and against the complete Wikipedia revision history in order to retrieve further Wikipedia articles relevant to the topic and to analyze Wikipedia's referencing patterns. We explore to what extent the selection of referenced literature of Wikipedia's central article on CRISPR adheres to scientific standards and inner-scientific perspectives by assessing its overlap with (1) the Web of Science (WoS) database, (2) a WoS-based field-delineated corpus, (3) highly-cited publications within this corpus, and (4) publications referenced by field-specific reviews. We develop a diachronic perspective on citation latency and compare the delays with which publications are cited in relevant Wikipedia articles to the citation dynamics of these publications over time. Our results confirm that a combination of verbatim searches by title, DOI, and PMID is sufficient and cannot be improved significantly by more elaborate search heuristics. We show that Wikipedia references a substantial amount of publications that are recognized by experts and highly cited, but that Wikipedia also cites less visible literature, and, to a certain degree, even not strictly scientific literature. Delays in occurrence on Wikipedia compared to the publication years show (most pronounced in case of the central CRISPR article) a dependence on the dynamics of both the field and the editor's reaction to it in terms of activity.}, }
@article {pmid37226848, year = {2023}, author = {Kayo, D and Kimura, S and Yamazaki, T and Naruse, K and Takeuchi, H and Ansai, S}, title = {Spatio-temporal control of targeted gene expression in combination with CRISPR/Cas and Tet-On systems in Medaka.}, journal = {Genesis (New York, N.Y. : 2000)}, volume = {}, number = {}, pages = {e23519}, doi = {10.1002/dvg.23519}, pmid = {37226848}, issn = {1526-968X}, abstract = {Spatial and temporal control of transgene expression is a powerful approach to understand gene functions in specific cells and tissues. The Tet-On system is a robust tool for controlling transgene expression spatially and temporally; however, few studies have examined whether this system can be applied to postembryonic stages of Medaka (Oryzias latipes) or other fishes. Here, we first improved a basal promoter sequence on the donor vector for a nonhomologous end joining (NHEJ)-based knock-in (KI) system. Next, using transgenic Medaka for establishing the Tet-On system by KI, we demonstrated that doxycycline administration for four or more days by feeding can be a stable and efficient method to achieve expression of the transduced reporter gene in adult fish. From these analyses, we propose an optimized approach for a spatio-temporal gene-expression system in the adult stage of Medaka and other small fishes.}, }
@article {pmid37226834, year = {2023}, author = {Walker-Sünderhauf, D and Klümper, U and Pursey, E and Westra, ER and Gaze, WH and van Houte, S}, title = {Removal of AMR plasmids using a mobile, broad host-range CRISPR-Cas9 delivery tool.}, journal = {Microbiology (Reading, England)}, volume = {169}, number = {5}, pages = {}, doi = {10.1099/mic.0.001334}, pmid = {37226834}, issn = {1465-2080}, support = {MR/N0137941/1/MRC_/Medical Research Council/United Kingdom ; BB/R010781/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/S017674/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; }, mesh = {Humans ; Animals ; Swine ; *CRISPR-Cas Systems ; Host Specificity ; *Bacteriophages ; Biological Transport ; Escherichia coli/genetics ; Plasmids/genetics ; }, abstract = {Antimicrobial resistance (AMR) genes are widely disseminated on plasmids. Therefore, interventions aimed at blocking plasmid uptake and transfer may curb the spread of AMR. Previous studies have used CRISPR-Cas-based technology to remove plasmids encoding AMR genes from target bacteria, using either phage- or plasmid-based delivery vehicles that typically have narrow host ranges. To make this technology feasible for removal of AMR plasmids from multiple members of complex microbial communities, an efficient, broad host-range delivery vehicle is needed. We engineered the broad host-range IncP1-plasmid pKJK5 to encode cas9 programmed to target an AMR gene. We demonstrate that the resulting plasmid pKJK5::csg has the ability to block the uptake of AMR plasmids and to remove resident plasmids from Escherichia coli. Furthermore, due to its broad host range, pKJK5::csg successfully blocked AMR plasmid uptake in a range of environmental, pig- and human-associated coliform isolates, as well as in isolates of two species of Pseudomonas. This study firmly establishes pKJK5::csg as a promising broad host-range CRISPR-Cas9 delivery tool for AMR plasmid removal, which has the potential to be applied in complex microbial communities to remove AMR genes from a broad range of bacterial species.}, }
@article {pmid37225346, year = {2023}, author = {Yue, Y and Wang, S and Jin, Q and An, N and Wu, L and Huang, H}, title = {A triple amplification strategy using GR-5 DNAzyme as a signal medium for ultrasensitive detection of trace Pb[2+] based on CRISPR/Cas12a empowered electrochemical biosensor.}, journal = {Analytica chimica acta}, volume = {1263}, number = {}, pages = {341241}, doi = {10.1016/j.aca.2023.341241}, pmid = {37225346}, issn = {1873-4324}, mesh = {Humans ; *CRISPR-Cas Systems ; *DNA, Catalytic ; Lead ; DNA, Single-Stranded ; }, abstract = {Lead ions (Pb[2+]) are a well-known toxic heavy metal that poses a significant threat to human health. Therefore, the development of a simple and ultrasensitive technique for detecting Pb[2+] is essential. With their trans-cleavage properties, the newly discovered CRISPR-V effectors have become a potential high-precision biometric tool. In this regard, a CRISPR/Cas12a-based electrochemical biosensor (E-CRISPR) has been developed, which is combined with the GR-5 DNAzyme that can specifically recognize Pb[2+]. In this strategy, the GR-5 DNAzyme acts as a signal-mediated intermediary, which can convert Pb[2+] into nucleic acid signals, thereby becoming single-stranded DNA that triggers strand displacement amplification (SDA) reaction. This is coupled with following activated CRISPR/Cas12a cleavage of the electrochemical signal probe, enabling cooperative signal amplification for ultrasensitive Pb[2+] detection. The proposed method has a detection limit as low as 0.02 pM. Therefore, we have developed an E-CRISPR detection platform with GR-5 DNAzyme as a signal medium (called SM-E-CRISPR biosensor). This provides a method for the CRISPR system to specifically detect non-nucleic substances by converting the signal using a medium.}, }
@article {pmid37223740, year = {2023}, author = {Miezner, G and Turgeman-Grott, I and Zatopek, KM and Gardner, AF and Reshef, L and Choudhary, DK and Alstetter, M and Allers, T and Marchfelder, A and Gophna, U}, title = {An archaeal Cas3 protein facilitates rapid recovery from DNA damage.}, journal = {microLife}, volume = {4}, number = {}, pages = {uqad007}, pmid = {37223740}, issn = {2633-6693}, abstract = {CRISPR-Cas systems provide heritable acquired immunity against viruses to archaea and bacteria. Cas3 is a CRISPR-associated protein that is common to all Type I systems, possesses both nuclease and helicase activities, and is responsible for degradation of invading DNA. Involvement of Cas3 in DNA repair had been suggested in the past, but then set aside when the role of CRISPR-Cas as an adaptive immune system was realized. Here we show that in the model archaeon Haloferax volcanii a cas3 deletion mutant exhibits increased resistance to DNA damaging agents compared with the wild-type strain, but its ability to recover quickly from such damage is reduced. Analysis of cas3 point mutants revealed that the helicase domain of the protein is responsible for the DNA damage sensitivity phenotype. Epistasis analysis indicated that cas3 operates with mre11 and rad50 in restraining the homologous recombination pathway of DNA repair. Mutants deleted for Cas3 or deficient in its helicase activity showed higher rates of homologous recombination, as measured in pop-in assays using non-replicating plasmids. These results demonstrate that Cas proteins act in DNA repair, in addition to their role in defense against selfish elements and are an integral part of the cellular response to DNA damage.}, }
@article {pmid37222810, year = {2023}, author = {Alinejad, T and Modarressi, S and Sadri, Z and Hao, Z and Chen, CS}, title = {Diagnostic applications and therapeutic option of Cascade CRISPR/Cas in the modulation of miRNA in diverse cancers: promises and obstacles.}, journal = {Journal of cancer research and clinical oncology}, volume = {}, number = {}, pages = {}, pmid = {37222810}, issn = {1432-1335}, abstract = {The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas technology is a molecular tool specific to sequences for engineering genomes. Among diverse clusters of Cas proteins, the class 2/type II CRISPR/Cas9 system, despite several challenges, such as off-target effects, editing efficiency, and efficient delivery, has shown great promise for driver gene mutation discovery, high-throughput gene screening, epigenetic modulation, nucleic acid detection, disease modeling, and more importantly for therapeutic purposes. CRISPR-based clinical and experimental methods have applications across a wide range of areas, especially for cancer research and, possibly, anticancer therapy. On the other hand, given the influential role of microRNAs (miRNAs) in the regulations of cellular division, carcinogenicity, tumorigenesis, migration/invasion, and angiogenesis in diverse normal and pathogenic cellular processes, in different stages of cancer, miRNAs are either oncogenes or tumor suppressors, according to what type of cancer they are involved in. Hence, these noncoding RNA molecules are conceivable biomarkers for diagnosis and therapeutic targets. Moreover, they are suggested to be adequate predictors for cancer prediction. Conclusive evidence proves that CRISPR/Cas system can be applied to target small non-coding RNAs. However, the majority of studies have highlighted the application of the CRISPR/Cas system for targeting protein-coding regions. In this review, we specifically discuss diverse applications of CRISPR-based tools for probing miRNA gene function and miRNA-based therapeutic involvement in different types of cancers.}, }
@article {pmid37222701, year = {2023}, author = {Engstler, M and Beneke, T}, title = {Gene editing and scalable functional genomic screening in Leishmania species using the CRISPR/Cas9 cytosine base editor toolbox LeishBASEedit.}, journal = {eLife}, volume = {12}, number = {}, pages = {}, pmid = {37222701}, issn = {2050-084X}, mesh = {*Leishmania/genetics ; CRISPR-Cas Systems ; Gene Editing ; Genomics ; Cytosine ; }, abstract = {CRISPR/Cas9 gene editing has revolutionised loss-of-function experiments in Leishmania, the causative agent of leishmaniasis. As Leishmania lack a functional non-homologous DNA end joining pathway however, obtaining null mutants typically requires additional donor DNA, selection of drug resistance-associated edits or time-consuming isolation of clones. Genome-wide loss-of-function screens across different conditions and across multiple Leishmania species are therefore unfeasible at present. Here, we report a CRISPR/Cas9 cytosine base editor (CBE) toolbox that overcomes these limitations. We employed CBEs in Leishmania to introduce STOP codons by converting cytosine into thymine and created http://www.leishbaseedit.net/ for CBE primer design in kinetoplastids. Through reporter assays and by targeting single- and multi-copy genes in L. mexicana, L. major, L. donovani, and L. infantum, we demonstrate how this tool can efficiently generate functional null mutants by expressing just one single-guide RNA, reaching up to 100% editing rate in non-clonal populations. We then generated a Leishmania-optimised CBE and successfully targeted an essential gene in a plasmid library delivered loss-of-function screen in L. mexicana. Since our method does not require DNA double-strand breaks, homologous recombination, donor DNA, or isolation of clones, we believe that this enables for the first time functional genetic screens in Leishmania via delivery of plasmid libraries.}, }
@article {pmid37222511, year = {2023}, author = {Lu, J and Wang, S}, title = {CRISPR/Cas9-Mediated Gene Knockout in Cells and Tissues Using Lentivirus.}, journal = {Current protocols}, volume = {3}, number = {5}, pages = {e772}, doi = {10.1002/cpz1.772}, pmid = {37222511}, issn = {2691-1299}, support = {/NH/NIH HHS/United States ; /DE/NIDCR NIH HHS/United States ; }, mesh = {Humans ; Animals ; Mice ; Mice, Knockout ; *Lentivirus/genetics ; CRISPR-Cas Systems/genetics ; Gene Knockout Techniques ; Cloning, Molecular ; *Craniocerebral Trauma ; }, abstract = {CRISPR-Cas9 has become a powerful and popular gene editing tool. However, successful application of this tool in the lab can still be quite daunting to many newcomers to molecular biology, mostly because it is a relatively lengthy process involving multiple steps with variations of each step. Here, we provide a reliable, stepwise, and newcomer-friendly protocol to knock out a target gene in wild-type human fibroblasts. This protocol involves sgRNA design using CRISPOR, construction of an "all-in-one" vector expressing both sgRNA and Cas9 using Golden Gate cloning, streamlined production of high-titer lentiviruses in 1 week after molecular cloning, and transduction of cells to generate a knockout cell pool. We further introduce a protocol for lentiviral transduction of ex vivo mouse embryonic salivary epithelial explants. In summary, our protocol is useful for new researchers to apply CRISPR-Cas9 to generate stable gene knockout cells and tissue explants using lentivirus. Published 2023. This article is a U.S. Government work and is in the public domain in the USA. Basic Protocol 1: sgRNA design Basic Protocol 2: Cloning sgRNA in plasmid vector containing Cas9 encoding sequence using golden gate cloning Basic Protocol 3: Lentivirus packaging Basic Protocol 4: Lentivirus transduction of cells Basic Protocol 5: Lentivirus transduction of salivary gland epithelial buds.}, }
@article {pmid37129004, year = {2023}, author = {Shi, Y and Kopparapu, N and Ohler, L and Dickinson, DJ}, title = {Efficient and rapid fluorescent protein knock-in with universal donors in mouse embryonic stem cells.}, journal = {Development (Cambridge, England)}, volume = {150}, number = {10}, pages = {}, doi = {10.1242/dev.201367}, pmid = {37129004}, issn = {1477-9129}, support = {R01 GM138443/GM/NIGMS NIH HHS/United States ; R01 GM138443/NH/NIH HHS/United States ; }, mesh = {Animals ; Mice ; *CRISPR-Cas Systems/genetics ; *Mouse Embryonic Stem Cells ; Proteins/genetics ; Gene Knock-In Techniques ; Gene Editing/methods ; Mammals/genetics ; }, abstract = {Fluorescent protein (FP) tagging is a key method for observing protein distribution, dynamics and interaction with other proteins in living cells. However, the typical approach using overexpression of tagged proteins can perturb cell behavior and introduce localization artifacts. To preserve native expression, fluorescent proteins can be inserted directly into endogenous genes. This approach has been widely used in yeast for decades, and more recently in invertebrate model organisms with the advent of CRISPR/Cas9. However, endogenous FP tagging has not been widely used in mammalian cells due to inefficient homology-directed repair. Recently, the CRISPaint system used non-homologous end joining for efficient integration of FP tags into native loci, but it only allows C-terminal knock-ins. Here, we have enhanced the CRISPaint system by introducing new universal donors for N-terminal insertion and for multi-color tagging with orthogonal selection markers. We adapted the procedure for mouse embryonic stem cells, which can be differentiated into diverse cell types. Our protocol is rapid and efficient, enabling live imaging in less than 2 weeks post-transfection. These improvements increase the versatility and applicability of FP knock-in in mammalian cells.}, }
@article {pmid37101010, year = {2023}, author = {Tanihara, F and Hirata, M and Namula, Z and Wittayarat, M and Do, LTK and Lin, Q and Takebayashi, K and Hara, H and Nagahara, M and Otoi, T}, title = {GHR-mutant pig derived from domestic pig and microminipig hybrid zygotes using CRISPR/Cas9 system.}, journal = {Molecular biology reports}, volume = {50}, number = {6}, pages = {5049-5057}, pmid = {37101010}, issn = {1573-4978}, mesh = {Male ; Humans ; Swine/genetics ; Animals ; Female ; *Zygote ; *CRISPR-Cas Systems/genetics ; Receptors, Somatotropin/genetics ; Swine, Miniature ; Oocytes ; }, abstract = {BACKGROUND: Pigs are excellent large animal models with several similarities to humans. They provide valuable insights into biomedical research that are otherwise difficult to obtain from rodent models. However, even if miniature pig strains are used, their large stature compared with other experimental animals requires a specific maintenance facility which greatly limits their usage as animal models. Deficiency of growth hormone receptor (GHR) function causes small stature phenotypes. The establishment of miniature pig strains via GHR modification will enhance their usage as animal models. Microminipig is an incredibly small miniature pig strain developed in Japan. In this study, we generated a GHR mutant pig using electroporation-mediated introduction of the CRISPR/Cas9 system into porcine zygotes derived from domestic porcine oocytes and microminipig spermatozoa.<br><br>METHODS AND RESULTS: First, we optimized the efficiency of five guide RNAs (gRNAs) designed to target GHR in zygotes. Embryos that had been electroporated with the optimized gRNAs and Cas9 were then transferred into recipient gilts. After embryo transfer, 10 piglets were delivered, and one carried a biallelic mutation in the GHR target region. The GHR biallelic mutant showed a remarkable growth-retardation phenotype. Furthermore, we obtained F1 pigs derived from the mating of GHR biallelic mutant with wild-type microminipig, and GHR biallelic mutant F2 pigs through sib-mating of F1 pigs.<br><br>CONCLUSIONS: We have successfully demonstrated the generation of biallelic GHR-mutant small-stature pigs. Backcrossing of GHR-deficient pig with microminipig will establish the smallest pig strain which can contribute significantly to the field of biomedical research.}, }
@article {pmid37016515, year = {2023}, author = {Hu, M and Liu, R and Qiu, Z and Cao, F and Tian, T and Lu, Y and Jiang, Y and Zhou, X}, title = {Light-Start CRISPR-Cas12a Reaction with Caged crRNA Enables Rapid and Sensitive Nucleic Acid Detection.}, journal = {Angewandte Chemie (International ed. in English)}, volume = {62}, number = {23}, pages = {e202300663}, doi = {10.1002/anie.202300663}, pmid = {37016515}, issn = {1521-3773}, mesh = {*CRISPR-Cas Systems/genetics ; *RNA, Guide, CRISPR-Cas Systems ; Agriculture ; Biological Assay ; Nucleotidyltransferases ; Nucleic Acid Amplification Techniques ; }, abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR) system is a promising platform for nucleic acid detection. Regulating the CRISPR reaction would be extremely useful to improve the detection efficiency and speed of CRISPR diagnostic applications. Here, we have developed a light-start CRISPR-Cas12a reaction by employing caged CRISPR RNA (crRNA). When combined with recombinase polymerase amplification, a robust photocontrolled one-pot assay is achieved. The photocontrolled one-pot assay is simpler and is 50-fold more sensitive than the conventional assay. This improved detection efficiency also facilitates the development of a faster CRISPR diagnostic method. The detection of clinical samples demonstrated that 10-20 min is sufficient for effective detection, which is much faster than the current gold-standard technique PCR. We expect this advance in CRISPR diagnostics to promote its widespread detection applications in biomedicine, agriculture, and food safety.}, }
@article {pmid36933474, year = {2023}, author = {Davachi, ND and Bartlewski, PM and Masoudi, R and Fallahi, R}, title = {Suitability of a universal electroporation device for genome editing and production of transgenic rats.}, journal = {Reproductive biology}, volume = {23}, number = {2}, pages = {100755}, doi = {10.1016/j.repbio.2023.100755}, pmid = {36933474}, issn = {2300-732X}, mesh = {Animals ; Female ; Male ; Rats ; *Gene Editing/methods ; Rats, Transgenic ; *CRISPR-Cas Systems ; Rats, Sprague-Dawley ; Electroporation/methods ; RNA, Messenger/genetics ; Mammals/genetics ; }, abstract = {Mammalian genome editing has utilized expensive and highly specialized electroporator devices. The "Gene Pulser XCell," a modular electroporation system for transfecting all cell types, has not been used extensively in mammalian embryo genome editing. The present experiment was undertaken to determine the usefulness of the Gene Pulser XCell for inserting the CRISPR/Cas9 system into intact zygotes in order to obtain the enhanced green fluorescent protein reporter rats (eGFP-R). An electroporation pulse response test using mCherry mRNA was performed to optimize the settings of the electroporator. Forty-five combinations of five pulse voltages (15, 25, 30, 35 and 40 V), three pulse durations (5, 10 and 25 ms), and three pulse frequencies (2, 5 and 6 pulses) applied at a constant 100-ms pulse interval and temperature of 37.5 °C were evaluated. The test revealed that the 35 V was the only voltage suitable for insertion of mCherry mRNA into intact rat zygotes and the only one that resulted in the production of embryos attaining the blastocyst stage. The incorporation of mCherry mRNA increased but the survival of the electroporated embryos declined with an increment in the number of pulses. Subsequent transfer of 1112 surviving Sprague Dawley rat embryos (after 8 h of incubating 1800 zygotes electroporated with the CRISPR/Cas9) resulted in the production of 287 offspring (25.8%). Ensuing PCR and phenotypic evaluation confirmed that twenty animals (6.96%) expressed eGFP in all body organs/tissues except for blood and blood vessels. The mortality of males and females before the attainment of puberty was 2 and 3 pups, respectively, and the final number/ratio of male to female of offspring was 9:11. All the surviving rats mated naturally and successfully transmitted the GFP transgene to their progeny. The Gene Pulser XCell total system with the settings predetermined in the present experiment can effectively be used to produce transgenic rats through the CRISPR/Cas9-mediated genome editing of zygotes.}, }
@article {pmid36473602, year = {2023}, author = {Li, X and Liu, X and Wei, J and Bu, S and Li, Z and Hao, Z and Zhang, W and Wan, J}, title = {Ultrasensitive detection of microRNAs based on click chemistry-terminal deoxynucleotidyl transferase combined with CRISPR/Cas12a.}, journal = {Biochimie}, volume = {208}, number = {}, pages = {38-45}, doi = {10.1016/j.biochi.2022.12.001}, pmid = {36473602}, issn = {1638-6183}, mesh = {*DNA Nucleotidylexotransferase ; CRISPR-Cas Systems ; Click Chemistry ; Coloring Agents ; DNA-Directed DNA Polymerase ; *MicroRNAs/genetics ; }, abstract = {The specificity and sensitivity of microRNA (miRNA) detection play a vital role in the early diagnosis of cancer and the treatment of various diseases. Here, we constructed a fluorescent biosensor based on click chemistry-terminal deoxynucleotidyl transferase (ccTdT) combined with the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas)12a cascade amplification system to achieve ultrasensitive miRNA-21 detection. Target miRNA-21 was employed as a template for click chemistry ligation of two nucleic acid probes, the product of which can be combined with magnetic microbeads (MBs). Then the 3'-end of the ligated nucleic acid and complementary strand miRNA-21 was extended by TdT. The extended poly-T tails activated the trans-cleavage ability of CRISPR/Cas12a, cleaving the reporter gene to generate the fluorescent signal. The proposed biosensor has a wide linear detection range, from 1 pM to 10[5] pM, with detection limits as low as 88 fM under optimal experimental conditions. Hence, this fluorescent biosensor enables simple, sensitive detection of miRNAs and offers a promising analytical platform for clinical diagnostics and biomedical research.}, }
@article {pmid37219967, year = {2023}, author = {}, title = {Correction to: Clonally Selected Lines After CRISPR-Cas Editing Are Not Isogenic by Panda et al. The CRISPR Journal, 2023;6(2):176-182; DOI: 10.1089/crispr.2022.0050.}, journal = {The CRISPR journal}, volume = {}, number = {}, pages = {}, doi = {10.1089/crispr.2022.0050.correx}, pmid = {37219967}, issn = {2573-1602}, }
@article {pmid37218113, year = {2023}, author = {Chen, D and Huang, W and Zhang, Y and Chen, B and Tan, J and Yang, Y and Yuan, Q}, title = {CRISPR-Mediated Profiling of Viral RNA at Single Nucleotide Resolution.}, journal = {Angewandte Chemie (International ed. in English)}, volume = {}, number = {}, pages = {e202304298}, doi = {10.1002/anie.202304298}, pmid = {37218113}, issn = {1521-3773}, abstract = {Mass pathogen screening is critical to preventing the outbreaks and spread of infectious diseases. The large-scale epidemic of COVID-19 and the rapid mutation of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus have put forward new requirements for virus detection and identification techniques. Here, we report a CRISPR-based Amplification-free Viral RNA Electrical Detection platform (CAVRED) for the rapid detection and identification of SARS-CoV-2 variants. A series of CRISPR RNA assays were designed to amplify the CRISPR-Cas system's ability to discriminate between mutant and wild RNA genomes with a single-nucleotide difference. The identified viral RNA information was converted into readable electrical signals through field-effect transistor biosensors for the achievement of highly sensitive detection of single-base mutations. CAVRED can detect the SARS-CoV-2 virus genome as low as 1 cp/µL within 20 mins without amplification, and this value is comparable to the detection limit of real-time quantitative polymerase chain reaction. Based on the excellent RNA mutation detection ability, an 8-in-1 CAVRED array was constructed and realized the rapid identification of 40 simulated throat swab samples of SARS-CoV-2 variants with a 95.0% accuracy. The advantages of accuracy, sensitivity, and fast speed of CAVRED promise its application in rapid and large-scale epidemic screening.}, }
@article {pmid37218074, year = {2023}, author = {Takebayashi, K and Wittayarat, M and Lin, Q and Torigoe, N and Liu, B and Hirata, M and Nagahara, M and Tanihara, F and Otoi, T}, title = {Efficiency of genetic modification in gene-knockout sperm-derived zygotes followed by electroporation of guide RNA targeting the same gene.}, journal = {Animal science journal = Nihon chikusan Gakkaiho}, volume = {94}, number = {1}, pages = {e13842}, doi = {10.1111/asj.13842}, pmid = {37218074}, issn = {1740-0929}, mesh = {Male ; Animals ; Swine ; *Gene Editing/veterinary ; *Zygote ; CRISPR-Cas Systems ; Semen ; Electroporation/veterinary ; RNA, Guide, CRISPR-Cas Systems ; }, abstract = {Genetic mosaicism is considered one of the main limitations of the electroporation method used to transfer CRISPR-Cas9/guide RNA (gRNA) into porcine zygotes. We hypothesized that fertilization of oocytes with sperm from gene-deficient boars, in combination with electroporation (EP) to target the same region of the gene in subsequent zygotes, would increase the gene modification efficiency. As myostatin (MSTN) and α1,3-galactosyltransferase (GGTA1) have beneficial effects on agricultural production and xenotransplantation, respectively, we used these two genes to test our hypothesis. Spermatozoa from gene-knockout boars were used for oocyte fertilization in combination with EP to transfer gRNAs targeting the same gene region to zygotes. No significant differences in the rates of cleavage and blastocyst formation as well as in the mutation rates of blastocysts were observed between the wild-type and gene-deficient spermatozoa groups, irrespective of the targeted gene. In conclusion, the combination of fertilization with gene-deficient spermatozoa and gene editing of the same targeted gene region using EP had no beneficial effects on embryo genetic modification, indicating that EP alone is a sufficient tool for genome modification.}, }
@article {pmid37217056, year = {2023}, author = {Lyu, M and Sun, Y and Yan, N and Chen, Q and Wang, X and Wei, Z and Zhang, Z and Xu, K}, title = {Efficient CRISPR/Cas9-mediated gene editing in mammalian cells by the novel selectable traffic light reporters.}, journal = {International journal of biological macromolecules}, volume = {}, number = {}, pages = {124926}, doi = {10.1016/j.ijbiomac.2023.124926}, pmid = {37217056}, issn = {1879-0003}, abstract = {CRISPR/Cas9 is a powerful tool for gene editing in various cell types and organisms. However, it is still challenging to screen genetically modified cells from an excess of unmodified cells. Our previous studies demonstrated that surrogate reporters can be used for efficient screening of genetically modified cells. Here, we developed two novel traffic light screening reporters, puromycin-mCherry-EGFP (PMG) based on single-strand annealing (SSA) and homology-directed repair (HDR), respectively, to measure the nuclease cleavage activity within transfected cells and to select genetically modified cells. We found that the two reporters could be self-repaired coupling the genome editing events driven by different CRISPR/Cas nucleases, resulting in a functional puromycin-resistance and EGFP selection cassette that can be afforded to screen genetically modified cells by puromycin selection or FACS enrichment. We further compared the novel reporters with different traditional reporters at several endogenous loci in different cell lines, for the enrichment efficiencies of genetically modified cells. The results indicated that the SSA-PMG reporter exhibited improvements in enriching gene knockout cells, while the HDR-PMG system was very useful in enriching knock-in cells. These results provide robust and efficient surrogate reporters for the enrichment of CRISPR/Cas9-mediated editing in mammalian cells, thereby advancing basic and applied research.}, }
@article {pmid37217031, year = {2023}, author = {Hamdi, I and Boni, F and Shen, Q and Moukendza, L and Peibo, LI and Jianping, X}, title = {Characteristics of subtype III-A CRISPR-Cas system in Mycobacterium tuberculosis: An overview.}, journal = {Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases}, volume = {}, number = {}, pages = {105445}, doi = {10.1016/j.meegid.2023.105445}, pmid = {37217031}, issn = {1567-7257}, abstract = {CRISPR-Cas systems are the only RNA- guided adaptive immunity pathways that trigger the detection and destruction of invasive phages and plasmids in bacteria and archaea. Due to its prevalence and mystery, the Class 1 CRISPR-Cas system has lately been the subject of several studies. This review highlights the specificity of CRISPR-Cas system III-A in Mycobacterium tuberculosis, the tuberculosis-causing pathogen, for over twenty years. We discuss the difference between the several subtypes of Type III and their defence mechanisms. The anti-CRISPRs (Acrs) recently described, the critical role of Reverse transcriptase (RT) and housekeeping nuclease for type III CRISPR-Cas systems, and the use of this cutting-edge technology, its impact on the search for novel anti-tuberculosis drugs.}, }
@article {pmid37119122, year = {2023}, author = {Fatehi, S and Marks, RM and Rok, MJ and Perillat, L and Ivakine, EA and Cohn, RD}, title = {Advances in CRISPR/Cas9 Genome Editing for the Treatment of Muscular Dystrophies.}, journal = {Human gene therapy}, volume = {34}, number = {9-10}, pages = {388-403}, doi = {10.1089/hum.2023.059}, pmid = {37119122}, issn = {1557-7422}, support = {//CIHR/Canada ; }, mesh = {Animals ; *Gene Editing/methods ; CRISPR-Cas Systems ; *Muscular Dystrophy, Duchenne/genetics ; Genetic Therapy/methods ; Dystrophin/genetics ; }, abstract = {Muscular dystrophies (MDs) comprise a diverse group of inherited disorders characterized by progressive muscle loss and weakness. Given the genetic etiology underlying MDs, researchers have explored the potential of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) genome editing as a therapeutic intervention, resulting in significant advances. Here, we review recent progress on the use of CRISPR/Cas9 genome editing as a potential therapy for MDs. Significant strides have been made in this realm, made possible through innovative techniques such as precision genetic editing by modified forms of CRISPR/Cas9. These approaches have shown varying degrees of success in animal models of MD, including Duchenne MD, congenital muscular dystrophy type 1A, and myotonic dystrophy type 1. Even so, there are several challenges facing the development of CRISPR/Cas9-based MD therapies, including the targeting of satellite cells, improved editing efficiency in skeletal and cardiac muscle tissue, delivery vehicle enhancements, and the host immunogenic response. Although more work is needed to advance CRISPR/Cas9 genome editing past the preclinical stages, its therapeutic potential for MD is extremely promising and justifies concentrated efforts to move into clinical trials.}, }
@article {pmid37060194, year = {2023}, author = {Chemello, F and Olson, EN and Bassel-Duby, R}, title = {CRISPR-Editing Therapy for Duchenne Muscular Dystrophy.}, journal = {Human gene therapy}, volume = {34}, number = {9-10}, pages = {379-387}, doi = {10.1089/hum.2023.053}, pmid = {37060194}, issn = {1557-7422}, mesh = {Mice ; Humans ; Animals ; *Muscular Dystrophy, Duchenne/genetics ; CRISPR-Cas Systems ; Genetic Therapy/methods ; Exons ; Dystrophin/genetics ; Gene Editing/methods ; Disease Models, Animal ; }, abstract = {Duchenne muscular dystrophy (DMD) is a debilitating genetic disorder that results in progressive muscle degeneration and premature death. DMD is caused by mutations in the gene encoding dystrophin protein, a membrane-associated protein required for maintenance of muscle structure and function. Although the genetic mutations causing the disease are well known, no curative therapies have been developed to date. The advent of genome-editing technologies provides new opportunities to correct the underlying mutations responsible for DMD. These mutations have been successfully corrected in human cells, mice, and large animal models through different strategies based on CRISPR-Cas9 gene editing. Ideally, CRISPR-editing could offer a one-time treatment for DMD by correcting the genetic mutations and enabling normal expression of the repaired gene. However, numerous challenges remain to be addressed, including optimization of gene editing, delivery of gene-editing components to all the muscles of the body, and the suppression of possible immune responses to the CRISPR-editing therapy. This review provides an overview of the recent advances toward CRISPR-editing therapy for DMD and discusses the opportunities and the remaining challenges in the path to clinical translation.}, }
@article {pmid36355853, year = {2023}, author = {Iyer, DN and Schimmer, AD and Chang, H}, title = {Applying CRISPR-Cas9 screens to dissect hematological malignancies.}, journal = {Blood advances}, volume = {7}, number = {10}, pages = {2252-2270}, doi = {10.1182/bloodadvances.2022008966}, pmid = {36355853}, issn = {2473-9537}, mesh = {Humans ; CRISPR-Cas Systems ; Gene Editing ; *Neoplasms/therapy ; *Hematologic Neoplasms/genetics ; Regulatory Sequences, Nucleic Acid ; }, abstract = {Bit by bit, over the last few decades, functional genomic tools have been piecing together the molecular puzzle driving tumorigenesis in human patients. Nevertheless, our understanding of the role of several genes and regulatory elements that drive critical cancer-associated physiological processes from disease development to progression to spread is very limited, which significantly affects our ability of applying these insights in the context of improved disease management. The recent advent of clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9)-based technology and its application in cancer genomics has, however, allowed the generation of a wealth of knowledge that has helped decipher several critical questions associated with translational cancer research. Precisely, the high-throughput capability coupled with a high level of technological plasticity associated with the CRISPR-Cas9 screens have expanded our horizons from a mere struggle to appreciate cancer as a genetic disease to observing the integrated genomic/epigenomic network of numerous malignancies and correlating it with our present knowledge of drugging strategies to develop innovative approaches for next-generation precision cancer medicine. Specifically, within blood cancers, current CRISPR screens have specifically focused on improving our understanding of drug resistance mechanisms, disease biology, the development of novel therapeutic approaches, and identifying the molecular mechanisms of current therapies, with an underlying aim of improving disease outcomes. Here, we review the development of the CRISPR-Cas9 genome-editing strategy, explicitly focusing on the recent advances in the CRISPR-Cas9-based screening approaches, its current capabilities, limitations, and future applications in the context of hematological malignancies.}, }
@article {pmid37216595, year = {2023}, author = {Zhou, F and Yu, X and Gan, R and Ren, K and Chen, C and Ren, C and Cui, M and Liu, Y and Gao, Y and Wang, S and Yin, M and Huang, T and Huang, Z and Zhang, F}, title = {CRISPRimmunity: an interactive web server for CRISPR-associated Important Molecular events and Modulators Used in geNome edIting Tool identifYing.}, journal = {Nucleic acids research}, volume = {}, number = {}, pages = {}, doi = {10.1093/nar/gkad425}, pmid = {37216595}, issn = {1362-4962}, abstract = {The CRISPR-Cas system is a highly adaptive and RNA-guided immune system found in bacteria and archaea, which has applications as a genome editing tool and is a valuable system for studying the co-evolutionary dynamics of bacteriophage interactions. Here introduces CRISPRimmunity, a new web server designed for Acr prediction, identification of novel class 2 CRISPR-Cas loci, and dissection of key CRISPR-associated molecular events. CRISPRimmunity is built on a suite of CRISPR-oriented databases providing a comprehensive co-evolutionary perspective of the CRISPR-Cas and anti-CRISPR systems. The platform achieved a high prediction accuracy of 0.997 for Acr prediction when tested on a dataset of 99 experimentally validated Acrs and 676 non-Acrs, outperforming other existing prediction tools. Some of the newly identified class 2 CRISPR-Cas loci using CRISPRimmunity have been experimentally validated for cleavage activity in vitro. CRISPRimmunity offers the catalogues of pre-identified CRISPR systems to browse and query, the collected resources or databases to download, a well-designed graphical interface, a detailed tutorial, multi-faceted information, and exportable results in machine-readable formats, making it easy to use and facilitating future experimental design and further data mining. The platform is available at http://www.microbiome-bigdata.com/CRISPRimmunity. Moreover, the source code for batch analysis are published on Github (https://github.com/HIT-ImmunologyLab/CRISPRimmunity).}, }
@article {pmid37216515, year = {2023}, author = {Han, W and Li, Z and Guo, Y and He, K and Li, W and Xu, C and Ge, L and He, M and Yin, X and Zhou, J and Li, C and Yao, D and Bao, J and Liang, H}, title = {Efficient precise integration of large DNA sequences with 3'-overhang dsDNA donors using CRISPR/Cas9.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {120}, number = {22}, pages = {e2221127120}, doi = {10.1073/pnas.2221127120}, pmid = {37216515}, issn = {1091-6490}, mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Base Sequence ; *Gene Editing/methods ; DNA/genetics ; Homologous Recombination ; Mammals/genetics ; }, abstract = {CRISPR/Cas9 genome-editing tools have tremendously boosted our capability of manipulating the eukaryotic genomes in biomedical research and innovative biotechnologies. However, the current approaches that allow precise integration of gene-sized large DNA fragments generally suffer from low efficiency and high cost. Herein, we developed a versatile and efficient approach, termed LOCK (Long dsDNA with 3'-Overhangs mediated CRISPR Knock-in), by utilizing specially designed 3'-overhang double-stranded DNA (odsDNA) donors harboring 50-nt homology arm. The length of the 3'-overhangs of odsDNA is specified by the five consecutive phosphorothioate modifications. Compared with existing methods, LOCK allows highly efficient targeted insertion of kilobase-sized DNA fragments into the mammalian genomes with low cost and low off-target effects, yielding >fivefold higher knock-in frequencies than conventional homologous recombination-based approaches. This newly designed LOCK approach based on homology-directed repair is a powerful tool suitable for gene-sized fragment integration that is urgently needed for genetic engineering, gene therapies, and synthetic biology.}, }
@article {pmid37215369, year = {2023}, author = {Srivastava, P and Prasad, D}, title = {Isothermal nucleic acid amplification and its uses in modern diagnostic technologies.}, journal = {3 Biotech}, volume = {13}, number = {6}, pages = {200}, pmid = {37215369}, issn = {2190-572X}, abstract = {Nucleic acids are prominent biomarkers for diagnosing infectious pathogens using nucleic acid amplification techniques (NAATs). PCR, a gold standard technique for amplifying nucleic acids, is widely used in scientific research and diagnosis. Efficient pathogen detection is a key to adequate food safety and hygiene. However, using bulky thermal cyclers and costly laboratory setup limits its uses in developing countries, including India. The isothermal amplification methods are exploited to develop miniaturized sensors against viruses, bacteria, fungi and other pathogenic organisms and have been applied for in situ diagnosis. Isothermal amplification techniques have been found suitable for POC techniques and follow WHO's ASSURED criteria. LAMP, NASBA, SDA, RCA and RPA are some of the isothermal amplification techniques which are preferable for POC diagnostics. Furthermore, methods such as WGA, CPA, HDA, EXPAR, SMART, SPIA and DAMP were introduced for even more accuracy and robustness. Using recombinant polymerases and other nucleic acid-modifying enzymes has dramatically broadened the detection range of target pathogens under the scanner. The coupling of isothermal amplification methods with advanced technologies such as CRISPR/Cas systems, fluorescence-based chemistries, microfluidics and paper-based sensors has significantly influenced the biosensing and diagnosis field. This review comprehensively analyzed isothermal nucleic acid amplification methods, emphasizing their advantages, disadvantages and limitations.}, }
@article {pmid37215153, year = {2023}, author = {Fichter, KM and Setayesh, T and Malik, P}, title = {Strategies for precise gene edits in mammalian cells.}, journal = {Molecular therapy. Nucleic acids}, volume = {32}, number = {}, pages = {536-552}, pmid = {37215153}, issn = {2162-2531}, abstract = {CRISPR-Cas technologies have the potential to revolutionize genetic medicine. However, work is still needed to make this technology clinically efficient for gene correction. A barrier to making precise genetic edits in the human genome is controlling how CRISPR-Cas-induced DNA breaks are repaired by the cell. Since error-prone non-homologous end-joining is often the preferred cellular repair pathway, CRISPR-Cas-induced breaks often result in gene disruption. Homology-directed repair (HDR) makes precise genetic changes and is the clinically desired pathway, but this repair pathway requires a homology donor template and cycling cells. Newer editing strategies, such as base and prime editing, can affect precise repair for relatively small edits without requiring HDR and circumvent cell cycle dependence. However, these technologies have limitations in the extent of genetic editing and require the delivery of bulky cargo. Here, we discuss the pros and cons of precise gene correction using CRISPR-Cas-induced HDR, as well as base and prime editing for repairing small mutations. Finally, we consider emerging new technologies, such as recombination and transposases, which can circumvent both cell cycle and cellular DNA repair dependence for editing the genome.}, }
@article {pmid37212595, year = {2023}, author = {Migliara, A and Cappelluti, MA and Giannese, F and Valsoni, S and Coglot, A and Merelli, I and Cittaro, D and Lombardo, A}, title = {In Vitro Selection of Engineered Transcriptional Repressors for Targeted Epigenetic Silencing.}, journal = {Journal of visualized experiments : JoVE}, volume = {}, number = {195}, pages = {}, doi = {10.3791/64403}, pmid = {37212595}, issn = {1940-087X}, mesh = {Humans ; *Gene Editing/methods ; *Epigenesis, Genetic ; Transcription Factors/metabolism ; Gene Silencing ; DNA/genetics ; CRISPR-Cas Systems ; }, abstract = {Gene inactivation is instrumental to study gene function and represents a promising strategy for the treatment of a broad range of diseases. Among traditional technologies, RNA interference suffers from partial target abrogation and the requirement for life-long treatments. In contrast, artificial nucleases can impose stable gene inactivation through induction of a DNA double strand break (DSB), but recent studies are questioning the safety of this approach. Targeted epigenetic editing via engineered transcriptional repressors (ETRs) may represent a solution, as a single administration of specific ETR combinations can lead to durable silencing without inducing DNA breaks. ETRs are proteins containing a programmable DNA-binding domain (DBD) and effectors from naturally occurring transcriptional repressors. Specifically, a combination of three ETRs equipped with the KRAB domain of human ZNF10, the catalytic domain of human DNMT3A and human DNMT3L, was shown to induce heritable repressive epigenetic states on the ETR-target gene. The hit-and-run nature of this platform, the lack of impact on the DNA sequence of the target, and the possibility to revert to the repressive state by DNA demethylation on demand, make epigenetic silencing a game-changing tool. A critical step is the identification of the proper ETRs' position on the target gene to maximize on-target and minimize off-target silencing. Performing this step in the final ex vivo or in vivo preclinical setting can be cumbersome. Taking the CRISPR/catalytically dead Cas9 system as a paradigmatic DBD for ETRs, this paper describes a protocol consisting of the in vitro screen of guide RNAs (gRNAs) coupled to the triple-ETR combination for efficient on-target silencing, followed by evaluation of the genome-wide specificity profile of top hits. This allows for reduction of the initial repertoire of candidate gRNAs to a short list of promising ones, whose complexity is suitable for their final evaluation in the therapeutically relevant setting of interest.}, }
@article {pmid37167072, year = {2023}, author = {Mao, G and Luo, X and Ye, S and Wang, X and He, J and Kong, J and Dai, J and Yin, W and Ma, Y}, title = {Fluorescence and Colorimetric Analysis of African Swine Fever Virus Based on the RPA-Assisted CRISPR/Cas12a Strategy.}, journal = {Analytical chemistry}, volume = {95}, number = {20}, pages = {8063-8069}, doi = {10.1021/acs.analchem.3c01033}, pmid = {37167072}, issn = {1520-6882}, mesh = {Animals ; Swine ; *Recombinases ; *African Swine Fever Virus/genetics ; CRISPR-Cas Systems/genetics ; Colorimetry ; Nucleotidyltransferases ; Alkaline Phosphatase ; Coloring Agents ; Nucleic Acid Amplification Techniques ; }, abstract = {It is well-established that different detection modes are necessary for corresponding applications, which can effectively reduce matrix interference and improve the detection accuracy. Here, we reported a magnetic separation method based on recombinase polymerase amplification (RPA)-assisted clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a for dual-mode analysis of African swine fever virus (ASFV) genes, including colorimetry and fluorescence. The ASFV gene was selected as the initial RPA template to generate the amplicon. The RPA amplicon was then recognized by CRISPR-associated RNA (crRNA), activating the trans-cleavage activity of Cas12a and leading to the nonspecific cleavage of ssDNA as well as a significant release of alkaline phosphatase (ALP) in the ALP-ssDNA modified magnetic bead. The released ALP can catalyze para-nitrophenyl phosphate to generate para-nitrophenol, resulting in substantial changes in absorbance and fluorescence, both of which can be used for detection with the naked eye. This strategy allows the sensitive detection of ASFV DNA, with a 20 copies/mL detection limit; no cross-reactivity with other viruses was observed. A good linear relationship was obtained in serum. In addition, this sensor displayed 100% specificity and sensitivity for clinical sample analysis. This method integrates the high sensitivity of fluorescence with easy readout of colorimetry and enables a simple, low-cost, and highly sensitive dual-mode detection of viral nucleic acid, thereby providing a broad prospect for the practical application in the diagnosis of virus infection.}, }
@article {pmid37156096, year = {2023}, author = {Xiang, X and Song, M and Xu, X and Lu, J and Chen, Y and Chen, S and He, Y and Shang, Y}, title = {Microfluidic Biosensor Integrated with Signal Transduction and Enhancement Mechanism for Ultrasensitive Noncompetitive Assay of Multiple Mycotoxins.}, journal = {Analytical chemistry}, volume = {95}, number = {20}, pages = {7993-8001}, doi = {10.1021/acs.analchem.3c00813}, pmid = {37156096}, issn = {1520-6882}, mesh = {Microfluidics ; Biological Assay ; Chromatography, High Pressure Liquid ; *Mycotoxins ; Signal Transduction ; *Biosensing Techniques ; CRISPR-Cas Systems ; }, abstract = {To achieve high-throughput ultrasensitive detection of mycotoxins in food, a functional DNA-guided transition-state CRISPR/Cas12a microfluidic biosensor (named FTMB) was successfully constructed. The signal transduction CRISPR/Cas12a strategy in FTMB has utilized DNA sequences with a specific recognition function and activators to form trigger switches. Meanwhile, the transition-state CRISPR/Cas12a system was constructed by adjusting the composition ratio of crRNA and activator to achieve a high response for low concentrations of target mycotoxins. On the other hand, the signal enhancement of FTMB has efficiently integrated the signal output of quantum dots (QDs) with the fluorescence enhancement effect of photonic crystals (PCs). The construction of universal QDs for the CRISPR/Cas12a system and PC films matching the photonic bandgap produced a significant signal enhancement by a factor of 45.6. Overall, FTMB exhibited a wide analytic range (10[-5]-10[1] ng·mL[-1]), low detection of limit (fg·mL[-1]), short detection period (∼40 min), high specificity, good precision (coefficients of variation <5%), and satisfactory practical sample analysis capacity (the consistency with HPLC at 88.76%-109.99%). It would provide a new and reliable solution for the rapid detection of multiple small molecules in the fields of clinical diagnosis and food safety.}, }
@article {pmid37146364, year = {2023}, author = {Gu, J and Sun, J and Tian, K and Bian, J and Peng, J and Xu, S and Zhao, L}, title = {Reversal of hepatic fibrosis by the co-delivery of drug and ribonucleoprotein-based genome editor.}, journal = {Biomaterials}, volume = {298}, number = {}, pages = {122133}, doi = {10.1016/j.biomaterials.2023.122133}, pmid = {37146364}, issn = {1878-5905}, mesh = {Mice ; Animals ; *CRISPR-Cas Systems/genetics ; *Ribonucleoproteins/genetics/metabolism ; Pharmaceutical Preparations ; Liver Cirrhosis/drug therapy ; Endonucleases/genetics/metabolism ; RNA, Messenger ; }, abstract = {Liver fibrosis is a chronic disease without effective treatment in the clinic. Gene editing systems such as the well-known CRISPR/Cas9 have shown great potential in the biomedical field. However, the delivery of the ribonucleoprotein is challenging due to the unstable RNA probe and the requirement for the entrance to the nucleus. Recently, a structure-guided endonuclease (SGN) has been reported as an effective gene-editing system composed of a nuclease and stable DNA probes, which can regulate the protein expression by targeting specific mRNA outside the nucleus. Here, we conjugated the SGN to a nanomicelle as the delivery system. In the resulting material, the chance of the collision between the endonuclease and the probe was raised due to the confinement of the two components within the 40-nm nanomicelle, thus the mRNA can be cleaved immediately after being captured by the probe, resulting in a space-induced nucleotide identification-cleavage acceleration effect. The delivery system was used to treat liver fibrosis via the co-delivery of SGN and a drug rosiglitazone to the hepatic stellate cells, which separately downregulated the expression of tissue inhibitor of metalloprotease-1 and inactivated the hepatic stellate cells. The system successfully reversed the liver fibrosis in mice through the bidirectional regulatory that simultaneously promoted the degradation and inhibited the production of the collagen, demonstrating the great potency of the SGN system as gene medicine.}, }
@article {pmid37214290, year = {2023}, author = {Fang, L and Yang, L and Han, M and Xu, H and Ding, W and Dong, X}, title = {CRISPR-cas technology: A key approach for SARS-CoV-2 detection.}, journal = {Frontiers in bioengineering and biotechnology}, volume = {11}, number = {}, pages = {1158672}, pmid = {37214290}, issn = {2296-4185}, abstract = {The CRISPR (Clustered Regularly Spaced Short Palindromic Repeats) system was first discovered in prokaryotes as a unique immune mechanism to clear foreign nucleic acids. It has been rapidly and extensively used in basic and applied research owing to its strong ability of gene editing, regulation and detection in eukaryotes. Hererin in this article, we reviewed the biology, mechanisms and relevance of CRISPR-Cas technology and its applications in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) diagnosis. CRISPR-Cas nucleic acid detection tools include CRISPR-Cas9, CRISPR-Cas12, CRISPR-Cas13, CRISPR-Cas14, CRISPR nucleic acid amplification detection technology, and CRISPR colorimetric readout detection system. The above CRISPR technologies have been applied to the nucleic acid detection, including SARS-CoV-2 detection. Common nucleic acid detection based on CRISPR derivation technology include SHERLOCK, DETECTR, and STOPCovid. CRISPR-Cas biosensing technology has been widely applied to point-of-care testing (POCT) by targeting recognition of both DNA molecules and RNA Molecules.}, }
@article {pmid37212293, year = {2023}, author = {Yang, X and Zeng, X and Chen, X and Huang, J and Wei, X and Ying, X and Tan, Q and Wang, Y and Li, S}, title = {Development of a CRISPR/Cas12a-recombinase polymerase amplification assay for visual and highly specific identification of the Congo Basin and West African strains of mpox virus.}, journal = {Journal of medical virology}, volume = {95}, number = {5}, pages = {e28757}, doi = {10.1002/jmv.28757}, pmid = {37212293}, issn = {1096-9071}, mesh = {Humans ; *Recombinases/genetics ; *CRISPR-Cas Systems ; Monkeypox virus ; Congo ; Nucleotidyltransferases ; Nucleic Acid Amplification Techniques ; }, abstract = {Human mpox is a zoonotic disease, similar to smallpox, caused by the mpox virus, which is further subdivided into Congo Basin and West African clades with different pathogenicity. In this study, a novel diagnostic protocol utilizing clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 12a nuclease (CRISPR/Cas12a)-mediated recombinase polymerase amplification (RPA) was developed to identify mpox in the Congo Basin and West Africa (CRISPR-RPA). Specific RPA primers targeting D14L and ATI were designed. CRISPR-RPA assay was performed using various target templates. In the designed CRISPR-RPA reaction system, the exponentially amplified RPA amplification products with a protospacer adjacent motif (PAM) site can locate the Cas12a/crRNA complex to its target regions, which successfully activates the CRISPR/Cas12a effector and achieves ultrafast trans-cleavage of a single-stranded DNA probe. The limit of detection for the CRISPR-RPA assay was 10 copies per reaction for D14L- and ATI-plasmids. No cross-reactivity was observed with non-mpox strains, confirming the high specificity of the CRISPR-RPA assay for distinguishing between the Congo Basin and West African mpox. The CRISPR-RPA assay can be completed within 45 min using real-time fluorescence readout. Moreover, the cleavage results were visualized under UV light or an imaging system, eliminating the need for a specialized apparatus. In summary, the developed CRISPR/RPA assay is a visual, rapid, sensitive, and highly specific detection technique that can be used as an attractive potential identification tool for Congo Basin and West African mpox in resource-limited laboratories.}, }
@article {pmid37210864, year = {2023}, author = {Chirco, KR and Martinez, C and Lamba, DA}, title = {Advancements in pre-clinical development of gene editing-based therapies to treat inherited retinal diseases.}, journal = {Vision research}, volume = {209}, number = {}, pages = {108257}, doi = {10.1016/j.visres.2023.108257}, pmid = {37210864}, issn = {1878-5646}, abstract = {One of the major goals in the inherited retinal disease (IRD) field is to develop an effective therapy that can be applied to as many patients as possible. Significant progress has already been made toward this end, with gene editing at the forefront. The advancement of gene editing-based tools has been a recent focus of many research groups around the world. Here, we provide an update on the status of CRISPR/Cas-derived gene editors, promising options for delivery of these editing systems to the retina, and animal models that aid in pre-clinical testing of new IRD therapeutics.}, }
@article {pmid37094742, year = {2023}, author = {Keppeke, GD and Diogenes, L and Gomes, K and Andrade, LEC}, title = {"Untargeting" autoantibodies using genome editing, a proof-of-concept study.}, journal = {Clinical immunology (Orlando, Fla.)}, volume = {251}, number = {}, pages = {109343}, doi = {10.1016/j.clim.2023.109343}, pmid = {37094742}, issn = {1521-7035}, mesh = {Humans ; *Gene Editing ; *CRISPR-Cas Systems/genetics ; Autoantibodies/genetics ; HEK293 Cells ; Genome ; }, abstract = {Autoantibodies (AAbs) are useful biomarkers and many have direct pathogenic role. Current standard therapies for elimination of specific B/plasma-cell clones are not fully efficient. We apply CRISPR/Cas9 genome-editing to knockout V(D)J rearrangements that produce pathogenic AAbs in vitro. HEK293T cell-lines were established stably expressing a humanized anti-dsDNA Ab (clone 3H9) and a human-derived anti-nAChR-α1 Ab (clone B12L). For each clone, five CRISPR/Cas9 heavy-chain's CDR2/3-targeting guided-RNAs (T-gRNAs) were designed. Non-Target-gRNA (NT-gRNA) was control. After editing, levels of secreted Abs were evaluated, as well as 3H9 anti-dsDNA and B12L anti-AChR reactivities. T-gRNAs editing decreased expression of heavy-chain genes to ∼50-60%, compared to >90% in NT-gRNA, although secreted Abs levels and reactivity to their respective antigens in T-gRNAs decreased ∼90% and ∼ 95% compared with NT-gRNA for 3H9 and B12L, respectively. Sequencing indicated indels at Cas9 cut-site, which could lead to codon jam, and consequently, knockout. Additionally, remaining secreted 3H9-Abs presented variable dsDNA reactivity among the five T-gRNA, suggesting the exact Cas9 cut-site and indels further interfere with antibody-antigen interaction. CRISPR/Cas9 genome-editing was very effective to knockout the Heavy-Chain-IgG genes, considerably affecting AAbs secretion and binding capacity, fostering application of this concept to in vivo models as a potential novel therapeutic approach for AAb-mediated diseases.}, }
@article {pmid37070192, year = {2023}, author = {Carusillo, A and Haider, S and Schäfer, R and Rhiel, M and Türk, D and Chmielewski, KO and Klermund, J and Mosti, L and Andrieux, G and Schäfer, R and Cornu, TI and Cathomen, T and Mussolino, C}, title = {A novel Cas9 fusion protein promotes targeted genome editing with reduced mutational burden in primary human cells.}, journal = {Nucleic acids research}, volume = {51}, number = {9}, pages = {4660-4673}, pmid = {37070192}, issn = {1362-4962}, mesh = {Animals ; Humans ; *Gene Editing ; *CRISPR-Associated Protein 9/genetics ; CRISPR-Cas Systems/genetics ; DNA Repair/genetics ; DNA Breaks, Double-Stranded ; Recombinational DNA Repair ; DNA End-Joining Repair/genetics ; Mammals/genetics ; }, abstract = {Precise genome editing requires the resolution of nuclease-induced DNA double strand breaks (DSBs) via the homology-directed repair (HDR) pathway. In mammals, this is typically outcompeted by non-homologous end-joining (NHEJ) that can generate potentially genotoxic insertion/deletion mutations at DSB sites. Because of higher efficacy, clinical genome editing has been restricted to imperfect but efficient NHEJ-based approaches. Hence, strategies that promote DSB resolution via HDR are essential to facilitate clinical transition of HDR-based editing strategies and increase safety. Here we describe a novel platform that consists of a Cas9 fused to DNA repair factors to synergistically inhibit NHEJ and favor HDR for precise repairing of Cas-induced DSBs. Compared to canonical CRISPR/Cas9, the increase in error-free editing ranges from 1.5-fold to 7-fold in multiple cell lines and in primary human cells. This novel CRISPR/Cas9 platform accepts clinically relevant repair templates, such as oligodeoxynucleotides (ODNs) and adeno-associated virus (AAV)-based vectors, and has a lower propensity to induce chromosomal translocations as compared to benchmark CRISPR/Cas9. The observed reduced mutational burden, resulting from diminished indel formation at on- and off-target sites, provides a remarkable gain in safety and advocates this novel CRISPR system as an attractive tool for therapeutic applications depending on precision genome editing.}, }
@article {pmid36999618, year = {2023}, author = {Byun, G and Yang, J and Seo, SW}, title = {CRISPRi-mediated tunable control of gene expression level with engineered single-guide RNA in Escherichia coli.}, journal = {Nucleic acids research}, volume = {51}, number = {9}, pages = {4650-4659}, pmid = {36999618}, issn = {1362-4962}, mesh = {*Escherichia coli/genetics/metabolism ; *RNA, Guide, CRISPR-Cas Systems ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Expression Regulation, Bacterial ; Gene Expression ; }, abstract = {Precise control of gene expression is essential for flux redistribution in metabolic pathways. Although the CRISPR interference (CRISPRi) system can effectively repress gene expression at the transcriptional level, it has still been difficult to precisely control the level without loss of specificity or an increase in cell toxicity. In this study, we developed a tunable CRISPRi system that performs transcriptional regulation at various levels. We constructed a single-guide RNA (sgRNA) library targeting repeat, tetraloop, and anti-repeat regions to modulate the binding affinity against dCas9. Each screened sgRNA could regulate the gene expression at a certain level between fully-repressing and non-repressing states (>45-fold). These sgRNAs also enabled modular regulation with various target DNA sequences. We applied this system to redistribute the metabolic flux to produce violacein derivatives in a predictable ratio and optimize lycopene production. This system would help accelerate the flux optimization processes in metabolic engineering and synthetic biology.}, }
@article {pmid36938898, year = {2023}, author = {Pan, C and Li, R and Shui, L and Xiao, Z and Wang, Y and Zhu, J and Wu, C and Zhang, L and Jia, J and Zheng, M}, title = {A new method to synthesize multiple gRNA libraries and functional mapping of mammalian H3K4me3 regions.}, journal = {Nucleic acids research}, volume = {51}, number = {9}, pages = {e50}, pmid = {36938898}, issn = {1362-4962}, mesh = {Animals ; *Genome ; *Histones/genetics ; Genomics ; DNA/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; CRISPR-Cas Systems ; Mammals/genetics ; }, abstract = {Genetic screening based on the clustered regularly interspaced palindromic repeat (CRISPR) system has been indicated to be a powerful tool for identifying regulatory genes or cis-elements. However, when applying CRISPR screens to pinpoint functional elements at particular loci, a large number of guide RNA (gRNA) spacers may be required to achieve saturated coverage. Here, we present a controlled template-dependent elongation (CTDE) method relying on reversible terminators to synthesize gRNA libraries with genomic regions of interest. By applying this approach to H3K4me3 chromatin immunoprecipitation (ChIP)-derived DNA of mammalian cells, mega-sized gRNA libraries were synthesized in a tissue-specific manner, with which we conducted screening experiments to annotate essential sites for cell proliferation. Additionally, we confirmed that an essential site within the intron of LINC00339 regulates its own mRNA and that LINC00339 is a novel regulator of the cell cycle that maintains HepG2 proliferation. The CTDE method has the potential to be automated with high efficiency at low cost, and will be widely used to identify functional elements in mammalian genomes.}, }
@article {pmid34923124, year = {2022}, author = {Rahman, K and Jamal, M and Chen, X and Zhou, W and Yang, B and Zou, Y and Xu, W and Lei, Y and Wu, C and Cao, X and Tyagi, R and Naeem, MA and Lin, D and Habib, Z and Peng, N and Fu, ZF and Cao, G}, title = {Reprogramming Mycobacterium tuberculosis CRISPR System for Gene Editing and Genome-wide RNA Interference Screening.}, journal = {Genomics, proteomics &amp; bioinformatics}, volume = {20}, number = {6}, pages = {1180-1196}, doi = {10.1016/j.gpb.2021.01.008}, pmid = {34923124}, issn = {2210-3244}, mesh = {Humans ; *Mycobacterium tuberculosis/genetics/metabolism ; Gene Editing ; RNA Interference ; *Tuberculosis/prevention &amp; control/genetics/microbiology ; Antitubercular Agents/metabolism ; CRISPR-Cas Systems ; }, abstract = {Mycobacterium tuberculosis is the causative agent of tuberculosis (TB), which is still the leading cause of mortality from a single infectious disease worldwide. The development of novel anti-TB drugs and vaccines is severely hampered by the complicated and time-consuming genetic manipulation techniques for M. tuberculosis. Here, we harnessed an endogenous type III-A CRISPR/Cas10 system of M. tuberculosis for efficient gene editing and RNA interference (RNAi). This simple and easy method only needs to transform a single mini-CRISPR array plasmid, thus avoiding the introduction of exogenous protein and minimizing proteotoxicity. We demonstrated that M. tuberculosis genes can be efficiently and specifically knocked in/out by this system as confirmed by DNA high-throughput sequencing. This system was further applied to single- and multiple-gene RNAi. Moreover, we successfully performed genome-wide RNAi screening to identify M. tuberculosis genes regulating in vitro and intracellular growth. This system can be extensively used for exploring the functional genomics of M. tuberculosis and facilitate the development of novel anti-TB drugs and vaccines.}, }
@article {pmid37210856, year = {2023}, author = {Wang, X and Jing, S and Wang, W and Wang, J}, title = {Direct and noninvasive fluorescence analysis of an RNA-protein interaction based on a CRISPR/Cas12a-powered assay.}, journal = {Spectrochimica acta. Part A, Molecular and biomolecular spectroscopy}, volume = {299}, number = {}, pages = {122884}, doi = {10.1016/j.saa.2023.122884}, pmid = {37210856}, issn = {1873-3557}, abstract = {RNA-protein interactions (RPIs) play critical roles in gene transcription and protein expression, but current analytical methods for RPIs are mainly performed in an invasive manner, involving special RNA/protein labeling, hampering access to intact and precise information on RPIs. In this work, we present the first CRISPR/Cas12a-based fluorescence assay for the direct analysis of RPIs without RNA/protein labeling steps. Select vascular endothelial growth factor 165 (VEGF165)/its RNA aptamer interaction as a model, the RNA sequence simultaneously serves as both the aptamer of VEGF165 and crRNA of CRISPR/Cas12a system, and the presence of VEGF165 facilitates VEGF165/its RNA aptamer interaction, thus prohibiting the formation of Cas12a-crRNA-DNA ternary complex along with low fluorescence signal. The assay showed a detection limit of 0.23 pg mL[-1], and good performance in serum-spiked samples with an RSD of 0.4 %-13.1 %. This simple and selective strategy opens the door for establishing CRISPR/Cas-based biosensors for gaining intact information on RPIs, and shows widespread potential for other RPIs analysis.}, }
@article {pmid37210555, year = {2023}, author = {Wu, J and Tao, Y and Deng, D and Meng, Z and Zhao, Y}, title = {The applications of CRISPR/Cas-mediated genome editing in genetic hearing loss.}, journal = {Cell &amp; bioscience}, volume = {13}, number = {1}, pages = {93}, pmid = {37210555}, issn = {2045-3701}, abstract = {Hearing loss (HL) can be caused by a number of different genetic factors. Non-syndromic HL refers that HL occurs as an isolated symptom in an individual, whereas syndromic HL refers that HL is associated with other symptoms or abnormalities. To date, more than 140 genes have been identified as being associated with non-syndromic HL, and approximately 400 genetic syndromes can include HL as one of the clinical symptoms. However, no gene therapeutic approaches are currently available to restore or improve hearing. Therefore, there is an urgent necessity to elucidate the possible pathogenesis of specific mutations in HL-associated genes and to investigate the promising therapeutic strategies for genetic HL. The development of the CRISPR/Cas system has revolutionized the field of genome engineering, which has become an efficacious and cost-effective tool to foster genetic HL research. Moreover, several in vivo studies have demonstrated the therapeutic efficacy of the CRISPR/Cas-mediated treatments for specific genetic HL. In this review, we briefly introduce the progress in CRISPR/Cas technique as well as the understanding of genetic HL, and then we detail the recent achievements of CRISPR/Cas technique in disease modeling and therapeutic strategies for genetic HL. Furthermore, we discuss the challenges for the application of CRISPR/Cas technique in future clinical treatments.}, }
@article {pmid37209468, year = {2023}, author = {Even-Zohar, N and Metin-Armagan, D and Ben-Shlomo, A and Sareen, D and Melmed, S}, title = {Generation of isogenic and homozygous MEN1 mutant cell lines from patient-derived iPSCs using CRISPR/Cas9.}, journal = {Stem cell research}, volume = {69}, number = {}, pages = {103124}, doi = {10.1016/j.scr.2023.103124}, pmid = {37209468}, issn = {1876-7753}, abstract = {MEN1, an autosomal dominant disorder caused by mutations in the tumor suppressor gene MEN1, manifests with co-occurrence of multiple endocrine/neuroendocrine neoplasms. An iPSC line derived from an index patient carrying the mutation c.1273C>T (p.Arg465*) was edited using a single multiplex CRISPR/Cas approach to create an isogenic control non-mutated line and a homozygous double mutant line. These cell lines will be useful for elucidating subcellular MEN1 pathophysiology and for screening to identify potential MEN1 therapeutic targets.}, }
@article {pmid37208532, year = {2023}, author = {Lee, K and Lee, C}, title = {Generation of CRISPR-Cas9-mediated knockin mutant models in mice and MEFs for studies of polymorphism in clock genes.}, journal = {Scientific reports}, volume = {13}, number = {1}, pages = {8109}, pmid = {37208532}, issn = {2045-2322}, support = {GM131283/GM/NIGMS NIH HHS/United States ; }, mesh = {Animals ; Mice ; *CRISPR-Cas Systems ; Fibroblasts/metabolism ; *Circadian Clocks/genetics ; Cell Culture Techniques ; Transcription Factors/metabolism ; Disease Models, Animal ; Circadian Rhythm/genetics ; }, abstract = {The creation of mutant mice has been invaluable for advancing biomedical science, but is too time- and resource-intensive for investigating the full range of mutations and polymorphisms. Cell culture models are therefore an invaluable complement to mouse models, especially for cell-autonomous pathways like the circadian clock. In this study, we quantitatively assessed the use of CRISPR to create cell models in mouse embryonic fibroblasts (MEFs) as compared to mouse models. We generated two point mutations in the clock genes Per1 and Per2 in mice and in MEFs using the same sgRNAs and repair templates for HDR and quantified the frequency of the mutations by digital PCR. The frequency was about an order of magnitude higher in mouse zygotes compared to that in MEFs. However, the mutation frequency in MEFs was still high enough for clonal isolation by simple screening of a few dozen individual cells. The Per mutant cells that we generated provide important new insights into the role of the PAS domain in regulating PER phosphorylation, a key aspect of the circadian clock mechanism. Quantification of the mutation frequency in bulk MEF populations provides a valuable basis for optimizing CRISPR protocols and time/resource planning for generating cell models for further studies.}, }
@article {pmid37207121, year = {2023}, author = {Wu, J and Xu, H and Hu, F and Jiang, Y and Fan, B and Khan, A and Sun, Y and Di, K and Gu, X and Shen, H and Li, Z}, title = {CRISPR-Cas and catalytic hairpin assembly technology for target-initiated amplification detection of pancreatic cancer specific tsRNAs.}, journal = {Frontiers in bioengineering and biotechnology}, volume = {11}, number = {}, pages = {1169424}, pmid = {37207121}, issn = {2296-4185}, abstract = {Transfer RNA-derived small RNAs (tsRNAs) tRF-LeuCAG-002 (ts3011a RNA) is a novel class of non-coding RNAs biomarker for pancreatic cancer (PC). Reverse transcription polymerase chain reaction (RT-qPCR) has been unfit for community hospitals that are short of specialized equipment or laboratory setups. It has not been reported whether isothermal technology can be used for detection, because the tsRNAs have rich modifications and secondary structures compared with other non-coding RNAs. Herein, we have employed a catalytic hairpin assembly (CHA) circuit and clustered regularly interspaced short palindromic repeats (CRISPR) to develop an isothermal and target-initiated amplification method for detecting ts3011a RNA. In the proposed assay, the presence of target tsRNA triggers the CHA circuit that transforms new DNA duplexes to activate collateral cleavage activity of CRISPR-associated proteins (CRISPR-Cas) 12a, achieving cascade signal amplification. This method showed a low detection limit of 88 aM at 37 °C within 2 h. Moreover, it was demonstrated for the first time that, this method is less likely to produce aerosol contamination than RT-qPCR by simulating aerosol leakage experiments. This method has good consistency with RT-qPCR in the detection of serum samples and showed great potential for PC-specific tsRNAs point-of-care testing (POCT).}, }
@article {pmid37204916, year = {2023}, author = {Shi, L and Su, J and Cho, MJ and Song, H and Dong, X and Liang, Y and Zhang, Z}, title = {Promoter editing for the genetic improvement of crops.}, journal = {Journal of experimental botany}, volume = {}, number = {}, pages = {}, doi = {10.1093/jxb/erad175}, pmid = {37204916}, issn = {1460-2431}, abstract = {The proper gene expression plays a fundamental role in the regulation of agronomically important traits in crop plants. The genetic manipulation of plant promoters through genome editing has emerged as an effective strategy to create favorable traits in crops by altering the expression pattern of the pertinent genes. Promoter editing can be applied in a directed manner, where nucleotide sequences associated with favorable traits are precisely generated. Alternatively, promoter editing can also be exploited as a random mutagenic approach to generate novel genetic variations within a designated promoter, from which elite alleles are selected based on their phenotypic effects. Pioneering studies have demonstrated the potential of promoter editing in engineering agronomically important traits as well as in mining novel promoter alleles valuable for plant breeding. In this review article, we update the progress in the application of promoter editing in crops for increased yield, enhanced tolerance to biotic and abiotic stresses, and improved quality. We also discuss several remaining technical bottlenecks and how this strategy may be better employed for the genetic improvement of crops in the future.}, }
@article {pmid37204621, year = {2023}, author = {Chaudhry, A and Hassan, AU and Khan, SH and Abbasi, A and Hina, A and Khan, MT and Abdelsalam, NR}, title = {The changing landscape of agriculture: role of precision breeding in developing smart crops.}, journal = {Functional &amp; integrative genomics}, volume = {23}, number = {2}, pages = {167}, pmid = {37204621}, issn = {1438-7948}, mesh = {Humans ; *CRISPR-Cas Systems ; Plants, Genetically Modified/genetics ; *Plant Breeding/methods ; Gene Editing/methods ; Crops, Agricultural/genetics ; Agriculture ; Genome, Plant ; }, abstract = {Food plants play a crucial role in human survival, providing them essential nutrients. However, traditional breeding methods have not been able to keep up with the demands of the growing population. The improvement of food plants aims to increase yield, quality, and resistance to biotic and abiotic stresses. With CRISPR/Cas9, researchers can identify and edit key genes conferring desirable qualities in agricultural plants, including increased yield, enhanced product quality attributes, and increased tolerance to biotic and abiotic challenges. These modifications have enabled the creation of "smart crops" that exhibit rapid climatic adaptation, resistance to extreme weather conditions and high yield and quality. The use of CRISPR/Cas9 combined with viral vectors or growth regulators has made it possible to produce more efficient modified plants with certain conventional breeding methods. However, ethical and regulatory aspects of this technology must be carefully considered. Proper regulation and application of genome editing technology can bring immense benefits to agriculture and food security. This article provides an overview of genetically modified genes and conventional as well as emerging tools, including CRISPR/Cas9, that have been utilized to enhance the quality of plants/fruits and their products. The review also discusses the challenges and prospects associated with these techniques.}, }
@article {pmid37204115, year = {2023}, author = {Zhang, J and Zhu, A and Mei, M and Qu, J and Huang, Y and Shi, Y and Xue, M and Zhang, J and Zhang, R and Zhou, B and Tan, X and Zhao, J and Wang, Y}, title = {Repurposing CRISPR/Cas to Discover SARS-CoV-2 Detecting and Neutralizing Aptamers.}, journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)}, volume = {}, number = {}, pages = {e2300656}, doi = {10.1002/advs.202300656}, pmid = {37204115}, issn = {2198-3844}, abstract = {RNA aptamers provide useful biological probes and therapeutic agents. New methodologies to screen RNA aptamers will be valuable by complementing the traditional Systematic Evolution of Ligands by Exponential Enrichment (SELEX). Meanwhile, repurposing clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated systems (Cas) has expanded their utility far beyond their native nuclease function. Here, CRISmers, a CRISPR/Cas-based novel screening system for RNA aptamers based on binding to a chosen protein of interest in a cellular context, is presented. Using CRISmers, aptamers are identified specifically targeting the receptor binding domain (RBD) of the spike glycoprotein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Two aptamer leads enable sensitive detection and potent neutralization of SARS-CoV-2 Delta and Omicron variants in vitro. Intranasal administration of one aptamer, further modified with 2'-fluoro pyrimidines (2'-F), 2'-O-methyl purines (2'-O), and conjugation with both cholesterol and polyethylene glycol of 40 kDa (PEG40K), achieves effective prophylactic and therapeutic antiviral activity against live Omicron BA.2 variants in vivo. The study concludes by demonstrating the robustness, consistency, and potential broad utility of CRISmers using two newly identified aptamers but switching CRISPR, selection marker, and host species.}, }
@article {pmid37202186, year = {2023}, author = {Ma, Y and Zou, L and Liang, Y and Liu, Q and Sun, Q and Pang, Y and Lin, H and Deng, X and Tang, S}, title = {[Rapid detection and genotyping of SARS-CoV-2 Omicron BA.4/5 variants using a RT-PCR and CRISPR-Cas12a-based assay].}, journal = {Nan fang yi ke da xue xue bao = Journal of Southern Medical University}, volume = {43}, number = {4}, pages = {516-526}, doi = {10.12122/j.issn.1673-4254.2023.04.03}, pmid = {37202186}, issn = {1673-4254}, mesh = {Humans ; *COVID-19 ; CRISPR-Cas Systems ; Genotype ; Reproducibility of Results ; Reverse Transcriptase Polymerase Chain Reaction ; SARS-CoV-2/genetics ; RNA ; COVID-19 Testing ; }, abstract = {OBJECTIVE: To establish a rapid detection and genotyping method for SARS-CoV-2 Omicron BA.4/5 variants using CRISPPR-Cas12a gene editing technology.<br><br>METHODS: We combined reverse transcription-polymerase chain reaction (RT-PCR) and CRISPR gene editing technology and designed a specific CRISPPR RNA (crRNA) with suboptimal protospacer adjacent motifs (PAM) for rapid detection and genotyping of SARS- CoV-2 Omicron BA.4/5 variants. The performance of this RT- PCR/ CRISPPR-Cas12a assay was evaluated using 43 clinical samples of patients infected by wild-type SARS-CoV-2 and the Alpha, Beta, Delta, Omicron BA. 1 and BA. 4/5 variants and 20 SARS- CoV- 2-negative clinical samples infected with 11 respiratory pathogens. With Sanger sequencing method as the gold standard, the specificity, sensitivity, concordance (Kappa) and area under the ROC curve (AUC) of RT-PCR/CRISPPR-Cas12a assay were calculated.<br><br>RESULTS: This assay was capable of rapid and specific detection of SARS- CoV-2 Omicron BA.4/5 variant within 30 min with the lowest detection limit of 10 copies/&#x3bc;L, and no cross-reaction was observed in SARS-CoV-2-negative clinical samples infected with 11 common respiratory pathogens. The two Omicron BA.4/5 specific crRNAs (crRNA-1 and crRNA-2) allowed the assay to accurately distinguish Omicron BA.4/5 from BA.1 sublineage and other major SARS-CoV-2 variants of concern. For detection of SARS-CoV-2 Omicron BA.4/5 variants, the sensitivity of the established assay using crRNA-1 and crRNA-2 was 97.83% and 100% with specificity of 100% and AUC of 0.998 and 1.000, respectively, and their concordance rate with Sanger sequencing method was 92.83% and 96.41%, respectively.<br><br>CONCLUSION: By combining RT-PCR and CRISPPR-Cas12a gene editing technology, we successfully developed a new method for rapid detection and identification of SARS-CoV-2 Omicron BA.4/5 variants with a high sensitivity, specificity and reproducibility, which allows rapid detection and genotyping of SARS- CoV-2 variants and monitoring of the emerging variants and their dissemination.}, }
@article {pmid37161551, year = {2023}, author = {Liu, C and Yao, X and Liu, C and You, S and Qi, W and Wang, M}, title = {Development and evaluation of RPA-NFO-LFT and RPA-Cas12a-LFT systems for the detection of Candida albicans.}, journal = {Analytical methods : advancing methods and applications}, volume = {15}, number = {19}, pages = {2355-2365}, doi = {10.1039/d3ay00259d}, pmid = {37161551}, issn = {1759-9679}, mesh = {*Candida albicans/genetics ; Sensitivity and Specificity ; CRISPR-Cas Systems ; *Candidiasis, Invasive ; DNA Primers ; }, abstract = {Recently, the growing number of medical interventions has led to the risk of invasive candidiasis. Among them, Candida albicans (C. albicans) infection has the highest incidence, which has led to great demand for developing early diagnosis methods. In this study, two lateral flow device based molecular assay systems, RPA-NFO-LFT and RPA-Cas12a-LFT, were established and optimized to achieve the detection of C. albicans. Firstly, efficient and specific primers for C. albicans detection were designed and screened, and the purification of amplification products was also explored. Then, many important conditions and issues for each system were investigated and discussed to improve the performances of the test strip devices in C. albicans detection. An evaluation study revealed that both systems showed favorable specificity and sensitivity in the detection of C. albicans samples with a lower detection limit of 10[3] CFU ml[-1], while RPA-Cas12a-LFT is more accurate for visual interpretation and more stable toward samples that exhibit serum nucleic acid interference. Finally, the performances of RPA-NFO-LFT and RPA-Cas12a-LFT were compared with that of the conventional qPCR method. This work might provide a reference for the development of molecular assay devices in practical candidiasis diagnosis.}, }
@article {pmid37147433, year = {2023}, author = {Foss, DV and Muldoon, JJ and Nguyen, DN and Carr, D and Sahu, SU and Hunsinger, JM and Wyman, SK and Krishnappa, N and Mendonsa, R and Schanzer, EV and Shy, BR and Vykunta, VS and Allain, V and Li, Z and Marson, A and Eyquem, J and Wilson, RC}, title = {Peptide-mediated delivery of CRISPR enzymes for the efficient editing of primary human lymphocytes.}, journal = {Nature biomedical engineering}, volume = {7}, number = {5}, pages = {647-660}, pmid = {37147433}, issn = {2157-846X}, support = {K08 AI153767/AI/NIAID NIH HHS/United States ; L40 AI140341/AI/NIAID NIH HHS/United States ; L30 AI140341/AI/NIAID NIH HHS/United States ; }, mesh = {Humans ; Mice ; Animals ; *CRISPR-Cas Systems ; *Gene Editing/methods ; T-Lymphocytes/metabolism ; Peptides/genetics ; Ribonucleoproteins ; }, abstract = {CRISPR-mediated genome editing of primary human lymphocytes is typically carried out via electroporation, which can be cytotoxic, cumbersome and costly. Here we show that the yields of edited primary human lymphocytes can be increased substantially by delivering a CRISPR ribonucleoprotein mixed with an amphiphilic peptide identified through screening. We evaluated the performance of this simple delivery method by knocking out genes in T cells, B cells and natural killer cells via the delivery of Cas9 or Cas12a ribonucleoproteins or an adenine base editor. We also show that peptide-mediated ribonucleoprotein delivery paired with an adeno-associated-virus-mediated homology-directed repair template can introduce a chimaeric antigen receptor gene at the T-cell receptor α constant locus, and that the engineered cells display antitumour potency in mice. The method is minimally perturbative, does not require dedicated hardware, and is compatible with multiplexed editing via sequential delivery, which minimizes the risk of genotoxicity. The peptide-mediated intracellular delivery of ribonucleoproteins may facilitate the manufacturing of engineered T cells.}, }
@article {pmid37141888, year = {2023}, author = {Guo, J and Yu, W and Li, M and Chen, H and Liu, J and Xue, X and Lin, J and Huang, S and Shu, W and Huang, X and Liu, Z and Wang, S and Qiao, Y}, title = {A DddA ortholog-based and transactivator-assisted nuclear and mitochondrial cytosine base editors with expanded target compatibility.}, journal = {Molecular cell}, volume = {83}, number = {10}, pages = {1710-1724.e7}, doi = {10.1016/j.molcel.2023.04.012}, pmid = {37141888}, issn = {1097-4164}, mesh = {Mice ; Animals ; *Gene Editing ; *Trans-Activators/metabolism ; Cytosine ; Mutation ; DNA, Mitochondrial/genetics ; CRISPR-Cas Systems ; }, abstract = {Bacterial double-stranded DNA (dsDNA) cytosine deaminase DddAtox-derived cytosine base editor (DdCBE) and its evolved variant, DddA11, guided by transcription-activator-like effector (TALE) proteins, enable mitochondrial DNA (mtDNA) editing at TC or HC (H = A, C, or T) sequence contexts, while it remains relatively unattainable for GC targets. Here, we identified a dsDNA deaminase originated from a Roseburia intestinalis interbacterial toxin (riDddAtox) and generated CRISPR-mediated nuclear DdCBEs (crDdCBEs) and mitochondrial CBEs (mitoCBEs) using split riDddAtox, which catalyzed C-to-T editing at both HC and GC targets in nuclear and mitochondrial genes. Moreover, transactivator (VP64, P65, or Rta) fusion to the tail of DddAtox- or riDddAtox-mediated crDdCBEs and mitoCBEs substantially improved nuclear and mtDNA editing efficiencies by up to 3.5- and 1.7-fold, respectively. We also used riDddAtox-based and Rta-assisted mitoCBE to efficiently stimulate disease-associated mtDNA mutations in cultured cells and in mouse embryos with conversion frequencies of up to 58% at non-TC targets.}, }
@article {pmid37080758, year = {2023}, author = {Sherkatghanad, Z and Abdar, M and Charlier, J and Makarenkov, V}, title = {Using traditional machine learning and deep learning methods for on- and off-target prediction in CRISPR/Cas9: a review.}, journal = {Briefings in bioinformatics}, volume = {24}, number = {3}, pages = {}, pmid = {37080758}, issn = {1477-4054}, mesh = {*CRISPR-Cas Systems ; *Deep Learning ; Gene Editing/methods ; Machine Learning ; }, abstract = {CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated protein 9) is a popular and effective two-component technology used for targeted genetic manipulation. It is currently the most versatile and accurate method of gene and genome editing, which benefits from a large variety of practical applications. For example, in biomedicine, it has been used in research related to cancer, virus infections, pathogen detection, and genetic diseases. Current CRISPR/Cas9 research is based on data-driven models for on- and off-target prediction as a cleavage may occur at non-target sequence locations. Nowadays, conventional machine learning and deep learning methods are applied on a regular basis to accurately predict on-target knockout efficacy and off-target profile of given single-guide RNAs (sgRNAs). In this paper, we present an overview and a comparative analysis of traditional machine learning and deep learning models used in CRISPR/Cas9. We highlight the key research challenges and directions associated with target activity prediction. We discuss recent advances in the sgRNA-DNA sequence encoding used in state-of-the-art on- and off-target prediction models. Furthermore, we present the most popular deep learning neural network architectures used in CRISPR/Cas9 prediction models. Finally, we summarize the existing challenges and discuss possible future investigations in the field of on- and off-target prediction. Our paper provides valuable support for academic and industrial researchers interested in the application of machine learning methods in the field of CRISPR/Cas9 genome editing.}, }
@article {pmid37069266, year = {2023}, author = {Everette, KA and Newby, GA and Levine, RM and Mayberry, K and Jang, Y and Mayuranathan, T and Nimmagadda, N and Dempsey, E and Li, Y and Bhoopalan, SV and Liu, X and Davis, JR and Nelson, AT and Chen, PJ and Sousa, AA and Cheng, Y and Tisdale, JF and Weiss, MJ and Yen, JS and Liu, DR}, title = {Ex vivo prime editing of patient haematopoietic stem cells rescues sickle-cell disease phenotypes after engraftment in mice.}, journal = {Nature biomedical engineering}, volume = {7}, number = {5}, pages = {616-628}, pmid = {37069266}, issn = {2157-846X}, support = {U01 AI142756/AI/NIAID NIH HHS/United States ; RM1 HG009490/HG/NHGRI NIH HHS/United States ; R35 GM118062/GM/NIGMS NIH HHS/United States ; R01 HL156647/HL/NHLBI NIH HHS/United States ; R01 HL136135/HL/NHLBI NIH HHS/United States ; }, mesh = {Adult ; Humans ; Mice ; Animals ; *Gene Editing ; CRISPR-Cas Systems ; beta-Globins/genetics ; *Anemia, Sickle Cell/therapy/genetics ; Hematopoietic Stem Cells ; Phenotype ; DNA ; }, abstract = {Sickle-cell disease (SCD) is caused by an A·T-to-T·A transversion mutation in the β-globin gene (HBB). Here we show that prime editing can correct the SCD allele (HBB[S]) to wild type (HBB[A]) at frequencies of 15%-41% in haematopoietic stem and progenitor cells (HSPCs) from patients with SCD. Seventeen weeks after transplantation into immunodeficient mice, prime-edited SCD HSPCs maintained HBB[A] levels and displayed engraftment frequencies, haematopoietic differentiation and lineage maturation similar to those of unedited HSPCs from healthy donors. An average of 42% of human erythroblasts and reticulocytes isolated 17 weeks after transplantation of prime-edited HSPCs from four SCD patient donors expressed HBB[A], exceeding the levels predicted for therapeutic benefit. HSPC-derived erythrocytes carried less sickle haemoglobin, contained HBB[A]-derived adult haemoglobin at 28%-43% of normal levels and resisted hypoxia-induced sickling. Minimal off-target editing was detected at over 100 sites nominated experimentally via unbiased genome-wide analysis. Our findings support the feasibility of a one-time prime editing SCD treatment that corrects HBB[S] to HBB[A], does not require any viral or non-viral DNA template and minimizes undesired consequences of DNA double-strand breaks.}, }
@article {pmid37068307, year = {2023}, author = {Guan, Z and Jiang, Z}, title = {Transformer-based anti-noise models for CRISPR-Cas9 off-target activities prediction.}, journal = {Briefings in bioinformatics}, volume = {24}, number = {3}, pages = {}, doi = {10.1093/bib/bbad127}, pmid = {37068307}, issn = {1477-4054}, mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; Base Sequence ; }, abstract = {The off-target effect occurring in the CRISPR-Cas9 system has been a challenging problem for the practical application of this gene editing technology. In recent years, various prediction models have been proposed to predict potential off-target activities. However, most of the existing prediction methods do not fully exploit guide RNA (gRNA) and DNA sequence pair information effectively. In addition, available prediction methods usually ignore the noise effect in original off-target datasets. To address these issues, we design a novel coding scheme, which considers the key features of mismatch type, mismatch location and the gRNA-DNA sequence pair information. Furthermore, a transformer-based anti-noise model called CrisprDNT is developed to solve the noise problem that exists in the off-target data. Experimental results of eight existing datasets demonstrate that the method with the inclusion of the anti-noise loss functions is superior to available state-of-the-art prediction methods. CrisprDNT is available at https://github.com/gzrgzx/CrisprDNT.}, }
@article {pmid37058502, year = {2023}, author = {Maestroni, L and Butti, P and Milanesi, R and Pagliari, S and Campone, L and Serra, I and Branduardi, P}, title = {Easy Modular Integrative fuSion-ready Expression (Easy-MISE) Toolkit for Fast Engineering of Heterologous Productions in Saccharomyces cerevisiae.}, journal = {ACS synthetic biology}, volume = {12}, number = {5}, pages = {1508-1519}, doi = {10.1021/acssynbio.3c00015}, pmid = {37058502}, issn = {2161-5063}, mesh = {*Saccharomyces cerevisiae/genetics/metabolism ; Fermentation ; *Metabolic Engineering ; CRISPR-Cas Systems ; }, abstract = {Nowadays, the yeast Saccharomyces cerevisiae is the platform of choice for demonstrating the proof of concept of the production of metabolites with a complex structure. However, introducing heterologous genes and rewiring the endogenous metabolism is still not standardized enough, affecting negatively the readiness-to-market of such metabolites. We developed the Easy Modular Integrative fuSion-ready Expression (Easy-MISE) toolkit, which is a novel combination of synthetic biology tools based on a single Golden Gate multiplasmid assembly meant to further ameliorate the rational predictability and flexibility of the process of yeast engineering. Thanks to an improved cloning screening strategy, double and independent transcription units are easily assembled and subsequently integrated into previously characterized loci. Moreover, the devices can be tagged for localization. This design allows for a higher degree of modularity and increases the flexibility of the engineering strategy. We show with a case study how the developed toolkit accelerates the construction and the analysis of the intermediate and the final engineered yeast strains, leaving space to better characterize the heterologous biosynthetic pathway in the final host and, overall, to improve the fermentation performances. Different S. cerevisiae strains were built harboring different versions of the biochemical pathway toward glucobrassicin (GLB) production, an indolyl-methyl glucosinolate. In the end, we could demonstrate that in the tested conditions the best-producing strain leads to a final concentration of GLB of 9.80 ± 0.267 mg/L, a titer 10-fold higher than the best result previously reported in the literature.}, }
@article {pmid37058298, year = {2023}, author = {Xiong, X and Tasan, I and Yang, C and Zhang, M and Hernandez Gonzalez, GA and Liu, S and Chaturvedi, P and Belmont, AS and Zhao, H}, title = {Imaging Method Using CRISPR/dCas9 and Engineered gRNA Scaffolds Can Perturb Replication Timing at the HSPA1 Locus.}, journal = {ACS synthetic biology}, volume = {12}, number = {5}, pages = {1424-1436}, doi = {10.1021/acssynbio.2c00433}, pmid = {37058298}, issn = {2161-5063}, mesh = {Animals ; *DNA Replication Timing ; *CRISPR-Cas Systems/genetics ; Chromosomes ; Genome ; DNA-Binding Proteins ; Molecular Chaperones ; Mammals ; }, abstract = {Fluorescence microscopy imaging of specific chromosomal sites is essential for genome architecture research. To enable visualization of endogenous loci in mammalian cells, programmable DNA-binding proteins such as TAL effectors and CRISPR/dCas9 are commonly utilized. In addition, site-specific insertion of a TetO repeat array, coupled with TetR-enhanced green fluorescent protein fusion protein expression, can be used for labeling nonrepetitive endogenous loci. Here, we performed a comparison of several live-cell chromosome tagging methods, including their effect on subnuclear positioning, expression of adjacent genes, and DNA replication timing. Our results showed that the CRISPR-based imaging method can delay DNA replication timing and sister chromatid resolution at certain region. However, subnuclear localization of the labeled locus and gene expression from adjacent loci were unaffected by either TetO/TetR or CRISPR-based methods, suggesting that CRISPR-based imaging could be used for applications that do not require DNA replication analysis.}, }
@article {pmid37037965, year = {2023}, author = {Kawamata, M and Suzuki, HI and Kimura, R and Suzuki, A}, title = {Optimization of Cas9 activity through the addition of cytosine extensions to single-guide RNAs.}, journal = {Nature biomedical engineering}, volume = {7}, number = {5}, pages = {672-691}, pmid = {37037965}, issn = {2157-846X}, mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *RNA, Guide, CRISPR-Cas Systems ; Cytosine ; Gene Editing ; Recombinational DNA Repair ; }, abstract = {The precise regulation of the activity of Cas9 is crucial for safe and efficient editing. Here we show that the genome-editing activity of Cas9 can be constrained by the addition of cytosine stretches to the 5'-end of conventional single-guide RNAs (sgRNAs). Such a 'safeguard sgRNA' strategy, which is compatible with Cas12a and with systems for gene activation and interference via CRISPR (clustered regularly interspaced short palindromic repeats), leads to the length-dependent inhibition of the formation of functional Cas9 complexes. Short cytosine extensions reduced p53 activation and cytotoxicity in human pluripotent stem cells, and enhanced homology-directed repair while maintaining bi-allelic editing. Longer extensions further decreased on-target activity yet improved the specificity and precision of mono-allelic editing. By monitoring indels through a fluorescence-based allele-specific system and computational simulations, we identified optimal windows of Cas9 activity for a number of genome-editing applications, including bi-allelic and mono-allelic editing, and the generation and correction of disease-associated single-nucleotide substitutions via homology-directed repair. The safeguard-sgRNA strategy may improve the safety and applicability of genome editing.}, }
@article {pmid36853024, year = {2023}, author = {Meng, J and Qiu, Y and Zhang, Y and Zhao, H and Shi, S}, title = {CMI: CRISPR/Cas9 Based Efficient Multiplexed Integration in Saccharomyces cerevisiae.}, journal = {ACS synthetic biology}, volume = {12}, number = {5}, pages = {1408-1414}, doi = {10.1021/acssynbio.2c00591}, pmid = {36853024}, issn = {2161-5063}, mesh = {*Saccharomyces cerevisiae/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; Synthetic Biology/methods ; Genomics ; Gene Editing/methods ; }, abstract = {Genomic integration is the preferred method for gene expression in microbial industrial production. However, traditional homologous recombination based multiplexed integration methods often suffer from low integration efficiency and complex experimental procedures. Here, we report a CRISPR/Cas9 based multiplexed integration (CMI) system in Saccharomyces cerevisiae, which can achieve quadruple integration at an individual locus without pre-engineering the host. A fused protein, Cas9-Brex27, was used as a bait to attract Rad51 recombinase to the proximity of the double-strand breaks introduced by the CRISPR/Cas9 system. The efficiency of quadruple integration was increased to 53.9% with 40 bp homology arms (HAs) and 78% with 100 bp HAs. CMI was applied to integrate a heterologous mogrol biosynthetic pathway consisting of four genes in a one-step transformation and offered an efficient solution for multiplexed integration. This method expands the synthetic biology toolbox of S. cerevisiae.}, }
@article {pmid36797418, year = {2023}, author = {Yan, S and Zheng, X and Lin, Y and Li, C and Liu, Z and Li, J and Tu, Z and Zhao, Y and Huang, C and Chen, Y and Li, J and Song, X and Han, B and Wang, W and Liang, W and Lai, L and Li, XJ and Li, S}, title = {Cas9-mediated replacement of expanded CAG repeats in a pig model of Huntington's disease.}, journal = {Nature biomedical engineering}, volume = {7}, number = {5}, pages = {629-646}, pmid = {36797418}, issn = {2157-846X}, mesh = {Animals ; Swine ; *Huntington Disease/genetics/therapy/metabolism ; Trinucleotide Repeat Expansion ; CRISPR-Cas Systems/genetics ; Genetic Engineering ; }, abstract = {The monogenic nature of Huntington's disease (HD) and other neurodegenerative diseases caused by the expansion of glutamine-encoding CAG repeats makes them particularly amenable to gene therapy. Here we show the feasibility of replacing expanded CAG repeats in the mutant HTT allele with a normal CAG repeat in genetically engineered pigs mimicking the selective neurodegeneration seen in patients with HD. A single intracranial or intravenous injection of adeno-associated virus encoding for Cas9, a single-guide RNA targeting the HTT gene, and donor DNA containing the normal CAG repeat led to the depletion of mutant HTT in the animals and to substantial reductions in the dysregulated expression and neurotoxicity of mutant HTT and in neurological symptoms. Our findings support the further translational development of virally delivered Cas9-based gene therapies for the treatment of genetic neurodegenerative diseases.}, }
@article {pmid37202505, year = {2023}, author = {Maier, CR and Hartmann, O and Prieto-Garcia, C and Al-Shami, KM and Schlicker, L and Vogel, FCE and Haid, S and Klann, K and Buck, V and Münch, C and Schmitz, W and Einig, E and Krenz, B and Calzado, MA and Eilers, M and Popov, N and Rosenfeldt, MT and Diefenbacher, ME and Schulze, A}, title = {USP28 controls SREBP2 and the mevalonate pathway to drive tumour growth in squamous cancer.}, journal = {Cell death and differentiation}, volume = {}, number = {}, pages = {}, pmid = {37202505}, issn = {1476-5403}, abstract = {SREBP2 is a master regulator of the mevalonate pathway (MVP), a biosynthetic process that drives the synthesis of dolichol, heme A, ubiquinone and cholesterol and also provides substrates for protein prenylation. Here, we identify SREBP2 as a novel substrate for USP28, a deubiquitinating enzyme that is frequently upregulated in squamous cancers. Our results show that silencing of USP28 reduces expression of MVP enzymes and lowers metabolic flux into this pathway. We also show that USP28 binds to mature SREBP2, leading to its deubiquitination and stabilisation. USP28 depletion rendered cancer cells highly sensitive to MVP inhibition by statins, which was rescued by the addition of geranyl-geranyl pyrophosphate. Analysis of human tissue microarrays revealed elevated expression of USP28, SREBP2 and MVP enzymes in lung squamous cell carcinoma (LSCC) compared to lung adenocarcinoma (LADC). Moreover, CRISPR/Cas-mediated deletion of SREBP2 selectively attenuated tumour growth in a KRas/p53/LKB1 mutant mouse model of lung cancer. Finally, we demonstrate that statins synergise with a dual USP28/25 inhibitor to reduce viability of SCC cells. Our findings suggest that combinatorial targeting of MVP and USP28 could be a therapeutic strategy for the treatment of squamous cell carcinomas.}, }
@article {pmid37201524, year = {2023}, author = {Kalamakis, G and Platt, RJ}, title = {CRISPR for neuroscientists.}, journal = {Neuron}, volume = {}, number = {}, pages = {}, doi = {10.1016/j.neuron.2023.04.021}, pmid = {37201524}, issn = {1097-4199}, abstract = {Genome engineering technologies provide an entry point into understanding and controlling the function of genetic elements in health and disease. The discovery and development of the microbial defense system CRISPR-Cas yielded a treasure trove of genome engineering technologies and revolutionized the biomedical sciences. Comprising diverse RNA-guided enzymes and effector proteins that evolved or were engineered to manipulate nucleic acids and cellular processes, the CRISPR toolbox provides precise control over biology. Virtually all biological systems are amenable to genome engineering-from cancer cells to the brains of model organisms to human patients-galvanizing research and innovation and giving rise to fundamental insights into health and powerful strategies for detecting and correcting disease. In the field of neuroscience, these tools are being leveraged across a wide range of applications, including engineering traditional and non-traditional transgenic animal models, modeling disease, testing genomic therapies, unbiased screening, programming cell states, and recording cellular lineages and other biological processes. In this primer, we describe the development and applications of CRISPR technologies while highlighting outstanding limitations and opportunities.}, }
@article {pmid37200846, year = {2023}, author = {Sheng, L and Madika, A and Lau, MSH and Zhang, Y and Minton, NP}, title = {Metabolic engineering for the production of acetoin and 2,3-butanediol at elevated temperature in Parageobacillus thermoglucosidasius NCIMB 11955.}, journal = {Frontiers in bioengineering and biotechnology}, volume = {11}, number = {}, pages = {1191079}, pmid = {37200846}, issn = {2296-4185}, abstract = {The current climate crisis has emphasised the need to achieve global net-zero by 2050, with countries being urged to set considerable emission reduction targets by 2030. Exploitation of a fermentative process that uses a thermophilic chassis can represent a way to manufacture chemicals and fuels through more environmentally friendly routes with a net reduction in greenhouse gas emissions. In this study, the industrially relevant thermophile Parageobacillus thermoglucosidasius NCIMB 11955 was engineered to produce 3-hydroxybutanone (acetoin) and 2,3-butanediol (2,3-BDO), organic compounds with commercial applications. Using heterologous acetolactate synthase (ALS) and acetolactate decarboxylase (ALD) enzymes, a functional 2,3-BDO biosynthetic pathway was constructed. The formation of by-products was minimized by the deletion of competing pathways surrounding the pyruvate node. Redox imbalance was addressed through autonomous overexpression of the butanediol dehydrogenase and by investigating appropriate aeration levels. Through this, we were able to produce 2,3-BDO as the predominant fermentation metabolite, with up to 6.6 g/L 2,3-BDO (0.33 g/g glucose) representing 66% of the theoretical maximum at 50°C. In addition, the identification and subsequent deletion of a previously unreported thermophilic acetoin degradation gene (acoB1) resulted in enhanced acetoin production under aerobic conditions, producing 7.6 g/L (0.38 g/g glucose) representing 78% of the theoretical maximum. Furthermore, through the generation of a ΔacoB1 mutant and by testing the effect of glucose concentration on 2,3-BDO production, we were able to produce 15.6 g/L of 2,3-BDO in media supplemented with 5% glucose, the highest titre of 2,3-BDO produced in Parageobacillus and Geobacillus species to date.}, }
@article {pmid37199299, year = {2022}, author = {Dholariya, S and Parchwani, D and Radadiya, M and Singh, RD and Sonagra, A and Patel, D and Sharma, G}, title = {CRISPR/Cas9: A Molecular Tool for Ovarian Cancer Management beyond Gene Editing.}, journal = {Critical reviews in oncogenesis}, volume = {27}, number = {4}, pages = {1-22}, doi = {10.1615/CritRevOncog.2022043814}, pmid = {37199299}, issn = {0893-9675}, mesh = {Female ; Humans ; *Gene Editing/methods ; CRISPR-Cas Systems/genetics ; *Ovarian Neoplasms/diagnosis/genetics/therapy ; Genetic Therapy/methods ; Carcinogenesis/genetics ; }, abstract = {Ovarian cancer manifests with early metastases and has an adverse outcome, impacting the health of women globally. Currently, this malignancy is often treated with cytoreductive surgery and platinum-based chemotherapy. This treatment option has a limited success rate due to tumor recurrence and chemoresistance. Consequently, the fundamental objective of ovarian cancer treatment is the development of novel treatment approaches. As a new robust tool, the CRISPR/Cas9 gene-editing system has shown immense promise in elucidating the molecular basis of all the facets of ovarian cancer. Due to the precise gene editing capabilities of CRISPR-Cas9, researchers have been able to conduct a more comprehensive investigation of the genesis of ovarian cancer. This gained knowledge can be translated into the development of novel diagnostic approaches and newer therapeutic targets for this dreadful malignancy. There is encouraging preclinical evidence that suggests that CRISPR/Cas9 is a powerful versatile tool for selectively targeting cancer cells and inhibiting tumor growth, establishing new signaling pathways involved in carcinogenesis, and verifying biomolecules as druggable targets. In this review, we analyzed the current research and progress made using CRISPR/Cas9-based engineering strategies in the diagnosis and treatment, as well as the challenges in bringing this method to clinics. This comprehensive analysis will lay the basis for subsequent research in the future for the treatment of ovarian cancer.}, }
@article {pmid37198545, year = {2023}, author = {Li, J and Liang, Q and Zhou, H and Zhou, M and Huang, H}, title = {Profiling the impact of the promoters on CRISPR-Cas12a system in human cells.}, journal = {Cellular &amp; molecular biology letters}, volume = {28}, number = {1}, pages = {41}, pmid = {37198545}, issn = {1689-1392}, mesh = {Animals ; Humans ; CRISPR-Cas Systems/genetics ; *CRISPR-Associated Proteins/genetics/metabolism ; Gene Editing/methods ; Genetic Vectors ; *Cytomegalovirus Infections/genetics ; Mammals/genetics ; }, abstract = {The plasmid vector platform is the most commonly used vector for the expression of the versatile CRISPR-Cas technique and the promoter is a crucial element for the expression vector, thus profiling the impact of the promoters on CRISPR editors provides the basic information for the gene-editing toolkits and can be a guideline for its design. Herein, we made a parallel comparison among four commonly used promoters (CAG, ~ 1700 bp; EF1a core, ~ 210 bp; CMV, ~ 500 bp; and PGK, ~ 500 bp) in CRISPR-Cas12a system in mammalian cells to explore the impact of promoters on this powerful tool. We found that without badly damaging targeting specificity, the CAG promoter-driving Cas12a editor exhibited the most active (efficiency takes as 100%, specificity index = ~ 75%) in genomic cleavage, multiplex editing, transcriptional activation, and base editing, followed by promoter CMV (efficiency = 70 ~ 90% (vs CAG), specificity index = ~ 78%), and then EF1a core and PGK (both efficiency = 40-60%, vs CAG) but with higher specificity (specificity index = ~ 84% and ~ 82%, respectively). Therefore, CAG is recommended in the CRISPR-Cas12a system for the applications that need a robust editing activity but without size limitation, CMV mostly can be an alternative for CAG when requiring a smaller space, EF1a is similar to PGK with relatively high specificity, but has a smaller size, thus is more suitable for in vivo therapeutic applications. The data outlined the properties of the widely used promoters in the CRISPR-Cas12a system, which can be a guide for its applications and can be a useful resource for the gene-editing field.}, }
@article {pmid37193354, year = {2022}, author = {Qiu, HY and Ji, RJ and Zhang, Y}, title = {Current advances of CRISPR-Cas technology in cell therapy.}, journal = {Cell insight}, volume = {1}, number = {6}, pages = {100067}, pmid = {37193354}, issn = {2772-8927}, abstract = {CRISPR-Cas is a versatile genome editing technology that has been broadly applied in both basic research and translation medicine. Ever since its discovery, the bacterial derived endonucleases have been engineered to a collection of robust genome-editing tools for introducing frameshift mutations or base conversions at site-specific loci. Since the initiation of first-in-human trial in 2016, CRISPR-Cas has been tested in 57 cell therapy trials, 38 of which focusing on engineered CAR-T cells and TCR-T cells for cancer malignancies, 15 trials of engineered hematopoietic stem cells treating hemoglobinopathies, leukemia and AIDS, and 4 trials of engineered iPSCs for diabetes and cancer. Here, we aim to review the recent breakthroughs of CRISPR technology and highlight their applications in cell therapy.}, }
@article {pmid37192099, year = {2023}, author = {Koonin, EV and Gootenberg, JS and Abudayyeh, OO}, title = {Discovery of Diverse CRISPR-Cas Systems and Expansion of the Genome Engineering Toolbox.}, journal = {Biochemistry}, volume = {}, number = {}, pages = {}, doi = {10.1021/acs.biochem.3c00159}, pmid = {37192099}, issn = {1520-4995}, abstract = {CRISPR systems mediate adaptive immunity in bacteria and archaea through diverse effector mechanisms and have been repurposed for versatile applications in therapeutics and diagnostics thanks to their facile reprogramming with RNA guides. RNA-guided CRISPR-Cas targeting and interference are mediated by effectors that are either components of multisubunit complexes in class 1 systems or multidomain single-effector proteins in class 2. The compact class 2 CRISPR systems have been broadly adopted for multiple applications, especially genome editing, leading to a transformation of the molecular biology and biotechnology toolkit. The diversity of class 2 effector enzymes, initially limited to the Cas9 nuclease, was substantially expanded via computational genome and metagenome mining to include numerous variants of Cas12 and Cas13, providing substrates for the development of versatile, orthogonal molecular tools. Characterization of these diverse CRISPR effectors uncovered many new features, including distinct protospacer adjacent motifs (PAMs) that expand the targeting space, improved editing specificity, RNA rather than DNA targeting, smaller crRNAs, staggered and blunt end cuts, miniature enzymes, promiscuous RNA and DNA cleavage, etc. These unique properties enabled multiple applications, such as harnessing the promiscuous RNase activity of the type VI effector, Cas13, for supersensitive nucleic acid detection. class 1 CRISPR systems have been adopted for genome editing, as well, despite the challenge of expressing and delivering the multiprotein class 1 effectors. The rich diversity of CRISPR enzymes led to rapid maturation of the genome editing toolbox, with capabilities such as gene knockout, base editing, prime editing, gene insertion, DNA imaging, epigenetic modulation, transcriptional modulation, and RNA editing. Combined with rational design and engineering of the effector proteins and associated RNAs, the natural diversity of CRISPR and related bacterial RNA-guided systems provides a vast resource for expanding the repertoire of tools for molecular biology and biotechnology.}, }
@article {pmid37128982, year = {2023}, author = {Kang, Y and Zhang, J and Zhao, L and Yan, H}, title = {Colorimetric miRNA detection based on self-primer-initiated CRISPR-Cas12a-assisted amplification.}, journal = {BioTechniques}, volume = {74}, number = {4}, pages = {172-178}, doi = {10.2144/btn-2023-0008}, pmid = {37128982}, issn = {1940-9818}, mesh = {Female ; Pregnancy ; Humans ; CRISPR-Cas Systems/genetics ; Colorimetry ; Fetus ; *MicroRNAs/genetics ; *Biosensing Techniques ; Nucleic Acid Amplification Techniques ; }, abstract = {miRNAs alter significantly throughout pregnancy to support the development of the fetus. However, sensitive detection of miRNA remains a challenge. Herein, a reliable miRNA detection approach integrating self-assembly-triggered signal amplification and CRISPR-Cas12a-system cleavage-based color generation is described. The colorimetric approach contains three signal amplification processes. The first signal amplification is formed by the released miRNA in a chain extension process. The produced sequence that is similar to the target miRNA initiates the second signal recycle. Finally, CRISPR-Cas12a-based transcleavage on linker sequences induces the third signal amplification. The method exhibits high sensitivity and a low limit of detection of 254 aM, showing promising prospects in disease diagnosis.}, }
@article {pmid37191877, year = {2023}, author = {Alalmaie, A and Diaf, S and Khashan, R}, title = {Insight into the molecular mechanism of the transposon-encoded type I-F CRISPR-Cas system.}, journal = {Journal, genetic engineering &amp; biotechnology}, volume = {21}, number = {1}, pages = {60}, pmid = {37191877}, issn = {2090-5920}, abstract = {CRISPR-Cas9 is a popular gene-editing tool that allows researchers to introduce double-strand breaks to edit parts of the genome. CRISPR-Cas9 system is used more than other gene-editing tools because it is simple and easy to customize. However, Cas9 may produce unintended double-strand breaks in DNA, leading to off-target effects. There have been many improvements in the CRISPR-Cas system to control the off-target effect and improve the efficiency. The presence of a nuclease-deficient CRISPR-Cas system in several bacterial Tn7-like transposons inspires researchers to repurpose to direct the insertion of Tn7-like transposons instead of cleaving the target DNA, which will eventually limit the risk of off-target effects. Two transposon-encoded CRISPR-Cas systems have been experimentally confirmed. The first system, found in Tn7 like-transposon (Tn6677), is associated with the variant type I-F CRISPR-Cas system. The second one, found in Tn7 like-transposon (Tn5053), is related to the variant type V-K CRISPR-Cas system. This review describes the molecular and structural mechanisms of DNA targeting by the transposon-encoded type I-F CRISPR-Cas system, from assembly around the CRISPR-RNA (crRNA) to the initiation of transposition.}, }
@article {pmid37191803, year = {2023}, author = {Rivera-Torres, N and Bialk, P and Kmiec, EB}, title = {CRISPR-Directed Gene Editing as a Method to Reduce Chemoresistance in Lung Cancer Cells.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2660}, number = {}, pages = {263-271}, pmid = {37191803}, issn = {1940-6029}, mesh = {Humans ; *Gene Editing/methods ; CRISPR-Cas Systems/genetics ; Drug Resistance, Neoplasm/genetics ; *Lung Neoplasms/genetics/therapy ; Lung ; }, abstract = {We are advancing a novel strategy for the treatment of solid tumors by employing CRISPR-directed gene editing to reduce levels of standard of care required to halt or reverse the progression of tumor growth. We intend to do this by utilizing a combinatorial approach in which CRISPR-directed gene editing is used to eliminate or significantly reduce the acquired resistance emerging from chemotherapy, radiation therapy, or immunotherapy. We will utilize CRISPR/Cas as a biomolecular tool to disable specific genes involved in the sustainability of resistance to cancer therapy. We have also developed a CRISPR/Cas molecule that can distinguish between the genome of a tumor cell in the genome of a normal cell, thereby conferring target selectivity onto this therapeutic approach. We envision delivering these molecules by direct injection into solid tumors for the treatment of squamous cell carcinomas of the lung, esophageal cancer, and head and neck cancer. We provide experimental details and methodology for utilizing CRISPR/Cas as a supplement to chemotherapy to destroy lung cancer cells.}, }
@article {pmid37190012, year = {2023}, author = {Bhokisham, N and Laudermilch, E and Traeger, LL and Bonilla, TD and Ruiz-Estevez, M and Becker, JR}, title = {CRISPR-Cas System: The Current and Emerging Translational Landscape.}, journal = {Cells}, volume = {12}, number = {8}, pages = {}, pmid = {37190012}, issn = {2073-4409}, mesh = {Animals ; Humans ; *CRISPR-Cas Systems/genetics ; *Gene Editing ; Ecosystem ; Genetic Therapy ; Epigenome ; }, abstract = {CRISPR-Cas technology has rapidly changed life science research and human medicine. The ability to add, remove, or edit human DNA sequences has transformative potential for treating congenital and acquired human diseases. The timely maturation of the cell and gene therapy ecosystem and its seamless integration with CRISPR-Cas technologies has enabled the development of therapies that could potentially cure not only monogenic diseases such as sickle cell anemia and muscular dystrophy, but also complex heterogenous diseases such as cancer and diabetes. Here, we review the current landscape of clinical trials involving the use of various CRISPR-Cas systems as therapeutics for human diseases, discuss challenges, and explore new CRISPR-Cas-based tools such as base editing, prime editing, CRISPR-based transcriptional regulation, CRISPR-based epigenome editing, and RNA editing, each promising new functionality and broadening therapeutic potential. Finally, we discuss how the CRISPR-Cas system is being used to understand the biology of human diseases through the generation of large animal disease models used for preclinical testing of emerging therapeutics.}, }
@article {pmid36357719, year = {2023}, author = {Neugebauer, ME and Hsu, A and Arbab, M and Krasnow, NA and McElroy, AN and Pandey, S and Doman, JL and Huang, TP and Raguram, A and Banskota, S and Newby, GA and Tolar, J and Osborn, MJ and Liu, DR}, title = {Evolution of an adenine base editor into a small, efficient cytosine base editor with low off-target activity.}, journal = {Nature biotechnology}, volume = {41}, number = {5}, pages = {673-685}, pmid = {36357719}, issn = {1546-1696}, support = {R35 GM118062/GM/NIGMS NIH HHS/United States ; U01 AI142756/AI/NIAID NIH HHS/United States ; R01 EB031172/EB/NIBIB NIH HHS/United States ; }, mesh = {Humans ; *CRISPR-Cas Systems ; *Cytosine ; Adenine ; Gene Editing ; DNA/genetics ; Deoxyadenosines ; Cytidine/genetics ; }, abstract = {Cytosine base editors (CBEs) are larger and can suffer from higher off-target activity or lower on-target editing efficiency than current adenine base editors (ABEs). To develop a CBE that retains the small size, low off-target activity and high on-target activity of current ABEs, we evolved the highly active deoxyadenosine deaminase TadA-8e to perform cytidine deamination using phage-assisted continuous evolution. Evolved TadA cytidine deaminases contain mutations at DNA-binding residues that alter enzyme selectivity to strongly favor deoxycytidine over deoxyadenosine deamination. Compared to commonly used CBEs, TadA-derived cytosine base editors (TadCBEs) offer similar or higher on-target activity, smaller size and substantially lower Cas-independent DNA and RNA off-target editing activity. We also identified a TadA dual base editor (TadDE) that performs equally efficient cytosine and adenine base editing. TadCBEs support single or multiplexed base editing at therapeutically relevant genomic loci in primary human T cells and primary human hematopoietic stem and progenitor cells. TadCBEs expand the utility of CBEs for precision gene editing.}, }
@article {pmid36357717, year = {2023}, author = {Chen, L and Zhu, B and Ru, G and Meng, H and Yan, Y and Hong, M and Zhang, D and Luan, C and Zhang, S and Wu, H and Gao, H and Bai, S and Li, C and Ding, R and Xue, N and Lei, Z and Chen, Y and Guan, Y and Siwko, S and Cheng, Y and Song, G and Wang, L and Yi, C and Liu, M and Li, D}, title = {Re-engineering the adenine deaminase TadA-8e for efficient and specific CRISPR-based cytosine base editing.}, journal = {Nature biotechnology}, volume = {41}, number = {5}, pages = {663-672}, pmid = {36357717}, issn = {1546-1696}, mesh = {Mice ; Animals ; *Gene Editing/methods ; *Cytosine/metabolism ; Aminohydrolases/metabolism ; RNA ; CRISPR-Cas Systems/genetics ; Cytidine Deaminase/genetics/metabolism ; }, abstract = {Cytosine base editors (CBEs) efficiently generate precise C·G-to-T·A base conversions, but the activation-induced cytidine deaminase/apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (AID/APOBEC) protein family deaminase component induces considerable off-target effects and indels. To explore unnatural cytosine deaminases, we repurpose the adenine deaminase TadA-8e for cytosine conversion. The introduction of an N46L variant in TadA-8e eliminates its adenine deaminase activity and results in a TadA-8e-derived C-to-G base editor (Td-CGBE) capable of highly efficient and precise C·G-to-G·C editing. Through fusion with uracil glycosylase inhibitors and further introduction of additional variants, a series of Td-CBEs was obtained either with a high activity similar to that of BE4max or with higher precision compared to other reported accurate CBEs. Td-CGBE/Td-CBEs show very low indel effects and a background level of Cas9-dependent or Cas9-independent DNA/RNA off-target editing. Moreover, Td-CGBE/Td-CBEs are more efficient in generating accurate edits in homopolymeric cytosine sites in cells or mouse embryos, suggesting their accuracy and safety for gene therapy and other applications.}, }
@article {pmid37189388, year = {2023}, author = {Vora, DS and Yadav, S and Sundar, D}, title = {Hybrid Multitask Learning Reveals Sequence Features Driving Specificity in the CRISPR/Cas9 System.}, journal = {Biomolecules}, volume = {13}, number = {4}, pages = {}, pmid = {37189388}, issn = {2218-273X}, mesh = {*CRISPR-Cas Systems/genetics ; *Machine Learning ; Genome ; }, abstract = {CRISPR/Cas9 technology is capable of precisely editing genomes and is at the heart of various scientific and medical advances in recent times. The advances in biomedical research are hindered because of the inadvertent burden on the genome when genome editors are employed-the off-target effects. Although experimental screens to detect off-targets have allowed understanding the activity of Cas9, that knowledge remains incomplete as the rules do not extrapolate well to new target sequences. Off-target prediction tools developed recently have increasingly relied on machine learning and deep learning techniques to reliably understand the complete threat of likely off-targets because the rules that drive Cas9 activity are not fully understood. In this study, we present a count-based as well as deep-learning-based approach to derive sequence features that are important in deciding on Cas9 activity at a sequence. There are two major challenges in off-target determination-the identification of a likely site of Cas9 activity and the prediction of the extent of Cas9 activity at that site. The hybrid multitask CNN-biLSTM model developed, named CRISP-RCNN, simultaneously predicts off-targets and the extent of activity on off-targets. Employing methods of integrated gradients and weighting kernels for feature importance approximation, analysis of nucleotide and position preference, and mismatch tolerance have been performed.}, }
@article {pmid37189371, year = {2023}, author = {Zuo, Y and Mei, X and Singson, A}, title = {CRISPR/Cas9 Mediated Fluorescent Tagging of Caenorhabditis elegans SPE-38 Reveals a Complete Localization Pattern in Live Spermatozoa.}, journal = {Biomolecules}, volume = {13}, number = {4}, pages = {}, pmid = {37189371}, issn = {2218-273X}, support = {R01 HD054681/NH/NIH HHS/United States ; }, mesh = {Animals ; Male ; *Caenorhabditis elegans/metabolism ; *Caenorhabditis elegans Proteins/metabolism ; CRISPR-Cas Systems/genetics ; Membrane Proteins/metabolism ; Semen/metabolism ; Spermatozoa/metabolism ; }, abstract = {The Caenorhabditis elegans spe-38 gene encodes a four-pass transmembrane molecule that is required in sperm for fertilization. In previous work, the localization of the SPE-38 protein was examined using polyclonal antibodies on spermatids and mature amoeboid spermatozoa. SPE-38 is localized to unfused membranous organelles (MOs) in nonmotile spermatids. Different fixation conditions revealed that SPE-38 either localized to fused MOs and the cell body plasma membrane or the pseudopod plasma membrane of mature sperm. To address this localization paradox in mature sperm, CRISPR/Cas9 genome editing was used to tag endogenous SPE-38 with fluorescent wrmScarlet-I. Homozygous male and hermaphrodite worms encoding SPE-38::wrmScarlet-I were fertile indicating the fluorescent tag does not interfere with SPE-38 function during sperm activation or fertilization. We found that SPE-38::wrmScarlet-I localized to MOs in spermatids consistent with previous antibody localization. In mature and motile spermatozoa we found SPE-38::wrmScarlet-I in fused MOs, the cell body plasma membrane, and the pseudopod plasma membrane. We conclude that the localization pattern observed with SPE-38::wrmScarlet-I represents the complete distribution of SPE-38 in mature spermatozoa and this localization pattern is consistent with a hypothesized role of SPE-38 directly in sperm-egg binding and/or fusion.}, }
@article {pmid37093234, year = {2023}, author = {Chin, S and Goyon, A and Zhang, K and Kurita, KL}, title = {Middle-out sequence confirmation of CRISPR/Cas9 single guide RNA (sgRNA) using DNA primers and ribonuclease T1 digestion.}, journal = {Analytical and bioanalytical chemistry}, volume = {415}, number = {14}, pages = {2809-2818}, pmid = {37093234}, issn = {1618-2650}, mesh = {Humans ; *CRISPR-Cas Systems ; *RNA, Guide, CRISPR-Cas Systems ; Ribonuclease T1/genetics ; DNA Primers ; Oligonucleotides ; Digestion ; }, abstract = {Accurate sequencing of single guide RNAs (sgRNAs) for CRISPR/Cas9 genome editing is critical for patient safety, as the sgRNA guides the Cas9 nuclease to target site-specific cleavages in DNA. An approach to fully sequence sgRNA using protective DNA primers followed by ribonuclease (RNase) T1 digestion was developed to facilitate the analysis of these larger molecules by hydrophilic interaction liquid chromatography coupled with high-resolution mass spectrometry (HILIC-HRMS). Without RNase digestion, top-down mass spectrometry alone struggles to properly fragment precursor ions in large RNA oligonucleotides to provide confidence in sequence coverage. With RNase T1 digestion of these larger oligonucleotides, however, bottom-up analysis cannot confirm full sequence coverage due to the presence of short, redundant digestion products. By combining primer protection with RNase T1 digestion, digestion products are large enough to prevent redundancy and small enough to provide base resolution by tandem mass spectrometry to allow for full sgRNA sequence coverage. An investigation into the general requirements for adequate primer protection of specific regions of the RNA was conducted, followed by the development of a generic protection and digestion strategy that may be applied to different sgRNA sequences. This middle-out technique has the potential to expedite accurate sequence confirmation of chemically modified sgRNA oligonucleotides.}, }
@article {pmid37040174, year = {2023}, author = {Feng, ZY and Yun, YF and Li, X and Zhang, J}, title = {Impact of Divalent Metal Ions on Regulation of Trans-Cleavage Activity of CRISPR-Cas13a: A Combined Experimental and Computational Study.}, journal = {Chembiochem : a European journal of chemical biology}, volume = {24}, number = {10}, pages = {e202300034}, doi = {10.1002/cbic.202300034}, pmid = {37040174}, issn = {1439-7633}, mesh = {*RNA ; *Manganese ; Calcium/metabolism ; Molecular Conformation ; Magnesium ; CRISPR-Cas Systems ; }, abstract = {CRISPR-LbuCas13a has emerged as a revolutionary tool for in vitro diagnosis. Similar to other Cas effectors, LbuCas13a requires Mg[2+] to maintain its nuclease activity. However, the effect of other divalent metal ions on its trans-cleavage activity remains less explored. Herein, we addressed this issue by combining experimental and molecular dynamics simulation analysis. In vitro studies showed that both Mn[2+] and Ca[2+] could replace Mg[2+] as cofactors of LbuCas13a. In contrast, Ni[2+] , Zn[2+] , Cu[2+] , or Fe[2+] inhibits the cis- and trans-cleavage activity, while Pb[2+] does not affect it. Importantly, molecular dynamics simulations confirmed that calcium, magnesium, and manganese hydrated ions have a strong affinity to nucleotide bases, thus stabilizing the conformation of crRNA repeat region and enhancing the trans-cleavage activity. Finally, we showed that combination of Mg[2+] and Mn[2+] can further enhance the trans-cleavage activity to allow amplified RNA detection, revealing its potential advantage for in vitro diagnosis.}, }
@article {pmid36823185, year = {2023}, author = {Li, L and Liu, W and Zhang, H and Cai, Q and Wen, D and Du, J and Sun, J and Li, L and Gao, C and Lin, P and Wu, M and Jiang, J}, title = {A New Method for Programmable RNA Editing Using CRISPR Effector Cas13X.1.}, journal = {The Tohoku journal of experimental medicine}, volume = {260}, number = {1}, pages = {51-61}, doi = {10.1620/tjem.2023.J011}, pmid = {36823185}, issn = {1349-3329}, abstract = {Type VI CRISPR-Cas13 is the only CRISPR system that can bind and cleave RNA without DNase activity. We used the newly discovered, smaller Cas13X.1 protein to construct an editing system in mammalian cells, aiming to break the delivery restrictions of CRISPR-Cas13 system in vivo and promote the application of Cas13X system in clinical therapy. We employed exogenous fluorescence reporter gene mCherry and endogenous gene transketolase (TKT) closely related to cancer cell metabolism as target genes to evaluate the Cas13X.1 system. The recombinant plasmids targeting exogenous gene mCherry and endogenous gene TKT were constructed based on Cas13X.1 backbone plasmid. The editing efficiency, protein expression level, downstream gene transcript level and safety of Cas13X.1 system were evaluated. Both TKT transcripts of endogenous genes and mCherry transcripts of exogenous genes were significantly degraded by Cas13X.1 system with a knockdown efficiency up to 50%. At the same time, Cas13X.1 down-regulated the expression of the corresponding protein level in the editing of transcripts. In addition, the transcripts of key metabolic enzymes related to TKT were also down-regulated synchronously, suggesting that the degradation of TKT transcripts by Cas13X.1 system affected the main metabolic pathways related to TKT. The morphology, RNA integrity and apoptosis of cells loaded with Cas13X.1 system were not affected. The Cas13X.1 system we constructed had strong RNA knockdown ability in mammalian cells with low cellular toxicity. Compared with other CRISPR-Cas13 systems, Cas13X.1 system with smaller molecular weight has more advantages in vivo delivery. The Cas13X.1 system targeting TKT transcripts also provides an alternative method for the study of anti-cancer therapy.}, }
@article {pmid36656467, year = {2023}, author = {Han, JL and Entcheva, E}, title = {Gene Modulation with CRISPR-based Tools in Human iPSC-Cardiomyocytes.}, journal = {Stem cell reviews and reports}, volume = {19}, number = {4}, pages = {886-905}, pmid = {36656467}, issn = {2629-3277}, mesh = {Animals ; Humans ; *CRISPR-Cas Systems/genetics ; *Induced Pluripotent Stem Cells ; Myocytes, Cardiac ; Gene Editing/methods ; Mammals ; }, abstract = {Precise control of gene expression (knock-out, knock-in, knockdown or overexpression) is at the heart of functional genomics - an approach to dissect the contribution of a gene/protein to the system's function. The development of a human in vitro system that can be patient-specific, induced pluripotent stem cells, iPSC, and the ability to obtain various cell types of interest, have empowered human disease modeling and therapeutic development. Scalable tools have been deployed for gene modulation in these cells and derivatives, including pharmacological means, DNA-based RNA interference and standard RNA interference (shRNA/siRNA). The CRISPR/Cas9 gene editing system, borrowed from bacteria and adopted for use in mammalian cells a decade ago, offers cell-specific genetic targeting and versatility. Outside genome editing, more subtle, time-resolved gene modulation is possible by using a catalytically "dead" Cas9 enzyme linked to an effector of gene transcription in combination with a guide RNA. The CRISPRi / CRISPRa (interference/activation) system evolved over the last decade as a scalable technology for performing functional genomics with libraries of gRNAs. Here, we review key developments of these approaches and their deployment in cardiovascular research. We discuss specific use with iPSC-cardiomyocytes and the challenges in further translation of these techniques.}, }
@article {pmid37189522, year = {2023}, author = {Altindiş, M and Kahraman Kilbaş, EP}, title = {Managing Viral Emerging Infectious Diseases via Current and Future Molecular Diagnostics.}, journal = {Diagnostics (Basel, Switzerland)}, volume = {13}, number = {8}, pages = {}, doi = {10.3390/diagnostics13081421}, pmid = {37189522}, issn = {2075-4418}, abstract = {Emerging viral infectious diseases have been a constant threat to global public health in recent times. In managing these diseases, molecular diagnostics has played a critical role. Molecular diagnostics involves the use of various technologies to detect the genetic material of various pathogens, including viruses, in clinical samples. One of the most commonly used molecular diagnostics technologies for detecting viruses is polymerase chain reaction (PCR). PCR amplifies specific regions of the viral genetic material in a sample, making it easier to detect and identify viruses. PCR is particularly useful for detecting viruses that are present in low concentrations in clinical samples, such as blood or saliva. Another technology that is becoming increasingly popular for viral diagnostics is next-generation sequencing (NGS). NGS can sequence the entire genome of a virus present in a clinical sample, providing a wealth of information about the virus, including its genetic makeup, virulence factors, and potential to cause an outbreak. NGS can also help identify mutations and discover new pathogens that could affect the efficacy of antiviral drugs and vaccines. In addition to PCR and NGS, there are other molecular diagnostics technologies that are being developed to manage emerging viral infectious diseases. One of these is CRISPR-Cas, a genome editing technology that can be used to detect and cut specific regions of viral genetic material. CRISPR-Cas can be used to develop highly specific and sensitive viral diagnostic tests, as well as to develop new antiviral therapies. In conclusion, molecular diagnostics tools are critical for managing emerging viral infectious diseases. PCR and NGS are currently the most commonly used technologies for viral diagnostics, but new technologies such as CRISPR-Cas are emerging. These technologies can help identify viral outbreaks early, track the spread of viruses, and develop effective antiviral therapies and vaccines.}, }
@article {pmid37189462, year = {2023}, author = {Ishikawa, K and Saitoh, S}, title = {Transcriptional Regulation Technology for Gene Perturbation in Fission Yeast.}, journal = {Biomolecules}, volume = {13}, number = {4}, pages = {}, doi = {10.3390/biom13040716}, pmid = {37189462}, issn = {2218-273X}, abstract = {Isolation and introduction of genetic mutations is the primary approach to characterize gene functions in model yeasts. Although this approach has proven very powerful, it is not applicable to all genes in these organisms. For example, introducing defective mutations into essential genes causes lethality upon loss of function. To circumvent this difficulty, conditional and partial repression of target transcription is possible. While transcriptional regulation techniques, such as promoter replacement and 3' untranslated region (3'UTR) disruption, are available for yeast systems, CRISPR-Cas-based technologies have provided additional options. This review summarizes these gene perturbation technologies, including recent advances in methods based on CRISPR-Cas systems for Schizosaccharomyces pombe. We discuss how biological resources afforded by CRISPRi can promote fission yeast genetics.}, }
@article {pmid37187674, year = {2023}, author = {Liu, J and Carmichael, C and Hasturk, H and Shi, W and Bor, B}, title = {Rapid specific detection of oral bacteria using Cas13-based SHERLOCK.}, journal = {Journal of oral microbiology}, volume = {15}, number = {1}, pages = {2207336}, pmid = {37187674}, issn = {2000-2297}, abstract = {Decades of ongoing research has established that oral microbial communities play a role in oral diseases such as periodontitis and caries. Yet the detection of oral bacteria and the profiling of oral polymicrobial communities currently rely on methods that are costly, slow, and technically complex, such as qPCR or next-generation sequencing. For the widescale screening of oral microorganisms suitable for point-of-care settings, there exists the need for a low-cost, rapid detection technique. Here, we tailored the novel CRISPR-Cas-based assay SHERLOCK for the species-specific detection of oral bacteria. We developed a computational pipeline capable of generating constructs suitable for SHERLOCK and experimentally validated the detection of seven oral bacteria. We achieved detection within the single-molecule range that remained specific in the presence of off-target DNA found within saliva. Further, we adapted the assay for detecting target sequences directly from unprocessed saliva samples. The results of our detection, when tested on 30 healthy human saliva samples, fully aligned with 16S rRNA sequencing. Looking forward, this method of detecting oral bacteria is highly scalable and can be easily optimized for implementation at point-of-care settings.}, }
@article {pmid37186473, year = {2023}, author = {Sahel, DK and Goswami, SG and Jatyan, R and Kumari, A and Mittal, A and Ramalingam, S and Chitkara, D}, title = {Lipopolymeric nanocarrier enables effective delivery of CRISPR/Cas9 expressing plasmid.}, journal = {Macromolecular rapid communications}, volume = {}, number = {}, pages = {e2300101}, doi = {10.1002/marc.202300101}, pmid = {37186473}, issn = {1521-3927}, abstract = {CRISPR/Cas9 has proven its accuracy and precision for gene editing by making a double-strand break at the predetermined nucleic acid sequence. Despite being a mainstream gene editing tool, CRISPR/Cas9 has limitations for its in vivo delivery due to the physico-chemical properties such as high molecular weight, supranegative charge, degradation in the presence of nucleases in the biological fluid, etc. Viral vector has been vastly used to deliver CRISPR/Cas components but possesses ample drawbacks and is challenging to translate. We hereby explored a cationic lipopolymer, i.e, mPEG b-(CB-{g-cationic chain; g-Chol; g-Morph}) for its efficiency in delivering CRISPR/Cas9 plasmid (pCas9) in vitro and in vivo. The polymer was utilized to form blank cationic nanoplexes having a zeta potential of +15.8 ± 0.7 mV. Being cationic, the blank nanoplexes were able to condense the pCas9 plasmid at a ratio of 1:20 with a complexation efficiency of ∼98% and showed a size and zeta potential of ∼141 ± 16 nm and 4.2 mV ± 0.7, respectively. The pCas9-loaded nanoplexes showed a transfection efficiency of ∼69% in ARPE-19 cells and showed ∼22% of indel frequency indicating the successful translation of Cas9 protein and guide RNA in the cytosol. Further, they were found to be stable under in vivo environment when given intravenously in Swiss albino mice. These lipopolymeric nanoplexes could be a potential carrier for CRISPR plasmids for genome editing applications. This article is protected by copyright. All rights reserved.}, }
@article {pmid37186287, year = {2023}, author = {Luelf, UJ and Böhmer, LM and Li, S and Urlacher, VB}, title = {Effect of chromosomal integration on catalytic performance of a multi-component P450 system in Escherichia coli.}, journal = {Biotechnology and bioengineering}, volume = {}, number = {}, pages = {}, doi = {10.1002/bit.28404}, pmid = {37186287}, issn = {1097-0290}, abstract = {Cytochromes P450 are useful biocatalysts in synthetic chemistry and important bio-bricks in synthetic biology. Almost all bacterial P450s require separate redox partners for their activity, which are often expressed in recombinant Escherichia coli using multiple plasmids. However, the application of CRISPR/Cas recombineering facilitated chromosomal integration of heterologous genes which enables more stable and tunable expression of multi-component P450 systems for whole-cell biotransformations. Herein, we compared three E. coli strains W3110, JM109, and BL21(DE3) harboring three heterologous genes encoding a P450 and two redox partners either on plasmids or after chromosomal integration in two genomic loci. Both loci proved to be reliable and comparable for the model regio- and stereoselective two-step oxidation of (S)-ketamine. Furthermore, the CRISPR/Cas-assisted integration of the T7 RNA polymerase gene enabled an easy extension of T7 expression strains. Higher titers of soluble active P450 were achieved in E. coli harboring a single chromosomal copy of the P450 gene compared to E. coli carrying a medium copy pET plasmid. In addition, improved expression of both redox partners after chromosomal integration resulted in up to 80% higher (S)-ketamine conversion and more than fourfold increase in total turnover numbers.}, }
@article {pmid37186230, year = {2023}, author = {Scrascia, M and Roberto, R and D'Addabbo, P and Ahmed, Y and Porcelli, F and Oliva, M and Calia, C and Marzella, A and Pazzani, C}, title = {Bioinformatic survey of CRISPR loci across 15 Serratia species.}, journal = {MicrobiologyOpen}, volume = {12}, number = {2}, pages = {e1339}, doi = {10.1002/mbo3.1339}, pmid = {37186230}, issn = {2045-8827}, abstract = {The Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated proteins (CRISPR-Cas) system of prokaryotes is an adaptative immune defense mechanism to protect themselves from invading genetic elements (e.g., phages and plasmids). Studies that describe the genetic organization of these prokaryotic systems have mainly reported on the Enterobacteriaceae family (now reorganized within the order of Enterobacterales). For some genera, data on CRISPR-Cas systems remain poor, as in the case of Serratia (now part of the Yersiniaceae family) where data are limited to a few genomes of the species marcescens. This study describes the detection, in silico, of CRISPR loci in 146 Serratia complete genomes and 336 high-quality assemblies available for the species ficaria, fonticola, grimesii, inhibens, liquefaciens, marcescens, nematodiphila, odorifera, oryzae, plymuthica, proteomaculans, quinivorans, rubidaea, symbiotica, and ureilytica. Apart from subtypes I-E and I-F1 which had previously been identified in marcescens, we report that of I-C and the I-E unique locus 1, I-E*, and I-F1 unique locus 1. Analysis of the genomic contexts for CRISPR loci revealed mdtN-phnP as the region mostly shared (grimesii, inhibens, marcescens, nematodiphila, plymuthica, rubidaea, and Serratia sp.). Three new contexts detected in genomes of rubidaea and fonticola (puu genes-mnmA) and rubidaea (osmE-soxG and ampC-yebZ) were also found. The plasmid and/or phage origin of spacers was also established.}, }
@article {pmid37184565, year = {2023}, author = {Ronai, I}, title = {How molecular techniques are developed from natural systems.}, journal = {Genetics}, volume = {}, number = {}, pages = {}, doi = {10.1093/genetics/iyad067}, pmid = {37184565}, issn = {1943-2631}, abstract = {A striking characteristic of the molecular techniques of genetics is that they are derived from natural occurring systems. RNA interference, for example, utilizes a mechanism that evolved in eukaryotes to destroy foreign nucleic acid. Other case studies I highlight are restriction enzymes, DNA sequencing, polymerase chain reaction, gene targeting, fluorescent proteins (such as, green fluorescent protein), induced pluripotent stem cells, and clustered regularly interspaced short palindromic repeats-CRISPR associated 9. The natural systems' strategy for technique development means that biologists utilize the activity of a mechanism's effector (protein or RNA) and exploit biological specificity (protein or nucleic acid can cause precise reactions). I also argue that the developmental trajectory of novel molecular techniques, such as RNA interference, has 4 characteristic phases. The first phase is discovery of a biological phenomenon. The second phase is identification of the biological mechanism's trigger(s): the effector and biological specificity. The third phase is the application of the trigger(s) as a technique. The final phase is the maturation and refinement of the technique. Developing new molecular techniques from nature is crucial for future genetic research.}, }
@article {pmid37183363, year = {2023}, author = {Comunanza, V and Gigliotti, C and Lamba, S and Doronzo, G and Vallariello, E and Martin, V and Isella, C and Medico, E and Bardelli, A and Sangiolo, D and Di Nicolantonio, F and Bussolino, F}, title = {Dual VEGFA/BRAF targeting boosts PD-1 blockade in melanoma through GM-CSF-mediated infiltration of M1 macrophages.}, journal = {Molecular oncology}, volume = {}, number = {}, pages = {}, doi = {10.1002/1878-0261.13450}, pmid = {37183363}, issn = {1878-0261}, abstract = {The introduction of targeted therapies represented one of the most significant advances in the treatment of BRAFV600E melanoma. However, the onset of acquired resistance remains a challenge. Previously, we showed in mouse xenografts that vascular endothelial growth factor (VEGFA) removal enhanced the antitumor effect of BRAF inhibition through the recruitment of M1 macrophages. In this work, we explored the strategy of VEGFA/BRAF inhibition in immunocompetent melanoma murine models. In BRAF mutant D4M melanoma tumors, VEGFA/BRAF targeting reshaped the tumor microenvironment, largely by stimulating infiltration of M1 macrophages and CD8[+] T cells, and sensitized tumors to immune checkpoint blockade (ICB). Further, we reported that the association of VEGFA/BRAF targeting with anti-PD-1 antibody (triple therapy) resulted in a durable response and enabled complete tumor eradication in 50% of the mice, establishing immunological memory. Neutralization and CRISPR-Cas-mediated editing of granulocyte-macrophage colony-stimulating factor (GM-CSF) abrogated anti-tumor response prompted by triple therapy and identified GM-CSF as the cytokine instrumental in M1-macrophage recruitment. Our data suggest that VEGFA/BRAF targeting in melanoma induces the activation of innate and adaptive immunity and prepares tumors for ICB. Our study contributes to understanding the tumor biology of BRAFV600E melanoma, and suggests VEGFA as therapeutic target.}, }
@article {pmid37183261, year = {2023}, author = {Mijiti, M and Maimaiti, A and Chen, X and Tuersun, M and Dilixiati, M and Dilixiati, Y and Zhu, G and Wu, H and Li, Y and Turhon, M and Abulaiti, A and Maimaitiaili, N and Yiming, N and Kasimu, M and Wang, Y}, title = {CRISPR-cas9 screening identified lethal genes enriched in Hippo kinase pathway and of predictive significance in primary low-grade glioma.}, journal = {Molecular medicine (Cambridge, Mass.)}, volume = {29}, number = {1}, pages = {64}, pmid = {37183261}, issn = {1528-3658}, mesh = {Humans ; Hippo Signaling Pathway ; CRISPR-Cas Systems/genetics ; Genes, Lethal ; *Glioma/genetics ; Oncogenes ; *Brain Neoplasms/genetics ; }, abstract = {BACKGROUND: Low-grade gliomas (LGG) are a type of brain tumor that can be lethal, and it is essential to identify genes that are correlated with patient prognosis. In this study, we aimed to use CRISPR-cas9 screening data to identify key signaling pathways and develop a genetic signature associated with high-risk, low-grade glioma patients.<br><br>METHODS: The study used CRISPR-cas9 screening data to identify essential genes correlated with cell survival in LGG. We used RNA-seq data to identify differentially expressed genes (DEGs) related to cell viability. Moreover, we used the least absolute shrinkage and selection operator (LASSO) method to construct a genetic signature for predicting overall survival in patients. We performed enrichment analysis to identify pathways mediated by DEGs, overlapping genes, and genes shared in the Weighted correlation network analysis (WGCNA). Finally, the study used western blot, qRT-PCR, and IHC to detect the expression of hub genes from signature in clinical samples.<br><br>RESULTS: The study identified 145 overexpressed oncogenes in low-grade gliomas using the TCGA database. These genes were intersected with lethal genes identified in the CRISPR-cas9 screening data from Depmap database, which are enriched in Hippo pathways. A total of 19 genes were used to construct a genetic signature, and the Hippo signaling pathway was found to be the predominantly enriched pathway. The signature effectively distinguished between low- and high-risk patients, with high-risk patients showing a shorter overall survival duration. Differences in hub gene expression were found in different clinical samples, with the protein and mRNA expression of REP65 being significantly up-regulated in tumor cells. The study suggests that the Hippo signaling pathway may be a critical regulator of viability and tumor proliferation and therefore is an innovative new target for treating cancerous brain tumors, including low-grade gliomas.<br><br>CONCLUSION: Our study identified a novel genetic signature associated with high-risk, LGG patients. We found that the Hippo signaling pathway was significantly enriched in this signature, indicating that it may be a critical regulator of tumor viability and proliferation in LGG. Targeting the Hippo pathway could be an innovative new strategy for treating LGG.}, }
@article {pmid37182461, year = {2023}, author = {Li, L and Hong, F and Pan, S and Ren, L and Xiao, R and Liu, P and Li, N and Wang, J and Chen, Y}, title = {"Lollipop" particle counting immunoassay based on antigen-powered CRISPR-Cas12a dual signal amplification for the sensitive detection of deoxynivalenol in the environment and food samples.}, journal = {Journal of hazardous materials}, volume = {455}, number = {}, pages = {131573}, doi = {10.1016/j.jhazmat.2023.131573}, pmid = {37182461}, issn = {1873-3336}, abstract = {Deoxynivalenol is one of the most widely distributed mycotoxins in cereals and poses tremendous threats to the agricultural environment and public health. Therefore, it is particularly important to develop sensitive and interference-resistant deoxynivalenol analysis methods. Here, we establish a "Lollipop" particle counting immunoassay (LPCI) based on antigen-powered CRISPR-Cas12a dual signal amplification. LPCI achieves high sensitivity and accuracy through antigen-powered CRISPR-Cas dual signal amplification combined with particle counting immunoassay. This strategy not only broadens the applicability of the CRISPR-Cas system in the field of non-nucleic acid target detection; it also improves the sensitivity of particle counting immunoassay. The introduction of a polystyrene "lollipop" immunoassay carrier further enables efficiently simultaneous pre-treatment of multiple samples and overcomes complex matrix interference in real samples. The linear detection range of LPCI for deoxynivalenol was 0.1-500 ng/mL with a detection limit of 0.061 ng/mL. The platform greatly broadens the scope of the CRISPR-Cas sensor for the detection of non-nucleic acid hazards in the environment and food samples.}, }
@article {pmid37179485, year = {2023}, author = {Thevendran, R and Maheswaran, S}, title = {Recognizing CRISPR as the New Age Disease-Modifying Drug: Strategies to Bioengineer CRISPR/Cas for Direct in vivo Delivery.}, journal = {Biotechnology journal}, volume = {}, number = {}, pages = {e2300077}, doi = {10.1002/biot.202300077}, pmid = {37179485}, issn = {1860-7314}, abstract = {CRISPR has established itself as a frontier technology in genetic engineering. Researchers have successfully used the CRISPR/Cas system as precise gene editing tools and have further expanded their scope beyond both imaging and diagnostic applications. The most prominent utility of CRISPR is its capacity for gene therapy, serving as the contemporary, disease-modifying drug at the genetic level of human medical disorders. Correcting these diseases using CRISPR-based gene editing has developed to the extent of preclinical trials and possible patient treatments. A major impediment in actualizing this is the complications associated with in vivo delivery of the CRISPR/Cas complex. Currently, only the viral vectors (e.g. lentivirus) and non-viral encapsulation (e.g. lipid particles, polymer-based and gold nanoparticles) techniques have been extensively reviewed, neglecting the efficiency of direct delivery. However, the direct delivery of CRISPR/Cas for in vivo gene editing therapies is an intricate process with numerous drawbacks. Hence, this paper discusses in detail both the need and the strategies that can potentially improve the direct delivery aspects of CRISPR/Cas biomolecules for gene therapy of human diseases. Here, we focus on enhancing the molecular and functional features of the CRISPR/Cas system for targeted in vivo delivery such as on-site localization, internalization, reduced immunogenicity, and better in vivo stability. We additionally emphasize the CRISPR/Cas complex as a multifaceted, biomolecular vehicle for co-delivery with therapeutic agents in targeted disease treatments. The delivery formats of efficient CRISPR/Cas systems for human gene editing are also briefly elaborated. This article is protected by copyright. All rights reserved.}, }
@article {pmid37176969, year = {2023}, author = {Ijaz, M and Khan, F and Zaki, HEM and Khan, MM and Radwan, KSA and Jiang, Y and Qian, J and Ahmed, T and Shahid, MS and Luo, J and Li, B}, title = {Recent Trends and Advancements in CRISPR-Based Tools for Enhancing Resistance against Plant Pathogens.}, journal = {Plants (Basel, Switzerland)}, volume = {12}, number = {9}, pages = {}, pmid = {37176969}, issn = {2223-7747}, abstract = {Targeted genome editing technologies are becoming the most important and widely used genetic tools in studies of phytopathology. The "clustered regularly interspaced short palindromic repeats (CRISPR)" and its accompanying proteins (Cas) have been first identified as a natural system associated with the adaptive immunity of prokaryotes that have been successfully used in various genome-editing techniques because of its flexibility, simplicity, and high efficiency in recent years. In this review, we have provided a general idea about different CRISPR/Cas systems and their uses in phytopathology. This review focuses on the benefits of knock-down technologies for targeting important genes involved in the susceptibility and gaining resistance against viral, bacterial, and fungal pathogens by targeting the negative regulators of defense pathways of hosts in crop plants via different CRISPR/Cas systems. Moreover, the possible strategies to employ CRISPR/Cas system for improving pathogen resistance in plants and studying plant-pathogen interactions have been discussed.}, }
@article {pmid37176948, year = {2023}, author = {Gajardo, HA and Gómez-Espinoza, O and Boscariol Ferreira, P and Carrer, H and Bravo, LA}, title = {The Potential of CRISPR/Cas Technology to Enhance Crop Performance on Adverse Soil Conditions.}, journal = {Plants (Basel, Switzerland)}, volume = {12}, number = {9}, pages = {}, pmid = {37176948}, issn = {2223-7747}, abstract = {Worldwide food security is under threat in the actual scenery of global climate change because the major staple food crops are not adapted to hostile climatic and soil conditions. Significant efforts have been performed to maintain the actual yield of crops, using traditional breeding and innovative molecular techniques to assist them. However, additional strategies are necessary to achieve the future food demand. Clustered regularly interspaced short palindromic repeat/CRISPR-associated protein (CRISPR/Cas) technology, as well as its variants, have emerged as alternatives to transgenic plant breeding. This novelty has helped to accelerate the necessary modifications in major crops to confront the impact of abiotic stress on agriculture systems. This review summarizes the current advances in CRISPR/Cas applications in crops to deal with the main hostile soil conditions, such as drought, flooding and waterlogging, salinity, heavy metals, and nutrient deficiencies. In addition, the potential of extremophytes as a reservoir of new molecular mechanisms for abiotic stress tolerance, as well as their orthologue identification and edition in crops, is shown. Moreover, the future challenges and prospects related to CRISPR/Cas technology issues, legal regulations, and customer acceptance will be discussed.}, }
@article {pmid37176822, year = {2023}, author = {Eckerstorfer, MF and Dolezel, M and Engelhard, M and Giovannelli, V and Grabowski, M and Heissenberger, A and Lener, M and Reichenbecher, W and Simon, S and Staiano, G and Wüst Saucy, AG and Zünd, J and Lüthi, C}, title = {Recommendations for the Assessment of Potential Environmental Effects of Genome-Editing Applications in Plants in the EU.}, journal = {Plants (Basel, Switzerland)}, volume = {12}, number = {9}, pages = {}, pmid = {37176822}, issn = {2223-7747}, abstract = {The current initiative of the European Commission (EC) concerning plants produced using certain new genomic techniques, in particular, targeted mutagenesis and cisgenesis, underlines that a high level of protection for human and animal health and the environment needs to be maintained when using such applications. The current EU biosafety regulation framework ensures a high level of protection with a mandatory environmental risk assessment (ERA) of genetically modified (GM) products prior to the authorization of individual GMOs for environmental release or marketing. However, the guidance available from the European Food Safety Authority (EFSA) for conducting such an ERA is not specific enough regarding the techniques under discussion and needs to be further developed to support the policy goals towards ERA, i.e., a case-by-case assessment approach proportionate to the respective risks, currently put forward by the EC. This review identifies important elements for the case-by-case approach for the ERA that need to be taken into account in the framework for a risk-oriented regulatory approach. We also discuss that the comparison of genome-edited plants with plants developed using conventional breeding methods should be conducted at the level of a scientific case-by-case assessment of individual applications rather than at a general, technology-based level. Our considerations aim to support the development of further specific guidance for the ERA of genome-edited plants.}, }
@article {pmid37175878, year = {2023}, author = {Karginov, AV and Tarutina, MG and Lapteva, AR and Pakhomova, MD and Galliamov, AA and Filkin, SY and Fedorov, AN and Agaphonov, MO}, title = {A Split-Marker System for CRISPR-Cas9 Genome Editing in Methylotrophic Yeasts.}, journal = {International journal of molecular sciences}, volume = {24}, number = {9}, pages = {}, pmid = {37175878}, issn = {1422-0067}, mesh = {*Gene Editing ; *CRISPR-Cas Systems/genetics ; Plasmids/genetics ; }, abstract = {Methylotrophic yeasts such as Ogataea polymorpha and Komagataella phaffii (sin. Hansenula polymorpha and Pichia pastoris, respectively) are commonly used in basic research and biotechnological applications, frequently those requiring genome modifications. However, the CRISPR-Cas9 genome editing approaches reported for these species so far are relatively complex and laborious. In this work we present an improved plasmid vector set for CRISPR-Cas9 genome editing in methylotrophic yeasts. This includes a plasmid encoding Cas9 with a nuclear localization signal and plasmids with a scaffold for the single guide RNA (sgRNA). Construction of a sgRNA gene for a particular target sequence requires only the insertion of a 24 bp oligonucleotide duplex into the scaffold. Prior to yeast transformation, each plasmid is cleaved at two sites, one of which is located within the selectable marker, so that the functional marker can be restored only via recombination of the Cas9-containing fragment with the sgRNA gene-containing fragment. This recombination leads to the formation of an autonomously replicating plasmid, which can be lost from yeast clones after acquisition of the required genome modification. The vector set allows the use of G418-resistance and LEU2 auxotrophic selectable markers. The functionality of this setup has been demonstrated in O. polymorpha, O. parapolymorpha, O. haglerorum and Komagataella phaffii.}, }
@article {pmid37175730, year = {2023}, author = {Sheng, X and Ai, Z and Tan, Y and Hu, Y and Guo, X and Liu, X and Sun, Z and Yu, D and Chen, J and Tang, N and Duan, M and Yuan, D}, title = {Novel Salinity-Tolerant Third-Generation Hybrid Rice Developed via CRISPR/Cas9-Mediated Gene Editing.}, journal = {International journal of molecular sciences}, volume = {24}, number = {9}, pages = {}, pmid = {37175730}, issn = {1422-0067}, mesh = {*Gene Editing ; CRISPR-Cas Systems/genetics ; *Oryza/genetics ; Salinity ; Plant Breeding/methods ; }, abstract = {Climate change has caused high salinity in many fields, particularly in the mud flats in coastal regions. The resulting salinity has become one of the most significant abiotic stresses affecting the world's rice crop productivity. Developing elite cultivars with novel salinity-tolerance traits is regarded as the most cost-effective and environmentally friendly approach for utilizing saline-alkali land. To develop a highly efficient green strategy and create novel rice germplasms for salt-tolerant rice breeding, this study aimed to improve rice salinity tolerance by combining targeted CRISPR/Cas9-mediated editing of the OsRR22 gene with heterosis utilization. The novel alleles of the genic male-sterility (GMS) and elite restorer line (733S[rr22]-T1447-1 and HZ[rr22]-T1349-3) produced 110 and 1 bp deletions at the third exon of OsRR22 and conferred a high level of salinity tolerance. Homozygous transgene-free progeny were identified via segregation in the T2 generation, with osrr22 showing similar agronomic performance to wild-type (733S and HZ). Furthermore, these two osrr22 lines were used to develop a new promising third-generation hybrid rice line with novel salinity tolerance. Overall, the results demonstrate that combining CRISPR/Cas9 targeted gene editing with the "third-generation hybrid rice system" approach allows for the efficient development of novel hybrid rice varieties that exhibit a high level of salinity tolerance, thereby ensuring improved cultivar stability and enhanced rice productivity.}, }
@article {pmid37175696, year = {2023}, author = {Muto, V and Benigni, F and Magliocca, V and Borghi, R and Flex, E and Pallottini, V and Rosa, A and Compagnucci, C and Tartaglia, M}, title = {CRISPR/Cas9 and piggyBac Transposon-Based Conversion of a Pathogenic Biallelic TBCD Variant in a Patient-Derived iPSC Line Allows Correction of PEBAT-Related Endophenotypes.}, journal = {International journal of molecular sciences}, volume = {24}, number = {9}, pages = {}, pmid = {37175696}, issn = {1422-0067}, mesh = {Animals ; Humans ; *CRISPR-Cas Systems/genetics ; *Induced Pluripotent Stem Cells ; Endophenotypes ; Cell Differentiation/genetics ; Gene Editing ; Mutation ; Microtubule-Associated Proteins/metabolism ; }, abstract = {Induced pluripotent stem cells (iPSCs) have been established as a reliable in vitro disease model system and represent a particularly informative tool when animal models are not available or do not recapitulate the human pathophenotype. The recognized limit in using this technology is linked to some degree of variability in the behavior of the individual patient-derived clones. The development of CRISPR/Cas9-based gene editing solves this drawback by obtaining isogenic iPSCs in which the genetic lesion is corrected, allowing a straightforward comparison with the parental patient-derived iPSC lines. Here, we report the generation of a footprint-free isogenic cell line of patient-derived TBCD-mutated iPSCs edited using the CRISPR/Cas9 and piggyBac technologies. The corrected iPSC line had no genetic footprint after the removal of the selection cassette and maintained its "stemness". The correction of the disease-causing TBCD missense substitution restored proper protein levels of the chaperone and mitotic spindle organization, as well as reduced cellular death, which were used as read-outs of the TBCD KO-related endophenotype. The generated line represents an informative in vitro model to understand the impact of pathogenic TBCD mutations on nervous system development and physiology.}, }
@article {pmid37174639, year = {2023}, author = {Lu, J and Ding, Y and Zhang, W and Qi, Y and Zhou, J and Xu, N and Zhang, Y and Xie, W}, title = {SQSTM1/p62 Knockout by Using the CRISPR/Cas9 System Inhibits Migration and Invasion of Hepatocellular Carcinoma.}, journal = {Cells}, volume = {12}, number = {9}, pages = {}, pmid = {37174639}, issn = {2073-4409}, mesh = {Animals ; Mice ; *Carcinoma, Hepatocellular/genetics/metabolism ; Kelch-Like ECH-Associated Protein 1/genetics/metabolism ; Sequestosome-1 Protein/genetics/metabolism ; NF-E2-Related Factor 2/genetics/metabolism ; Mice, Nude ; CRISPR-Cas Systems/genetics ; *Liver Neoplasms/genetics/metabolism ; Mice, Knockout ; Tumor Microenvironment ; }, abstract = {Migration and invasion play crucial roles in the progression of hepatocellular carcinoma (HCC), but the underlying mechanisms are not clear. Analysis of clinical samples indicates that SQSTM1/p62 is highly expressed in HCC and seriously affects the prognosis of patients. Subsequently, we showed that SQSTM1/p62 knockout using the CRISPR/Cas9 system led to impaired migration and invasion of HCC, upregulated Keap1, and promoted the inhibitory effect of Keap1 on Nrf2. Then, the inactivation of Nrf2 inhibited the expression of matrix metalloproteinases (MMPs), thus attenuating the migration and invasion of HCC. We also found that SQSTM1/p62 knockout significantly inhibited migration and invasion in a lung metastasis model of nude mice with HCC. Furthermore, we found that cisplatin not only significantly inhibited the expression of SQSTM1/p62 but also slowed down the migration and invasion of HCC, while the inflammatory microenvironment accelerated the migration and invasion of HCC. These results suggest for the first time that SQSTM1/p62 knockout inhibits the migration and invasion of HCC through the Keap1/Nrf2/MMP2 signaling pathway. SQSTM1/p62 may be developed into a key drug target to regulate the migration and invasion of HCC cells.}, }
@article {pmid37173533, year = {2023}, author = {Perk, EA and Arruebarrena Di Palma, A and Colman, S and Mariani, O and Cerrudo, I and D'Ambrosio, JM and Robuschi, L and Pombo, MA and Rosli, HG and Villareal, F and Laxalt, AM}, title = {CRISPR/Cas9-mediated phospholipase C 2 knock-out tomato plants are more resistant to Botrytis cinerea.}, journal = {Planta}, volume = {257}, number = {6}, pages = {117}, pmid = {37173533}, issn = {1432-2048}, mesh = {*Type C Phospholipases/metabolism ; *Solanum lycopersicum/genetics ; CRISPR-Cas Systems ; Plant Proteins/genetics/metabolism ; Reactive Oxygen Species/metabolism ; Oxylipins/metabolism ; Plant Breeding ; Botrytis/metabolism ; Phospholipases/genetics/metabolism ; Plant Diseases/genetics/microbiology ; Disease Resistance/genetics ; Gene Expression Regulation, Plant ; }, abstract = {CRISPR/Cas9-mediated Phospholipase C2 knock-out tomato plants are more resistant to Botrytis cinerea than wild-type plants, with less ROS and an increase and reduction of (JA) and (SA)-response marker genes, respectively. Genome-editing technologies allow non-transgenic site-specific mutagenesis of crops, offering a viable alternative to traditional breeding methods. In this study we used CRISPR/Cas9 to inactivate the tomato Phospholipase C2 gene (SlPLC2). Plant PLC activation is one of the earliest responses triggered by different pathogens regulating plant responses that, depending on the plant-pathogen interaction, result in plant resistance or susceptibility. The tomato (Solanum lycopersicum) PLC gene family has six members, named from SlPLC1 to SlPLC6. We previously showed that SlPLC2 transcript levels increased upon xylanase infiltration (fungal elicitor) and that SlPLC2 participates in plant susceptibility to Botrytis cinerea. An efficient strategy to control diseases caused by pathogens is to disable susceptibility genes that facilitate infection. We obtained tomato SlPLC2-knock-out lines with decreased ROS production upon B. cinerea challenge. Since this fungus requires ROS-induced cell death to proliferate, SlPLC2-knock-out plants showed an enhanced resistance with smaller necrotic areas and reduced pathogen proliferation. Thus, we obtained SlPLC2 loss-of-function tomato lines more resistant to B. cinerea by means of CRISPR/Cas9 genome editing technology.}, }
@article {pmid37172090, year = {2023}, author = {Hany, D and Vafeiadou, V and Picard, D}, title = {CRISPR-Cas9 screen reveals a role of purine synthesis for estrogen receptor α activity and tamoxifen resistance of breast cancer cells.}, journal = {Science advances}, volume = {9}, number = {19}, pages = {eadd3685}, pmid = {37172090}, issn = {2375-2548}, mesh = {Humans ; Female ; *Tamoxifen/pharmacology ; *Breast Neoplasms/drug therapy/genetics/metabolism ; Estrogen Receptor alpha/genetics/metabolism ; CRISPR-Cas Systems ; Drug Resistance, Neoplasm/genetics ; Estrogens ; Purines/pharmacology ; Cell Line, Tumor ; Gene Expression Regulation, Neoplastic ; }, abstract = {In breast cancer, resistance to endocrine therapies that target estrogen receptor α (ERα), such as tamoxifen and fulvestrant, remains a major clinical problem. Whether and how ERα[+] breast cancers switch from being estrogen-dependent to estrogen-independent remains unclear. With a genome-wide CRISPR-Cas9 knockout screen, we identified previously unknown biomarkers and potential therapeutic targets of endocrine resistance. We demonstrate that high levels of PAICS, an enzyme involved in the de novo biosynthesis of purines, can shift the balance of ERα activity to be more estrogen-independent and tamoxifen-resistant. We find that this may be due to elevated activities of cAMP-activated protein kinase A and mTOR, kinases known to phosphorylate ERα specifically and to stimulate its activity. Genetic or pharmacological targeting of PAICS sensitizes tamoxifen-resistant cells to tamoxifen. Addition of purines renders them more resistant. On the basis of these findings, we propose the combined targeting of PAICS and ERα as a new, effective, and potentially safe therapeutic regimen.}, }
@article {pmid37172086, year = {2023}, author = {Bu, W and Creighton, CJ and Heavener, KS and Gutierrez, C and Dou, Y and Ku, AT and Zhang, Y and Jiang, W and Urrutia, J and Jiang, W and Yue, F and Jia, L and Ibrahim, AA and Zhang, B and Huang, S and Li, Y}, title = {Efficient cancer modeling through CRISPR-Cas9/HDR-based somatic precision gene editing in mice.}, journal = {Science advances}, volume = {9}, number = {19}, pages = {eade0059}, pmid = {37172086}, issn = {2375-2548}, mesh = {Animals ; Mice ; Humans ; *Gene Editing/methods ; CRISPR-Cas Systems/genetics ; *Neoplasms/genetics/therapy ; Recombinational DNA Repair ; Disease Models, Animal ; }, abstract = {CRISPR-Cas9 has been used successfully to introduce indels in somatic cells of rodents; however, precise editing of single nucleotides has been hampered by limitations of flexibility and efficiency. Here, we report technological modifications to the CRISPR-Cas9 vector system that now allows homology-directed repair-mediated precise editing of any proto-oncogene in murine somatic tissues to generate tumor models with high flexibility and efficiency. Somatic editing of either Kras or Pik3ca in both normal and hyperplastic mammary glands led to swift tumorigenesis. The resulting tumors shared some histological, transcriptome, and proteome features with tumors induced by lentivirus-mediated expression of the respective oncogenes, but they also exhibited some distinct characteristics, particularly showing less intertumor variation, thus potentially offering more consistent models for cancer studies and therapeutic development. Therefore, this technological advance fills a critical gap between the power of CRISPR technology and high-fidelity mouse models for studying human tumor evolution and preclinical drug testing.}, }
@article {pmid37170124, year = {2023}, author = {Karapurkar, JK and Kim, MS and Colaco, JC and Suresh, B and Sarodaya, N and Kim, DH and Park, CH and Hong, SH and Kim, KS and Ramakrishna, S}, title = {CRISPR/Cas9-based genome-wide screening of the deubiquitinase subfamily identifies USP3 as a protein stabilizer of REST blocking neuronal differentiation and promotes neuroblastoma tumorigenesis.}, journal = {Journal of experimental &amp; clinical cancer research : CR}, volume = {42}, number = {1}, pages = {121}, pmid = {37170124}, issn = {1756-9966}, mesh = {Humans ; Animals ; Mice ; *Transcription Factors/metabolism ; CRISPR-Cas Systems ; Ubiquitination ; Ubiquitin-Specific Proteases/metabolism ; Ubiquitin-Protein Ligases/metabolism ; *Neuroblastoma/genetics ; Cell Transformation, Neoplastic/genetics ; Cell Differentiation/genetics ; }, abstract = {BACKGROUND: The repressor element-1 silencing transcription factor (REST), a master transcriptional repressor, is essential for maintenance, self-renewal, and differentiation in neuroblastoma. An elevated expression of REST is associated with impaired neuronal differentiation, which results in aggressive neuroblastoma formation. E3 ligases are known to regulate REST protein abundance through the 26 S proteasomal degradation pathway in neuroblastoma. However, deubiquitinating enzymes (DUBs), which counteract the function of E3 ligase-mediated REST protein degradation and their impact on neuroblastoma tumorigenesis have remained unexplored.<br><br>METHODS: We employed a CRISPR/Cas9 system to perform a genome-wide knockout of ubiquitin-specific proteases (USPs) and used western blot analysis to screen for DUBs that regulate REST protein abundance. The interaction between USP3 and REST was confirmed by immunoprecipitation and Duolink in situ proximity assays. The deubiquitinating effect of USP3 on REST protein degradation, half-life, and neuronal differentiation was validated by immunoprecipitation, in vitro deubiquitination, protein-turnover, and immunostaining assays. The correlation between USP3 and REST expression was assessed using patient neuroblastoma datasets. The USP3 gene knockout in neuroblastoma cells was performed using CRISPR/Cas9, and the clinical relevance of USP3 regulating REST-mediated neuroblastoma tumorigenesis was confirmed by in vitro and in vivo oncogenic experiments.<br><br>RESULTS: We identified a deubiquitinase USP3 that interacts with, stabilizes, and increases the half-life of REST protein by counteracting its ubiquitination in neuroblastoma. An in silico analysis showed a correlation between USP3 and REST in multiple neuroblastoma cell lines and identified USP3 as a prognostic marker for overall survival in neuroblastoma patients. Silencing of USP3 led to a decreased self-renewal capacity and promoted retinoic acid-induced differentiation in neuroblastoma. A loss of USP3 led to attenuation of REST-mediated neuroblastoma tumorigenesis in a mouse xenograft model.<br><br>CONCLUSION: The findings of this study indicate that USP3 is a critical factor that blocks neuronal differentiation, which can lead to neuroblastoma. We envision that targeting USP3 in neuroblastoma tumors might provide an effective therapeutic differentiation strategy for improved survival rates of neuroblastoma patients.}, }
@article {pmid37166670, year = {2023}, author = {Cheng, S and Park, M and Yong, J}, title = {RNA and Protein Interactomes of an RNA-Binding Protein Tagged with FLAG Epitopes Using Combinatory Approaches of Genome Engineering and Stable Transfection.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2666}, number = {}, pages = {247-263}, pmid = {37166670}, issn = {1940-6029}, mesh = {*RNA/genetics ; Epitopes/genetics ; *RNA-Binding Proteins/metabolism ; Transfection ; Gene Editing/methods ; CRISPR-Cas Systems/genetics ; }, abstract = {To study the function of RNA-binding proteins (RBPs), an overexpression or knockout approach is generally used. However, as many RBPs are essential to cellular functions, the complete knockout of these proteins may be lethal to the cell. Overexpression of RBPs, on the other hand, may create an altered transcriptome and aberrant phenotypes that can mask their physiological function. Additionally, biochemical characterization of RBP often requires highly specific antibodies for efficient immunoprecipitation for downstream mass spectrometry or RNA footprinting profiling. To overcome these hurdles, we have developed a strategy to generate cellular systems either using a CRISPR-Cas9-mediated epitope tag knock-in approach or a two-step workflow to first stably express an exogenous Flag-tagged RBP and subsequently knockout the endogenous RBP using CRISPR-Cas9 gene editing. The generation of these cell lines will be beneficial for downstream RNA footprinting studies and mass spectrometry-mediated interactome studies.}, }
@article {pmid37166646, year = {2023}, author = {Blanco-Fernandez, J and Jourdain, AA}, title = {Dead-Seq: Discovering Synthetic Lethal Interactions from Dead Cells Genomics.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2661}, number = {}, pages = {329-342}, pmid = {37166646}, issn = {1940-6029}, mesh = {*Genomics/methods ; *Genome ; Genetic Testing/methods ; CRISPR-Cas Systems ; }, abstract = {Pooled genetic screens have revolutionized the field of functional genomics, yet perturbations that decrease fitness, such as those leading to synthetic lethality, have remained difficult to quantify at the genomic level. We and colleagues previously developed "death screening," a protocol based on the purification of dead cells in genetic screens, and used it to identify a set of genes necessary for mitochondrial gene expression, translation, and oxidative phosphorylation (OXPHOS), thus offering new possibilities for the diagnosis of mitochondrial disorders. Here, we describe Dead-Seq, a refined protocol for death screening that is compatible with most pooled screening protocols, including genome-wide CRISPR/Cas9 screening. Dead-Seq converts negative-selection screens into positive-selection screens and generates high-quality data directly from dead cells, at limited sequencing costs.}, }
@article {pmid37141850, year = {2023}, author = {Chen, X and Moran Torres, JP and Li, Y and Lugones, LG and Wösten, HAB}, title = {Inheritable CRISPR based epigenetic modification in a fungus.}, journal = {Microbiological research}, volume = {272}, number = {}, pages = {127397}, doi = {10.1016/j.micres.2023.127397}, pmid = {37141850}, issn = {1618-0623}, mesh = {Humans ; *CRISPR-Cas Systems ; *Epigenesis, Genetic ; DNA Methylation ; Gene Silencing ; Fungi/genetics ; Gene Editing/methods ; Mixed Function Oxygenases/genetics ; Proto-Oncogene Proteins/genetics ; }, abstract = {The CRISPRoff system was recently introduced as a programmable epigenetic memory writer that can be used to silence genes in human cells. The system makes use of a dead Cas9 protein (dCas9) that is fused with the ZNF10 KRAB, Dnmt3A, and Dnmt3L protein domains. The DNA methylation resulting from the CRISPRoff system can be removed by the CRISPRon system that consists of dCas9 fused to the catalytic domain of Tet1. Here, the CRISPRoff and CRISPRon systems were applied for the first time in a fungus. The CRISPRoff system resulted in an inactivation up to 100 % of the target genes flbA and GFP in Aspergillus niger. Phenotypes correlated with the degree of gene silencing in the transformants and were stable when going through a conidiation cycle, even when the CRISPRoff plasmid was removed from the flbA silenced strain. Introducing the CRISPRon system in a strain in which the CRISPRoff plasmid was removed fully reactivated flbA showing a phenotype similar to that of the wildtype. Together, the CRISPRoff and CRISPRon systems can be used to study gene function in A. niger.}, }
@article {pmid37119812, year = {2023}, author = {Yu, G and Kim, HK and Park, J and Kwak, H and Cheong, Y and Kim, D and Kim, J and Kim, J and Kim, HH}, title = {Prediction of efficiencies for diverse prime editing systems in multiple cell types.}, journal = {Cell}, volume = {186}, number = {10}, pages = {2256-2272.e23}, doi = {10.1016/j.cell.2023.03.034}, pmid = {37119812}, issn = {1097-4172}, mesh = {*Knowledge ; *Gene Editing ; CRISPR-Cas Systems ; }, abstract = {Applications of prime editing are often limited due to insufficient efficiencies, and it can require substantial time and resources to determine the most efficient pegRNAs and prime editors (PEs) to generate a desired edit under various experimental conditions. Here, we evaluated prime editing efficiencies for a total of 338,996 pairs of pegRNAs including 3,979 epegRNAs and target sequences in an error-free manner. These datasets enabled a systematic determination of factors affecting prime editing efficiencies. Then, we developed computational models, named DeepPrime and DeepPrime-FT, that can predict prime editing efficiencies for eight prime editing systems in seven cell types for all possible types of editing of up to 3 base pairs. We also extensively profiled the prime editing efficiencies at mismatched targets and developed a computational model predicting editing efficiencies at such targets. These computational models, together with our improved knowledge about prime editing efficiency determinants, will greatly facilitate prime editing applications.}, }
@article {pmid37116617, year = {2023}, author = {Wang, G and Wang, C and Chu, T and Wu, X and Anderson, CM and Huang, D and Li, J}, title = {Deleting specific residues from the HNH linkers creates a CRISPR-SpCas9 variant with high fidelity and efficiency.}, journal = {Journal of biotechnology}, volume = {368}, number = {}, pages = {42-52}, doi = {10.1016/j.jbiotec.2023.04.008}, pmid = {37116617}, issn = {1873-4863}, mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; DNA/genetics ; }, abstract = {Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) systems are immunological defenses used in archaea and bacteria to recognize and destroy DNA from external invaders. The CRISPR-SpCas9 system harnessed from Streptococcus pyogenes (SpCas9) has become the most widely utilized genome editing tool and shows promise for clinical application. However, the off-target effect is still the major challenge for the genome editing of CRISPR-SpCas9. Based on analysis of the structure and cleavage procedures, we proposed two strategies to modify the SpCas9 structure and reduce off-target effects. Shortening the HNH or REC3 linkers (Strategy #1) aimed to move the primary position of HNH or REC3 far away from the single-guide RNA (sgRNA)/DNA hybrid (hybrid), while elongating the helix around the sgRNA (Strategy #2) aimed to strengthen the contacts between SpCas9 and the sgRNA/DNA. We designed 11 SpCas9 variants (variant No.1- variant No.11) and verified their efficiencies on the classic genome site EMX1-1, EMX1-1-OT1, and EMX1-1-OT2. The top three effective SpCas9 variants, variant No.1, variant No.2, and variant No.5, were additionally validated on other genome sites. The further selected variant No.1 was compared with two previous SpCas9 variants, HypaCas9 (a hyper-accurate Cas9 variant released in 2017) and eSpCas9 (1.1) (an "enhanced specificity" SpCas9 variant released in 2016), on two genome sites, EMX1-1 and FANCF-1. The results revealed that the deletion of Thr769 and Gly906 could substantially decrease off-target effects, while maintaining robust on-target efficiency in most of the selected genome sites.}, }
@article {pmid36948241, year = {2023}, author = {Pham, NN and Chang, CW and Chang, YH and Tu, Y and Chou, JY and Wang, HY and Hu, YC}, title = {Rational genome and metabolic engineering of Candida viswanathii by split CRISPR to produce hundred grams of dodecanedioic acid.}, journal = {Metabolic engineering}, volume = {77}, number = {}, pages = {76-88}, doi = {10.1016/j.ymben.2023.03.007}, pmid = {36948241}, issn = {1096-7184}, mesh = {*Metabolic Engineering ; *Candida/genetics/metabolism ; Dicarboxylic Acids/metabolism ; CRISPR-Cas Systems ; }, abstract = {Candida viswanathii is a promising cell factory for producing dodecanedioic acid (DDA) and other long chain dicarboxylic acids. However, metabolic engineering of C. viswanathii is difficult partly due to the lack of synthetic biology toolkits. Here we developed CRISPR-based approaches for rational genome and metabolic engineering of C. viswanathii. We first optimized the CRISPR system and protocol to promote the homozygous gene integration efficiency to >60%. We also designed a split CRISPR system for one-step integration of multiple genes into C. viswanathii. We uncovered that co-expression of CYP52A19, CPRb and FAO2 that catalyze different steps in the biotransformation enhances DDA production and abolishes accumulation of intermediates. We also unveiled that co-expression of additional enzyme POS5 further promotes DDA production and augments cell growth. We harnessed the split CRISPR system to co-integrate these 4 genes (13.6 kb) into C. viswanathii and generated a stable strain that doubles the DDA titer (224 g/L), molar conversion (83%) and productivity (1.87 g/L/h) when compared with the parent strain. This study altogether identifies appropriate enzymes/promoters to augment dodecane conversion to DDA and implicates the potential of split CRISPR system for metabolic engineering of C. viswanathii.}, }
@article {pmid36864663, year = {2023}, author = {Rojek, JB and Basavaraju, Y and Nallapareddy, S and Bulté, DB and Baumgartner, R and Schoffelen, S and Grav, LM and Goletz, S and Pedersen, LE}, title = {Expanding the CRISPR toolbox for Chinese hamster ovary cells with comprehensive tools for Mad7 genome editing.}, journal = {Biotechnology and bioengineering}, volume = {120}, number = {6}, pages = {1478-1491}, doi = {10.1002/bit.28367}, pmid = {36864663}, issn = {1097-0290}, mesh = {Cricetinae ; Animals ; *Gene Editing ; Cricetulus ; CHO Cells ; *CRISPR-Cas Systems/genetics ; Gene Knockout Techniques ; Endonucleases/genetics ; }, abstract = {The production of high-value biopharmaceuticals is dominated by mammalian production cells, particularly Chinese hamster ovary (CHO) cells, which have been widely used and preferred in manufacturing processes. The discovery of CRISPR-Cas9 significantly accelerated cell line engineering advances, allowing for production yield and quality improvements. Since then, several other CRISPR systems have become appealing genome editing tools, such as the Cas12a nucleases, which provide broad editing capabilities while utilizing short guide RNAs (gRNAs) that reduce the complexity of the editing systems. One of these is the Mad7 nuclease, which has been shown to efficiently convey targeted gene disruption and insertions in several different organisms. In this study, we demonstrate that Mad7 can generate indels for gene knockout of host cell proteins in CHO cells. We found that the efficiency of Mad7 depends on the addition of protein nuclear localization signals and the gRNAs employed for genome targeting. Moreover, we provide computational tools to design Mad7 gRNAs against any genome of choice and for automated indel detection analysis from next-generation sequencing data. In summary, this paper establishes the application of Mad7 in CHO cells, thereby improving the CRISPR toolbox versatility for research and cell line engineering.}, }
@article {pmid36762520, year = {2023}, author = {Navaridas, R and Vidal-Sabanés, M and Ruiz-Mitjana, A and Perramon-Güell, A and Megino-Luque, C and Llobet-Navas, D and Matias-Guiu, X and Egea, J and Encinas, M and Bardia, L and Colombelli, J and Dolcet, X}, title = {Transient and DNA-free in vivo CRISPR/Cas9 genome editing for flexible modeling of endometrial carcinogenesis.}, journal = {Cancer communications (London, England)}, volume = {43}, number = {5}, pages = {620-624}, pmid = {36762520}, issn = {2523-3548}, mesh = {Humans ; *Gene Editing ; *CRISPR-Cas Systems ; DNA ; }, }
@article {pmid36734182, year = {2023}, author = {Cao, Y and Li, L and Ren, X and Mao, B and Yang, Y and Mi, H and Guan, Y and Li, S and Zhou, S and Guan, X and Yang, T and Zhao, X}, title = {CRISPR/Cas9 correction of a dominant cis-double-variant in COL1A1 isolated from a patient with osteogenesis imperfecta increases the osteogenic capacity of induced pluripotent stem cells.}, journal = {Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research}, volume = {38}, number = {5}, pages = {719-732}, doi = {10.1002/jbmr.4783}, pmid = {36734182}, issn = {1523-4681}, mesh = {Humans ; *Osteogenesis Imperfecta/genetics/therapy/pathology ; *Induced Pluripotent Stem Cells/pathology ; CRISPR-Cas Systems/genetics ; Mutation ; Collagen Type I/genetics ; }, abstract = {Osteogenesis imperfecta (OI) is a hereditary skeletal disorder that is mainly caused by variants in COL1A1/2. So far, no specific treatment has been developed to correct its underlying etiology. We aimed to gain a better understanding of the pathological mechanisms of OI and develop gene therapies to correct OI-causing variants. A de novel cis-double-variant c.[175C>T; 187T>A] in COL1A1 was identified from a 5-year-old OI patient by whole-exome sequencing (WES). Three peptide nucleic acids (PNAs) were designed and then transfected patient-derived fibroblasts. PNA2 affected the translational strand and induced an optimal interfering effect at 0.25μM concentration, proved by Sanger sequencing, qPCR, Western blot, and immunostaining. Additionally, induced pluripotent stem cells (iPSCs) were cultured from patient-derived fibroblasts. Clones of iPSCs with c.187T>A variant and those with both variants largely restored their osteogenic capacities after CRISPR/Cas9 gene editing, which corrected the variants. Importantly, correcting c.187T>A variant alone in CRISPR-edited iPSCs was sufficient to alleviate OI phenotypes, as indicated by increased levels of COL1A1, COL1A2, ALP mRNAs, and COL1A1 protein. Our findings suggest that c.187T>A is the dominant variant of cis-double-variant in COL1A1 that led to OI, and PNA interference and CRISPR/Cas9 gene editing may be new therapeutic tools for OI treatment. © 2023 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).}, }
@article {pmid36658005, year = {2023}, author = {Strobbe, S and Wesana, J and Van Der Straeten, D and De Steur, H}, title = {Public acceptance and stakeholder views of gene edited foods: a global overview.}, journal = {Trends in biotechnology}, volume = {41}, number = {6}, pages = {736-740}, doi = {10.1016/j.tibtech.2022.12.011}, pmid = {36658005}, issn = {1879-3096}, mesh = {*Gene Editing ; Plants, Genetically Modified/genetics ; *Crops, Agricultural/genetics ; Genome, Plant ; CRISPR-Cas Systems ; }, abstract = {The increasing popularity of gene editing in plants has prompted research on stakeholder views. Gene edited foods are often more accepted than genetically modified foods, though differences occur within target groups, regions, and products. Nevertheless, marketing challenges related to a lack of familiarity with the technology, labeling, and risk perception remain.}, }
@article {pmid37175977, year = {2023}, author = {Wettengel, JM and Hansen-Palmus, L and Yusova, S and Rust, L and Biswas, S and Carson, J and Ryu, J and Bimber, BN and Hennebold, JD and Burwitz, BJ}, title = {A Multifunctional and Highly Adaptable Reporter System for CRISPR/Cas Editing.}, journal = {International journal of molecular sciences}, volume = {24}, number = {9}, pages = {}, pmid = {37175977}, issn = {1422-0067}, support = {P51 OD011092/NH/NIH HHS/United States ; U24 OD026631-01/NH/NIH HHS/United States ; R21 AI108401-01/NH/NIH HHS/United States ; }, abstract = {CRISPR/Cas systems are some of the most promising tools for therapeutic genome editing. The use of these systems is contingent on the optimal designs of guides and homology-directed repair (HDR) templates. While this design can be achieved in silico, validation and further optimization are usually performed with the help of reporter systems. Here, we describe a novel reporter system, termed BETLE, that allows for the fast, sensitive, and cell-specific detection of genome editing and template-specific HDR by encoding multiple reporter proteins in different open-reading frames. Out-of-frame non-homologous end joining (NHEJ) leads to the expression of either secretable NanoLuc luciferase, enabling a highly sensitive and low-cost analysis of editing, or fluorescent mTagBFP2, allowing for the enumeration and tissue-specific localization of genome-edited cells. BETLE includes a site to validate CRISPR/Cas systems for a sequence-of-interest, making it broadly adaptable. We evaluated BETLE using a defective moxGFP with a 39-base-pair deletion and showed spCas9, saCas9, and asCas12a editing as well as sequence-specific HDR and the repair of moxGFP in cell lines with single and multiple reporter integrants. Taken together, these data show that BETLE allows for the rapid detection and optimization of CRISPR/Cas genome editing and HDR in vitro and represents a state-of the art tool for future applications in vivo.}, }
@article {pmid37175850, year = {2023}, author = {Glinšek, K and Bozovičar, K and Bratkovič, T}, title = {CRISPR Technologies in Chinese Hamster Ovary Cell Line Engineering.}, journal = {International journal of molecular sciences}, volume = {24}, number = {9}, pages = {}, pmid = {37175850}, issn = {1422-0067}, abstract = {The Chinese hamster ovary (CHO) cell line is a well-established platform for the production of biopharmaceuticals due to its ability to express complex therapeutic proteins with human-like glycopatterns in high amounts. The advent of CRISPR technology has opened up new avenues for the engineering of CHO cell lines for improved protein production and enhanced product quality. This review summarizes recent advances in the application of CRISPR technology for CHO cell line engineering with a particular focus on glycosylation modulation, productivity enhancement, tackling adventitious agents, elimination of problematic host cell proteins, development of antibiotic-free selection systems, site-specific transgene integration, and CRISPR-mediated gene activation and repression. The review highlights the potential of CRISPR technology in CHO cell line genome editing and epigenetic engineering for the more efficient and cost-effective development of biopharmaceuticals while ensuring the safety and quality of the final product.}, }
@article {pmid37174743, year = {2023}, author = {Melo-Silva, CR and Knudson, CJ and Tang, L and Kafle, S and Springer, LE and Choi, J and Snyder, CM and Wang, Y and Kim, SV and Sigal, LJ}, title = {Multiple and Consecutive Genome Editing Using i-GONAD and Breeding Enrichment Facilitates the Production of Genetically Modified Mice.}, journal = {Cells}, volume = {12}, number = {9}, pages = {}, doi = {10.3390/cells12091343}, pmid = {37174743}, issn = {2073-4409}, abstract = {Genetically modified (GM) mice are essential tools in biomedical research. Traditional methods for generating GM mice are expensive and require specialized personnel and equipment. The use of clustered regularly interspaced short palindromic repeats (CRISPR) coupled with improved-Genome editing via Oviductal Nucleic Acids Delivery (i-GONAD) has highly increased the feasibility of producing GM mice in research laboratories. However, genetic modification in inbred mouse strains of interest such as C57BL/6 (B6) is still challenging because of their low fertility and embryo fragility. We have successfully generated multiple novel GM mouse strains in the B6 background while attempting to optimize i-GONAD. We found that i-GONAD reduced the litter size in superovulated pregnant females but did not impact pregnancy rates. Natural mating or low-hormone dose did not increase the low fertility rate observed in superovulated B6 females. However, diet enrichment had a positive effect on pregnancy success. We also optimized breeding conditions to increase the survival of small litters by co-housing i-GONAD-treated pregnant B6 females with synchronized pregnant FVB/NJ companion mothers. Thus, GM mice generation was increased by an enriched diet and shared pup rearing with highly fertile females such as FVB/NJ. In the present study, we generated 16 GM mice using a CRISPR/Cas system to target individual and multiple loci simultaneously or consecutively. We also compared homology-directed repair efficiency using different methods for LoxP insertion for conditional knockout mouse production. We found that a two-step serial LoxP insertion, in which each LoxP sequence was inserted individually in different i-GONAD procedures, was a low-risk high-efficiency method for generating floxed mice.}, }
@article {pmid37172271, year = {2023}, author = {Fang, T and Zhang, L and Ding, W and Liu, Y and Li, P and Wang, W and Xiang, W and Wang, B and Sun, W}, title = {Point-of-Care Testing for Norovirus Typing Using CRISPR/Cas12a Combined with Reverse Transcription Recombinase Polymerase Amplification.}, journal = {Bioconjugate chemistry}, volume = {}, number = {}, pages = {}, doi = {10.1021/acs.bioconjchem.3c00181}, pmid = {37172271}, issn = {1520-4812}, abstract = {Noroviruses (NoVs) are one of the leading causes of acute gastroenteritis in humans. This study combined reverse transcription recombinase polymerase amplification (RT-RPA) with a clustered regularly interspaced short palindromic repeat/CRISPR-associated protein (CRISPR/Cas) nucleic acid detection system to develop a point-of-care testing (POCT) technology for typing NoVs. The detection can be completed within 35 min at 37 °C, covering each genotype of genogroup I (GI) and II (GII) NoVs. The sensitivity of this method is 10 copies/μL for GI and 1 copy/μL for GII NoV plasmids. For the detection of clinical samples, the detection results of this method for NoV infected samples are consistent with the RT-qPCR detection method in the laboratory, and this detection method has no cross-reactivity with rotavirus and adenovirus. Therefore, the detection method established in this study enables the diagnosis and screening of suspected patients and close contacts by POCT, which is important for the timely identification and control of NoV outbreaks. In addition, the typing detection of GI and GII NoVs can achieve a precise diagnosis and treatment of patients infected with NoVs.}, }
@article {pmid37170319, year = {2023}, author = {Fu, L and Ma, E and Okada, M and Shibata, Y and Shi, YB}, title = {Competitive PCR with dual fluorescent primers enhances the specificity and reproducibility of genotyping animals generated from genome editing.}, journal = {Cell &amp; bioscience}, volume = {13}, number = {1}, pages = {83}, pmid = {37170319}, issn = {2045-3701}, abstract = {Targeted genome editing is a powerful tool for studying gene function in almost every aspect of biological and pathological processes. The most widely used genome editing approach is to introduce engineered endonucleases or CRISPR/Cas system into cells or fertilized eggs to generate double-strand DNA breaks within the targeted region, leading to DNA repair through homologous recombination or non-homologous end joining (NHEJ). DNA repair through NHEJ mechanism is an error-prone process that often results in point mutations or stretches of indels (insertions and deletions) within the targeted region. Such mutations in embryos are germline transmissible, thus providing an easy means to generate organisms with gene mutations. However, point mutations and short indels present difficulty for genotyping, often requiring labor intensive sequencing to obtain reliable results. Here, we developed a single-tube competitive PCR assay with dual fluorescent primers that allowed simple and reliable genotyping. While we used Xenopus tropicalis as a model organism, the approach should be applicable to genotyping of any organisms.}, }
@article {pmid37169661, year = {2023}, author = {Hołubowicz, R and Palczewski, K}, title = {Saving eyesight, one gene at a time.}, journal = {Trends in molecular medicine}, volume = {}, number = {}, pages = {}, doi = {10.1016/j.molmed.2023.04.008}, pmid = {37169661}, issn = {1471-499X}, abstract = {Kai Yao's group used prime editing to repair a blindness-causing mutation in the Pde6b gene in the mouse retina. This breakthrough was made possible by a Cas9 nickase that is not constrained by a protospacer adjacent motif (PAM) sequence requirement. This innovation brings prime editing technology one step closer to correcting disease-causing mutations at will.}, }
@article {pmid37168121, year = {2023}, author = {Tao, S and Zhou, D and Chen, H and Li, N and Zheng, L and Fang, Y and Xu, Y and Jiang, Q and Liang, W}, title = {Analysis of genetic structure and function of clustered regularly interspaced short palindromic repeats loci in 110 Enterococcus strains.}, journal = {Frontiers in microbiology}, volume = {14}, number = {}, pages = {1177841}, pmid = {37168121}, issn = {1664-302X}, abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR) and their CRISPR-associated proteins (Cas) are an adaptive immune system involved in specific defenses against the invasion of foreign mobile genetic elements, such as plasmids and phages. This study aims to analyze the gene structure and to explore the function of the CRISPR system in the Enterococcus genome, especially with regard to drug resistance. The whole genome information of 110 enterococci was downloaded from the NCBI database to analyze the distribution and the structure of the CRISPR-Cas system including the Cas gene, repeat sequences, and spacer sequence of the CRISPR-Cas system by bioinformatics methods, and to find drug resistance-related genes and analyze the relationship between them and the CRISPR-Cas system. Multilocus sequence typing (MLST) of enterococci was performed against the reference MLST database. Information on the drug resistance of Enterococcus was retrieved from the CARD database, and its relationship to the presence or absence of CRISPR was statistically analyzed. Among the 110 Enterococcus strains, 39 strains (35.45%) contained a complete CRISPR-Cas system, 87 CRISPR arrays were identified, and 62 strains contained Cas gene clusters. The CRISPR system in the Enterococcus genome was mainly type II-A (59.68%), followed by type II-C (33.87%). The phylogenetic analysis of the cas1 gene sequence was basically consistent with the typing of the CRISPR-Cas system. Of the 74 strains included in the study for MLST typing, only 19 (25.68%) were related to CRISPR-Cas typing, while the majority of the strains (74.32%) of MLST typing were associated with the untyped CRISPR system. Additionally, the CRISPR-Cas system may only be related to the carrying rate of some drug-resistant genes and the drug-resistant phenotype. In conclusion, the distribution of the enterococcus CRISPR-Cas system varies greatly among different species and the presence of CRISPR loci reduces the horizontal transfer of some drug resistance genes.}, }
@article {pmid37166088, year = {2023}, author = {Ali, A and Zafar, MM and Farooq, Z and Ahmed, SR and Ijaz, A and Anwar, Z and Abbas, H and Tariq, MS and Tariq, H and Mustafa, M and Bajwa, MH and Shaukat, F and Razzaq, A and Maozhi, R}, title = {Breakthrough in CRISPR/Cas system: Current and future directions and challenges.}, journal = {Biotechnology journal}, volume = {}, number = {}, pages = {e2200642}, doi = {10.1002/biot.202200642}, pmid = {37166088}, issn = {1860-7314}, abstract = {Targeted genome editing (GE) technology has brought a significant revolution in fictional genomic research and given hope to plant scientists to develop desirable varieties. This technology involves inducing site-specific DNA perturbations that can be repaired through DNA repair pathways. GE products currently include CRISPR-associated nuclease DNA breaks, prime editors generated DNA flaps, single nucleotide-modifications, transposases, and recombinases. The discovery of double-strand breaks, site-specific nucleases, and repair mechanisms paved the way for targeted genome editing, and the first-generation genome editing tools, ZFNs and TALENs, were successfully utilized in plant genome editing. However, CRISPR-Cas has now become the preferred tool for genome editing due to its speed, reliability, and cost-effectiveness. Plant functional genomics has benefited significantly from the widespread use of CRISPR technology for advancements and developments. This review highlights the progress made in CRISPR technology, including multiplex editing, base editing, and prime editing, as well as the challenges and potential delivery mechanisms. This article is protected by copyright. All rights reserved.}, }
@article {pmid37164010, year = {2023}, author = {Fong, JHC and Chu, HY and Zhou, P and Wong, ASL}, title = {Parallel engineering and activity profiling of a base editor system.}, journal = {Cell systems}, volume = {}, number = {}, pages = {}, doi = {10.1016/j.cels.2023.03.007}, pmid = {37164010}, issn = {2405-4720}, abstract = {Selecting the most suitable existing base editors and engineering new variants for installing specific base conversions with maximal efficiency and minimal undesired edits are pivotal for precise genome editing applications. Here, we present a platform for creating and analyzing a library of engineered base editor variants to enable head-to-head evaluation of their editing performance at scale. Our comprehensive comparison provides quantitative measures on each variant's editing efficiency, purity, motif preference, and bias in generating single and multiple base conversions, while uncovering undesired higher indel generation rate and noncanonical base conversion for some of the existing base editors. In addition to engineering the base editor protein, we further applied this platform to investigate a hitherto underexplored engineering route and created guide RNA scaffold variants that augment the editor's base-editing activity. With the unknown performance and compatibility of the growing number of engineered parts including deaminase, CRISPR-Cas enzyme, and guide RNA scaffold variants for assembling the expanding collection of base editor systems, our platform addresses the unmet need for an unbiased, scalable method to benchmark their editing outcomes and accelerate the engineering of next-generation precise genome editors.}, }
@article {pmid37114454, year = {2023}, author = {Wang, Y and Qi, Y and Guo, X and Zhang, K and Yu, B and Xu, FJ}, title = {Mannose-functionalized star polycation mediated CRISPR/Cas9 delivery for lung cancer therapy.}, journal = {Journal of materials chemistry. B}, volume = {11}, number = {18}, pages = {4121-4130}, doi = {10.1039/d2tb02274e}, pmid = {37114454}, issn = {2050-7518}, mesh = {Humans ; *Mannose ; Survivin ; CRISPR-Cas Systems/genetics ; DNA ; *Lung Neoplasms/genetics/therapy ; }, abstract = {The survivin gene, highly expressed in most cancer cells, is closely associated with inhibiting apoptosis. Therefore, gene editing for the survivin gene has great potential in tumor therapy. However, it is difficult for plasmid DNA (pDNA) to be taken up directly by cells, and thus the construction of gene vectors is a key step for successful gene editing. Ethanolamine-functionalized polyglycidyl methacrylate (PGEA) has been proved to facilitate the transfection of pDNA into cells in both in vivo and in vitro experiments. However, PGEA does not specifically recognize tumor cells. Some tumor cells express more mannose receptor (MR) than healthy cells. To achieve efficient target and transfection, we designed mannose-functionalized four-arm PGEA cationic polymers (P(GEA-co-ManMA), GM) with different molecular weights. GM was combined with pCas9-survivin. The mannose unit of GM/pCas9-survivin was identified by MR to selectively enter lung cancer cells. In vitro experiments showed that GM not only had excellent biocompatibility, gene transfection performance, and targeted ability, but also significantly inhibited the proliferation of tumor cells when used in combination with pCas9-survivin. At the same time, we also studied the relationship between the molecular weight and therapeutic effect.}, }
@article {pmid37163044, year = {2023}, author = {Taylor, D and Schwartz, E and Bravo, J and Ahsan, M and Macias, L and McCafferty, C and Dangerfield, T and Walker, J and Brodbelt, J and Palermo, G and Fineran, P and Fagerlund, R}, title = {Type III CRISPR-Cas effectors act as protein-assisted ribozymes during RNA cleavage.}, journal = {Research square}, volume = {}, number = {}, pages = {}, doi = {10.21203/rs.3.rs-2837968/v1}, pmid = {37163044}, abstract = {CRISPR-Cas systems are an adaptive immune system in bacteria and archaea that utilize CRISPR RNA-guided surveillance complexes to target complementary RNA or DNA for destruction. Target RNA cleavage at regular intervals is characteristic of type III effector complexes; however, the mechanism has remained enigmatic. Here, we determine the structures of the Synechocystis type III-Dv complex, an evolutionary intermediate in type III effectors, in pre- and post-cleavage states, which show metal ion coordination in the active sites. Using structural, biochemical, and quantum/classical molecular dynamics simulation, we reveal the structure and dynamics of the three catalytic sites, where a 2'-OH of the ribose on the target RNA acts as a nucleophile for in line self-cleavage of the upstream scissile phosphate. Strikingly, the arrangement at the catalytic residues of most type III complexes resembles the active site of ribozymes, including the hammerhead, pistol, and Varkud satellite ribozymes. Thus, type III CRISPR-Cas complexes function as protein-assisted ribozymes, and their programmable nature has important implications for how these complexes could be repurposed for applications.}, }
@article {pmid37162202, year = {2023}, author = {Huang, Z and Lyon, CJ and Wang, J and Lu, S and Hu, TY}, title = {CRISPR Assays for Disease Diagnosis: Progress to and Barriers Remaining for Clinical Applications.}, journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)}, volume = {}, number = {}, pages = {e2301697}, doi = {10.1002/advs.202301697}, pmid = {37162202}, issn = {2198-3844}, support = {U01CA252965/NH/NIH HHS/United States ; R01HD090927/NH/NIH HHS/United States ; R01AI144168/NH/NIH HHS/United States ; R01HD103511/NH/NIH HHS/United States ; R21NS130542/NH/NIH HHS/United States ; }, abstract = {Numerous groups have employed the special properties of CRISPR/Cas systems to develop platforms that have broad potential applications for sensitive and specific detection of nucleic acid (NA) targets. However, few of these approaches have progressed to commercial or clinical applications. This review summarizes the properties of known CRISPR/Cas systems and their applications, challenges associated with the development of such assays, and opportunities to improve their performance or address unmet assay needs using nano-/micro-technology platforms. These include rapid and efficient sample preparation, integrated single-tube, amplification-free, quantifiable, multiplex, and non-NA assays. Finally, this review discusses the current outlook for such assays, including remaining barriers for clinical or point-of-care applications and their commercial development.}, }
@article {pmid37162093, year = {2023}, author = {Ishikawa, K and Soejima, S and Saitoh, S}, title = {Genetic knockdown of genes that are obscure, conserved and essential using CRISPR interference methods in the fission yeast S. pombe.}, journal = {Journal of cell science}, volume = {136}, number = {9}, pages = {}, doi = {10.1242/jcs.261186}, pmid = {37162093}, issn = {1477-9137}, mesh = {Animals ; *Schizosaccharomyces/genetics ; Cell Proliferation ; Gene Knockdown Techniques ; Phenotype ; Mammals ; }, abstract = {Characterizing functions of essential genes is challenging, as perturbing them is generally lethal. Conditional gene perturbation, including use of temperature-sensitive mutants, has been widely utilized to reveal functions of essential genes in the fission yeast Schizosaccharomyces pombe. However, recently we implemented a systematic and less time-consuming knockdown method, CRISPR interference (CRISPRi), in this organism using catalytically inactive Cas9 (dCas9). This technology has been expected to facilitate characterization of essential genes in S. pombe, although this still has not occurred. Here, CRISPRi was harnessed to study uncharacterized essential genes that are evolutionally conserved from yeasts to mammals. Transcription of these genes, which we call conserved essential obscure (ceo) genes, was repressed using conventional dCas9-mediated CRISPRi and by implementing technologies that enhance repression efficiency or alleviate limitations on small guide RNA (sgRNA) design. These CRISPRi methods successfully reduced transcription of target genes and allowed us to characterize resulting phenotypes. Knockdown of ceo genes inhibited cell proliferation and altered cellular morphology. Thus, dCas9-based CRISPRi methods utilized in this study enhanced accessibility of genetic analyses targeting essential genes in S. pombe.}, }
@article {pmid37161535, year = {2023}, author = {Wenger, A and Karlsson, I and Kling, T and Carén, H}, title = {CRISPR-Cas9 knockout screen identifies novel treatment targets in childhood high-grade glioma.}, journal = {Clinical epigenetics}, volume = {15}, number = {1}, pages = {80}, pmid = {37161535}, issn = {1868-7083}, mesh = {Humans ; Child ; *CRISPR-Cas Systems ; DNA Methylation ; *Glioma/genetics ; Genes, Regulator ; Histones/genetics ; }, abstract = {BACKGROUND: Brain tumours are the leading cause of cancer-related death in children, and there is no effective treatment. A growing body of evidence points to deregulated epigenetics as a tumour driver, particularly in paediatric cancers as they have relatively few genomic alterations, and key driver mutations have been identified in histone 3 (H3). Cancer stem cells (CSC) are implicated in tumour development, relapse and therapy resistance and thus particularly important to target. We therefore aimed to identify novel epigenetic treatment targets in CSC derived from H3-mutated high-grade glioma (HGG) through a CRISPR-Cas9 knockout screen.<br><br>RESULTS: The knockout screen identified more than 100 novel genes essential for the growth of CSC derived from paediatric HGG with H3K27M mutation. We successfully validated 12 of the 13 selected hits by individual knockout in the same two CSC lines, and for the top six hits we included two additional CSC lines derived from H3 wild-type paediatric HGG. Knockout of these genes led to a significant decrease in CSC growth, and altered stem cell and differentiation markers.<br><br>CONCLUSIONS: The screen robustly identified essential genes known in the literature, but also many novel genes essential for CSC growth in paediatric HGG. Six of the novel genes (UBE2N, CHD4, LSM11, KANSL1, KANSL3 and EED) were validated individually thus demonstrating their importance for CSC growth in H3-mutated and wild-type HGG. These genes should be further studied and evaluated as novel treatment targets in paediatric HGG.}, }
@article {pmid37160974, year = {2023}, author = {Du, S and Tong, X and Lai, ACK and Chan, CK and Mason, CE and Lee, PKH}, title = {Highly host-linked viromes in the built environment possess habitat-dependent diversity and functions for potential virus-host coevolution.}, journal = {Nature communications}, volume = {14}, number = {1}, pages = {2676}, pmid = {37160974}, issn = {2041-1723}, mesh = {Virome ; Built Environment ; *Microbiota/genetics ; *Alkanesulfonic Acids ; }, abstract = {Viruses in built environments (BEs) raise public health concerns, yet they are generally less studied than bacteria. To better understand viral dynamics in BEs, this study assesses viromes from 11 habitats across four types of BEs with low to high occupancy. The diversity, composition, metabolic functions, and lifestyles of the viromes are found to be habitat dependent. Caudoviricetes species are ubiquitous on surface habitats in the BEs, and some of them are distinct from those present in other environments. Antimicrobial resistance genes are identified in viruses inhabiting surfaces frequently touched by occupants and in viruses inhabiting occupants' skin. Diverse CRISPR/Cas immunity systems and anti-CRISPR proteins are found in bacterial hosts and viruses, respectively, consistent with the strongly coupled virus-host links. Evidence of viruses potentially aiding host adaptation in a specific-habitat manner is identified through a unique gene insertion. This work illustrates that virus-host interactions occur frequently in BEs and that viruses are integral members of BE microbiomes.}, }
@article {pmid37159673, year = {2023}, author = {Chen, Y and Meng, L and Lang, B and Li, L and Liu, J and Wang, Y and Huang, Z and Tian, X and Tian, R and Hu, Z}, title = {A Cas12a ortholog with distinct TTNA PAM enables sensitive detection of HPV16/18.}, journal = {Cell reports methods}, volume = {3}, number = {4}, pages = {100444}, pmid = {37159673}, issn = {2667-2375}, mesh = {Humans ; *CRISPR-Cas Systems/genetics ; Human papillomavirus 16/genetics ; Human papillomavirus 18/genetics ; *Papillomavirus Infections/diagnosis ; Biological Assay ; Capsid Proteins ; Papillomaviridae ; }, abstract = {CRISPR-associated (Cas) nucleases are multifunctional tools for gene editing. Cas12a possesses several advantages, including the requirement of a single guide RNA and high fidelity of gene editing. Here, we tested three Cas12a orthologs from human gut samples and identified a LtCas12a that utilizes a TTNA protospacer adjacent motif (PAM) distinct from the canonical TTTV PAM but with equivalent cleavage ability and specificity. These features significantly broadened the targeting scope of Cas12a family. Furthermore, we developed a sensitive, accurate, and rapid human papillomavirus (HPV) 16/18 gene detection platform based on LtCas12a DNA endonuclease-targeted CRISPR trans reporter (DETECTR) and lateral flow assay (LFA). LtCas12a showed comparable sensitivity to quantitative polymerase chain reaction (qPCR) and no cross-reaction with 13 other high-risk HPV genotypes in detecting the HPV16/18 L1 gene. Taken together, LtCas12a can broaden the applications of the CRISPR-Cas12a family and serve as a promising next-generation tool for therapeutic application and molecular diagnosis.}, }
@article {pmid37160120, year = {2023}, author = {Nguyen, LT and Rananaware, SR and Yang, LG and Macaluso, NC and Ocana-Ortiz, JE and Meister, KS and Pizzano, BLM and Sandoval, LSW and Hautamaki, RC and Fang, ZR and Joseph, SM and Shoemaker, GM and Carman, DR and Chang, L and Rakestraw, NR and Zachary, JF and Guerra, S and Perez, A and Jain, PK}, title = {Engineering highly thermostable Cas12b via de novo structural analyses for one-pot detection of nucleic acids.}, journal = {Cell reports. Medicine}, volume = {}, number = {}, pages = {101037}, doi = {10.1016/j.xcrm.2023.101037}, pmid = {37160120}, issn = {2666-3791}, abstract = {CRISPR-Cas-based diagnostics have the potential to elevate nucleic acid detection. CRISPR-Cas systems can be combined with a pre-amplification step in a one-pot reaction to simplify the workflow and reduce carryover contamination. Here, we report an engineered Cas12b with improved thermostability that falls within the optimal temperature range (60°C-65°C) of reverse transcription-loop-mediated isothermal amplification (RT-LAMP). Using de novo structural analyses, we introduce mutations to wild-type BrCas12b to tighten its hydrophobic cores, thereby enhancing thermostability. The one-pot detection assay utilizing the engineered BrCas12b, called SPLENDID (single-pot LAMP-mediated engineered BrCas12b for nucleic acid detection of infectious diseases), exhibits robust trans-cleavage activity up to 67°C in a one-pot setting. We validate SPLENDID clinically in 80 serum samples for hepatitis C virus (HCV) and 66 saliva samples for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with high specificity and accuracy. We obtain results in as little as 20 min, and with the extraction process, the entire assay can be performed within an hour.}, }
@article {pmid37158910, year = {2023}, author = {Bahrulolum, H and Tarrahimofrad, H and Rouzbahani, FN and Nooraei, S and Sameh, MM and Hajizade, A and Ahmadian, G}, title = {Potential of CRISPR/Cas system as emerging tools in the detection of viral hepatitis infection.}, journal = {Virology journal}, volume = {20}, number = {1}, pages = {91}, pmid = {37158910}, issn = {1743-422X}, abstract = {Viral hepatitis, the most common cause of inflammatory liver disease, affects hundreds of millions of people worldwide. It is most commonly associated with one of the five nominal hepatitis viruses (hepatitis A-E viruses). HBV and HCV can cause acute infections and lifelong, persistent chronic infections, while HAV and HEV cause self-limiting acute infections. HAV and HEV are predominantly transmitted through the fecal-oral route, while diseases transmitted by the other forms are blood-borne diseases. Despite the success in the treatment of viral hepatitis and the development of HAV and HBV vaccines, there is still no accurate diagnosis at the genetic level for these diseases. Timely diagnosis of viral hepatitis is a prerequisite for efficient therapeutic intervention. Due to the specificity and sensitivity of clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated sequences (Cas) technology, it has the potential to meet critical needs in the field of diagnosis of viral diseases and can be used in versatile point-of-care (POC) diagnostic applications to detect viruses with both DNA and RNA genomes. In this review, we discuss recent advances in CRISPR-Cas diagnostics tools and assess their potential and prospects in rapid and effective strategies for the diagnosis and control of viral hepatitis infection.}, }
@article {pmid37158576, year = {2023}, author = {Yang, B and Khatri, M and Zheng, J and Deogun, J and Yin, Y}, title = {Genome mining for anti-CRISPR operons using machine learning.}, journal = {Bioinformatics (Oxford, England)}, volume = {}, number = {}, pages = {}, doi = {10.1093/bioinformatics/btad309}, pmid = {37158576}, issn = {1367-4811}, abstract = {MOTIVATION: Encoded by (pro-)viruses, anti-CRISPR (Acr) proteins inhibit the CRISPR-Cas immune system of their prokaryotic hosts. As a result, Acr proteins can be employed to develop more controllable CRISPR-Cas genome editing tools. Recent studies revealed that known acr genes often coexist with other acr genes and with phage structural genes within the same operon. For example, we found that 47 of 98 known acr genes (or their homologs) co-exist in the same operons. None of the current Acr prediction tools have considered this important genomic context feature. We have developed a new software tool AOminer to facilitate the improved discovery of new Acrs by fully exploiting the genomic context of known acr genes and their homologs.<br><br>RESULTS: AOminer is the first machine learning based tool focused on the discovery of Acr operons (AOs). A two-state HMM (hidden Markov model) was trained to learn the conserved genomic context of operons that contain known acr genes or their homologs, and the learnt features could distinguish AOs and non-AOs. AOminer allows automated mining for potential AOs from query genomes or operons. AOminer outperformed all existing Acr prediction tools with an accuracy = 0.85. AOminer will facilitate the discovery of novel anti-CRISPR operons.<br><br>AVAILABILITY: The webserver is available at: http://aca.unl.edu/AOminer/AOminer_APP/. The python program is at: https://github.com/boweny920/AOminer.<br><br>SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.}, }
@article {pmid37154329, year = {2023}, author = {Gao, S and Fang, G and Zhang, Y and DU, J}, title = {[Generation of Mlk3 KO mice by CRISPR/Cas9 and its effect on blood pressure].}, journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology}, volume = {39}, number = {4}, pages = {1644-1654}, doi = {10.13345/j.cjb.221022}, pmid = {37154329}, issn = {1872-2075}, mesh = {Animals ; Mice ; Mice, Knockout ; *CRISPR-Cas Systems ; Blood Pressure ; Gene Knockout Techniques ; *Zygote ; }, abstract = {To explore the effect of Mlk3 (mixed lineage kinase 3) deficiency on blood pressure, Mlk3 gene knockout (Mlk3KO) mice were generated. Activities of sgRNAs targeted Mlk3 gene were evaluated by T7 endonuclease I (T7E1) assay. CRISPR/Cas9 mRNA and sgRNA were obtained by in vitro transcription, microinjected into zygote, followed by transferring into a foster mother. Genotyping and DNA sequencing confirmed the deletion of Mlk3 gene. Real- time PCR (RT-PCR), Western blotting or immunofluorescence analysis showed that Mlk3KO mice had an undetectable expression of Mlk3 mRNA or Mlk3 protein. Mlk3KO mice exhibited an elevated systolic blood pressure compared with wild-type mice as measured by tail-cuff system. Immunohistochemistry and Western blotting analysis showed that the phosphorylation of MLC (myosin light chain) was significantly increased in aorta isolated from Mlk3KO mice. Together, Mlk3KO mice was successfully generated by CRISPR/Cas9 system. MLK3 functions in maintaining blood pressure homeostasis by regulating MLC phosphorylation. This study provides an animal model for exploring the mechanism by which Mlk3 protects against the development of hypertension and hypertensive cardiovascular remodeling.}, }
@article {pmid37154311, year = {2023}, author = {Hong, T and Luo, Q}, title = {[Advances in the RNA-targeting CRISPR-Cas systems].}, journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology}, volume = {39}, number = {4}, pages = {1363-1373}, doi = {10.13345/j.cjb.220633}, pmid = {37154311}, issn = {1872-2075}, mesh = {*CRISPR-Cas Systems/genetics ; *RNA/genetics ; Bacteria/genetics ; Gene Editing ; Archaea ; }, abstract = {The CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR associated proteins) system is an adaptive immune system of bacteria and archaea against phages, plasmids and other exogenous genetic materials. The system uses a special RNA (CRISPR RNA, crRNA) guided endonuclease to cut the exogenous genetic materials complementary to crRNA, thus blocking the infection of exogenous nucleic acid. According to the composition of the effector complex, CRISPR-Cas system can be divided into two categories: class 1 (including type Ⅰ, Ⅳ, and Ⅲ) and class 2 (including type Ⅱ, Ⅴ, and Ⅵ). Several CRISPR-Cas systems have been found to have very strong ability to specifically target RNA editing, such as type Ⅵ CRISPR-Cas13 system and type Ⅲ CRISPR-Cas7-11 system. Recently, several systems have been widely used in the field of RNA editing, making them a powerful tool for gene editing. Understanding the composition, structure, molecular mechanism and potential application of RNA-targeting CRISPR-Cas systems will facilitate the mechanistic research of this system and provide new ideas for developing gene editing tools.}, }
@article {pmid37153079, year = {2023}, author = {Hendel, A and Bak, RO}, title = {Editorial: CRISPR and beyond: Cutting-edge technologies for gene correction in therapeutic applications.}, journal = {Frontiers in genome editing}, volume = {5}, number = {}, pages = {1203864}, pmid = {37153079}, issn = {2673-3439}, }
@article {pmid37120835, year = {2023}, author = {Xie, TJ and Xie, JL and Luo, YJ and Mao, K and Huang, CZ and Li, YF and Zhen, SJ}, title = {CRISPR-Cas12a Coupled with DNA Nanosheet-Amplified Fluorescence Anisotropy for Sensitive Detection of Biomolecules.}, journal = {Analytical chemistry}, volume = {95}, number = {18}, pages = {7237-7243}, doi = {10.1021/acs.analchem.3c00156}, pmid = {37120835}, issn = {1520-6882}, mesh = {Humans ; *COVID-19 ; SARS-CoV-2 ; DNA ; DNA, Single-Stranded ; *MicroRNAs ; *Biosensing Techniques ; CRISPR-Cas Systems/genetics ; }, abstract = {DNA nanosheets (DNSs) have been utilized effectively as a fluorescence anisotropy (FA) amplifier for biosensing. But, their sensitivity needs to be further improved. Herein, CRISPR-Cas12a with strong trans-cleavage activity was utilized to enhance the FA amplification ability of DNSs for the sensitive detection of miRNA-155 (miR-155) as a proof-of-principle target. In this method, the hybrid of the recognition probe of miR-155 (T1) and a blocker sequence (T2) was immobilized on the surface of magnetic beads (MBs). In the presence of miR-155, T2 was released by a strand displacement reaction, which activated the trans-cleavage activity of CRISPR-Cas12a. The single-stranded DNA (ssDNA) probe modified with a carboxytetramethylrhodamine (TAMRA) fluorophore was cleaved in large quantities and could not bind to the handle chain on DNSs, inducing a low FA value. In contrast, in the absence of miR-155, T2 could not be released and the trans-cleavage activity of CRISPR-Cas12a could not be activated. The TAMRA-modified ssDNA probe remained intact and was complementary to the handle chain on the DNSs, and a high FA value was obtained. Thus, miR-155 was detected through the obviously decreased FA value with a low limit of detection (LOD) of 40 pM. Impressively, the sensitivity of this method was greatly improved about 322 times by CRISPR-Cas12a, confirming the amazing signal amplification ability of CRISPR-Cas12a. At the same time, the SARS-CoV-2 nucleocapsid protein was detected by the strategy successfully, indicating that this method was general. Moreover, this method has been applied in the analysis of miR-155 in human serum and the lysates of cells, which provides a new avenue for the sensitive determination of biomarkers in biochemical research and disease diagnosis.}, }
@article {pmid37119146, year = {2023}, author = {Li, L and Yu, S and Wu, J and Ju, H}, title = {Regulation of Target-activated CRISPR/Cas12a on Surface Binding of Polymer Dots for Sensitive Electrochemiluminescence DNA Analysis.}, journal = {Analytical chemistry}, volume = {95}, number = {18}, pages = {7396-7402}, doi = {10.1021/acs.analchem.3c01521}, pmid = {37119146}, issn = {1520-6882}, mesh = {Humans ; *Quantum Dots ; Gold ; Polymers ; Reproducibility of Results ; CRISPR-Cas Systems/genetics ; Electrochemical Techniques/methods ; Luminescent Measurements/methods ; *Metal Nanoparticles ; DNA/analysis ; Limit of Detection ; *Biosensing Techniques/methods ; }, abstract = {Polymer dots (Pdots) have emerged as a type of attractive electrochemiluminescence (ECL) emitter. However, the low ECL efficiency severely limits their practicability. In this work, we develop a sensitive ECL biosensing strategy for the detection of human papilloma virus subtype (HPV-16) DNA by using target-activated CRISPR/Cas12a to regulate the binding of Pdots-DNA to biosensor and local surface plasmon resonance (LSPR) effect of electrochemically deposited Au nanoparticles (depAuNPs) to enhance the ECL emission of Pdots bound on biosensor. The biosensor is prepared by simply assembling hairpin DNA on depAuNPs modified electrode. In the presence of target DNA, the designed specific CRISPR/Cas12a can be activated to digest single-stranded assistant DNA, which decreases the amount of hairpin DNA opened by assistant DNA to bind Pdots-DNA on the biosensor surface, thus reduces the ECL emission. The integration of target DNA-triggered catalysis and the LSPR effect of depAuNPs greatly improves the sensitivity of ECL analysis. Using HPV-16 DNA as a target model, the proposed method shows a limit of detection (LOD) of 3.2 fM at a signal-to-noise ratio of 3 and a detectable concentration range of 5.0 fM to 50 pM. The high sensitivity, excellent selectivity, good testing stability, and acceptable fabrication reproducibility of the designed ECL biosensing strategy demonstrate its potential application in DNA bioanalysis.}, }
@article {pmid37039522, year = {2023}, author = {Hadi, J and Rapp, D and Dhawan, S and Gupta, SK and Gupta, TB and Brightwell, G}, title = {Molecular detection and characterization of foodborne bacteria: Recent progresses and remaining challenges.}, journal = {Comprehensive reviews in food science and food safety}, volume = {22}, number = {3}, pages = {2433-2464}, doi = {10.1111/1541-4337.13153}, pmid = {37039522}, issn = {1541-4337}, mesh = {*CRISPR-Cas Systems ; *Genome-Wide Association Study ; Bacteria/genetics ; Food Safety ; }, abstract = {The global food demand is expected to increase in the coming years, along with challenges around climate change and food security. Concomitantly, food safety risks, particularly those related to bacterial pathogens, may also increase. Thus, the food sector needs to innovate to rise to this challenge. Here, we discuss recent advancements in molecular techniques that can be deployed within various foodborne bacteria surveillance systems across food settings. To start with, we provide updates on nucleic acid-based detection, with a focus on polymerase chain reaction (PCR)-based technologies and loop-mediated isothermal amplification (LAMP). These include descriptions of novel genetic markers for several foodborne bacteria and progresses in multiplex PCR and droplet digital PCR. The next section provides an overview of the development of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins systems, such as CRISPR-Cas9, CRISPR-Cas12a, and CRISPR-Cas13a, as tools for enhanced sensitive and specific detection of foodborne pathogens. The final section describes utilizations of whole genome sequencing for accurate characterization of foodborne bacteria, ranging from epidemiological surveillance to model-based predictions of bacterial phenotypic traits through genome-wide association studies or machine learning.}, }
@article {pmid36800529, year = {2023}, author = {Li, M and Zhao, Y and Xue, X and Zhong, J and Lin, J and Zhou, J and Yu, W and Chen, J and Qiao, Y}, title = {Cas9-orthologue-mediated cytosine and adenine base editors recognizing NNAAAA PAM sequences.}, journal = {Biotechnology journal}, volume = {18}, number = {5}, pages = {e2200533}, doi = {10.1002/biot.202200533}, pmid = {36800529}, issn = {1860-7314}, mesh = {Mice ; Animals ; Humans ; *CRISPR-Cas Systems/genetics ; *Gene Editing ; Adenine/metabolism ; Cytosine/metabolism ; Nucleotides ; }, abstract = {CRISPR/Cas9 system has been applied as an effective genome-targeting technology. By fusing deaminases with Cas9 nickase (nCas9), various cytosine and adenine base editors (CBEs and ABEs) have been successfully developed that can efficiently induce nucleotide conversions and install pathogenic single nucleotide variants (SNVs) in cultured cells and animal models. However, the applications of BEs are frequently limited by the specific protospacer adjacent motif (PAM) sequences and protein sizes. To expand the toolbox for BEs that can recognize novel PAM sequences, we cloned a Cas9 ortholog from Streptococcus sinensis (named as SsiCas9) with a smaller size and constructed it into APOBEC1- or APOBEC3A-composed CBEs and TadA or TadA*-composed ABEs, which yield high editing efficiencies, low off-targeting activities, and low indel rates in human cells. Compared to PAMless SpRY Cas9-composed BE4max, SsiCas9-mediated BE4max displayed higher editing efficiencies for targets with "NNAAAA" PAM sequences. Moreover, SsiCas9-mediated BE4max induced highly efficient C-to-T conversions in the mouse Ar gene (R841C) to introduce a human androgen resistance syndrome-related mutation (AR R820C) in early mouse embryos. Thus, we developed novel BEs mediated by SsiCas9, expanded the toolbox for base conversions, and broadened the range of editable genomes in vitro and in vivo.}, }
@article {pmid37153078, year = {2023}, author = {Pascher, K and Hainz-Renetzeder, C and Jagersberger, M and Kneissl, K and Gollmann, G and Schneeweiss, GM}, title = {Contamination of imported kernels by unapproved genome-edited varieties poses a major challenge for monitoring and traceability during transport and handling on a global scale: inferences from a study on feral oilseed rape in Austria.}, journal = {Frontiers in genome editing}, volume = {5}, number = {}, pages = {1176290}, pmid = {37153078}, issn = {2673-3439}, abstract = {Novel techniques such as CRISPR/Cas are increasingly being applied for the development of modern crops. However, the regulatory framework for production, labelling and handling of genome-edited organisms varies worldwide. Currently, the European Commission is raising the question whether genome-edited organisms should still be regulated as genetically modified organisms in the future or whether a deregulation should be implemented. In our paper, based on the outcome of a 2-year case study on oilseed rape in Austria, we show that seed spillage during import and subsequent transport and handling activities is a key factor for the unintended dispersal of seeds into the environment, the subsequent emergence of feral oilseed rape populations and their establishment and long-term persistence in natural habitats. These facts must likewise be considered in case of genome-edited oilseed rape contaminants that might be accidentally introduced with conventional kernels. We provide evidence that in Austria a high diversity of oilseed rape genotypes, including some with alleles not known from cultivated oilseed rape in Austria, exists at sites with high seed spillage and low weed management, rendering these sites of primary concern with respect to possible escape of genome-edited oilseed rape varieties into the environment. Since appropriate detection methods for single genome-edited oilseed rape events have only recently started to be successfully developed and the adverse effects of these artificial punctate DNA exchanges remain largely unknown, tracing the transmission and spread of these genetic modifications places high requirements on their monitoring, identification, and traceability.}, }
@article {pmid37152166, year = {2023}, author = {Ye, S and Ding, W and Bai, W and Lu, J and Zhou, L and Ma, X and Zhu, Q}, title = {Application of a novel strong promoter from Chinese fir (Cunninghamia lanceolate) in the CRISPR/Cas mediated genome editing of its protoplasts and transgenesis of rice and poplar.}, journal = {Frontiers in plant science}, volume = {14}, number = {}, pages = {1179394}, pmid = {37152166}, issn = {1664-462X}, abstract = {Novel constitutive promoters are essential for plant biotechnology. Although in angiosperms, a number of promoters were applied in monocots or dicots genetic engineering, only a few promoters were used in gymnosperm. Here we identified two strong promoters (Cula11 and Cula08) from Chinese fir (C. lanceolate) by screening the transcriptomic data and preliminary promoter activity assays in tobacco. By using the newly established Chinese fir protoplast transient expression technology that enables in vivo molecular biology studies in its homologous system, we compared the activities of Cula11 and Cula08 with that of the commonly used promoters in genetic engineering of monocots or dicots, such as CaM35S, CmYLCV, and ZmUbi, and our results revealed that Cula11 and Cula08 promoters have stronger activities in Chinese fir protoplasts. Furthermore, the vector containing Cas gene driven by Cula11 promoter and sgRNA driven by the newly isolated CulaU6b polyIII promoters were introduced into Chinese fir protoplasts, and CRISPR/Cas mediated gene knock-out event was successfully achieved. More importantly, compared with the commonly used promoters in the genetic engineering in angiosperms, Cula11 promoter has much stronger activity than CaM35S promoter in transgenic poplar, and ZmUbi promoter in transgenic rice, respectively, indicating its potential application in poplar and rice genetic engineering. Overall, the novel putative constitutive gene promoters reported here will have great potential application in gymnosperm and angiosperm biotechnology, and the transient gene expression system established here will serve as a useful tool for the molecular and genetic analyses of Chinese fir genes.}, }
@article {pmid37149686, year = {2023}, author = {Cetin, R and Wegner, M and Luwisch, L and Saud, S and Achmedov, T and Süsser, S and Vera-Guapi, A and Müller, K and Matthess, Y and Quandt, E and Schaubeck, S and Beisel, CL and Kaulich, M}, title = {Optimized metrics for orthogonal combinatorial CRISPR screens.}, journal = {Scientific reports}, volume = {13}, number = {1}, pages = {7405}, pmid = {37149686}, issn = {2045-2322}, support = {865973/ERC_/European Research Council/International ; }, mesh = {*CRISPR-Cas Systems ; *Benchmarking ; Gene Editing ; Endonucleases/genetics ; }, abstract = {CRISPR-based gene perturbation enables unbiased investigations of single and combinatorial genotype-to-phenotype associations. In light of efforts to map combinatorial gene dependencies at scale, choosing an efficient and robust CRISPR-associated (Cas) nuclease is of utmost importance. Even though SpCas9 and AsCas12a are widely used for single, combinatorial, and orthogonal screenings, side-by-side comparisons remain sparse. Here, we systematically compared combinatorial SpCas9, AsCas12a, and CHyMErA in hTERT-immortalized retinal pigment epithelial cells and extracted performance-critical parameters for combinatorial and orthogonal CRISPR screens. Our analyses identified SpCas9 to be superior to enhanced and optimized AsCas12a, with CHyMErA being largely inactive in the tested conditions. Since AsCas12a contains RNA processing activity, we used arrayed dual-gRNAs to improve AsCas12a and CHyMErA applications. While this negatively influenced the effect size range of combinatorial AsCas12a applications, it enhanced the performance of CHyMErA. This improved performance, however, was limited to AsCas12a dual-gRNAs, as SpCas9 gRNAs remained largely inactive. To avoid the use of hybrid gRNAs for orthogonal applications, we engineered the multiplex SpCas9-enAsCas12a approach (multiSPAS) that avoids RNA processing for efficient orthogonal gene editing.}, }
@article {pmid37014013, year = {2023}, author = {Pan, J and Mabuchi, M and Robb, GB}, title = {DNA rehybridization drives product release from Cas9 ribonucleoprotein to enable multiple-turnover cleavage.}, journal = {Nucleic acids research}, volume = {51}, number = {8}, pages = {3903-3917}, pmid = {37014013}, issn = {1362-4962}, mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; Ribonucleoproteins/genetics/metabolism ; CRISPR-Associated Protein 9/metabolism ; DNA/chemistry ; Streptococcus pyogenes ; DNA Cleavage ; }, abstract = {The RNA-guided Cas9 endonuclease from Staphylococcus aureus (SauCas9) can catalyze multiple-turnover reactions whereas Cas9 from Streptococcus pyogenes (SpyCas9) is a single-turnover enzyme. Here we dissect the mechanism of multiple-turnover catalysis by SauCas9 and elucidate its molecular basis. We show that the multiple-turnover catalysis does not require more than stoichiometric RNA guides to Cas9 nuclease. Rather, the RNA-guide loaded ribonucleoprotein (RNP) is the reactive unity that is slowly released from product and recycled in the subsequent reaction. The mechanism that RNP is recycled for multiple-turnover reaction entails the unwinding of the RNA:DNA duplex in the R-loop. We argue that DNA rehybridization is required for RNP release by supplementing the energy cost in the process. Indeed, turnover is arrested when DNA rehybridization is suppressed. Further, under higher salt conditions, both SauCas9 and SpyCas9 showed increased turnover, and engineered SpyCas9 nucleases that form fewer direct or hydrogen bonding interactions with target DNA became multiple-turnover enzymes. Thus, these results indicate that for both SpyCas9 and SauCas9, turnover is determined by the energetic balance of the post-chemistry RNP-DNA interaction. Due to the conserved protein core folds, the mechanism underpinning turnover we establish here is likely operant in all Cas9 nucleases.}, }
@article {pmid36919598, year = {2023}, author = {Lee, RRQ and Cher, WY and Wang, J and Chen, Y and Chae, E}, title = {Generating minimum set of gRNA to cover multiple targets in multiple genomes with MINORg.}, journal = {Nucleic acids research}, volume = {51}, number = {8}, pages = {e43}, pmid = {36919598}, issn = {1362-4962}, mesh = {Humans ; Polymerase Chain Reaction ; Gene Knockout Techniques ; *Software ; *CRISPR-Cas Systems ; }, abstract = {MINORg is an offline gRNA design tool that generates the smallest possible combination of gRNA capable of covering all desired targets in multiple non-reference genomes. As interest in pangenomic research grows, so does the workload required for large screens in multiple individuals. MINORg aims to lessen this workload by capitalising on sequence homology to favour multi-target gRNA while simultaneously screening multiple genetic backgrounds in order to generate reusable gRNA panels. We demonstrated the practical application of MINORg by knocking out 11 homologous genes tandemly arrayed in a multi-gene cluster in two Arabidopsis thaliana lineages using three gRNA output by MINORg. We also described a new PCR-free modular cloning system for multiplexing gRNA, and used it to knockout three tandemly arrayed genes in another multi-gene cluster with gRNA designed by MINORg. Source code is freely available at https://github.com/rlrq/MINORg.}, }
@article {pmid36912089, year = {2023}, author = {Deng, H and Xu, H and Wang, Y and Jia, R and Ma, X and Feng, Y and Chen, H}, title = {G-quadruplex-based CRISPR photoswitch for spatiotemporal control of genomic modulation.}, journal = {Nucleic acids research}, volume = {51}, number = {8}, pages = {4064-4077}, pmid = {36912089}, issn = {1362-4962}, mesh = {*Gene Editing/methods ; *Genomics ; Genome ; Gene Expression Regulation ; CRISPR-Cas Systems/genetics ; }, abstract = {CRISPR (clustered regularly interspaced short palindromic repeats) technology holds tremendous promise for gene regulation and editing. However, precise control of CRISPR editing is essential to overcome its uncontrollable reaction process and excessive activity that leads to off-target editing. To overcome this problem, we engineered a photoswitch on G-quadruplex gRNA (GqRNA) for precisely controlled gene editing and expression by embedding dicationic azobenzene derivatives (AZD++). Our results demonstrated that rational design of the G-quadruplex onto crRNA conferred higher stability and sequence recognition specificity than unmodified single guide (sgRNA). Light-induced isomerization of AZD++ quickly transformed the on state of GqRNA, which facilitated rapid activation of ribonucleoprotein activity for genome editing of on-target sites in cells with excellent editing efficiency. In turn, AZD++-GqRNA promptly refolded to an off state to inhibit genomic cleavage, and limited the generation of off-target effects and by-products. Therefore, the proposed strategy of a photo-reversible modality presents a new opportunity for CRISPR-Cas9 modulation to improve its safety and applicability.}, }
@article {pmid36241547, year = {2023}, author = {Jo, DH and Bae, S and Kim, HH and Kim, JS and Kim, JH}, title = {In vivo application of base and prime editing to treat inherited retinal diseases.}, journal = {Progress in retinal and eye research}, volume = {94}, number = {}, pages = {101132}, doi = {10.1016/j.preteyeres.2022.101132}, pmid = {36241547}, issn = {1873-1635}, mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Gene Editing ; Genome ; *Retinal Diseases/genetics/therapy ; }, abstract = {Inherited retinal diseases (IRDs) are vision-threatening retinal disorders caused by pathogenic variants of genes related to visual functions. Genomic analyses in patients with IRDs have revealed pathogenic variants which affect vision. However, treatment options for IRDs are limited to nutritional supplements regardless of genetic variants or gene-targeting approaches based on antisense oligonucleotides and adeno-associated virus vectors limited to targeting few genes. Genome editing, particularly that involving clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 technologies, can correct pathogenic variants and provide additional treatment opportunities. Recently developed base and prime editing platforms based on CRISPR-Cas9 technologies are promising for therapeutic genome editing because they do not employ double-stranded breaks (DSBs), which are associated with P53 activation, large deletions, and chromosomal translocations. Instead, using attached deaminases and reverse transcriptases, base and prime editing efficiently induces specific base substitutions and intended genetic changes (substitutions, deletions, or insertions), respectively, without DSBs. In this review, we will discuss the recent in vivo application of CRISPR-Cas9 technologies, focusing on base and prime editing, in animal models of IRDs.}, }
@article {pmid37148798, year = {2023}, author = {Kang, Y and Wang, J and Zhang, W and Xu, Y and Xu, B and Qu, G and Yu, Y and Yan, B and Su, G}, title = {RNA extraction-free workflow integrated with a single-tube CRISPR-Cas-based colorimetric assay for rapid SARS-CoV-2 detection in different environmental matrices.}, journal = {Journal of hazardous materials}, volume = {454}, number = {}, pages = {131487}, pmid = {37148798}, issn = {1873-3336}, abstract = {On-site environmental surveillance of viruses is increasingly important for infection prevention and pandemic control. Herein, we report a facile single-tube colorimetric assay for detecting severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) from environmental compartments. Using glycerol as the phase separation additive, reverse transcription recombinase polymerase amplification (RT-RPA), CRISPR-Cas system activation, G-quadruplex (G4) cleavage, and G4-based colorimetric reaction were performed in a single tube. To further simplify the test, viral RNA genomes used for the one-tube assay were obtained via acid/base treatment without further purification. The whole assay from sampling to visual readout was completed within 30 min at a constant temperature without the need for sophisticated instruments. Coupling the RT-RPA to CRISPR-Cas improved the reliability by avoiding false positive results. Non-labeled cost-effective G4-based colorimetric systems are highly sensitive to CRISPR-Cas cleavage events, and the proposed assay reached the limit of detection of 0.84 copies/µL. Moreover, environmental samples from contaminated surfaces and wastewater were analyzed using this facile colorimetric assay. Given its simplicity, sensitivity, specificity, and cost-effectiveness, our proposed colorimetric assay is highly promising for applications in on-site environmental surveillance of viruses.}, }
@article {pmid37148478, year = {2023}, author = {Liu, Y and Jing, P and Zhou, Y and Zhang, J and Shi, J and Zhang, M and Yang, H and Fei, J}, title = {The effects of length and sequence of gRNA on Cas13b and Cas13d activity in vitro and in vivo.}, journal = {Biotechnology journal}, volume = {}, number = {}, pages = {e2300002}, doi = {10.1002/biot.202300002}, pmid = {37148478}, issn = {1860-7314}, abstract = {Cas13 are the only CRISPR/Cas systems found so far, which target RNA strand while preserving chromosomal integrity. Cas13b or Cas13d cleaves RNA by the crRNA guidance. However, the effect of the characteristics of the spacer sequences, such as the length and sequence preference, on the activity of Cas13b and Cas13d remains unclear. Our study shows that neither Cas13b nor Cas13d has a particular preference for the sequence composition of gRNA, including the sequence of crRNA and its flanking sites on target RNA. However, the crRNA, complementary to the middle part of the target RNA, seems to show higher cleavage efficiency for both Cas13b and Cas13d. As for the length of crRNAs, the most appropriate crRNA length for Cas13b is 22-25 nt and crRNA as short as 15 nt is still functional. Whereas, Cas13d requires longer crRNA, and 22-30 nt crRNA can achieve good effect. Both Cas13b and Cas13d show the ability to process precursor crRNAs. Our study suggests that Cas13b may have a stronger precursor processing ability than Cas13d. There are few in vivo studies on the application of Cas13b or Cas13d in mammals. With the methods of transgenic mice and hydrodynamic injection via tail vein, our study showed that both of them had high knock-down efficiency against target RNA in vivo. These results indicate that Cas13b and Cas13d have great potential for in vivo RNA operation and disease treatment without damaging genomic DNA. This article is protected by copyright. All rights reserved.}, }
@article {pmid37147507, year = {2023}, author = {Sonnenburg, A and Stahlmann, R and Kreutz, R and Peiser, M}, title = {A new cell line based coculture system for skin sensitisation testing in one single assay using T cells, aryl hydrocarbon receptor knockout, and co-inhibitory blockage.}, journal = {Archives of toxicology}, volume = {}, number = {}, pages = {}, pmid = {37147507}, issn = {1432-0738}, abstract = {Established in vitro assays for regulatory testing of skin sensitisation partly suffer from only moderate sensitivity, specificity, and predictivity when testing specific groups of chemicals. This may be due to limited biomarker response in vitro in cell types that interact as crucial players of in vivo skin sensitisation pathogenesis. Here, we propose a molecular approach to overcome this limitation. In our model, we apply genome editing and blocking of immunoregulatory molecules to increase the range of biomarker modulation by sensitising chemicals. To this end, aryl hydrocarbon receptor (AhR) knockout was done by CRISPR/Cas9 technology in THP-1 cells and combined with Programmed Cell Death-Ligand (PD-L)1 blockade. AhR-knockout THP-1 in coculture with HaCaT keratinocytes showed increased CD54 expression compared to wild type cells after stimulation with 10 µmol/L dinitrochlorobenzene (DNCB) that was further enhanced by anti-PD-L1. After stimulation of AhR-knockout THP-1 with 200 µmol/L mercaptobenzothiazol or 10 µmol/L DNCB, cocultivated Jurkat T cells significantly increased expression of T cell receptor-associated CD3. No such increase was detected after prior treatment of THP-1 with 150 µmol/L of irritant sodium lauryl sulphate. Additionally, higher levels of inflammatory cytokines MIP-3α, MIP-1β, TNF-α, and IL-8 were found in supernatants of enhanced loose-fit co-culture based sensitisation assay (eLCSA) after substance treatment. Hence, eLCSA allowed to discriminate between sensitisers and non-sensitisers. Thus, inhibition of immunoinhibitory pathway signalling by combining AhR knockout and PD-L1 antibody blockage into an assay involving main acting cell types in skin sensitisation may increase sensitivity and specificity of such assays and allow potency derivation.}, }
@article {pmid37147050, year = {2023}, author = {Wu, X and Ju, T and Li, Z and Li, J and Zhai, X and Han, K}, title = {Target-independent hybridization chain reaction-fluorescence resonance energy transfer for sensitive assay of ctDNA based on Cas12a.}, journal = {Analytica chimica acta}, volume = {1261}, number = {}, pages = {341170}, doi = {10.1016/j.aca.2023.341170}, pmid = {37147050}, issn = {1873-4324}, mesh = {*Fluorescence Resonance Energy Transfer ; CRISPR-Cas Systems ; Nucleic Acid Hybridization ; DNA/genetics ; *Biosensing Techniques/methods ; }, abstract = {Circulating tumor DNA (ctDNA) is a noninvasive biomarker which offer valuable information for cancer diagnosis and prognosis. In this study, a target-independent fluorescent signal system, Hybridization chain reaction-Fluorescence resonance energy transfer (HCR-FRET) system, is designed and optimized. Combined with CRISPR/Cas12a system, a fluorescent biosensing protocol was developed for sensing assay of T790 M. When the target is absent, the initiator remains intact, opens the fuel hairpins and triggers the following HCR-FRET. At presence of the target, the Cas12a/crRNA binary complex specifically recognizes the target, and the Cas12a trans-cleavage activity is activated. As a result, the initiator is cleaved and subsequent HCR responses and FRET processes are attenuated. This method showed detection range from 1 pM to 400 pM with a detection limit of 316 fM. The target independent property of the HCR-FRET system endows this protocol a promising potential to transplant to the assay of other DNA target in parallel.}, }
@article {pmid37146580, year = {2023}, author = {Ferrari, S and Valeri, E and Conti, A and Scala, S and Aprile, A and Di Micco, R and Kajaste-Rudnitski, A and Montini, E and Ferrari, G and Aiuti, A and Naldini, L}, title = {Genetic engineering meets hematopoietic stem cell biology for next-generation gene therapy.}, journal = {Cell stem cell}, volume = {30}, number = {5}, pages = {549-570}, doi = {10.1016/j.stem.2023.04.014}, pmid = {37146580}, issn = {1875-9777}, mesh = {*CRISPR-Cas Systems ; Prospective Studies ; *Hematopoietic Stem Cells ; Gene Editing ; Genetic Therapy ; Biology ; }, abstract = {The growing clinical success of hematopoietic stem/progenitor cell (HSPC) gene therapy (GT) relies on the development of viral vectors as portable "Trojan horses" for safe and efficient gene transfer. The recent advent of novel technologies enabling site-specific gene editing is broadening the scope and means of GT, paving the way to more precise genetic engineering and expanding the spectrum of diseases amenable to HSPC-GT. Here, we provide an overview of state-of-the-art and prospective developments of the HSPC-GT field, highlighting how advances in biological characterization and manipulation of HSPCs will enable the design of the next generation of these transforming therapeutics.}, }
@article {pmid37144477, year = {2023}, author = {Shariq, M and Khan, MF and Raj, R and Ahsan, N and Singh, R and Kumar, P}, title = {CRISPR‑based diagnostic approaches: Implications for rapid management of future pandemics (Review).}, journal = {Molecular medicine reports}, volume = {27}, number = {6}, pages = {}, doi = {10.3892/mmr.2023.13005}, pmid = {37144477}, issn = {1791-3004}, mesh = {Humans ; *COVID-19 ; SARS-CoV-2/genetics ; CRISPR-Cas Systems/genetics ; Pandemics ; Bacteria/genetics ; COVID-19 Testing ; }, abstract = {Sudden viral outbreaks have increased in the early part of the 21st century, such as those of severe acute respiratory syndrome coronavirus (SARS‑CoV), Middle East respiratory syndrome corona virus, and SARS‑CoV‑2, owing to increased human access to wildlife habitats. Therefore, the likelihood of zoonotic transmission of human‑associated viruses has increased. The emergence of severe acute respiratory syndrome coronavirus 2 in China and its spread worldwide within months have highlighted the need to be ready with advanced diagnostic and antiviral approaches to treat newly emerging diseases with minimal harm to human health. The gold‑standard molecular diagnostic approaches currently used are time‑consuming, require trained personnel and sophisticated equipment, and therefore cannot be used as point‑of‑care devices for widespread monitoring and surveillance. Clustered regularly interspaced short palindromic repeats (CRISPR)‑associated (Cas) systems are widespread and have been reported in bacteria, archaea and bacteriophages. CRISPR‑Cas systems are organized into CRISPR arrays and adjacent Cas proteins. The detection and in‑depth biochemical characterization of class 2 type V and VI CRISPR‑Cas systems and orthologous proteins such as Cas12 and Cas13 have led to the development of CRISPR‑based diagnostic approaches, which have been used to detect viral diseases and distinguish between serotypes and subtypes. CRISPR‑based diagnostic approaches detect human single nucleotide polymorphisms in samples from patients with cancer and are used as antiviral agents to detect and destroy viruses that contain RNA as a genome. CRISPR‑based diagnostic approaches are likely to improve disease detection methods in the 21st century owing to their ease of development, low cost, reduced turnaround time, multiplexing and ease of deployment. The present review discusses the biochemical properties of Cas12 and Cas13 orthologs in viral disease detection and other applications. The present review expands the scope of CRISPR‑based diagnostic approaches to detect diseases and fight viruses as antivirals.}, }
@article {pmid37066613, year = {2023}, author = {Zhao, F and Wang, P and Wang, H and Liu, S and Sohail, M and Zhang, X and Li, B and Huang, H}, title = {CRISPR/Cas12a-mediated ultrasensitive and on-site monkeypox viral testing.}, journal = {Analytical methods : advancing methods and applications}, volume = {15}, number = {17}, pages = {2105-2113}, doi = {10.1039/d2ay01998a}, pmid = {37066613}, issn = {1759-9679}, mesh = {Humans ; *Monkeypox virus/genetics ; *Monkeypox/diagnosis/genetics ; CRISPR-Cas Systems/genetics ; Recombinases/genetics ; Africa, Western ; }, abstract = {The spread of the monkeypox virus (MPXV) from Central and West Africa to previously non-endemic regions has caused a global panic. In this context, the rapid, specific, and ultrasensitive detection of MPXV is crucial to contain its spread, though such technology has seldom been reported. Herein, we proposed an MPXV assay combining recombinase-aided amplification (RAA) and CRISPR/Cas12a. This assay targeted the highly conserved MPXV F3L gene and demonstrates a low detection limit (LOD) of 10[1] copies per μL. By leveraging the high specificity nature of RAA and CRISPR/Cas12a, we rationally optimized probes and conditions to achieve high selectivity that differentiates MPXV from other orthopox viruses and current high-profile viruses. To facilitate on-site screening of potential MPXV carriers, a kit integrating lateral flow strips was developed, enabling naked-eye MPXV detection with a LOD of 10[4] copies per μL. Our RAA-Cas12a-MPXV assay was able to detect MPXV without the need for sophisticated operation and expensive equipment. We believe that this assay can be rapidly deployed in emerging viral outbreaks for on-site surveillance of MPXV.}, }
@article {pmid37024675, year = {2023}, author = {Villanueva, MT}, title = {Base editing treats SMA in mice.}, journal = {Nature reviews. Drug discovery}, volume = {22}, number = {5}, pages = {353}, doi = {10.1038/d41573-023-00057-1}, pmid = {37024675}, issn = {1474-1784}, mesh = {Animals ; Mice ; *Gene Editing ; *CRISPR-Cas Systems/genetics ; Biotechnology ; }, }
@article {pmid37012339, year = {2023}, author = {Kingwell, K}, title = {First CRISPR therapy seeks landmark approval.}, journal = {Nature reviews. Drug discovery}, volume = {22}, number = {5}, pages = {339-341}, doi = {10.1038/d41573-023-00050-8}, pmid = {37012339}, issn = {1474-1784}, mesh = {Humans ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *CRISPR-Cas Systems/genetics ; Genetic Therapy ; }, }
@article {pmid37003192, year = {2023}, author = {Yang, M and Jiao, Y and Yan, Y and Li, L and Hu, X and Jiao, Z and Li, M and Chen, Y and Shi, Y and Shen, Z and Peng, G}, title = {Safety and immunogenicity of a TK/ gI/gE gene-deleted feline herpesvirus-1 mutant constructed via CRISPR/Cas9 in feline.}, journal = {Veterinary microbiology}, volume = {281}, number = {}, pages = {109728}, doi = {10.1016/j.vetmic.2023.109728}, pmid = {37003192}, issn = {1873-2542}, mesh = {Cats ; Animals ; CRISPR-Cas Systems ; *Varicellovirus ; *Herpesviridae Infections/prevention &amp; control/veterinary ; Antibodies, Neutralizing/genetics ; *Viral Vaccines ; *Cat Diseases/prevention &amp; control ; }, abstract = {Feline herpesvirus-1 (FHV-1) is the aetiological agent of feline viral rhinotracheitis, which accounts for approximately 50 % of all viral upper respiratory diseases in cats. Commercially available modified live vaccines containing FHV-1 are generally safe and effective, but these FHV-1 vaccines retain full virulence genes and can establish latency and reactivate to cause infectious rhinotracheitis in vaccine recipients, raising safety concerns. To address this shortcoming, we constructed a novel TK/gI/gE -gene-deleted recombinant FHV-1 (WH2020-ΔTK/gI/gE) through CRISPR/Cas9-mediated homologous recombination. The growth kinetics of WH2020-ΔTK/gI/gE were slightly delayed compared to those of the parent strain WH2020. Recombinant FHV-1 had severely impaired pathogenicity in cats. Felines immunized with WH2020-ΔTK/gI/gE produced high levels of gB-specific antibodies, neutralizing antibodies and IFN-β. Additionally, WH2020-ΔTK/gI/gE provided greater protection against challenge with FHV-1 field strain WH2020 than did the commercial modified live vaccine. After challenge, the cats vaccinated with WH2020-ΔTK/gI/gE showed significantly fewer clinical signs, pathological changes, viral shedding, and viral loads in the lung and trigeminal ganglia than those vaccinated with the commercial vaccine or unvaccinated. Our results suggest that WH2020-ΔTK/gI/gE is a promising candidate as a safer and more efficacious live FHV-1 vaccine, with a decreased risk of vaccine-related complications, and could inform the design of other herpesvirus vaccines.}, }
@article {pmid36917276, year = {2023}, author = {Zhang, C and Ren, Z and Gong, Z}, title = {Generation of Albino Phenotype in Ornamental Fish by CRISPR/Cas9-Mediated Genome Editing of slc45a2 Gene.}, journal = {Marine biotechnology (New York, N.Y.)}, volume = {25}, number = {2}, pages = {281-290}, pmid = {36917276}, issn = {1436-2236}, mesh = {Animals ; *Gene Editing/methods ; CRISPR-Cas Systems ; Zebrafish/genetics ; Phylogeny ; *Albinism/genetics ; }, abstract = {Albinism is the most common color variation described in fish and is a fascinating trait of some ornamental fish species. Albino mutants can be generated by knocking out core genes affecting melanin synthesis like slc45a2 in several fish species. However, genetic mutation remains challenging for species with unknown genome information. In this study, we generated albino mutants in two selected ornamental fish species, royal farlowella (Sturisoma panamense), and redhead cichlid (Vieja melanura). For this purpose, we carried out phylogenetic analyses of fish slc45a2 sequences and identified a highly conserved region among different fish species. A pair of degenerate primers spanning this region was designed and used to amplify a conserved slc45a2 fragment of 340 bp from the two fish species. Based on the amplified sequences, a target site in the 6[th] exon was used for designing guide RNA and this targeted site was first verified by the CRISPR/Cas9 system in the zebrafish (Danio rerio) model for the effectiveness. Then, specific guide RNAs were designed for the two ornamental fish species and tested. Most of the injected larvae completely lost black pigment over the whole body and eyes. DNA sequencing confirmed a high degree of mutation at the targeted site. Overall, we described a fast and efficient method to generate albino phenotype in fish species by targeting the conserved 6[th] exon of slc45a2 gene for genome editing via CRISPR/Cas9 and this approach could be a new genetic tool to generate desirable albino ornamental fish.}, }
@article {pmid36896650, year = {2023}, author = {Carneiro, SP and Greco, A and Chiesa, E and Genta, I and Merkel, OM}, title = {Shaping the future from the small scale: dry powder inhalation of CRISPR-Cas9 lipid nanoparticles for the treatment of lung diseases.}, journal = {Expert opinion on drug delivery}, volume = {20}, number = {4}, pages = {471-487}, doi = {10.1080/17425247.2023.2185220}, pmid = {36896650}, issn = {1744-7593}, mesh = {Humans ; Powders ; CRISPR-Cas Systems ; Administration, Inhalation ; *Nanoparticles ; *Lung Diseases/drug therapy ; Lung ; Dry Powder Inhalers ; Particle Size ; }, abstract = {INTRODUCTION: Most lung diseases are serious conditions resulting from genetic and environmental causes associated with high mortality and severe symptoms. Currently, treatments available have a palliative effect and many targets are still considered undruggable. Gene therapy stands as an attractive approach to offering innovative therapeutic solutions. CRISPRCas9 has established a remarkable potential for genome editing with high selectivity to targeted mutations. To ensure high efficacy with minimum systemic exposure, the delivery and administration route are key components that must be investigated.<br><br>AREAS COVERED: This review is focused on the delivery of CRISPRCas9 to the lungs, taking advantage of lipid nanoparticles (LNPs), the most clinically advanced nucleic acid carriers. We also aim to highlight the benefits of pulmonary administration as a local delivery route and the use of spray drying to prepare stable nucleic-acid-based dry powder formulations that can overcome multiple lung barriers.<br><br>EXPERT OPINION: Exploring the pulmonary administration to deliver CRISPRCas9 loaded in LNPs as a dry powder increases the chances to achieve high efficacy and reduced adverse effects. CRISPRCas9 loaded in LNP-embedded microparticles has not yet been reported in the literature but has the potential to reach and accumulate in target cells in the lung, thus, enhancing overall efficacy and safety.}, }
@article {pmid36861495, year = {2023}, author = {Fujii, T and Inoue, N and Nobeyama, T and Inoue, J and Ogasawara, S and Otani, Y and Fujii, S and Ito, C and Fukunaga, R}, title = {A simple, rapid, and efficient method for generating multigene-knockout culture cells by the CRISPR/Cas9 system.}, journal = {Genes to cells : devoted to molecular &amp; cellular mechanisms}, volume = {28}, number = {5}, pages = {390-397}, doi = {10.1111/gtc.13021}, pmid = {36861495}, issn = {1365-2443}, mesh = {Humans ; *CRISPR-Cas Systems/genetics ; HeLa Cells ; Gene Knockout Techniques ; Plasmids ; Transfection ; *Gene Editing/methods ; }, abstract = {We evaluated the efficacy of simultaneous multiple-gene knockout in human culture cells. By simple co-transfection of HeLa cells with a mixture of pX330-based targeting plasmids together with a puromycin resistance plasmid, followed by transient selection of puromycin-resistant cells, Cas9/single-guide RNA (sgRNA)-transduced polyclonal cell populations were selected and grown. Western blot analyses revealed co-transfection of up to seven targeting plasmids for p38α, p38β, JNK1, JNK2, Mnk1, ERK1, and mLST8 genes, drastically reduced protein expression of these genes in the polyclonal population. Analyses of a randomly isolated group of 25 clones revealed knockout efficiencies for the seven targeted genes ranging between 68% and 100%, and in six clones (24%), all targeted genes were disrupted. Deep sequencing analyses of the individual target sites revealed that, in most cases, Cas9/sgRNA-induced nonhomologous end joining resulted in deletion or insertion of only a few base pairs at the break points. These results demonstrate that simple co-transfection-based simultaneous targeting offers an easy, rapid, and efficient method to generate multiplex gene-knockout cell lines.}, }
@article {pmid36840757, year = {2023}, author = {Galindo-Sotomonte, L and Jozefkowicz, C and Gómez, C and Stritzler, M and Frare, R and Bottero, E and Tajima, H and Blumwald, E and Ayub, N and Soto, G}, title = {CRISPR/Cas9-mediated knockout of a polyester synthase-like gene delays flowering time in alfalfa.}, journal = {Plant cell reports}, volume = {42}, number = {5}, pages = {953-956}, pmid = {36840757}, issn = {1432-203X}, mesh = {*Medicago sativa/genetics ; CRISPR-Cas Systems/genetics ; Flowers/genetics ; *Arabidopsis/genetics ; }, abstract = {T-DNA and CRISPR/Cas9-mediated knockout of polyester synthase-like genes delays flowering time in Arabidopsis thaliana and Medicago sativa (alfalfa). Thus, we here present the first report of edited alfalfa with delayed flowering.}, }
@article {pmid36756818, year = {2023}, author = {Zhang, X and Guo, B and Yang, L and Zhao, C and Wang, Y and Tang, Y and Yang, G and Wang, P and Gao, S}, title = {CRISPR/Cas12a combined with recombinase polymerase amplification for rapid and sensitive detection of Vibrio vulnificus in one tube.}, journal = {Acta biochimica et biophysica Sinica}, volume = {55}, number = {2}, pages = {322-326}, pmid = {36756818}, issn = {1745-7270}, mesh = {*Recombinases/genetics ; *Vibrio vulnificus/genetics ; CRISPR-Cas Systems/genetics ; Nucleic Acid Amplification Techniques ; }, }
@article {pmid36734409, year = {2023}, author = {Kurzhagen, JT and Noel, S and Lee, K and Sadasivam, M and Gharaie, S and Ankireddy, A and Lee, SA and Newman-Rivera, A and Gong, J and Arend, LJ and Hamad, ARA and Reddy, SP and Rabb, H}, title = {T Cell Nrf2/Keap1 Gene Editing Using CRISPR/Cas9 and Experimental Kidney Ischemia-Reperfusion Injury.}, journal = {Antioxidants &amp; redox signaling}, volume = {38}, number = {13-15}, pages = {959-973}, doi = {10.1089/ars.2022.0058}, pmid = {36734409}, issn = {1557-7716}, support = {R01 HL136946/HL/NHLBI NIH HHS/United States ; R01 DK111209/DK/NIDDK NIH HHS/United States ; }, mesh = {Mice ; Animals ; Kelch-Like ECH-Associated Protein 1/genetics/metabolism ; *Antioxidants/metabolism ; NF-E2-Related Factor 2/genetics/metabolism ; CRISPR-Cas Systems ; Glutamate-Cysteine Ligase/genetics/metabolism ; Gene Editing ; Kidney/metabolism ; *Reperfusion Injury/genetics/therapy/metabolism ; Oxidative Stress ; }, abstract = {Aims: T cells play pathophysiologic roles in kidney ischemia-reperfusion injury (IRI), and the nuclear factor erythroid 2-related factor 2/kelch-like ECH-associated protein 1 (Nrf2/Keap1) pathway regulates T cell responses. We hypothesized that clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated Keap1-knockout (KO) augments Nrf2 antioxidant potential of CD4+ T cells, and that Keap1-KO CD4+ T cell immunotherapy protects from kidney IRI. Results: CD4+ T cell Keap1-KO resulted in significant increase of Nrf2 target genes NAD(P)H quinone dehydrogenase 1, heme oxygenase 1, glutamate-cysteine ligase catalytic subunit, and glutamate-cysteine ligase modifier subunit. Keap1-KO cells displayed no signs of exhaustion, and had significantly lower levels of interleukin 2 (IL2) and IL6 in normoxic conditions, but increased interferon gamma in hypoxic conditions in vitro. In vivo, adoptive transfer of Keap1-KO CD4+ T cells before IRI improved kidney function in T cell-deficient nu/nu mice compared with mice receiving unedited control CD4+ T cells. Keap1-KO CD4+ T cells isolated from recipient kidneys 24 h post IR were less activated compared with unedited CD4+ T cells, isolated from control kidneys. Innovation: Editing Nrf2/Keap1 pathway in murine T cells using CRISPR/Cas9 is an innovative and promising immunotherapy approach for kidney IRI and possibly other solid organ IRI. Conclusion: CRISPR/Cas9-mediated Keap1-KO increased Nrf2-regulated antioxidant gene expression in murine CD4+ T cells, modified responses to in vitro hypoxia and in vivo kidney IRI. Gene editing targeting the Nrf2/Keap1 pathway in T cells is a promising approach for immune-mediated kidney diseases.}, }
@article {pmid37144131, year = {2023}, author = {Singh, C and Kumar, R and Sehgal, H and Bhati, S and Singhal, T and Gayacharan,  and Nimmy, MS and Yadav, R and Gupta, SK and Abdallah, NA and Hamwieh, A and Kumar, R}, title = {Unclasping potentials of genomics and gene editing in chickpea to fight climate change and global hunger threat.}, journal = {Frontiers in genetics}, volume = {14}, number = {}, pages = {1085024}, pmid = {37144131}, issn = {1664-8021}, abstract = {Genomics and genome editing promise enormous opportunities for crop improvement and elementary research. Precise modification in the specific targeted location of a genome has profited over the unplanned insertional events which are generally accomplished employing unadventurous means of genetic modifications. The advent of new genome editing procedures viz; zinc finger nucleases (ZFNs), homing endonucleases, transcription activator like effector nucleases (TALENs), Base Editors (BEs), and Primer Editors (PEs) enable molecular scientists to modulate gene expressions or create novel genes with high precision and efficiency. However, all these techniques are exorbitant and tedious since their prerequisites are difficult processes that necessitate protein engineering. Contrary to first generation genome modifying methods, CRISPR/Cas9 is simple to construct, and clones can hypothetically target several locations in the genome with different guide RNAs. Following the model of the application in crop with the help of the CRISPR/Cas9 module, various customized Cas9 cassettes have been cast off to advance mark discrimination and diminish random cuts. The present study discusses the progression in genome editing apparatuses, and their applications in chickpea crop development, scientific limitations, and future perspectives for biofortifying cytokinin dehydrogenase, nitrate reductase, superoxide dismutase to induce drought resistance, heat tolerance and higher yield in chickpea to encounter global climate change, hunger and nutritional threats.}, }
@article {pmid37143874, year = {2023}, author = {Kumar, M and Prusty, MR and Pandey, MK and Singh, PK and Bohra, A and Guo, B and Varshney, RK}, title = {Application of CRISPR/Cas9-mediated gene editing for abiotic stress management in crop plants.}, journal = {Frontiers in plant science}, volume = {14}, number = {}, pages = {1157678}, pmid = {37143874}, issn = {1664-462X}, abstract = {Abiotic stresses, including drought, salinity, cold, heat, and heavy metals, extensively reducing global agricultural production. Traditional breeding approaches and transgenic technology have been widely used to mitigate the risks of these environmental stresses. The discovery of engineered nucleases as genetic scissors to carry out precise manipulation in crop stress-responsive genes and associated molecular network has paved the way for sustainable management of abiotic stress conditions. In this context, the clustered regularly interspaced short palindromic repeat-Cas (CRISPR/Cas)-based gene-editing tool has revolutionized due to its simplicity, accessibility, adaptability, flexibility, and wide applicability. This system has great potential to build up crop varieties with enhanced tolerance against abiotic stresses. In this review, we summarize the latest findings on understanding the mechanism of abiotic stress response in plants and the application of CRISPR/Cas-mediated gene-editing system towards enhanced tolerance to a multitude of stresses including drought, salinity, cold, heat, and heavy metals. We provide mechanistic insights on the CRISPR/Cas9-based genome editing technology. We also discuss applications of evolving genome editing techniques such as prime editing and base editing, mutant library production, transgene free and multiplexing to rapidly deliver modern crop cultivars adapted to abiotic stress conditions.}, }
@article {pmid37142716, year = {2023}, author = {Huang, D and Yuan, MM and Chen, J and Zheng, X and Wong, D and Alvarez, PJJ and Yu, P}, title = {The association of prokaryotic antiviral systems and symbiotic phage communities in drinking water microbiomes.}, journal = {ISME communications}, volume = {3}, number = {1}, pages = {46}, pmid = {37142716}, issn = {2730-6151}, abstract = {Prokaryotic antiviral systems are important mediators for prokaryote-phage interactions, which have significant implications for the survival of prokaryotic community. However, the prokaryotic antiviral systems under environmental stress are poorly understood, limiting the understanding of microbial adaptability. Here, we systematically investigated the profile of the prokaryotic antiviral systems at the community level and prokaryote-phage interactions in the drinking water microbiome. Chlorine disinfectant was revealed as the main ecological driver for the difference in prokaryotic antiviral systems and prokaryote-phage interactions. Specifically, the prokaryotic antiviral systems in the microbiome exhibited a higher abundance, broader antiviral spectrum, and lower metabolic burden under disinfectant stress. Moreover, significant positive correlations were observed between phage lysogenicity and enrichment of antiviral systems (e.g., Type IIG and IV restriction-modification (RM) systems, and Type II CRISPR-Cas system) in the presence of disinfection, indicating these antiviral systems might be more compatible with lysogenic phages and prophages. Accordingly, there was a stronger prokaryote-phage symbiosis in disinfected microbiome, and the symbiotic phages carried more auxiliary metabolic genes (AMGs) related to prokaryotic adaptability as well as antiviral systems, which might further enhance prokaryote survival in drinking water distribution systems. Overall, this study demonstrates that the prokaryotic antiviral systems had a close association with their symbiotic phages, which provides novel insights into prokaryote-phage interactions and microbial environmental adaptation.}, }
@article {pmid37142704, year = {2023}, author = {Gencay, YE and Jasinskytė, D and Robert, C and Semsey, S and Martínez, V and Petersen, A&#xd8; and Brunner, K and de Santiago Torio, A and Salazar, A and Turcu, IC and Eriksen, MK and Koval, L and Takos, A and Pascal, R and Schou, TS and Bayer, L and Bryde, T and Johansen, KC and Bak, EG and Smrekar, F and Doyle, TB and Satlin, MJ and Gram, A and Carvalho, J and Jessen, L and Hallström, B and Hink, J and Damholt, B and Troy, A and Grove, M and Clube, J and Grøndahl, C and Haaber, JK and van der Helm, E and Zdravkovic, M and Sommer, MOA}, title = {Engineered phage with antibacterial CRISPR-Cas selectively reduce E. coli burden in mice.}, journal = {Nature biotechnology}, volume = {}, number = {}, pages = {}, pmid = {37142704}, issn = {1546-1696}, abstract = {Antibiotic treatments have detrimental effects on the microbiome and lead to antibiotic resistance. To develop a phage therapy against a diverse range of clinically relevant Escherichia coli, we screened a library of 162 wild-type (WT) phages, identifying eight phages with broad coverage of E. coli, complementary binding to bacterial surface receptors, and the capability to stably carry inserted cargo. Selected phages were engineered with tail fibers and CRISPR-Cas machinery to specifically target E. coli. We show that engineered phages target bacteria in biofilms, reduce the emergence of phage-tolerant E. coli and out-compete their ancestral WT phages in coculture experiments. A combination of the four most complementary bacteriophages, called SNIPR001, is well tolerated in both mouse models and minipigs and reduces E. coli load in the mouse gut better than its constituent components separately. SNIPR001 is in clinical development to selectively kill E. coli, which may cause fatal infections in hematological cancer patients.}, }
@article {pmid37140042, year = {2023}, author = {Johnson, MC and Laderman, E and Huiting, E and Zhang, C and Davidson, A and Bondy-Denomy, J}, title = {Core defense hotspots within Pseudomonas aeruginosa are a consistent and rich source of anti-phage defense systems.}, journal = {Nucleic acids research}, volume = {}, number = {}, pages = {}, doi = {10.1093/nar/gkad317}, pmid = {37140042}, issn = {1362-4962}, support = {R01GM127489/NH/NIH HHS/United States ; }, abstract = {Bacteria use a diverse arsenal of anti-phage immune systems, including CRISPR-Cas and restriction enzymes. Recent advances in anti-phage system discovery and annotation tools have unearthed many unique systems, often encoded in horizontally transferred defense islands, which can be horizontally transferred. Here, we developed Hidden Markov Models (HMMs) for defense systems and queried microbial genomes on the NCBI database. Out of the 30 species with >200 completely sequenced genomes, our analysis found Pseudomonas aeruginosa exhibits the greatest diversity of anti-phage systems, as measured by Shannon entropy. Using network analysis to identify the common neighbors of anti-phage systems, we identified two core defense hotspot loci (cDHS1 and cDHS2). cDHS1 is up to 224 kb (median: 26 kb) with varied arrangements of more than 30 distinct immune systems across isolates, while cDHS2 has 24 distinct systems (median: 6 kb). Both cDHS regions are occupied in a majority of P. aeruginosa isolates. Most cDHS genes are of unknown function potentially representing new anti-phage systems, which we validated by identifying a novel anti-phage system (Shango) commonly encoded in cDHS1. Identifying core genes flanking immune islands could simplify immune system discovery and may represent popular landing spots for diverse MGEs carrying anti-phage systems.}, }
@article {pmid37132114, year = {2023}, author = {Gao, TT and Oh, TJ and Mehta, K and Huang, YA and Camp, T and Fan, H and Han, JW and Barnes, CM and Zhang, K}, title = {The clinical potential of optogenetic interrogation of pathogenesis.}, journal = {Clinical and translational medicine}, volume = {13}, number = {5}, pages = {e1243}, doi = {10.1002/ctm2.1243}, pmid = {37132114}, issn = {2001-1326}, support = {R01GM132438/GM/NIGMS NIH HHS/United States ; R01MH124827/MH/NIMH NIH HHS/United States ; }, mesh = {*Light ; *Optogenetics/methods ; CRISPR-Cas Systems ; }, abstract = {BACKGROUND: Opsin-based optogenetics has emerged as a powerful biomedical tool using light to control protein conformation. Such capacity has been initially demonstrated to control ion flow across the cell membrane, enabling precise control of action potential in excitable cells such as neurons or muscle cells. Further advancement in optogenetics incorporates a greater variety of photoactivatable proteins and results in flexible control of biological processes, such as gene expression and signal transduction, with commonly employed light sources such as LEDs or lasers in optical microscopy. Blessed by the precise genetic targeting specificity and superior spatiotemporal resolution, optogenetics offers new biological insights into physiological and pathological mechanisms underlying health and diseases. Recently, its clinical potential has started to be capitalized, particularly for blindness treatment, due to the convenient light delivery into the eye.<br><br>AIMS AND METHODS: This work summarizes the progress of current clinical trials and provides a brief overview of basic structures and photophysics of commonly used photoactivable proteins. We highlight recent achievements such as optogenetic control of the chimeric antigen receptor, CRISPR-Cas system, gene expression, and organelle dynamics. We discuss conceptual innovation and technical challenges faced by current optogenetic research.<br><br>CONCLUSION: In doing so, we provide a framework that showcases ever-growing applications of optogenetics in biomedical research and may inform novel precise medicine strategies based on this enabling technology.}, }
@article {pmid37127628, year = {2023}, author = {Shabbir, S and Wang, W and Nawaz, M and Boruah, P and Kulyar, MF and Chen, M and Wu, B and Liu, P and Dai, Y and Sun, L and Gou, Q and Liu, R and Hu, G and Younis, T and He, M}, title = {Molecular mechanism of engineered Zymomonas mobilis to furfural and acetic acid stress.}, journal = {Microbial cell factories}, volume = {22}, number = {1}, pages = {88}, pmid = {37127628}, issn = {1475-2859}, mesh = {*Acetic Acid/metabolism ; *Zymomonas/genetics ; Furaldehyde/metabolism ; DNA Shuffling ; Fermentation ; Ethanol/metabolism ; }, abstract = {Acetic acid and furfural (AF) are two major inhibitors of microorganisms during lignocellulosic ethanol production. In our previous study, we successfully engineered Zymomonas mobilis 532 (ZM532) strain by genome shuffling, but the molecular mechanisms of tolerance to inhibitors were still unknown. Therefore, this study investigated the responses of ZM532 and its wild-type Z. mobilis (ZM4) to AF using multi-omics approaches (transcriptomics, genomics, and label free quantitative proteomics). Based on RNA-Seq data, two differentially expressed genes, ZMO_RS02740 (up-regulated) and ZMO_RS06525 (down-regulated) were knocked out and over-expressed through CRISPR-Cas technology to investigate their roles in AF tolerance. Overall, we identified 1865 and 14 novel DEGs in ZM532 and wild-type ZM4. In contrast, 1532 proteins were identified in ZM532 and wild-type ZM4. Among these, we found 96 important genes in ZM532 involving acid resistance mechanisms and survival rates against stressors. Furthermore, our knockout results demonstrated that growth activity and glucose consumption of mutant strains ZM532∆ZMO_RS02740 and ZM4∆ZMO_RS02740 decreased with increased fermentation time from 42 to 55 h and ethanol production up to 58% in ZM532 than that in ZM532∆ZMO_RS02740. Hence, these findings suggest ZMO_RS02740 as a protective strategy for ZM ethanol production under stressful conditions.}, }
@article {pmid36930400, year = {2023}, author = {Zhang, G and Wang, J and Li, Y and Shang, G}, title = {CRISPR/Cas9-assisted ssDNA recombineering for site-directed mutagenesis and saturation mutagenesis of plasmid-encoded genes.}, journal = {Biotechnology letters}, volume = {45}, number = {5-6}, pages = {629-637}, pmid = {36930400}, issn = {1573-6776}, mesh = {*CRISPR-Cas Systems/genetics ; Plasmids/genetics ; Mutagenesis, Site-Directed ; Mutagenesis ; Mutation ; *DNA, Single-Stranded/genetics ; Escherichia coli/genetics ; Gene Editing/methods ; }, abstract = {Site-directed and saturation mutagenesis are critical DNA methodologies for studying protein structure and function. For plasmid-based gene mutation, PCR and overlap-extension PCR involve tedious cloning steps. When the plasmid size is large, PCR yield may be too low for cloning; and for saturation mutagenesis of a single codon, one experiment may not enough to generate all twenty coding variants. Oligo-mediated recombineering sidesteps the complicated cloning process by homologous recombination between a mutagenic oligo and its target site. However, the low recombineering efficiency and inability to select for the recombinant makes it necessary to screen a large number of clones. Herein, we describe two plasmid-based mutagenic strategies: CRISPR/Cas9-assisted ssDNA recombineering for site-directed mutagenesis (CRM) and saturation mutagenesis (CRSM). CRM and CRSM involve co-electroporation of target plasmid, sgRNA expression plasmid and mutagenic oligonucleotide into Escherichia coli cells with induced expression of λ-Red recombinase and Cas9, followed by plasmid extraction and characterization. We established CRM and CRSM via ampicillin resistance gene repair and mutagenesis of N-acetyl‑D‑neuraminic acid aldolase. The mutational efficiency was between 80 and 100% and all twenty amino acid coding variants were obtained at a target site via a single CRSM strategy. CRM and CRSM have the potential to be general plasmid-based gene mutagenesis tools.}, }
@article {pmid36847422, year = {2023}, author = {Wang, Z and Huang, C and Wei, S and Zhu, P and Li, Y and Fu, W and Zhang, Y}, title = {A CRISPR/Cas12a-Mediated Sensitive DNA Detection System for Gene-Edited Rice.}, journal = {Journal of AOAC International}, volume = {106}, number = {3}, pages = {558-567}, doi = {10.1093/jaoacint/qsad022}, pmid = {36847422}, issn = {1944-7922}, mesh = {*Gene Editing/methods ; CRISPR-Cas Systems/genetics ; *Oryza/genetics ; Crops, Agricultural/genetics ; Plants, Genetically Modified/genetics ; DNA ; }, abstract = {BACKGROUND: In recent years, genome editing technology represented by clustered regularly interspaced short palindromic repeat/CRISPR-associated nuclease 9 （CRISPR/Cas9） has been developed and applied in transgenic research and development, and transgenic products have been developed for a variety of applications. Gene editing products, unlike traditional genetically modified crops, which are generally obtained by target gene deletion, insertion, base mutation, etc., may not differ significantly at the gene level from conventional crops, which increases the complexity of testing.<br><br>OBJECTIVE: We established a specific and sensitive CRISPR/Cas12a-mediated gene editing system to detect target fragments in a variety of transgenic rice lines and commercial rice-based processing products.<br><br>METHODS: In this study, the CRISPR/Cas12a visible detection system was optimized for the visualization of nucleic acid detection in gene-edited rice. The fluorescence signals were detected by both gel electrophoresis and fluorescence-based methods.<br><br>RESULTS: The detection limit of the CRISPR/Cas12a detection system established in this study was more precise, especially&#xa0;for low-concentration samples. In addition to achieving single-base detection in gene-edited rice, we showed that different base mutations in the target sequence have different detection efficiencies by sitewise variant compact analysis. The CRISPR/Cas12a system was verified via a common transgenic rice strain and commercial rice sources. The results proved that the detection method could not only be tested in samples with multiple mutation types but could also effectively detect target fragments in commercial rice products.<br><br>CONCLUSION: We have developed a set of efficient detection methods with CRISPR/Cas12a for gene-edited rice detection to provide a new technical basis for rapid field detection of gene-edited rice.<br><br>HIGHLIGHTS: The CRISPR/Cas12a-mediated visual detection method used to detect gene-edited rice was evaluated for its specificity, sensitivity, and robustness.}, }
@article {pmid37127441, year = {2023}, author = {Vink, JNA and Hayhurst, M and Gerth, ML}, title = {Harnessing CRISPR-Cas for oomycete genome editing.}, journal = {Trends in microbiology}, volume = {}, number = {}, pages = {}, doi = {10.1016/j.tim.2023.03.017}, pmid = {37127441}, issn = {1878-4380}, abstract = {Oomycetes are a group of microorganisms that include pathogens responsible for devastating diseases in plants and animals worldwide. Despite their importance, the development of genome editing techniques for oomycetes has progressed more slowly than for model microorganisms. Here, we review recent breakthroughs in clustered regularly interspaced short palindromic repeats (CRISPR)-Cas technologies that are expanding the genome editing toolbox for oomycetes - from the original Cas9 study to Cas12a editing, ribonucleoprotein (RNP) delivery, and complementation. We also discuss some of the challenges to applying CRISPR-Cas in oomycetes and potential ways to overcome them. Advances in CRISPR-Cas technologies are being used to illuminate the biology of oomycetes, which ultimately can guide the development of tools for managing oomycete diseases.}, }
@article {pmid37124148, year = {2023}, author = {Balke-Want, H and Keerthi, V and Cadinanos-Garai, A and Fowler, C and Gkitsas, N and Brown, AK and Tunuguntla, R and Abou-El-Enein, M and Feldman, SA}, title = {Non-viral chimeric antigen receptor (CAR) T cells going viral.}, journal = {Immuno-oncology technology}, volume = {18}, number = {}, pages = {100375}, pmid = {37124148}, issn = {2590-0188}, abstract = {Chimeric antigen receptor (CAR) T cell therapy has made significant strides in the treatment of B-cell malignancies, but its application in treating solid tumors still poses significant challenges. Particularly, the widespread use of viral vectors to deliver CAR transgenes into T cells comes with limitations, including high costs and regulatory restrictions, which hinder the translation of novel genetic engineering concepts into clinical applications. Non-viral methods, such as transposon/transposase and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas systems, offer promising alternatives for stable transgene insertion in CAR-T cells. These methods offer the potential to increase accessibility and efficiency in the development and delivery of CAR-T cell therapies. The main challenge in using non-viral methods, however, is their low knock-in efficiency, which leads to low transgene expression levels. In this review, we discuss recent developments in non-viral approaches for CAR-T cell production, the manufacturing requirements for clinical-grade production of non-viral CAR-T cells, and the adjustments needed in quality control for proper characterization of genomic features and evaluation of potential genotoxicity.}, }
@article {pmid37122009, year = {2023}, author = {Zhang, L and Li, G and Zhang, Y and Cheng, Y and Roberts, N and Glenn, SE and DeZwaan-McCabe, D and Rube, HT and Manthey, J and Coleman, G and Vakulskas, CA and Qi, Y}, title = {Boosting genome editing efficiency in human cells and plants with novel LbCas12a variants.}, journal = {Genome biology}, volume = {24}, number = {1}, pages = {102}, pmid = {37122009}, issn = {1474-760X}, mesh = {Animals ; Humans ; *Gene Editing/methods ; CRISPR-Cas Systems ; Escherichia coli/genetics ; Mutagenesis ; Endonucleases/genetics/metabolism ; *Oryza/genetics/metabolism ; Genome, Plant ; Mammals/genetics ; }, abstract = {BACKGROUND: Cas12a (formerly known as Cpf1), the class II type V CRISPR nuclease, has been widely used for genome editing in mammalian cells and plants due to its distinct characteristics from Cas9. Despite being one of the most robust Cas12a nucleases, LbCas12a in general is less efficient than SpCas9 for genome editing in human cells, animals, and plants.<br><br>RESULTS: To improve the editing efficiency of LbCas12a, we conduct saturation mutagenesis in E. coli and identify 1977 positive point mutations of LbCas12a. We selectively assess the editing efficiency of 56 LbCas12a variants in human cells, identifying an optimal LbCas12a variant (RVQ: G146R/R182V/E795Q) with the most robust editing activity. We further test LbCas12a-RV, LbCas12a-RRV, and LbCas12a-RVQ in plants and find LbCas12a-RV has robust editing activity in rice and tomato protoplasts. Interestingly, LbCas12a-RRV, resulting from the stacking of RV and D156R, displays improved editing efficiency in stably transformed rice and poplar plants, leading to up to 100% editing efficiency in T0 plants of both plant species. Moreover, this high-efficiency editing occurs even at the non-canonical TTV PAM sites.<br><br>CONCLUSIONS: Our results demonstrate that LbCas12a-RVQ is a powerful tool for genome editing in human cells while LbCas12a-RRV confers robust genome editing in plants. Our study reveals the tremendous potential of these LbCas12a variants for advancing precision genome editing applications across a wide range of organisms.}, }
@article {pmid36938935, year = {2023}, author = {Park, J and Kang, SJ and Go, S and Lee, J and An, J and Chung, HS and Jeong, C and Ahn, DR}, title = {Split-tracrRNA as an efficient tracrRNA system with an improved potential of scalability.}, journal = {Biomaterials science}, volume = {11}, number = {9}, pages = {3241-3251}, doi = {10.1039/d2bm01901a}, pmid = {36938935}, issn = {2047-4849}, mesh = {*RNA, Guide, CRISPR-Cas Systems ; *Gene Editing ; RNA/genetics ; }, abstract = {Due to the relatively long sequence, tracrRNAs are chemically less synthesizable than crRNAs, leading to limited scalability of RNA guides for CRISPR-Cas9 systems. To develop shortened versions of RNA guides with improved cost-effectiveness, we have developed a split-tracrRNA system by nicking the 67-mer tracrRNA (tracrRNA(67)). Cellular gene editing assays and in vitro DNA cleavage assays revealed that the position of the nick is critical for maintaining the activity of tracrRNA(67). TracrRNA(41 + 23), produced by nicking in stem loop 2, showed gene editing efficiency and specificity comparable to those of tracrRNA(67). Removal of the loop of stem loop 2 was further possible without compromising the efficiency and specificity when the stem duplex was stabilized via a high GC content. Binding assays and single-molecule experiments suggested that efficient split-tracrRNAs could be engineered as long as their binding affinity to Cas9 and their reaction kinetics are similar to those of tracrRNA(67).}, }
@article {pmid36780174, year = {2023}, author = {Ma, T and Chen, X and Wang, M}, title = {Intracellular Delivery of mRNA for Cell-Selective CRISPR/Cas9 Genome Editing using Lipid Nanoparticles.}, journal = {Chembiochem : a European journal of chemical biology}, volume = {24}, number = {9}, pages = {e202200801}, doi = {10.1002/cbic.202200801}, pmid = {36780174}, issn = {1439-7633}, mesh = {Humans ; Gene Editing ; CRISPR-Cas Systems/genetics ; Gene Transfer Techniques ; RNA, Messenger/genetics ; *COVID-19/genetics ; *Nanoparticles ; }, abstract = {Messenger RNA (mRNA) is being used as part of an emerging class of biotherapeutics with great promise for preventing and treating a wide range of diseases, as well as encoding programmable nucleases for genome editing. However, mRNA's low stability and immunogenicity, as well as the impermeability of the cell membrane to mRNA greatly limit mRNA's potential for therapeutic use. Lipid nanoparticles (LNPs) are currently one of the most extensively studied nanocarriers for mRNA delivery and have recently been clinically approved for developing mRNA-based vaccines to prevent COVID-19. In this review, we summarize the latest advances in designing ionizable lipids and formulating LNPs for intracellular and tissue-targeted mRNA delivery. Furthermore, we discuss the progress of intracellular mRNA delivery for spatiotemporally controlled CRISPR/Cas9 genome editing by using LNPs. Finally, we provide a perspective on the future of LNP-based mRNA delivery for CRISPR/Cas9 genome editing and the treatment of genetic disorders.}, }
@article {pmid36757014, year = {2023}, author = {Liu, Y and Yuan, G and Hyden, B and Tuskan, GA and Abraham, PE and Yang, X}, title = {Expanding the application of anti-CRISPR proteins in plants for tunable genome editing.}, journal = {Plant physiology}, volume = {192}, number = {1}, pages = {60-64}, pmid = {36757014}, issn = {1532-2548}, mesh = {*Gene Editing ; *Plants/genetics ; CRISPR-Cas Systems/genetics ; Genome, Plant/genetics ; }, abstract = {Anti-CRISPR proteins are very efficient for inhibiting CRISPR/Cas9-based genome editing tools in both herbaceous and woody plant species.}, }
@article {pmid36083715, year = {2023}, author = {Nold, SP and Sych, K and Imre, G and Fuhrmann, DC and Pfeilschifter, J and Vutukuri, R and Schnutgen, F and Wittig, I and Meisterknecht, J and Frank, S and Goren, I}, title = {Reciprocal abrogation of PKM isoforms: contradictory outcomes and differing impact of splicing signal on CRISPR/Cas9 mediates gene editing in keratinocytes.}, journal = {The FEBS journal}, volume = {290}, number = {9}, pages = {2338-2365}, doi = {10.1111/febs.16625}, pmid = {36083715}, issn = {1742-4658}, mesh = {*Gene Editing ; *CRISPR-Cas Systems/genetics ; Protein Isoforms/metabolism ; RNA Splicing ; Carrier Proteins/genetics/metabolism ; Pyruvate Kinase/genetics/metabolism ; }, abstract = {The healing of wounded skin is a highly organized process involving a massive cell in- and outflux, proliferation and tissue remodelling. It is well accepted that metabolic constraints such as diabetes mellitus, overweight or anorexia impairs wound healing. Indeed, wound inflammation involves a boost of overall metabolic changes. As wound healing converges inflammatory processes that are also common to transformation, we investigate the functional role of the pro-neoplastic factor pyruvate kinase (PK) M2 and its metabolic active splice variant PKM1 in keratinocytes. Particularly, we challenge the impact of reciprocal ablation of PKM1 or two expression. Here, CRISPR/Cas9 genome editing of the PKM gene in HaCaT reveals an unexpected mutational bias at the 3'SS of exon 9, whereas no preference for any particular kind of mutation at exon 10 3' splice, despite the close vicinity (400 nucleotides apart) and sequence similarity between the two sites. Furthermore, as opposed to transient silencing of PKM2, exclusion splicing of PKM2 via genome editing mutually increases PKM1 mRNA and protein expression and compensates for the absence of PKM2, whereas the reciprocal elimination of PKM1 splicing reduces PKM2 expression and impedes cell proliferation, thus unveiling an essential role for PKM1 in growth and metabolic balance of HaCaT keratinocytes.}, }
@article {pmid37119838, year = {2023}, author = {Pal, P and Anand, U and Saha, SC and Sundaramurthy, S and Okeke, ES and Kumar, M and Radha,  and Bontempi, E and Albertini, E and Dey, A and Di Maria, F}, title = {Novel CRISPR/Cas technology in the realm of algal bloom biomonitoring: Recent trends and future perspectives.}, journal = {Environmental research}, volume = {}, number = {}, pages = {115989}, doi = {10.1016/j.envres.2023.115989}, pmid = {37119838}, issn = {1096-0953}, abstract = {In conjunction with global climate change, progressive ocean warming, and acclivity in pollution and anthropogenic eutrophication, the incidence of harmful algal blooms (HABs) and cyanobacterial harmful algal blooms (CHABs) continue to expand in distribution, frequency, and magnitude. Algal bloom-related toxins have been implicated in human health disorders and ecological dysfunction and are detrimental to the national and global economy. Biomonitoring programs based on traditional monitoring protocols were characterised by some limitations that can be efficiently overdone using the CRISPR/Cas technology. In the present review, the potential and challenges of exploiting the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas technology for early detection of HABs and CHABs-associated toxigenic species were analysed. Based on more than 30 scientific papers, the main results indicate the great potential of CRISPR/Cas technology for this issue, even if the high sensitivity detected for the Cas12 and Cas13 platforms represents a possible interference risk.}, }
@article {pmid37079720, year = {2023}, author = {Zhu, F and Zhao, Q}, title = {Sensitive CRISPR-Cas12a-Assisted Immunoassay for Small Molecule Detection in Homogeneous Solution.}, journal = {Analytical chemistry}, volume = {95}, number = {17}, pages = {6769-6774}, doi = {10.1021/acs.analchem.3c00218}, pmid = {37079720}, issn = {1520-6882}, mesh = {*CRISPR-Cas Systems ; *Antibodies ; Biotin ; Catalysis ; Immunoassay ; }, abstract = {Sensitive detection of small molecules is crucial for many applications, like biomedical diagnosis, food safety, and environmental analysis. Here, we describe a sensitive CRISPR-Cas12a-assisted immunoassay for small molecule detection in homogeneous solution. An active DNA (acDNA) modified with a specific small molecule serves as a competitor for antibody binding and an activator of CRISPR-Cas12a. Large-sized antibody binding with this acDNA probe inactivates the collateral cleavage activity of CRISPR-Cas12a due to a steric effect. When free small molecule target exists, it replaces the small molecule-modified acDNA from antibody, triggering catalytic cleavage of DNA reporters by CRISPR-Cas12a, and strong fluorescence is generated. With this strategy, we achieved detection of three important small molecules as models, biotin, digoxin, and folic acid, at picomolar levels by using streptavidin or antibody as recognition elements. With the progress of DNA-encoded small molecules and antibody, the proposed strategy provides a powerful toolbox for detection of small molecules in wide applications.}, }
@article {pmid36805659, year = {2023}, author = {Malaiwong, N and Porta-de-la-Riva, M and Krieg, M}, title = {FLInt: single shot safe harbor transgene integration via Fluorescent Landmark Interference.}, journal = {G3 (Bethesda, Md.)}, volume = {13}, number = {5}, pages = {}, pmid = {36805659}, issn = {2160-1836}, support = {P40 OD010440/OD/NIH HHS/United States ; }, mesh = {Animals ; Transgenes ; Animals, Genetically Modified ; *Genome ; *Chromosomes ; Phenotype ; CRISPR-Cas Systems ; }, abstract = {The stable incorporation of transgenes and recombinant DNA material into the host genome is a bottleneck in many bioengineering applications. Due to the low efficiency, identifying the transgenic animals is often a needle in the haystack. Thus, optimal conditions require efficient screening procedures, but also known and safe landing sites that do not interfere with host expression, low input material and strong expression from the new locus. Here, we leverage an existing library of ≈300 different loci coding for fluorescent markers that are distributed over all 6 chromosomes in Caenorhabditis elegans as safe harbors for versatile transgene integration sites using CRISPR/Cas9. We demonstrated that a single crRNA was sufficient for cleavage of the target region and integration of the transgene of interest, which can be easily followed by loss of the fluorescent marker. The same loci can also be used for extrachromosomal landing sites and as co-CRISPR markers without affecting body morphology or animal behavior. Thus, our method overcomes the uncertainty of transgene location during random mutagenesis, facilitates easy screening through fluorescence interference and can be used as co-CRISPR markers without further influence in phenotypes.}, }
@article {pmid37119416, year = {2023}, author = {Jogam, P and Sandhya, D and Alok, A and Peddaboina, V and Singh, SP and Abbagani, S and Zhang, B and Allini, VR}, title = {Editing of TOM1 gene in tobacco using CRISPR/Cas9 confers resistance to Tobacco mosaic virus.}, journal = {Molecular biology reports}, volume = {}, number = {}, pages = {}, pmid = {37119416}, issn = {1573-4978}, abstract = {BACKGROUND: Genome editing technology has become one of the excellent tools for precise plant breeding to develop novel plant germplasm. The Tobacco mosaic virus (TMV) is the most prominent pathogen that infects several Solanaceae plants, such as tobacco, tomato, and capsicum, which requires critical host factors for infection and replication of its genomic RNA in the host. The Tobamovirus multiplication (TOM) genes, such as TOM1, TOM2A, TOM2B, and TOM3, are involved in the multiplication of Tobamoviruses. TOM1 is a transmembrane protein necessary for efficient TMV multiplication in several plant species. The TOM genes are crucial recessive resistance genes that act against the tobamoviruses in various plant species.<br><br>METHODS AND RESULTS: The single guided RNA (sgRNA) was designed to target the first exon of the NtTOM1 gene and cloned into the pHSE401 vector. The pHSE401-NtTOM1 vector was introduced into Agrobacterium tumefaciens strain LBA4404 and then transformed into tobacco plants. The analysis on T0 transgenic plants showed the presence of the hptII and Cas9 transgenes. The sequence analysis of the NtTOM1 from T0 plants showed the indels. Genotypic evaluation of the NtTOM1 mutant lines displayed the stable inheritance of the mutations in the subsequent generations of tobacco plants. The NtTOM1 mutant lines successfully conferred resistance to TMV.<br><br>CONCLUSIONS: CRISPR/Cas genome editing is a reliable tool for investigating gene function and precision breeding across different plant species, especially the species in the Solanaceae family.}, }
@article {pmid37030292, year = {2023}, author = {Stokar-Avihail, A and Fedorenko, T and Hör, J and Garb, J and Leavitt, A and Millman, A and Shulman, G and Wojtania, N and Melamed, S and Amitai, G and Sorek, R}, title = {Discovery of phage determinants that confer sensitivity to bacterial immune systems.}, journal = {Cell}, volume = {186}, number = {9}, pages = {1863-1876.e16}, doi = {10.1016/j.cell.2023.02.029}, pmid = {37030292}, issn = {1097-4172}, mesh = {*Bacteria/genetics/virology ; *Bacteriophages/genetics ; CRISPR-Cas Systems ; Viral Proteins/metabolism ; Mutation ; Bacterial Physiological Phenomena ; }, abstract = {Over the past few years, numerous anti-phage defense systems have been discovered in bacteria. Although the mechanism of defense for some of these systems is understood, a major unanswered question is how these systems sense phage infection. To systematically address this question, we isolated 177 phage mutants that escape 15 different defense systems. In many cases, these escaper phages were mutated in the gene sensed by the defense system, enabling us to map the phage determinants that confer sensitivity to bacterial immunity. Our data identify specificity determinants of diverse retron systems and reveal phage-encoded triggers for multiple abortive infection systems. We find general themes in phage sensing and demonstrate that mechanistically diverse systems have converged to sense either the core replication machinery of the phage, phage structural components, or host takeover mechanisms. Combining our data with previous findings, we formulate key principles on how bacterial immune systems sense phage invaders.}, }
@article {pmid37014765, year = {2023}, author = {Wang, P and Guo, B and Zhang, X and Wang, Y and Yang, G and Shen, H and Gao, S and Zhang, L}, title = {One-Pot Molecular Diagnosis of Acute Hepatopancreatic Necrosis Disease by Recombinase Polymerase Amplification and CRISPR/Cas12a with Specially Designed crRNA.}, journal = {Journal of agricultural and food chemistry}, volume = {71}, number = {16}, pages = {6490-6498}, doi = {10.1021/acs.jafc.2c08689}, pmid = {37014765}, issn = {1520-5118}, mesh = {Humans ; *Recombinases ; *CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems ; Nucleotidyltransferases ; Acute Disease ; Necrosis ; Nucleic Acid Amplification Techniques ; }, abstract = {Acute hepatopancreatic necrosis disease (AHPND) is one of the most devastating diseases in aquaculture, causing significant economic losses in seafood supplies worldwide. Early detection is critical for its prevention, which requires reliable and fast-responding diagnosis tools with point-of-care testing (POCT) capacity. Recombinase polymerase amplification (RPA) has been combined with CRISPR/Cas12a for AHPND diagnosis with a two-step procedure, but the operation is inconvenient and has the risk of carryover contamination. Here, we develop an RPA-CRISPR one-pot assay that integrates RPA and CRISPR/Cas12a cleavage into simultaneous reactions. Using the special design of crRNA, which is based on suboptimal protospacer adjacent motifs (PAM), RPA and Cas12a are made compatible in one pot. The assay is highly specific with a good sensitivity of 10[2] copies/reaction. This study provides a new choice for AHPND diagnosis with a POCT facility and sets a good example for developing RPA-CRISPR one-pot molecular diagnosis assays.}, }
@article {pmid36848135, year = {2023}, author = {Pathak, N and Patino, CA and Ramani, N and Mukherjee, P and Samanta, D and Ebrahimi, SB and Mirkin, CA and Espinosa, HD}, title = {Cellular Delivery of Large Functional Proteins and Protein-Nucleic Acid Constructs via Localized Electroporation.}, journal = {Nano letters}, volume = {23}, number = {8}, pages = {3653-3660}, doi = {10.1021/acs.nanolett.2c04374}, pmid = {36848135}, issn = {1530-6992}, mesh = {*CRISPR-Cas Systems/genetics ; *Nucleic Acids ; Gene Editing ; Electroporation ; Proteins/genetics ; }, abstract = {Delivery of proteins and protein-nucleic acid constructs into live cells enables a wide range of applications from gene editing to cell-based therapies and intracellular sensing. However, electroporation-based protein delivery remains challenging due to the large sizes of proteins, their low surface charge, and susceptibility to conformational changes that result in loss of function. Here, we use a nanochannel-based localized electroporation platform with multiplexing capabilities to optimize the intracellular delivery of large proteins (β-galactosidase, 472 kDa, 75.38% efficiency), protein-nucleic acid conjugates (protein spherical nucleic acids (ProSNA), 668 kDa, 80.25% efficiency), and Cas9-ribonucleoprotein complex (160 kDa, ∼60% knock-out and ∼24% knock-in) while retaining functionality post-delivery. Importantly, we delivered the largest protein to date using a localized electroporation platform and showed a nearly 2-fold improvement in gene editing efficiencies compared to previous reports. Furthermore, using confocal microscopy, we observed enhanced cytosolic delivery of ProSNAs, which may expand opportunities for detection and therapy.}, }
@article {pmid36775840, year = {2023}, author = {Zhang, YC and Gao, Y and Ye, WN and Peng, YX and Zhu, KY and Gao, CF}, title = {CRISPR/Cas9-mediated knockout of NlCYP6CS1 gene reveals its role in detoxification of insecticides in Nilaparvata lugens (Hemiptera: Delphacidae).}, journal = {Pest management science}, volume = {79}, number = {6}, pages = {2239-2246}, doi = {10.1002/ps.7404}, pmid = {36775840}, issn = {1526-4998}, mesh = {Animals ; *Insecticides/pharmacology ; *Hemiptera/genetics ; CRISPR-Cas Systems ; Neonicotinoids/pharmacology ; Thiamethoxam ; Nitro Compounds/pharmacology ; *Chlorpyrifos/pharmacology ; Insecticide Resistance/genetics ; }, abstract = {BACKGROUND: The brown planthopper (Nilaparvata lugens) is one of the major rice insect pests in Asia. Recently, high levels of insecticide resistance have been frequently reported and cytochrome P450 monooxygenase (P450)-mediated metabolic detoxification is a common resistance mechanism in N. lugens. However, there has been no persuasive genetic method to prove the role of P450s in insecticide resistance in N. lugens.<br><br>RESULTS: Here, CRISPR/Cas9 system was used to disrupt the P450 gene NlCYP6CS1 to elucidate its role in insecticide resistance in field populations of N. lugens. We successfully constructed a homozygous strain (Nl6CS1-KO) with a 5-bp deletion and 1-bp insertion mutation of NlCYP6CS1. Compared with a background resistant strain (Nl-R), the susceptibility of knockout strain Nl6CS1-KO to imidacloprid, nitenpyram, thiamethoxam, dinotefuran, and pymetrozine was increased by 2.3-, 3.4-, 7.0-, 4.2- and 3.9-fold, respectively, but not significantly changed to triflumezopyrim, chlorpyrifos and buprofezin. Life table analysis demonstrated that the Nl6CS1-KO strain resembled the Nl-R strain in terms of egg and nymph developmental duration and adult lifespan, but differed from the Nl-R strain in the survival rate of eggs and nymphs, reproduction, and body weight.<br><br>CONCLUSIONS: Our study demonstrates the effect of functional deletion of NlCYP6CS1 on multiple insecticide resistance in N. lugens. For the first time, we applied CRISPR/Cas9 system to reveal the mechanism of insecticide resistance in N. lugens, which may shed light on similar studies in other hemipteran insects. &#xa9; 2023 Society of Chemical Industry.}, }
@article {pmid35831613, year = {2022}, author = {Li, S and Wu, H and Chen, LL}, title = {Screening circular RNAs with functional potential using the RfxCas13d/BSJ-gRNA system.}, journal = {Nature protocols}, volume = {17}, number = {9}, pages = {2085-2107}, pmid = {35831613}, issn = {1750-2799}, support = {55008728/HHMI/Howard Hughes Medical Institute/United States ; }, mesh = {Humans ; RNA/genetics/metabolism ; RNA Splicing ; *RNA, Circular ; RNA, Messenger/genetics/metabolism ; Sequence Analysis, RNA/methods ; RNA, Guide, CRISPR-Cas Systems ; }, abstract = {Circular RNAs (circRNAs) are covalently enclosed, single-stranded RNAs produced by back-splicing of pre-mRNA exons that have recently emerged as an important class of molecules in gene expression regulation. circRNAs share overlapping sequences with their cognate linear mRNAs except the back-splicing junction (BSJ) sites. This feature makes it difficult to discriminate between the functions of circRNAs and their cognate mRNAs. We previously reported that the programmable RNA-guided, RNA-targeting CRISPR-Cas13 (RfxCas13d) system effectively and specifically discriminates circRNAs from mRNAs by using guide RNAs (gRNAs) targeting sequences across BSJ sites. Here, we describe a detailed protocol based on this RfxCas13d/BSJ-gRNA system for large-scale functional circRNA screening in human cell lines. The protocol includes gRNA library design, construction and transduction, analysis of screening results and validation of functional circRNA candidates. In total, it takes ~3-4 months of collaborative work between a well-trained molecular biologist and a bioinformatic expert. This protocol can be applied both in cells and in vivo to identify highly expressed circRNAs affecting cell growth, either in unperturbed conditions or under environmental stimulation, without disturbing their cognate linear mRNAs.}, }
@article {pmid35023328, year = {2022}, author = {Bozoglu, T and Lee, S and Ziegler, T and Jurisch, V and Maas, S and Baehr, A and Hinkel, R and Hoenig, A and Hariharan, A and Kim, CI and Decker, S and Sami, H and Koppara, T and Oellinger, R and Müller, OJ and Frank, D and Megens, R and Nelson, P and Weber, C and Schnieke, A and Sperandio, M and Santamaria, G and Rad, R and Moretti, A and Laugwitz, KL and Soehnlein, O and Ogris, M and Kupatt, C}, title = {Endothelial Retargeting of AAV9 In Vivo.}, journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)}, volume = {9}, number = {7}, pages = {e2103867}, pmid = {35023328}, issn = {2198-3844}, support = {/ERC_/European Research Council/International ; }, mesh = {Animals ; Blood Pressure ; *Dependovirus/genetics ; *Endothelial Cells ; Mice ; Mice, Transgenic ; Swine ; RNA, Guide, CRISPR-Cas Systems ; }, abstract = {Adeno-associated viruses (AAVs) are frequently used for gene transfer and gene editing in vivo, except for endothelial cells, which are remarkably resistant to unmodified AAV-transduction. AAVs are retargeted here toward endothelial cells by coating with second-generation polyamidoamine dendrimers (G2) linked to endothelial-affine peptides (CNN). G2[CNN] AAV9-Cre (encoding Cre recombinase) are injected into mTmG-mice or mTmG-pigs, cell-specifically converting red to green fluorescence upon Cre-activity. Three endothelial-specific functions are assessed: in vivo quantification of adherent leukocytes after systemic injection of - G2[CNN] AAV9 encoding 1) an artificial adhesion molecule (S1FG) in wildtype mice (day 10) or 2) anti-inflammatory Annexin A1 (Anxa1) in ApoE[-/-] mice (day 28). Moreover, 3) in Cas9-transgenic mice, blood pressure is monitored till day 56 after systemic application of G2[CNN] AAV9-gRNAs, targeting exons 6-10 of endothelial nitric oxide synthase (eNOS), a vasodilatory enzyme. G2[CNN] AAV9-Cre transduces microvascular endothelial cells in mTmG-mice or mTmG-pigs. Functionally, G2[CNN] AAV9-S1FG mediates S1FG-leukocyte adhesion, whereas G2[CNN] AAV9-Anxa1-application reduces long-term leukocyte recruitment. Moreover, blood pressure increases in Cas9-expressing mice subjected to G2[CNN] AAV9-gRNA[eNOS] . Therefore, G2[CNN] AAV9 may enable gene transfer in vascular and atherosclerosis models.}, }
@article {pmid37118221, year = {2023}, author = {Kushwaha, SK and Kumar, AA and Gupta, H and Marathe, SA}, title = {The Phylogenetic Study of the CRISPR-Cas System in Enterobacteriaceae.}, journal = {Current microbiology}, volume = {80}, number = {6}, pages = {196}, pmid = {37118221}, issn = {1432-0991}, mesh = {*Enterobacteriaceae/genetics ; Phylogeny ; *CRISPR-Cas Systems ; }, abstract = {The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated (Cas) system is a bacterial and archaeal adaptive immune system undergoing rapid multifaceted evolution. This evolution plausibly occurs due to the genetic exchanges of complete loci or individual entities. Here, we systematically investigate the evolutionary framework of the CRISPR-Cas system in six Enterobacteriaceae species and its evolutionary association with housekeeping genes as determined by the gyrB phenogram. The strains show high variability in the cas3 gene and the CRISPR1 locus among the closely related Enterobacteriaceae species, hinting at a series of genetic exchanges. The CRISPR leader is conserved, especially toward the distal end, and could be a core region of the leader. The spacers are conserved within the strains of most species, while some strains show unique sets of spacers. However, inter-species spacer conservation was rarely observed. For a considerable proportion of these spacers, protospacer sources were not detected. These results advance our understanding of the dynamics of the CRISPR-Cas system; however, the biological functions are yet to be characterised.}, }
@article {pmid37116358, year = {2023}, author = {Zhao, D and Tang, J and Tan, Q and Xie, X and Zhao, X and Xing, D}, title = {CRISPR/Cas13a-triggered Cas12a biosensing method for ultrasensitive and specific miRNA detection.}, journal = {Talanta}, volume = {260}, number = {}, pages = {124582}, doi = {10.1016/j.talanta.2023.124582}, pmid = {37116358}, issn = {1873-3573}, abstract = {Constructing an ultrasensitive CRISPR/Cas-based biosensing strategy is highly significant for the detection of trace targets. Here we presented a dual-amplified biosensing method based on CRISPR/Cas13a-triggered Cas12a, namely, Cas13a-12a amplification. As proof-of-principle, the developed strategy was used for miRNA-155 detection. The target bound to the Cas13a-crRNA complex and activated the cleavage activity of Cas13a for cleaving uracil ribonucleotides (rU) in the bulge structure of blocker strand (BS), resulting in the release of primer strand (PS) from the BS modified on magnetic beads. Then, the released PS activated the cleavage activity of Cas12a to cleave single-strand DNA reporter probes, producing a significantly increased fluorescent signal. The detection limit of the Cas13a-12a amplification using synthetic miRNA-155 was as low as 0.35 fM, which was much lower than that of the only Cas13a-based assay. The applied performance of this amplification strategy was verified by accurately quantifying miRNA-155 expression levels in different cancer patients. Therefore, the developed strategy offers a supersensitive and highly specific miRNAs sensing platform for clinical application.}, }
@article {pmid37114789, year = {2023}, author = {F Saad, J and A Saad, F}, title = {Gene Therapy for Alzheimer and Parkinson Diseases.}, journal = {Current gene therapy}, volume = {}, number = {}, pages = {}, doi = {10.2174/1566523223666230419101023}, pmid = {37114789}, issn = {1875-5631}, abstract = {Alzheimer and Parkinson diseases are associated with cholinergic neuron loss and deterioration of bone mineral density. Gene therapy through either gene transfer, CRISPR gene editing, or CRISPR gene modulation holds the potential to cure Alzheimer and Parkinson diseases. The emerging role of weight-bearing exercise in the prevention of, and care for, osteoporosis, obesity, and diabetes has been previously recognized. Moreover, endurance exercise offers a viable alternative to reduce amyloid peptides deposits while increasing bone mineral density in Alzheimer and Parkinson patients. β-amyloid peptides, α-synuclein, and tau aggregates start building up two decades before the onset of Alzheimer and Parkinson diseases. Therefore, an early intervention program for the detection of these deposits is required to prevent or delay the onset of these diseases. This article spots light on the potential of gene therapy for Alzheimer and Parkinson diseases.}, }
@article {pmid37112797, year = {2023}, author = {Teng, M and Liu, JL and Luo, Q and Zheng, LP and Yao, Y and Nair, V and Zhang, GP and Luo, J}, title = {Efficient Cross-Screening and Characterization of Monoclonal Antibodies against Marek's Disease Specific Meq Oncoprotein Using CRISPR/Cas9-Gene-Edited Viruses.}, journal = {Viruses}, volume = {15}, number = {4}, pages = {}, pmid = {37112797}, issn = {1999-4915}, support = {BBS/OS/NW/000007/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; }, mesh = {Animals ; *Marek Disease ; Gene Editing ; CRISPR-Cas Systems ; Antibodies, Monoclonal/metabolism ; *Herpesvirus 2, Gallid/genetics ; Oncogene Proteins/metabolism ; Chickens ; *Poultry Diseases ; *Oncogene Proteins, Viral/genetics ; }, abstract = {Marek's disease (MD) caused by pathogenic Marek's disease virus type 1 (MDV-1) is one of the most important neoplastic diseases of poultry. MDV-1-encoded unique Meq protein is the major oncoprotein and the availability of Meq-specific monoclonal antibodies (mAbs) is crucial for revealing MDV pathogenesis/oncogenesis. Using synthesized polypeptides from conserved hydrophilic regions of the Meq protein as immunogens, together with hybridoma technology and primary screening by cross immunofluorescence assay (IFA) on Meq-deleted MDV-1 viruses generated by CRISPR/Cas9-gene editing, a total of five positive hybridomas were generated. Four of these hybridomas, namely 2A9, 5A7, 7F9 and 8G11, were further confirmed to secrete specific antibodies against Meq as confirmed by the IFA staining of 293T cells overexpressing Meq. Confocal microscopic analysis of cells stained with these antibodies confirmed the nuclear localization of Meq in MDV-infected CEF cells and MDV-transformed MSB-1 cells. Furthermore, two mAb hybridoma clones, 2A9-B12 and 8G11-B2 derived from 2A9 and 8G11, respectively, displayed high specificity for Meq proteins of MDV-1 strains with diverse virulence. Our data presented here, using synthesized polypeptide immunization combined with cross IFA staining on CRISPR/Cas9 gene-edited viruses, has provided a new efficient approach for future generation of specific mAbs against viral proteins.}, }
@article {pmid37108761, year = {2023}, author = {Major, L and McClements, ME and MacLaren, RE}, title = {A Review of CRISPR Tools for Treating Usher Syndrome: Applicability, Safety, Efficiency, and In Vivo Delivery.}, journal = {International journal of molecular sciences}, volume = {24}, number = {8}, pages = {}, pmid = {37108761}, issn = {1422-0067}, support = {MR/V029924/1/MRC_/Medical Research Council/United Kingdom ; }, mesh = {Humans ; *Gene Editing ; CRISPR-Cas Systems/genetics ; *Usher Syndromes/genetics/therapy ; Genetic Therapy ; Epigenesis, Genetic ; }, abstract = {This review considers research into the treatment of Usher syndrome, a deaf-blindness syndrome inherited in an autosomal recessive manner. Usher syndrome mutations are markedly heterogeneous, involving many different genes, and research grants are limited due to minimal patient populations. Furthermore, gene augmentation therapies are impossible in all but three Usher syndromes as the cDNA sequence exceeds the 4.7 kb AAV packaging limit. It is, therefore, vital to focus research efforts on alternative tools with the broadest applicability. The CRISPR field took off in recent years following the discovery of the DNA editing activity of Cas9 in 2012. New generations of CRISPR tools have succeeded the original CRISPR/Cas9 model to enable more sophisticated genomic amendments such as epigenetic modification and precise sequence alterations. This review will evaluate the most popular CRISPR tools to date: CRISPR/Cas9, base editing, and prime editing. It will consider these tools in terms of applicability (in relation to the ten most prevalent USH2A mutations), safety, efficiency, and in vivo delivery potential with the intention of guiding future research investment.}, }
@article {pmid37108214, year = {2023}, author = {Allemailem, KS and Almatroodi, SA and Almatroudi, A and Alrumaihi, F and Al Abdulmonem, W and Al-Megrin, WAI and Aljamaan, AN and Rahmani, AH and Khan, AA}, title = {Recent Advances in Genome-Editing Technology with CRISPR/Cas9 Variants and Stimuli-Responsive Targeting Approaches within Tumor Cells: A Future Perspective of Cancer Management.}, journal = {International journal of molecular sciences}, volume = {24}, number = {8}, pages = {}, pmid = {37108214}, issn = {1422-0067}, mesh = {Humans ; *CRISPR-Cas Systems/genetics ; CRISPR-Associated Protein 9/metabolism ; Gene Editing ; *Neoplasms/genetics/therapy ; Technology ; }, abstract = {The innovative advances in transforming clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR/Cas9) into different variants have taken the art of genome-editing specificity to new heights. Allosteric modulation of Cas9-targeting specificity by sgRNA sequence alterations and protospacer adjacent motif (PAM) modifications have been a good lesson to learn about specificity and activity scores in different Cas9 variants. Some of the high-fidelity Cas9 variants have been ranked as Sniper-Cas9, eSpCas9 (1.1), SpCas9-HF1, HypaCas9, xCas9, and evoCas9. However, the selection of an ideal Cas9 variant for a given target sequence remains a challenging task. A safe and efficient delivery system for the CRISPR/Cas9 complex at tumor target sites faces considerable challenges, and nanotechnology-based stimuli-responsive delivery approaches have significantly contributed to cancer management. Recent innovations in nanoformulation design, such as pH, glutathione (GSH), photo, thermal, and magnetic responsive systems, have modernized the art of CRISPR/Cas9 delivery approaches. These nanoformulations possess enhanced cellular internalization, endosomal membrane disruption/bypass, and controlled release. In this review, we aim to elaborate on different CRISPR/Cas9 variants and advances in stimuli-responsive nanoformulations for the specific delivery of this endonuclease system. Furthermore, the critical constraints of this endonuclease system on clinical translations towards the management of cancer and prospects are described.}, }
@article {pmid37107664, year = {2023}, author = {Chapman, B and Han, JH and Lee, HJ and Ruud, I and Kim, TH}, title = {Targeted Modulation of Chicken Genes In Vitro Using CRISPRa and CRISPRi Toolkit.}, journal = {Genes}, volume = {14}, number = {4}, pages = {}, pmid = {37107664}, issn = {2073-4425}, mesh = {Animals ; *Chickens/genetics ; *CRISPR-Cas Systems ; Gene Expression Regulation ; Transcriptional Activation ; Cell Line ; }, abstract = {Engineering of clustered regularly interspaced short palindromic repeats (CRISPR) and the CRISPR-associated protein 9 (Cas9) system has enabled versatile applications of CRISPR beyond targeted DNA cleavage. Combination of nuclease-deactivated Cas9 (dCas9) and transcriptional effector domains allows activation (CRISPRa) or repression (CRISPRi) of target loci. To demonstrate the effectiveness of the CRISPR-mediated transcriptional regulation in chickens, three CRISPRa (VP64, VPR, and p300) and three CRISPRi (dCas9, dCas9-KRAB, and dCas9-KRAB-MeCP2) systems were tested in chicken DF-1 cells. By introducing guide RNAs (gRNAs) targeting near the transcription start site (TSS) of each gene in CRISPRa and CRISPRi effector domain-expressing chicken DF-1 cell lines, significant gene upregulation was induced in dCas9-VPR and dCas9-VP64 cells, while significant downregulation was observed with dCas9 and dCas9-KRAB. We further investigated the effect of gRNA positions across TSS and discovered that the location of gRNA is an important factor for targeted gene regulation. RNA sequencing analysis of IRF7 CRISPRa and CRISPRi- DF-1 cells revealed the specificity of CRISPRa and CRISPRi-based targeted transcriptional regulation with minimal off-target effects. These findings suggest that the CRISPRa and CRISPRi toolkits are an effective and adaptable platform for studying the chicken genome by targeted transcriptional modulation.}, }
@article {pmid37107564, year = {2023}, author = {Yang, L and Li, H and Han, Y and Song, Y and Wei, M and Fang, M and Sun, Y}, title = {CRISPR/Cas9 Gene Editing System Can Alter Gene Expression and Induce DNA Damage Accumulation.}, journal = {Genes}, volume = {14}, number = {4}, pages = {}, pmid = {37107564}, issn = {2073-4425}, mesh = {*Gene Editing ; *CRISPR-Cas Systems/genetics ; Genome ; DNA Damage/genetics ; Gene Expression ; }, abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR) and the associated protein (Cas) gene editing can induce P53 activation, large genome fragment deletions, and chromosomal structural variations. Here, gene expression was detected in host cells using transcriptome sequencing following CRISPR/Cas9 gene editing. We found that the gene editing reshaped the gene expression, and the number of differentially expressed genes was correlated with the gene editing efficiency. Moreover, we found that alternative splicing occurred at random sites and that targeting a single site for gene editing may not result in the formation of fusion genes. Further, gene ontology and KEGG enrichment analysis showed that gene editing altered the fundamental biological processes and pathways associated with diseases. Finally, we found that cell growth was not affected; however, the DNA damage response protein-γH2AX-was activated. This study revealed that CRISPR/Cas9 gene editing may induce cancer-related changes and provided basic data for research on the safety risks associated with the use of the CRISPR/Cas9 system.}, }
@article {pmid37106426, year = {2023}, author = {Frank, E and Cailleret, M and Nelep, C and Fragner, P and Polentes, J and Herardot, E and El Kassar, L and Giraud-Triboult, K and Monville, C and Ben M'Barek, K}, title = {Semi-automated optimized method to isolate CRISPR/Cas9 edited human pluripotent stem cell clones.}, journal = {Stem cell research &amp; therapy}, volume = {14}, number = {1}, pages = {110}, pmid = {37106426}, issn = {1757-6512}, mesh = {Humans ; *CRISPR-Cas Systems/genetics ; Gene Editing/methods ; *Pluripotent Stem Cells/metabolism ; Mutation ; Clone Cells ; }, abstract = {BACKGROUND: CRISPR/Cas9 editing systems are currently used to generate mutations in a particular gene to mimic a genetic disorder in vitro. Such "disease in a dish" models based on human pluripotent stem cells (hPSCs) offer the opportunity to have access to virtually all cell types of the human body. However, the generation of mutated hPSCs remains fastidious. Current CRISPR/Cas9 editing approaches lead to a mixed cell population containing simultaneously non-edited and a variety of edited cells. These edited hPSCs need therefore to be isolated through manual dilution cloning, which is time-consuming, labor intensive and tedious.<br><br>METHODS: Following CRISPR/Cas9 edition, we obtained a mixed cell population with various edited cells. We then used a semi-automated robotic platform to isolate single cell-derived clones.<br><br>RESULTS: We optimized CRISPR/Cas9 editing to knock out a representative gene and developed a semi-automated method for the clonal isolation of edited hPSCs. This method is faster and more reliable than current manual approaches.<br><br>CONCLUSIONS: This novel method of hPSC clonal isolation will greatly improve and upscale the generation of edited hPSCs required for downstream applications including disease modeling and drug screening.}, }
@article {pmid37105976, year = {2023}, author = {Yang, C and Ma, Z and Wang, K and Dong, X and Huang, M and Li, Y and Zhu, X and Li, J and Cheng, Z and Bi, C and Zhang, X}, title = {HMGN1 enhances CRISPR-directed dual-function A-to-G and C-to-G base editing.}, journal = {Nature communications}, volume = {14}, number = {1}, pages = {2430}, pmid = {37105976}, issn = {2041-1723}, mesh = {*Gene Editing/methods ; CRISPR-Cas Systems/genetics ; *HMGN1 Protein/genetics ; Chromatin ; Genome ; Transcription Factors/genetics ; }, abstract = {C-to-G base editors have been successfully constructed recently, but limited work has been done on concurrent C-to-G and A-to-G base editing. In addition, there is also limited data on how chromatin-associated factors affect the base editing. Here, we test a series of chromatin-associated factors, and chromosomal protein HMGN1 was found to enhance the efficiency of both C-to-G and A-to-G base editing. By fusing HMGN1, GBE and ABE to Cas9, we develop a CRISPR-based dual-function A-to-G and C-to-G base editor (GGBE) which is capable of converting simultaneous A and C to G conversion with substantial editing efficiency. Accordingly, the HMGN1 role shown in this work and the resulting GGBE tool further broaden the genome manipulation capacity of CRISPR-directed base editors.}, }
@article {pmid37104211, year = {2023}, author = {Xiong, L and Liu, Z and Li, J and Yao, S and Li, Z and Chen, X and Shen, L and Zhang, Z and Li, Y and Hou, Q and Zhang, Y and You, M and Yuchi, Z and You, S}, title = {Analysis of the Effect of Plutella xylostella Polycalin and ABCC2 Transporter on Cry1Ac Susceptibility by CRISPR/Cas9-Mediated Knockout.}, journal = {Toxins}, volume = {15}, number = {4}, pages = {}, pmid = {37104211}, issn = {2072-6651}, mesh = {Animals ; *Moths ; *Bacillus thuringiensis/genetics/metabolism ; Bacillus thuringiensis Toxins/metabolism ; CRISPR-Cas Systems ; Endotoxins/genetics/pharmacology/metabolism ; Larva ; Multidrug Resistance-Associated Protein 2 ; Hemolysin Proteins/genetics/pharmacology/metabolism ; Bacterial Proteins/genetics/pharmacology/metabolism ; Insecticide Resistance/genetics ; Insect Proteins/metabolism ; }, abstract = {Many insects, including the Plutella xylostella (L.), have developed varying degrees of resistance to many insecticides, including Bacillus thuringiensis (Bt) toxins, the bioinsecticides derived from Bt. The polycalin protein is one of the potential receptors for Bt toxins, and previous studies have confirmed that the Cry1Ac toxin can bind to the polycalin protein of P. xylostella, but whether polycalin is associated with the resistance of Bt toxins remains controversial. In this study, we compared the midgut of larvae from Cry1Ac-susceptible and -resistant strains, and found that the expression of the Pxpolycalin gene was largely reduced in the midgut of the resistant strains. Moreover, the spatial and temporal expression patterns of Pxpolycalin showed that it was mainly expressed in the larval stage and midgut tissue. However, genetic linkage experiments showed that the Pxpolycalin gene and its transcript level were not linked to Cry1Ac resistance, whereas both the PxABCC2 gene and its transcript levels were linked to Cry1Ac resistance. The larvae fed on a diet containing the Cry1Ac toxin showed no significant change in the expression of the Pxpolycalin gene in a short term. Furthermore, the knockout of polycalin and ATP-binding cassette transporter subfamily C2 (ABCC2) genes separately by CRISPR/Cas9 technology resulted in resistance to decreased susceptibility to Cry1Ac toxin. Our results provide new insights into the potential role of polycalin and ABCC2 proteins in Cry1Ac resistance and the mechanism underlying the resistance of insects to Bt toxins.}, }
@article {pmid37088039, year = {2023}, author = {Xiao, Y and Ren, H and Wang, H and Zou, D and Liu, Y and Li, H and Hu, P and Li, Y and Liu, Z and Lu, S}, title = {A rapid and inexpensive nucleic acid detection platform for Listeria monocytogenes based on the CRISPR/Cas12a system.}, journal = {Talanta}, volume = {259}, number = {}, pages = {124558}, doi = {10.1016/j.talanta.2023.124558}, pmid = {37088039}, issn = {1873-3573}, mesh = {Cattle ; Animals ; CRISPR-Cas Systems ; *Listeria monocytogenes/genetics ; Biological Assay ; Drug Contamination ; Recombinases ; *Nucleic Acids ; Nucleic Acid Amplification Techniques ; }, abstract = {Listeria monocytogenes (LM) is an important foodborne pathogen that is associated with a high mortality rate. Currently, there is an urgent need for an inexpensive and rapid assay for the large-scale diagnosis and monitoring of LM. To meet these requirements, we designed a one-step, low-cost platform for the simultaneous amplification and detection of LM based on the CRISPR/Cas12a system with a micro-amplification (named Cas12a-MA). This method utilizes a combination of CRISPR/Cas12a and recombinase polymerase amplification (RPA) in the same vessel to provide a contamination-free platform for rapid nucleic acid detection with high specificity and ultra-sensitivity. In this study, we screened for three specific genes and selected the hly gene in LM as the final target. Our data showed that the number of amplification products plays a crucial role in the function of the CRISPR/Cas12a system. Our method was then further optimized for the specific detection of target DNA on 4.4 CFU/g in 25min. These assays successfully detected LM in spiked pork samples and natural meat samples (pork, beef, and mutton). All results indicate that Cas12a-MA shows great promise for foodborne pathogen detection.}, }
@article {pmid37086563, year = {2023}, author = {Tian, M and Zhang, R and Li, J}, title = {Emergence of CRISPR/Cas9-mediated bioimaging: A new dawn of in-situ detection.}, journal = {Biosensors &amp; bioelectronics}, volume = {232}, number = {}, pages = {115302}, doi = {10.1016/j.bios.2023.115302}, pmid = {37086563}, issn = {1873-4235}, mesh = {*CRISPR-Cas Systems/genetics ; *Biosensing Techniques ; In Situ Hybridization, Fluorescence ; Gene Editing/methods ; }, abstract = {In-situ detection provides deep insights into the function of genes and their relationship with diseases by directly visualizing their spatiotemporal behavior. As an emerging in-situ imaging tool, clustered regularly interspaced short palindromic repeats (CRISPR)-mediated bioimaging can localize targets in living and fixed cells. CRISPR-mediated bioimaging has inherent advantages over the gold standard of fluorescent in-situ hybridization (FISH), including fast imaging, cost-effectiveness, and ease of preparation. Existing reviews have provided a detailed classification and overview of the principles of CRISPR-mediated bioimaging. However, the exploitation of potential clinical applicability of this bioimaging technique is still limited. Therefore, analyzing the potential value of CRISPR-mediated in-situ imaging is of great significance to the development of bioimaging. In this review, we initially discuss the available CRISPR-mediated imaging systems from the following aspects: summary of imaging substances, the design and optimization of bioimaging strategies, and factors influencing CRISPR-mediated in-situ detection. Subsequently, we highlight the potential of CRISPR-mediated bioimaging for application in biomedical research and clinical practice. Furthermore, we outline the current bottlenecks and future perspectives of CRISPR-based bioimaging. We believe that this review will facilitate the potential integration of bioimaging-related research with current clinical workflow.}, }
@article {pmid37027291, year = {2023}, author = {Weng, Z and You, Z and Li, H and Wu, G and Song, Y and Sun, H and Fradlin, A and Neal-Harris, C and Lin, M and Gao, X and Zhang, Y}, title = {CRISPR-Cas12a Biosensor Array for Ultrasensitive Detection of Unamplified DNA with Single-Nucleotide Polymorphic Discrimination.}, journal = {ACS sensors}, volume = {8}, number = {4}, pages = {1489-1499}, doi = {10.1021/acssensors.2c02495}, pmid = {37027291}, issn = {2379-3694}, mesh = {Humans ; *CRISPR-Cas Systems/genetics ; Polymorphism, Single Nucleotide ; Reproducibility of Results ; DNA/genetics ; DNA, Single-Stranded/genetics ; Escherichia coli/genetics ; *Graphite ; Nucleotides ; }, abstract = {Quantitative polymerase chain reaction as a powerful tool for DNA detection has been pivotal to a vast range of applications, including disease screening, food safety assessment, environmental monitoring, and many others. However, the essential target amplification step in combination with fluorescence readout poses a significant challenge to rapid and streamlined analysis. The discovery and engineering of the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) technology have recently paved the way for a novel approach to nucleic acid detection, but the majority of current CRISPR-mediated DNA detection platforms are limited by insufficient sensitivity and still require target preamplification. Herein, we report a CRISPR-Cas12a-mediated graphene field-effect transistor (gFET) array, named CRISPR Cas12a-gFET, for amplification-free, ultrasensitive, and reliable detection of both single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) targets. CRISPR Cas12a-gFET leverages the multiturnover trans-cleavage activity of CRISPR Cas12a for intrinsic signal amplification and ultrasensitivity of gFET. As demonstrated, CRISPR Cas12a-gFET achieves a limit of detection of 1 aM for the ssDNA human papillomavirus 16 synthetic target and 10 aM for the dsDNA Escherichia coli plasmid target without target preamplification. In addition, an array of 48 sensors on a single 1.5 cm × 1.5 cm chip is employed to improve data reliability. Finally, Cas12a-gFET demonstrates the capability to discriminate single-nucleotide polymorphisms. Together, the CRISPR Cas12a-gFET biosensor array provides a detection tool for amplification-free, ultrasensitive, reliable, and highly specific DNA detections.}, }
@article {pmid36800642, year = {2023}, author = {Li, C and Georgakopoulou, A and Newby, GA and Chen, PJ and Everette, KA and Paschoudi, K and Vlachaki, E and Gil, S and Anderson, AK and Koob, T and Huang, L and Wang, H and Kiem, HP and Liu, DR and Yannaki, E and Lieber, A}, title = {In vivo HSC prime editing rescues sickle cell disease in a mouse model.}, journal = {Blood}, volume = {141}, number = {17}, pages = {2085-2099}, doi = {10.1182/blood.2022018252}, pmid = {36800642}, issn = {1528-0020}, mesh = {Mice ; Animals ; *Gene Editing/methods ; CRISPR-Cas Systems ; *Anemia, Sickle Cell/genetics/therapy ; Hematopoietic Stem Cells ; Hemoglobin, Sickle/genetics ; }, abstract = {Sickle cell disease (SCD) is a monogenic disease caused by a nucleotide mutation in the β-globin gene. Current gene therapy studies are mainly focused on lentiviral vector-mediated gene addition or CRISPR/Cas9-mediated fetal globin reactivation, leaving the root cause unfixed. We developed a vectorized prime editing system that can directly repair the SCD mutation in hematopoietic stem cells (HSCs) in vivo in a SCD mouse model (CD46/Townes mice). Our approach involved a single intravenous injection of a nonintegrating, prime editor-expressing viral vector into mobilized CD46/Townes mice and low-dose drug selection in vivo. This procedure resulted in the correction of ∼40% of βS alleles in HSCs. On average, 43% of sickle hemoglobin was replaced by adult hemoglobin, thereby greatly mitigating the SCD phenotypes. Transplantation in secondary recipients demonstrated that long-term repopulating HSCs were edited. Highly efficient target site editing was achieved with minimal generation of insertions and deletions and no detectable off-target editing. Because of its simplicity and portability, our in vivo prime editing approach has the potential for application in resource-poor countries where SCD is prevalent.}, }
@article {pmid36738269, year = {2023}, author = {Brower-Toland, B and Shyu, C and Vega-Sanchez, ME and Slewinski, TL}, title = {Pedigree or identity? How genome editing can fundamentally change the path for crop development.}, journal = {Journal of experimental botany}, volume = {74}, number = {9}, pages = {2794-2798}, pmid = {36738269}, issn = {1460-2431}, mesh = {*Gene Editing ; *CRISPR-Cas Systems ; Genome, Plant/genetics ; Plant Breeding ; }, }
@article {pmid36111891, year = {2023}, author = {Nguyen, NHK and Rafiee, R and Tagmount, A and Sobh, A and Loguinov, A and de Jesus Sosa, AK and Elsayed, AH and Gbadamosi, M and Seligson, N and Cogle, CR and Rubnitz, J and Ribeiro, R and Downing, J and Cao, X and Pounds, SB and Vulpe, CD and Lamba, JK}, title = {Genome-wide CRISPR/Cas9 screen identifies etoposide response modulators associated with clinical outcomes in pediatric AML.}, journal = {Blood advances}, volume = {7}, number = {9}, pages = {1769-1783}, doi = {10.1182/bloodadvances.2022007934}, pmid = {36111891}, issn = {2473-9537}, mesh = {Humans ; Child ; Etoposide/pharmacology/therapeutic use ; *CRISPR-Cas Systems ; *Leukemia, Myeloid, Acute/drug therapy/genetics/pathology ; Cell Line ; DNA Helicases/genetics ; }, abstract = {Etoposide is used to treat a wide range of malignant cancers, including acute myeloid leukemia (AML) in children. Despite the use of intensive chemotherapeutic regimens containing etoposide, a significant proportion of pediatric patients with AML become resistant to treatment and relapse, leading to poor survival. This poses a pressing clinical challenge to identify mechanisms underlying drug resistance to enable effective pharmacologic targeting. We performed a genome-wide CRISPR/Cas9 synthetic-lethal screening to identify functional modulators of etoposide response in leukemic cell line and integrated results from CRISPR-screen with gene expression and clinical outcomes in pediatric patients with AML treated with etoposide-containing regimen. Our results confirmed the involvement of well-characterized genes, including TOP2A and ABCC1, as well as identified novel genes such as RAD54L2, PRKDC, and ZNF451 that have potential to be novel drug targets. This study demonstrates the ability for leveraging CRISPR/Cas9 screening in conjunction with clinically relevant endpoints to make meaningful discoveries for the identification of prognostic biomarkers and novel therapeutic targets to overcome treatment resistance.}, }
@article {pmid37111880, year = {2023}, author = {Yuan, G and Liu, Y and Yao, T and Muchero, W and Chen, JG and Tuskan, GA and Yang, X}, title = {eYGFPuv-Assisted Transgenic Selection in Populus deltoides WV94 and Multiplex Genome Editing in Protoplasts of P. trichocarpa × P. deltoides Clone '52-225'.}, journal = {Plants (Basel, Switzerland)}, volume = {12}, number = {8}, pages = {}, pmid = {37111880}, issn = {2223-7747}, abstract = {Although CRISPR/Cas-based genome editing has been widely used for plant genetic engineering, its application in the genetic improvement of trees has been limited, partly because of challenges in Agrobacterium-mediated transformation. As an important model for poplar genomics and biotechnology research, eastern cottonwood (Populus deltoides) clone WV94 can be transformed by A. tumefaciens, but several challenges remain unresolved, including the relatively low transformation efficiency and the relatively high rate of false positives from antibiotic-based selection of transgenic events. Moreover, the efficacy of CRISPR-Cas system has not been explored in P. deltoides yet. Here, we first optimized the protocol for Agrobacterium-mediated stable transformation in P. deltoides WV94 and applied a UV-visible reporter called eYGFPuv in transformation. Our results showed that the transgenic events in the early stage of transformation could be easily recognized and counted in a non-invasive manner to narrow down the number of regenerated shoots for further molecular characterization (at the DNA or mRNA level) using PCR. We found that approximately 8.7% of explants regenerated transgenic shoots with green fluorescence within two months. Next, we examined the efficacy of multiplex CRISPR-based genome editing in the protoplasts derived from P. deltoides WV94 and hybrid poplar clone '52-225' (P. trichocarpa × P. deltoides clone '52-225'). The two constructs expressing the Trex2-Cas9 system resulted in mutation efficiency ranging from 31% to 57% in hybrid poplar clone 52-225, but no editing events were observed in P. deltoides WV94 transient assay. The eYGFPuv-assisted plant transformation and genome editing approach demonstrated in this study has great potential for accelerating the genome editing-based breeding process in poplar and other non-model plants species and point to the need for additional CRISPR work in P. deltoides.}, }
@article {pmid37111505, year = {2023}, author = {Majumdar, A and Sharma, A and Belludi, R}, title = {Natural and Engineered Resistance Mechanisms in Plants against Phytoviruses.}, journal = {Pathogens (Basel, Switzerland)}, volume = {12}, number = {4}, pages = {}, pmid = {37111505}, issn = {2076-0817}, abstract = {Plant viruses, as obligate intracellular parasites, rely exclusively on host machinery to complete their life cycle. Whether a virus is pathogenic or not depends on the balance between the mechanisms used by both plants and viruses during the intense encounter. Antiviral defence mechanisms in plants can be of two types, i.e., natural resistance and engineered resistance. Innate immunity, RNA silencing, translational repression, autophagy-mediated degradation, and resistance to virus movement are the possible natural defence mechanisms against viruses in plants, whereas engineered resistance includes pathogen-derived resistance along with gene editing technologies. The incorporation of various resistance genes through breeding programmes, along with gene editing tools such as CRISPR/Cas technologies, holds great promise in developing virus-resistant plants. In this review, different resistance mechanisms against viruses in plants along with reported resistance genes in major vegetable crops are discussed.}, }
@article {pmid37110466, year = {2023}, author = {Moreira, POL and Nogueira, PM and Monte-Neto, RL}, title = {Next-Generation Leishmanization: Revisiting Molecular Targets for Selecting Genetically Engineered Live-Attenuated Leishmania.}, journal = {Microorganisms}, volume = {11}, number = {4}, pages = {}, pmid = {37110466}, issn = {2076-2607}, abstract = {Despite decades of research devoted to finding a vaccine against leishmaniasis, we are still lacking a safe and effective vaccine for humans. Given this scenario, the search for a new prophylaxis alternative for controlling leishmaniasis should be a global priority. Inspired by leishmanization-a first generation vaccine strategy where live L. major parasites are inoculated in the skin to protect against reinfection-live-attenuated Leishmania vaccine candidates are promising alternatives due to their robust elicited protective immune response. In addition, they do not cause disease and could provide long-term protection upon challenge with a virulent strain. The discovery of a precise and easy way to perform CRISPR/Cas-based gene editing allowed the selection of safer null mutant live-attenuated Leishmania parasites obtained by gene disruption. Here, we revisited molecular targets associated with the selection of live-attenuated vaccinal strains, discussing their function, their limiting factors and the ideal candidate for the next generation of genetically engineered live-attenuated Leishmania vaccines to control leishmaniasis.}, }
@article {pmid37108063, year = {2023}, author = {Gunitseva, N and Evteeva, M and Borisova, A and Patrushev, M and Subach, F}, title = {RNA-Dependent RNA Targeting by CRISPR-Cas Systems: Characterizations and Applications.}, journal = {International journal of molecular sciences}, volume = {24}, number = {8}, pages = {}, pmid = {37108063}, issn = {1422-0067}, abstract = {Genome editing technologies that are currently available and described have a fundamental impact on the development of molecular biology and medicine, industrial and agricultural biotechnology and other fields. However, genome editing based on detection and manipulation of the targeted RNA is a promising alternative to control the gene expression at the spatiotemporal transcriptomic level without complete elimination. The innovative CRISPR-Cas RNA-targeting systems changed the conception of biosensing systems and also allowed the RNA effectors to be used in various applications; for example, genomic editing, effective virus diagnostic tools, biomarkers, transcription regulations. In this review, we discussed the current state-of-the-art of specific CRISPR-Cas systems known to bind and cleave RNA substrates and summarized potential applications of the versatile RNA-targeting systems.}, }
@article {pmid37107608, year = {2023}, author = {He, Y and Yan, W and Long, L and Dong, L and Ma, Y and Li, C and Xie, Y and Liu, N and Xing, Z and Xia, W and Li, F}, title = {The CRISPR/Cas System: A Customizable Toolbox for Molecular Detection.}, journal = {Genes}, volume = {14}, number = {4}, pages = {}, doi = {10.3390/genes14040850}, pmid = {37107608}, issn = {2073-4425}, abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR) and their associated proteins (Cas) are promising molecular diagnostic tools for rapidly and precisely elucidating the structure and function of genomes due to their high specificity, programmability, and multi-system compatibility in nucleic acid recognition. Multiple parameters limit the ability of a CRISPR/Cas system to detect DNA or RNA. Consequently, it must be used in conjunction with other nucleic acid amplification techniques or signal detection techniques, and the reaction components and reaction conditions should be modified and optimized to maximize the detection performance of the CRISPR/Cas system against various targets. As the field continues to develop, CRISPR/Cas systems have the potential to become an ultra-sensitive, convenient, and accurate biosensing platform for the detection of specific target sequences. The design of a molecular detection platform employing the CRISPR/Cas system is asserted on three primary strategies: (1) Performance optimization of the CRISPR/Cas system; (2) enhancement of the detection signal and its interpretation; and (3) compatibility with multiple reaction systems. This article focuses on the molecular characteristics and application value of the CRISPR/Cas system and reviews recent research progress and development direction from the perspectives of principle, performance, and method development challenges to provide a theoretical foundation for the development and application of the CRISPR/CAS system in molecular detection technology.}, }
@article {pmid37105997, year = {2023}, author = {LaBauve, AE and Saada, EA and Jones, IKA and Mosesso, R and Noureddine, A and Techel, J and Gomez, A and Collette, N and Sherman, MB and Serda, RE and Butler, KS and Brinker, CJ and Schoeniger, JS and Sasaki, D and Negrete, OA}, title = {Lipid-coated mesoporous silica nanoparticles for anti-viral applications via delivery of CRISPR-Cas9 ribonucleoproteins.}, journal = {Scientific reports}, volume = {13}, number = {1}, pages = {6873}, pmid = {37105997}, issn = {2045-2322}, abstract = {Emerging and re-emerging viral pathogens present a unique challenge for anti-viral therapeutic development. Anti-viral approaches with high flexibility and rapid production times are essential for combating these high-pandemic risk viruses. CRISPR-Cas technologies have been extensively repurposed to treat a variety of diseases, with recent work expanding into potential applications against viral infections. However, delivery still presents a major challenge for these technologies. Lipid-coated mesoporous silica nanoparticles (LCMSNs) offer an attractive delivery vehicle for a variety of cargos due to their high biocompatibility, tractable synthesis, and amenability to chemical functionalization. Here, we report the use of LCMSNs to deliver CRISPR-Cas9 ribonucleoproteins (RNPs) that target the Niemann-Pick disease type C1 gene, an essential host factor required for entry of the high-pandemic risk pathogen Ebola virus, demonstrating an efficient reduction in viral infection. We further highlight successful in vivo delivery of the RNP-LCMSN platform to the mouse liver via systemic administration.}, }
@article {pmid37105947, year = {2023}, author = {Zhou, J and He, M and Yin, X and Yu, Y and Yao, D and Liang, H}, title = {Unexploited Performance of NLS in the dCas9-VPR-Mediated Transcriptional Activation.}, journal = {ACS chemical biology}, volume = {}, number = {}, pages = {}, doi = {10.1021/acschembio.3c00195}, pmid = {37105947}, issn = {1554-8937}, abstract = {Nuclear localization signal (NLS) is a short peptide guiding the nuclear transport process, recognized as playing an important role in constructing clustered regularly interspaced short palindromic repeats-Cas (CRISPR-Cas) activators. Here, we investigate the effect of the position and number of the NLS on transcriptional activation based on the dCas9-VPR activator. Our results not only demonstrate that the position of the SV40 NLS could have different degrees of influence on activation efficiency but also, surprisingly, we find that the SV40 NLS plays a detrimental role. Complete deletion of the NLS from the system could increase the transcriptional activation efficiency by 2 to 4 times compared with the original dCas9-VPR. This finding is also supported by some typical first- and third-generation activators. Our work should be beneficial to the design of the NLS-based system.}, }
@article {pmid37104586, year = {2023}, author = {VanderWal, AR and Park, JU and Polevoda, B and Nicosia, JK and Molina Vargas, AM and Kellogg, EH and O'Connell, MR}, title = {Csx28 is a membrane pore that enhances CRISPR-Cas13b-dependent antiphage defense.}, journal = {Science (New York, N.Y.)}, volume = {380}, number = {6643}, pages = {410-415}, doi = {10.1126/science.abm1184}, pmid = {37104586}, issn = {1095-9203}, abstract = {Type VI CRISPR-Cas systems use RNA-guided ribonuclease (RNase) Cas13 to defend bacteria against viruses, and some of these systems encode putative membrane proteins that have unclear roles in Cas13-mediated defense. We show that Csx28, of type VI-B2 systems, is a transmembrane protein that assists to slow cellular metabolism upon viral infection, increasing antiviral defense. High-resolution cryo-electron microscopy reveals that Csx28 forms an octameric pore-like structure. These Csx28 pores localize to the inner membrane in vivo. Csx28's antiviral activity in vivo requires sequence-specific cleavage of viral messenger RNAs by Cas13b, which subsequently results in membrane depolarization, slowed metabolism, and inhibition of sustained viral infection. Our work suggests a mechanism by which Csx28 acts as a downstream, Cas13b-dependent effector protein that uses membrane perturbation as an antiviral defense strategy.}, }
@article {pmid37100946, year = {2023}, author = {}, title = {The gene-therapy revolution risks stalling if we don't talk about drug pricing.}, journal = {Nature}, volume = {616}, number = {7958}, pages = {629-630}, doi = {10.1038/d41586-023-01389-z}, pmid = {37100946}, issn = {1476-4687}, mesh = {*Drug Costs ; *Genetic Therapy ; Gene Editing ; CRISPR-Cas Systems ; }, }
@article {pmid37100808, year = {2023}, author = {Seda, M and Crespo, B and Corcelli, M and Osborn, DP and Jenkins, D}, title = {A CRISPR/Cas9-generated mutation in the zebrafish orthologue of PPP2R3B causes idiopathic scoliosis.}, journal = {Scientific reports}, volume = {13}, number = {1}, pages = {6783}, pmid = {37100808}, issn = {2045-2322}, mesh = {Animals ; Adolescent ; Humans ; *Zebrafish/genetics/metabolism ; *Scoliosis/genetics ; CRISPR-Cas Systems ; Zebrafish Proteins/genetics/metabolism ; Protein Phosphatase 2/genetics/metabolism ; Mutation ; }, abstract = {Idiopathic scoliosis (IS) is the deformation and/or abnormal curvature of the spine that develops progressively after birth. It is a very common condition, affecting approximately 4% of the general population, yet the genetic and mechanistic causes of IS are poorly understood. Here, we focus on PPP2R3B, which encodes a protein phosphatase 2A regulatory subunit. We found that PPP2R3B is expressed at sites of chondrogenesis within human foetuses, including the vertebrae. We also demonstrated prominent expression in myotome and muscle fibres in human foetuses, and zebrafish embryos and adolescents. As there is no rodent orthologue of PPP2R3B, we used CRIPSR/Cas9-mediated gene-editing to generate a series of frameshift mutations in zebrafish ppp2r3b. Adolescent zebrafish that were homozygous for this mutation exhibited a fully penetrant kyphoscoliosis phenotype which became progressively worse over time, mirroring IS in humans. These defects were associated with reduced mineralisation of vertebrae, resembling osteoporosis. Electron microscopy demonstrated abnormal mitochondria adjacent to muscle fibres. In summary, we report a novel zebrafish model of IS and reduced bone mineral density. In future, it will be necessary to delineate the aetiology of these defects in relation to bone, muscle, neuronal and ependymal cilia function.}, }
@article {pmid37100476, year = {2023}, author = {Deng, F and Li, Y and Hall, T and Vesey, G and Goldys, EM}, title = {Bi-functional antibody-CRISPR/Cas12a ribonucleoprotein conjugate for improved immunoassay performance.}, journal = {Analytica chimica acta}, volume = {1259}, number = {}, pages = {341211}, doi = {10.1016/j.aca.2023.341211}, pmid = {37100476}, issn = {1873-4324}, mesh = {Humans ; CRISPR-Cas Systems ; *Biosensing Techniques ; *Cryptosporidiosis ; *Cryptosporidium ; Immunoassay ; *Immunoconjugates ; Antibodies ; Ribonucleoproteins ; }, abstract = {Protein conjugates are commonly used in biochemistry, including diagnostic platforms such as antibody-based immunoassays. Antibodies can be bound to a variety of molecules creating conjugates with desirable functions, particularly for imaging and signal amplification. Cas12a is a recently discovered programable nuclease with the remarkable capability to amplify assay signals due to its trans-cleavage property. In this study, we directly conjugated antibody with Cas12a/gRNA ribonucleoprotein without the loss of function in either constituent. The conjugated antibody was suitable for immunoassays and the conjugated Cas12a was capable of amplifying the signal produced in an immunosensor without the need to change the original assay protocol. We applied the bi-functional antibody-Cas12a/gRNA conjugate to successfully detect two different types of targets, a whole pathogenic microorganism, Cryptosporidium, and a small protein, cytokine IFN-γ, with sensitivity reaching one single microorganism per sample and 10 fg/mL for IFN-γ, respectively. With simple substitution of the antibody conjugated with the Cas12a/gRNA RNP, this approach can potentially be applied to increase sensitivity of a variety of immunoassays for a broad range of different analytes.}, }
@article {pmid37100169, year = {2023}, author = {Kraus, C and Sontheimer, EJ}, title = {Applications of Anti-CRISPR Proteins in Genome Editing and Biotechnology.}, journal = {Journal of molecular biology}, volume = {}, number = {}, pages = {168120}, doi = {10.1016/j.jmb.2023.168120}, pmid = {37100169}, issn = {1089-8638}, abstract = {In the ten years since the discovery of the first anti-CRISPR (Acr) proteins, the number of validated Acrs has expanded rapidly, as has our understanding of the diverse mechanisms they employ to suppress natural CRISPR-Cas immunity. Many, though not all, function via direct, specific interaction with Cas protein effectors. The abilities of Acr proteins to modulate the activities and properties of CRISPR-Cas effectors have been exploited for an ever-increasing spectrum of biotechnological uses, most of which involve the establishment of control over genome editing systems. This control can be used to minimize off-target editing, restrict editing based on spatial, temporal, or conditional cues, limit the spread of gene drive systems, and select for genome-edited bacteriophages. Anti-CRISPRs have also been developed to overcome bacterial immunity, facilitate viral vector production, control synthetic gene circuits, and other purposes. The impressive and ever-growing diversity of Acr inhibitory mechanisms will continue to allow the tailored applications of Acrs.}, }
@article {pmid37098485, year = {2023}, author = {Park, HM and Won, J and Park, Y and Anzaku, ET and Vankerschaver, J and Van Messem, A and De Neve, W and Shim, H}, title = {CRISPR-Cas-Docker: web-based in silico docking and machine learning-based classification of crRNAs with Cas proteins.}, journal = {BMC bioinformatics}, volume = {24}, number = {1}, pages = {167}, pmid = {37098485}, issn = {1471-2105}, mesh = {*CRISPR-Cas Systems ; *RNA/genetics ; Internet ; }, abstract = {BACKGROUND: CRISPR-Cas-Docker is a web server for in silico docking experiments with CRISPR RNAs (crRNAs) and Cas proteins. This web server aims at providing experimentalists with the optimal crRNA-Cas pair predicted computationally when prokaryotic genomes have multiple CRISPR arrays and Cas systems, as frequently observed in metagenomic data.<br><br>RESULTS: CRISPR-Cas-Docker provides two methods to predict the optimal Cas protein given a particular crRNA sequence: a structure-based method (in silico docking) and a sequence-based method (machine learning classification). For the structure-based method, users can either provide experimentally determined 3D structures of these macromolecules or use an integrated pipeline to generate 3D-predicted structures for in silico docking experiments.<br><br>CONCLUSION: CRISPR-Cas-Docker addresses the need of the CRISPR-Cas community to predict RNA-protein interactions in silico by optimizing multiple stages of computation and evaluation, specifically for CRISPR-Cas systems. CRISPR-Cas-Docker is available at www.crisprcasdocker.org as a web server, and at https://github.com/hshimlab/CRISPR-Cas-Docker as an open-source tool.}, }
@article {pmid36932303, year = {2023}, author = {Lu, PJ and Zhang, P and Liu, YC and Jing, N and Guo, YN and Wang, PS and Su, LL and Guo, Q and Ma, Q and Xu, YM and Zhang, ST}, title = {Gene Repair of iPSC Line with GARS (G294R) Mutation of CMT2D Disease by CRISPR/Cas9.}, journal = {Current medical science}, volume = {43}, number = {2}, pages = {261-267}, pmid = {36932303}, issn = {2523-899X}, mesh = {Humans ; *Charcot-Marie-Tooth Disease/genetics/therapy/metabolism ; CRISPR-Cas Systems/genetics ; *Induced Pluripotent Stem Cells/metabolism ; Mutation ; }, abstract = {OBJECTIVE: Charcot-Marie-Tooth disease (CMT) severely affects patient activity, and may cause disability. However, no clinical treatment is available to reverse the disease course. The combination of CRISPR/Cas9 and iPSCs may have therapeutic potential against nervous diseases, such as CMT.<br><br>METHODS: In the present study, the skin fibroblasts of CMT type 2D (CMT2D) patients with the c.880G>A heterozygous nucleotide mutation in the GARS gene were reprogrammed into iPSCs using three plasmids (pCXLE-hSK, pCXLE-hUL and pCXLE-hOCT3/4-shp5-F). Then, CRISPR/Cas9 technology was used to repair the mutated gene sites at the iPSC level.<br><br>RESULTS: An iPSC line derived from the GARS (G294R) family with fibular atrophy was successfully induced, and the mutated gene loci were repaired at the iPSC level using CRISPR/Cas9 technology. These findings lay the foundation for future research on drug screening and cell therapy.<br><br>CONCLUSION: iPSCs can differentiate into different cell types, and originate from autologous cells. Therefore, they are promising for the development of autologous cell therapies for degenerative diseases. The combination of CRISPR/Cas9 and iPSCs may open a new avenue for the treatment of nervous diseases, such as CMT.}, }
@article {pmid36693779, year = {2023}, author = {Zhang, C and Wang, X and Liu, G and Ren, H and Liu, J and Jiang, Z and Zhang, Y}, title = {CRISPR/Cas9 and Chlorophyll Coordination Micelles for Cancer Treatment by Genome Editing and Photodynamic Therapy.}, journal = {Small (Weinheim an der Bergstrasse, Germany)}, volume = {19}, number = {17}, pages = {e2206981}, doi = {10.1002/smll.202206981}, pmid = {36693779}, issn = {1613-6829}, mesh = {Mice ; Animals ; Micelles ; Gene Editing ; Chlorophyll ; CRISPR-Cas Systems/genetics ; NF-E2-Related Factor 2 ; *Photochemotherapy ; *Neoplasms/genetics/therapy ; }, abstract = {CRISPR/Cas9-based gene therapy and photodynamic therapy both show promise for cancer treatment but still have their drawbacks limited by tumor microenvironment and long treatment duration. Herein, CRISPR/Cas9 genome editing and photodynamic strategy for a synergistic anti-tumor therapeutic modality is merged. Chlorophyll (Chl) extracted from natural green vegetables is encapsulated in Pluronic F127 (F127) micelles and Histidine-tagged Cas9 can be effectively chelated onto micelles via metal coordination by simple incubation, affording Cas9-Chl@F127 micelles. Mg[2+] acts as an enzyme cofactor to correlatively enhance Cas9 gene-editing activity. Upon laser irradiation, Chl as an effective photosensitizer generates reactive oxygen species (ROS) to kill tumor cells. Meanwhile, CRISPR/Cas9, mediated by dual deliberately designed gRNAs of APE1 and NRF2, can reprogram the tumor microenvironment by increasing the intracellular oxygen accumulation and impairing the oxidative defense system of tumor cells. Cas9-Chl@F127 micelles can responsively release Cas9 in the presence of abundant ATP or low pH in tumor cells. In a murine tumor model, Cas9-Chl@F127 complexed with dual gRNAs including APE1 and NRF2 significantly inhibits the tumor growth. Taken together, Cas9-Chl@F127 micelles, representing the first Chl-based green biomaterial for the delivery of Cas9, show great promise for the synergistic anti-tumor treatment by PDT and gene editing.}, }
@article {pmid37097167, year = {2023}, author = {Wang, L and Huang, X and Jin, Q and Tang, J and Zhang, H and Zhang, JR and Wu, H}, title = {Two-Component Response Regulator OmpR Regulates Mucoviscosity through Energy Metabolism in Klebsiella pneumoniae.}, journal = {Microbiology spectrum}, volume = {}, number = {}, pages = {e0054423}, doi = {10.1128/spectrum.00544-23}, pmid = {37097167}, issn = {2165-0497}, abstract = {Hypermucoviscosity is a hallmark of hypervirulent Klebsiella pneumoniae (hvKP). However, the molecular basis of its regulation is largely unknown. We hypothesize that hypermucoviscosity is modulated via two-component signal transduction systems (TCSs). In-frame deletion mutants of all 33 response regulators of hvKP ATCC43816 were generated using CRISPR/CAS and evaluated for their impacts on hypermucoviscosity. The response regulator OmpR is required for hypermucoviscosity in vitro and virulence in vivo in a mouse pneumonia model. The ΔompR mutant lost its mucoidy but retained its capsule level and comparable rmpADC expression, so transcriptomic analysis by RNA-Seq was performed to identify differentially expressed genes (DEGs) in ΔompR mutant. The top 20 Gene Ontology terms of 273 DEGs belong to purine ribonucleotide triphosphate biosynthetic and metabolic process, transmembrane transport, and amino acid metabolism. Among the overexpressed genes in the ΔompR mutant, the atp operon encoding F-type ATP synthase and the gcvTHP encoding glycine cleavage system were characterized further as overexpression of either operon reduced the mucoviscosity and increased the production of ATP. Furthermore, OmpR directly bound the promoter region of the atp operon, not the gcvTHP, suggesting that OmpR regulates the expression of the atp operon directly and gcvTHP indirectly. Hence, the loss of OmpR led to the overexpression of F-type ATP synthase and glycine cleavage system, which altered the energetic status of ΔompR cells and contributed to the subsequent reduction in the mucoviscosity. Our study has uncovered a previously unknown regulation of bacterial metabolism by OmpR and its influence on hypermucoviscosity. IMPORTANCE Hypermucoviscosity is a critical virulent factor for Klebsiella pneumoniae infections, and its regulation remains poorly understood at the molecular level. This study aims to address this knowledge gap by investigating the role of response regulators in mediating hypermucoviscosity in K. pneumoniae. We screened 33 response regulators and found that OmpR is essential for hypermucoviscosity and virulence of K. pneumoniae in a mouse pneumonia model. Transcriptomic analysis uncovered that genes involved in energy production and metabolism are highly upregulated in the ΔompR mutant, suggesting a potential link between bacterial energy status and hypermucoviscosity. Overexpression of those genes increased production of ATP and reduced mucoviscosity, recapitulating the ΔompR mutant phenotype. Our findings provide new insights into the regulation of K. pneumoniae hypermucoviscosity by a two-component signal transduction system, highlighting the previously unknown role of OmpR in regulating bacterial energy status and its influence on hypermucoviscosity.}, }
@article {pmid37097160, year = {2023}, author = {Esquerra-Ruvira, B and Baquedano, I and Ruiz, R and Fernandez, A and Montoliu, L and Mojica, FJM}, title = {Identification of the EH CRISPR-Cas9 system on a metagenome and its application to genome engineering.}, journal = {Microbial biotechnology}, volume = {}, number = {}, pages = {}, doi = {10.1111/1751-7915.14266}, pmid = {37097160}, issn = {1751-7915}, abstract = {Non-coding RNAs (crRNAs) produced from clustered regularly interspaced short palindromic repeats (CRISPR) loci and CRISPR-associated (Cas) proteins of the prokaryotic CRISPR-Cas systems form complexes that interfere with the spread of transmissible genetic elements through Cas-catalysed cleavage of foreign genetic material matching the guide crRNA sequences. The easily programmable targeting of nucleic acids enabled by these ribonucleoproteins has facilitated the implementation of CRISPR-based molecular biology tools for in vivo and in vitro modification of DNA and RNA targets. Despite the diversity of DNA-targeting Cas nucleases so far identified, native and engineered derivatives of the Streptococcus pyogenes SpCas9 are the most widely used for genome engineering, at least in part due to their catalytic robustness and the requirement of an exceptionally short motif (5'-NGG-3' PAM) flanking the target sequence. However, the large size of the SpCas9 variants impairs the delivery of the tool to eukaryotic cells and smaller alternatives are desirable. Here, we identify in a metagenome a new CRISPR-Cas9 system associated with a smaller Cas9 protein (EHCas9) that targets DNA sequences flanked by 5'-NGG-3' PAMs. We develop a simplified EHCas9 tool that specifically cleaves DNA targets and is functional for genome editing applications in prokaryotes and eukaryotic cells.}, }
@article {pmid37096163, year = {2023}, author = {McMahon, MA and Rahdar, M and Mukhopadhyay, S and Bui, HH and Hart, C and Damle, S and Courtney, M and Baughn, MW and Cleveland, DW and Bennett, CF}, title = {GOLGA8 increases bulk antisense oligonucleotide uptake and activity in mammalian cells.}, journal = {Molecular therapy. Nucleic acids}, volume = {32}, number = {}, pages = {289-301}, pmid = {37096163}, issn = {2162-2531}, abstract = {Antisense oligonucleotides (ASOs) are short synthetic nucleic acids that recognize and bind to complementary RNA to modulate gene expression. It is well established that single-stranded, phosphorothioate-modified ASOs enter cells independent of carrier molecules, primarily via endocytic pathways, but that only a small portion of internalized ASO is released into the cytosol and/or nucleus, rendering the majority of ASO inaccessible to the targeted RNA. Identifying pathways that can increase the available ASO pool is valuable as a research tool and therapeutically. Here, we conducted a functional genomic screen for ASO activity by engineering GFP splice reporter cells and applying genome-wide CRISPR gene activation. The screen can identify factors that enhance ASO splice modulation activity. Characterization of hit genes uncovered GOLGA8, a largely uncharacterized protein, as a novel positive regulator enhancing ASO activity by ∼2-fold. Bulk ASO uptake is 2- to 5-fold higher in GOLGA8-overexpressing cells where GOLGA8 and ASOs are observed in the same intracellular compartments. We find GOLGA8 is highly localized to the trans-Golgi and readily detectable at the plasma membrane. Interestingly, overexpression of GOLGA8 increased activity for both splice modulation and RNase H1-dependent ASOs. Taken together, these results support a novel role for GOLGA8 in productive ASO uptake.}, }
@article {pmid37010434, year = {2023}, author = {Tian, X and Zhou, Y and Wang, S and Gao, M and Xia, Y and Li, Y and Zhong, Y and Xu, W and Bai, L and Fu, B and Zhou, Y and Lee, HR and Deng, H and Lan, K and Feng, P and Zhang, J}, title = {Genome-Wide CRISPR-Cas9 Screen Identifies SMCHD1 as a Restriction Factor for Herpesviruses.}, journal = {mBio}, volume = {14}, number = {2}, pages = {e0054923}, pmid = {37010434}, issn = {2150-7511}, mesh = {Mice ; Animals ; *Virus Replication/genetics ; DNA Replication ; CRISPR-Cas Systems ; DNA, Viral/genetics ; *Herpesvirus 8, Human/physiology ; Gene Expression Regulation, Viral ; Mammals/metabolism ; Chromosomal Proteins, Non-Histone/genetics ; }, abstract = {Intrinsic immunity is the frontline of host defense against invading pathogens. To combat viral infection, mammalian hosts deploy cell-intrinsic effectors to block viral replication prior to the onset of innate and adaptive immunity. In this study, SMCHD1 is identified as a pivotal cellular factor that restricts Kaposi's sarcoma-associated herpesvirus (KSHV) lytic reactivation through a genome-wide CRISPR-Cas9 knockout screen. Genome-wide chromatin profiling revealed that SMCHD1 associates with the KSHV genome, most prominently the origin of lytic DNA replication (ORI-Lyt). SMCHD1 mutants defective in DNA binding could not bind ORI-Lyt and failed to restrict KSHV lytic replication. Moreover, SMCHD1 functioned as a pan-herpesvirus restriction factor that potently suppressed a wide range of herpesviruses, including alpha, beta, and gamma subfamilies. SMCHD1 deficiency facilitated the replication of a murine herpesvirus in vivo. These findings uncovered SMCHD1 as a restriction factor against herpesviruses, and this could be harnessed for the development of antiviral therapies to limit viral infection. IMPORTANCE Intrinsic immunity represents the frontline of host defense against invading pathogens. However, our understanding of cell-intrinsic antiviral effectors remains limited. In this study, we identified SMCHD1 as a cell-intrinsic restriction factor that controlled KSHV lytic reactivation. Moreover, SMCHD1 restricted the replication of a wide range of herpesviruses by targeting the origins of viral DNA replication (ORIs), and SMCHD1 deficiency facilitated the replication of a murine herpesvirus in vivo. This study helps us to better understand intrinsic antiviral immunity, which may be harnessed to develop new therapeutics for the treatment of herpesvirus infection and the related diseases.}, }
@article {pmid36929689, year = {2023}, author = {Mund, M and Weber, W and Degreif, D and Schiklenk, C}, title = {A MAD7-based genome editing system for Escherichia coli.}, journal = {Microbial biotechnology}, volume = {16}, number = {5}, pages = {1000-1010}, pmid = {36929689}, issn = {1751-7915}, mesh = {*Gene Editing ; *CRISPR-Cas Systems ; Escherichia coli/genetics ; Genetic Engineering ; Plasmids ; }, abstract = {A broad variety of biomolecules is industrially produced in bacteria and yeasts. These microbial expression hosts can be optimized through genetic engineering using CRISPR tools. Here, we designed and characterized such a modular genome editing system based on the Cas12a-like RNA-guided nuclease MAD7 in Escherichia coli. This system enables the efficient generation of single nucleotide polymorphisms (SNPs) or gene deletions and can directly be used with donor DNA from benchtop DNA assembly to increase throughput. We combined multiple edits to engineer an E. coli strain with reduced overflow metabolism and increased plasmid yield, highlighting the versatility and industrial applicability of this approach.}, }
@article {pmid36880749, year = {2023}, author = {Homberger, C and Hayward, RJ and Barquist, L and Vogel, J}, title = {Improved Bacterial Single-Cell RNA-Seq through Automated MATQ-Seq and Cas9-Based Removal of rRNA Reads.}, journal = {mBio}, volume = {14}, number = {2}, pages = {e0355722}, pmid = {36880749}, issn = {2150-7511}, mesh = {*CRISPR-Cas Systems ; *Single-Cell Gene Expression Analysis ; High-Throughput Nucleotide Sequencing/methods ; RNA/genetics ; RNA, Ribosomal ; Gene Expression Profiling/methods ; Bacteria/genetics ; Sequence Analysis, RNA/methods ; RNA-Directed DNA Polymerase/genetics ; Single-Cell Analysis/methods ; }, abstract = {Bulk RNA sequencing technologies have provided invaluable insights into host and bacterial gene expression and associated regulatory networks. Nevertheless, the majority of these approaches report average expression across cell populations, hiding the true underlying expression patterns that are often heterogeneous in nature. Due to technical advances, single-cell transcriptomics in bacteria has recently become reality, allowing exploration of these heterogeneous populations, which are often the result of environmental changes and stressors. In this work, we have improved our previously published bacterial single-cell RNA sequencing (scRNA-seq) protocol that is based on multiple annealing and deoxycytidine (dC) tailing-based quantitative scRNA-seq (MATQ-seq), achieving a higher throughput through the integration of automation. We also selected a more efficient reverse transcriptase, which led to reduced cell loss and higher workflow robustness. Moreover, we successfully implemented a Cas9-based rRNA depletion protocol into the MATQ-seq workflow. Applying our improved protocol on a large set of single Salmonella cells sampled over different growth conditions revealed improved gene coverage and a higher gene detection limit compared to our original protocol and allowed us to detect the expression of small regulatory RNAs, such as GcvB or CsrB at a single-cell level. In addition, we confirmed previously described phenotypic heterogeneity in Salmonella in regard to expression of pathogenicity-associated genes. Overall, the low percentage of cell loss and high gene detection limit makes the improved MATQ-seq protocol particularly well suited for studies with limited input material, such as analysis of small bacterial populations in host niches or intracellular bacteria. IMPORTANCE Gene expression heterogeneity among isogenic bacteria is linked to clinically relevant scenarios, like biofilm formation and antibiotic tolerance. The recent development of bacterial single-cell RNA sequencing (scRNA-seq) enables the study of cell-to-cell variability in bacterial populations and the mechanisms underlying these phenomena. Here, we report a scRNA-seq workflow based on MATQ-seq with increased robustness, reduced cell loss, and improved transcript capture rate and gene coverage. Use of a more efficient reverse transcriptase and the integration of an rRNA depletion step, which can be adapted to other bacterial single-cell workflows, was instrumental for these improvements. Applying the protocol to the foodborne pathogen Salmonella, we confirmed transcriptional heterogeneity across and within different growth phases and demonstrated that our workflow captures small regulatory RNAs at a single-cell level. Due to low cell loss and high transcript capture rates, this protocol is uniquely suited for experimental settings in which the starting material is limited, such as infected tissues.}, }
@article {pmid37095570, year = {2023}, author = {Glaser, V and Flugel, C and Kath, J and Du, W and Drosdek, V and Franke, C and Stein, M and Pruß, A and Schmueck-Henneresse, M and Volk, HD and Reinke, P and Wagner, DL}, title = {Combining different CRISPR nucleases for simultaneous knock-in and base editing prevents translocations in multiplex-edited CAR T cells.}, journal = {Genome biology}, volume = {24}, number = {1}, pages = {89}, pmid = {37095570}, issn = {1474-760X}, mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; T-Lymphocytes ; DNA Breaks, Double-Stranded ; Genome ; }, abstract = {BACKGROUND: Multiple genetic modifications may be required to develop potent off-the-shelf chimeric antigen receptor (CAR) T cell therapies. Conventional CRISPR-Cas nucleases install sequence-specific DNA double-strand breaks (DSBs), enabling gene knock-out or targeted transgene knock-in. However, simultaneous DSBs provoke a high rate of genomic rearrangements which may impede the safety of the edited cells.<br><br>RESULTS: Here, we combine a non-viral CRISPR-Cas9 nuclease-assisted knock-in and Cas9-derived base editing technology for DSB free knock-outs within a single intervention. We demonstrate efficient insertion of a CAR into the T cell receptor alpha constant (TRAC) gene, along with two knock-outs that silence major histocompatibility complexes (MHC) class I and II expression. This approach reduces translocations to 1.4% of edited cells. Small insertions and deletions at the base editing target sites indicate guide RNA exchange between the editors. This is overcome by using CRISPR enzymes of distinct evolutionary origins. Combining Cas12a Ultra for CAR knock-in and a Cas9-derived base editor enables the efficient generation of triple-edited CAR T cells with a translocation frequency comparable to unedited T cells. Resulting TCR- and MHC-negative CAR T cells resist allogeneic T cell targeting in vitro.<br><br>CONCLUSIONS: We outline a solution for non-viral CAR gene transfer and efficient gene silencing using different CRISPR enzymes for knock-in and base editing to prevent translocations. This single-step procedure may enable safer multiplex-edited cell products and demonstrates a path towards off-the-shelf CAR therapeutics.}, }
@article {pmid35606905, year = {2023}, author = {Kouhen, M and García-Caparrós, P and Twyman, RM and Abdelly, C and Mahmoudi, H and Schillberg, S and Debez, A}, title = {Improving environmental stress resilience in crops by genome editing: insights from extremophile plants.}, journal = {Critical reviews in biotechnology}, volume = {43}, number = {4}, pages = {559-574}, doi = {10.1080/07388551.2022.2042481}, pmid = {35606905}, issn = {1549-7801}, mesh = {*Gene Editing/methods ; *Extremophiles ; CRISPR-Cas Systems ; Plants, Genetically Modified/genetics ; Crops, Agricultural/genetics ; Genome, Plant ; }, abstract = {In basic and applied sciences, genome editing has become an indispensable tool, especially the versatile and adaptable CRISPR/Cas9 system. Using CRISPR/Cas9 in plants has enabled modifications of many valuable traits, including environmental stress tolerance, an essential aspect when it comes to ensuring food security under climate change pressure. The CRISPR toolbox enables faster and more precise plant breeding by facilitating: multiplex gene editing, gene pyramiding, and de novo domestication. In this paper, we discuss the most recent advances in CRISPR/Cas9 and alternative CRISPR-based systems, along with the technical challenges that remain to be overcome. A revision of the latest proof-of-concept and functional characterization studies has indeed provided more insight into the quantitative traits affecting crop yield and stress tolerance. Additionally, we focus on the applications of CRISPR/Cas9 technology in regard to extremophile plants, due to their significance on: industrial, ecological and economic levels. These still unexplored genetic resources could provide the means to harden our crops against the threat of climate change, thus ensuring food security over the next century.}, }
@article {pmid37095348, year = {2023}, author = {Zhang, Z and Baxter, AE and Ren, D and Qin, K and Chen, Z and Collins, SM and Huang, H and Komar, CA and Bailer, PF and Parker, JB and Blobel, GA and Kohli, RM and Wherry, EJ and Berger, SL and Shi, J}, title = {Efficient engineering of human and mouse primary cells using peptide-assisted genome editing.}, journal = {Nature biotechnology}, volume = {}, number = {}, pages = {}, pmid = {37095348}, issn = {1546-1696}, abstract = {Simple, efficient and well-tolerated delivery of CRISPR genome editing systems into primary cells remains a major challenge. Here we describe an engineered Peptide-Assisted Genome Editing (PAGE) CRISPR-Cas system for rapid and robust editing of primary cells with minimal toxicity. The PAGE system requires only a 30-min incubation with a cell-penetrating Cas9 or Cas12a and a cell-penetrating endosomal escape peptide to achieve robust single and multiplex genome editing. Unlike electroporation-based methods, PAGE gene editing has low cellular toxicity and shows no significant transcriptional perturbation. We demonstrate rapid and efficient editing of primary cells, including human and mouse T cells, as well as human hematopoietic progenitor cells, with editing efficiencies upwards of 98%. PAGE provides a broadly generalizable platform for next-generation genome engineering in primary cells.}, }
@article {pmid37095220, year = {2023}, author = {Hao, L and Zhao, RT and Welch, NL and Tan, EKW and Zhong, Q and Harzallah, NS and Ngambenjawong, C and Ko, H and Fleming, HE and Sabeti, PC and Bhatia, SN}, title = {CRISPR-Cas-amplified urinary biomarkers for multiplexed and portable cancer diagnostics.}, journal = {Nature nanotechnology}, volume = {}, number = {}, pages = {}, pmid = {37095220}, issn = {1748-3395}, abstract = {Synthetic biomarkers, bioengineered sensors that generate molecular reporters in diseased microenvironments, represent an emerging paradigm in precision diagnostics. Despite the utility of DNA barcodes as a multiplexing tool, their susceptibility to nucleases in vivo has limited their utility. Here we exploit chemically stabilized nucleic acids to multiplex synthetic biomarkers and produce diagnostic signals in biofluids that can be 'read out' via CRISPR nucleases. The strategy relies on microenvironmental endopeptidase to trigger the release of nucleic acid barcodes and polymerase-amplification-free, CRISPR-Cas-mediated barcode detection in unprocessed urine. Our data suggest that DNA-encoded nanosensors can non-invasively detect and differentiate disease states in transplanted and autochthonous murine cancer models. We also demonstrate that CRISPR-Cas amplification can be harnessed to convert the readout to a point-of-care paper diagnostic tool. Finally, we employ a microfluidic platform for densely multiplexed, CRISPR-mediated DNA barcode readout that can potentially evaluate complex human diseases rapidly and guide therapeutic decisions.}, }
@article {pmid37094126, year = {2023}, author = {Zhang, M and Peng, R and Peng, Q and Liu, S and Li, Z and Zhang, Y and Song, H and Yang, J and Xing, X and Wang, P and Qi, J and Gao, GF}, title = {Mechanistic insights into DNA binding and cleavage by a compact type I-F CRISPR-Cas system in bacteriophage.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {120}, number = {18}, pages = {e2215098120}, doi = {10.1073/pnas.2215098120}, pmid = {37094126}, issn = {1091-6490}, abstract = {CRISPR-Cas systems are widespread adaptive antiviral systems used in prokaryotes. Some phages, in turn, although have small genomes can economize the use of genetic space to encode compact or incomplete CRISPR-Cas systems to inhibit the host and establish infection. Phage ICP1, infecting Vibrio cholerae, encodes a compact type I-F CRISPR-Cas system to suppress the antiphage mobile genetic element in the host genome. However, the mechanism by which this compact system recognizes the target DNA and executes interference remains elusive. Here, we present the electron cryo-microscopy (cryo-EM) structures of both apo- and DNA-bound ICP1 surveillance complexes (Aka Csy complex). Unlike most other type I surveillance complexes, the ICP1 Csy complex lacks the Cas11 subunit or a structurally homologous domain, which is crucial for dsDNA binding and Cas3 activation in other type I CRISPR-Cas systems. Structural and functional analyses revealed that the compact ICP1 Csy complex alone is inefficient in binding to dsDNA targets, presumably stalled at a partial R-loop conformation. The presence of Cas2/3 facilitates dsDNA binding and allows effective dsDNA target cleavage. Additionally, we found that Pseudomonas aeruginosa Cas2/3 efficiently cleaved the dsDNA target presented by the ICP1 Csy complex, but not vice versa. These findings suggest a unique mechanism for target dsDNA binding and cleavage by the compact phage-derived CRISPR-Cas system.}, }
@article {pmid37093294, year = {2023}, author = {Hunt, JMT and Samson, CA and Rand, AD and Sheppard, HM}, title = {Unintended CRISPR-Cas9 editing outcomes: a review of the detection and prevalence of structural variants generated by gene-editing in human cells.}, journal = {Human genetics}, volume = {}, number = {}, pages = {}, pmid = {37093294}, issn = {1432-1203}, abstract = {Genome editing using the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein (Cas) gene-editing system (CRISPR-Cas) is a valuable tool for fundamental and applied research applications. Significant improvements in editing efficacy have advanced genome editing strategies into phase 3 human clinical trials. However, recent studies suggest that our understanding of editing outcomes has lagged behind the developments made in generating the edits themselves. While many researchers have analyzed on- and off-target events through the lens of small insertions or deletions at predicted sites, screens for larger structural variants (SVs) and chromosomal abnormalities are not routinely performed. Full and comprehensive validation of on- and off-target effects is required to ensure reproducibility and to accurately assess the safety of future editing applications. Here we review SVs associated with CRISPR-editing in cells of human origin and highlight the methods used to detect and avoid them.}, }
@article {pmid37085898, year = {2023}, author = {Marinov, GK and Kim, SH and Bagdatli, ST and Higashino, SI and Trevino, AE and Tycko, J and Wu, T and Bintu, L and Bassik, MC and He, C and Kundaje, A and Greenleaf, WJ}, title = {CasKAS: direct profiling of genome-wide dCas9 and Cas9 specificity using ssDNA mapping.}, journal = {Genome biology}, volume = {24}, number = {1}, pages = {85}, pmid = {37085898}, issn = {1474-760X}, support = {P50 HG007735/HG/NHGRI NIH HHS/United States ; U19 AI057266/AI/NIAID NIH HHS/United States ; UM1 HG009442/HG/NHGRI NIH HHS/United States ; UM1 HG009436/HG/NHGRI NIH HHS/United States ; U01 HG009431/HG/NHGRI NIH HHS/United States ; F99 DK126120/DK/NIDDK NIH HHS/United States ; T32 GM007365/GM/NIGMS NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; }, mesh = {*CRISPR-Cas Systems ; *DNA, Single-Stranded/genetics ; Genome ; CRISPR-Associated Protein 9/genetics ; Epigenome ; Gene Editing/methods ; }, abstract = {Detecting and mitigating off-target activity is critical to the practical application of CRISPR-mediated genome and epigenome editing. While numerous methods have been developed to map Cas9 binding specificity genome-wide, they are generally time-consuming and/or expensive, and not applicable to catalytically dead CRISPR enzymes. We have developed CasKAS, a rapid, inexpensive, and facile assay for identifying off-target CRISPR enzyme binding and cleavage by chemically mapping the unwound single-stranded DNA structures formed upon binding of a sgRNA-loaded Cas9 protein. We demonstrate this method in both in vitro and in vivo contexts.}, }
@article {pmid37084235, year = {2023}, author = {Li, Y and Zhi, S and Wu, T and Chen, HX and Kang, R and Ma, DZ and Songyang, Z and He, C and Liang, P and Luo, GZ}, title = {Systematic identification of CRISPR off-target effects by CROss-seq.}, journal = {Protein &amp; cell}, volume = {14}, number = {4}, pages = {299-303}, pmid = {37084235}, issn = {1674-8018}, mesh = {*Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *CRISPR-Cas Systems/genetics ; Gene Editing ; }, }
@article {pmid37083429, year = {2023}, author = {Parker, J}, title = {How has CRISPR transformed therapeutic drug discovery?.}, journal = {BioTechniques}, volume = {74}, number = {3}, pages = {119-121}, doi = {10.2144/btn-2023-0020}, pmid = {37083429}, issn = {1940-9818}, mesh = {Humans ; *Gene Editing/methods ; CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Neurodegenerative Diseases/drug therapy/genetics ; Drug Discovery ; }, abstract = {The genome is the blueprint for life, and over the past decade, CRISPR has become a very powerful method for editing our genetic makeup. In this article, we will explore the importance of CRISPR in developing breakthrough therapies for monogenic conditions and neurodegenerative diseases, and for enhancing the effectiveness of immuno-oncology.}, }
@article {pmid36928106, year = {2023}, author = {Wang, Q and Liu, J and Janssen, JM and Gonçalves, MAFV}, title = {Precise homology-directed installation of large genomic edits in human cells with cleaving and nicking high-specificity Cas9 variants.}, journal = {Nucleic acids research}, volume = {51}, number = {7}, pages = {3465-3484}, pmid = {36928106}, issn = {1362-4962}, mesh = {Animals ; Humans ; *CRISPR-Cas Systems/genetics ; DNA Breaks, Double-Stranded ; *Induced Pluripotent Stem Cells ; Gene Editing/methods ; Genomics ; Mammals ; }, abstract = {Homology-directed recombination (HDR) between donor constructs and acceptor genomic sequences cleaved by programmable nucleases, permits installing large genomic edits in mammalian cells in a precise fashion. Yet, next to precise gene knock-ins, programmable nucleases yield unintended genomic modifications resulting from non-homologous end-joining processes. Alternatively, in trans paired nicking (ITPN) involving tandem single-strand DNA breaks at target loci and exogenous donor constructs by CRISPR-Cas9 nickases, fosters seamless and scarless genome editing. In the present study, we identified high-specificity CRISPR-Cas9 nucleases capable of outperforming parental CRISPR-Cas9 nucleases in directing genome editing through homologous recombination (HR) and homology-mediated end joining (HMEJ) with donor constructs having regular and 'double-cut' designs, respectively. Additionally, we explored the ITPN principle by demonstrating its compatibility with orthogonal and high-specificity CRISPR-Cas9 nickases and, importantly, report that in human induced pluripotent stem cells (iPSCs), in contrast to high-specificity CRISPR-Cas9 nucleases, neither regular nor high-specificity CRISPR-Cas9 nickases activate P53 signaling, a DNA damage-sensing response linked to the emergence of gene-edited cells with tumor-associated mutations. Finally, experiments in human iPSCs revealed that differently from HR and HMEJ genome editing based on high-specificity CRISPR-Cas9 nucleases, ITPN involving high-specificity CRISPR-Cas9 nickases permits editing allelic sequences associated with essentiality and recurrence in the genome.}, }
@article {pmid36718951, year = {2023}, author = {Develtere, W and Waegneer, E and Debray, K and De Saeger, J and Van Glabeke, S and Maere, S and Ruttink, T and Jacobs, TB}, title = {SMAP design: a multiplex PCR amplicon and gRNA design tool to screen for natural and CRISPR-induced genetic variation.}, journal = {Nucleic acids research}, volume = {51}, number = {7}, pages = {e37}, pmid = {36718951}, issn = {1362-4962}, support = {833866/ERC_/European Research Council/International ; }, mesh = {*Multiplex Polymerase Chain Reaction ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Genome ; Genetic Variation ; CRISPR-Cas Systems ; }, abstract = {Multiplex amplicon sequencing is a versatile method to identify genetic variation in natural or mutagenized populations through eco-tilling or multiplex CRISPR screens. Such genotyping screens require reliable and specific primer designs, combined with simultaneous gRNA design for CRISPR screens. Unfortunately, current tools are unable to combine multiplex gRNA and primer design in a high-throughput and easy-to-use manner with high design flexibility. Here, we report the development of a bioinformatics tool called SMAP design to overcome these limitations. We tested SMAP design on several plant and non-plant genomes and obtained designs for more than 80-90% of the target genes, depending on the genome and gene family. We validated the designs with Illumina multiplex amplicon sequencing and Sanger sequencing in Arabidopsis, soybean, and maize. We also used SMAP design to perform eco-tilling by tilling PCR amplicons across nine candidate genes putatively associated with haploid induction in Cichorium intybus. We screened 60 accessions of chicory and witloof and identified thirteen knockout haplotypes and their carriers. SMAP design is an easy-to-use command-line tool that generates highly specific gRNA and/or primer designs for any number of loci for CRISPR or natural variation screens and is compatible with other SMAP modules for seamless downstream analysis.}, }
@article {pmid37091050, year = {2023}, author = {Waitkus, J and Chang, Y and Liu, L and Puttaswamy, SV and Chung, T and Vargas, AMM and Dollery, SJ and O'Connell, MR and Cai, H and Tobin, GJ and Bhalla, N and Du, K}, title = {Gold Nanoparticle Enabled Localized Surface Plasmon Resonance on Unique Gold Nanomushroom Structures for On-Chip CRISPR-Cas13a Sensing.}, journal = {Advanced materials interfaces}, volume = {10}, number = {1}, pages = {}, pmid = {37091050}, issn = {2196-7350}, abstract = {A novel localized surface plasmon resonance (LSPR) system based on the coupling of gold nanomushrooms (AuNMs) and gold nanoparticles (AuNPs) is developed to enable a significant plasmonic resonant shift. The AuNP size, surface chemistry, and concentration are characterized to maximize the LSPR effect. A 31 nm redshift is achieved when the AuNMs are saturated by the AuNPs. This giant redshift also increases the full width of the spectrum and is explained by the 3D finite-difference time-domain (FDTD) calculation. In addition, this LSPR substrate is packaged in a microfluidic cell and integrated with a CRISPR-Cas13a RNA detection assay for the detection of the SARS-CoV-2 RNA targets. Once activated by the target, the AuNPs are cleaved from linker probes and randomly deposited on the AuNM substrate, demonstrating a large redshift. The novel LSPR chip using AuNP as an indicator is simple, specific, isothermal, and label-free; and thus, provides a new opportunity to achieve the next generation multiplexing and sensitive molecular diagnostic system.}, }
@article {pmid37090614, year = {2023}, author = {Shmakov, SA and Barth, ZK and Makarova, KS and Wolf, YI and Brover, V and Peters, JE and Koonin, EV}, title = {Widespread CRISPR repeat-like RNA regulatory elements in CRISPR-Cas systems.}, journal = {bioRxiv : the preprint server for biology}, volume = {}, number = {}, pages = {}, doi = {10.1101/2023.03.03.530964}, pmid = {37090614}, abstract = {CRISPR- cas loci typically contain CRISPR arrays with unique spacers separating direct repeats. Spacers along with portions of adjacent repeats are transcribed and processed into CRISPR(cr) RNAs that target complementary sequences (protospacers) in mobile genetic elements, resulting in cleavage of the target DNA or RNA. Additional, standalone repeats in some CRISPR- cas loci produce distinct cr-like RNAs implicated in regulatory or other functions. We developed a computational pipeline to systematically predict crRNA-like elements by scanning for standalone repeat sequences that are conserved in closely related CRISPR- cas loci. Numerous crRNA-like elements were detected in diverse CRISPR-Cas systems, mostly, of type I, but also subtype V-A. Standalone repeats often form mini-arrays containing two repeat-like sequence separated by a spacer that is partially complementary to promoter regions of cas genes, in particular cas8 , or cargo genes located within CRISPR-Cas loci, such as toxins-antitoxins. We show experimentally that a mini-array from a type I-F1 CRISPR-Cas system functions as a regulatory guide. We also identified mini-arrays in bacteriophages that could abrogate CRISPR immunity by inhibiting effector expression. Thus, recruitment of CRISPR effectors for regulatory functions via spacers with partial complementarity to the target is a common feature of diverse CRISPR-Cas systems.}, }
@article {pmid37085666, year = {2023}, author = {Pan, C and Qi, Y}, title = {CRISPR-Combo-mediated orthogonal genome editing and transcriptional activation for plant breeding.}, journal = {Nature protocols}, volume = {}, number = {}, pages = {}, pmid = {37085666}, issn = {1750-2799}, abstract = {CRISPR-Cas nuclease systems, base editors, and CRISPR activation have greatly advanced plant genome engineering. However, the combinatorial approaches for multiplexed orthogonal genome editing and transcriptional regulation were previously unexploited in plants. We have recently established a single Cas9 protein-based CRISPR-Combo platform, enabling efficient multiplexed orthogonal genome editing (double-strand break-mediated genome editing or base editing) and transcriptional activation in plants via engineering the single guide RNA (sgRNA) structure. Here, we provide step-by-step instructions for constructing CRISPR-Combo systems for speed breeding of transgene-free, genome-edited Arabidopsis plants and enhancing rice regeneration with more heritable targeted mutations in a hormone-free manner. We also provide guidance on designing efficient sgRNA, Agrobacterium-mediated transformation of Arabidopsis and rice, rice regeneration without exogenous plant hormones, gene editing evaluation and visual identification of transgene-free Arabidopsis plants with high editing activity. With the use of this protocol, it takes ~2 weeks to establish the CRISPR-Combo systems, 4 months to obtain transgene-free genome-edited Arabidopsis plants and 4 months to obtain rice plants with enrichment of heritable targeted mutations by hormone-free tissue culture.}, }
@article {pmid37084259, year = {2023}, author = {Li, Y and Wei, Y and Xu, S and Tan, Q and Zong, L and Wang, J and Wang, Y and Chen, J and Hong, L and Li, Y}, title = {AcrNET: Predicting anti-CRISPR with Deep Learning.}, journal = {Bioinformatics (Oxford, England)}, volume = {}, number = {}, pages = {}, doi = {10.1093/bioinformatics/btad259}, pmid = {37084259}, issn = {1367-4811}, abstract = {MOTIVATION: As an important group of proteins discovered in phages, anti-CRISPR inhibits the activity of the immune system of bacteria (i.e., CRISPR-Cas), offering promise for gene editing and phage therapy. However, the prediction and discovery of anti-CRISPR are challenging due to their high variability and fast evolution. Existing biological studies rely on known CRISPR and anti-CRISPR pairs, which may not be practical considering the huge number. Computational methods struggle with prediction performance. To address these issues, we propose a novel deep neural network for anti-CRISPR analysis (AcrNET), which achieves significant performance.<br><br>RESULTS: On both the cross-fold and cross-dataset validation, our method outperforms the state-of-the-art methods. Notably, AcrNET improves the prediction performance by at least 15% regarding the F1 score for the cross-dataset test problem comparing with state-of-art Deep Learning method. Moreover, AcrNET is the first computational method to predict the detailed anti-CRISPR classes, which may help illustrate the anti-CRISPR mechanism. Taking advantage of a Transformer protein language model ESM-1b, which was pre-trained on 250 million protein sequences, AcrNET overcomes the data scarcity problem. Extensive experiments and analysis suggest that the Transformer model feature, evolutionary feature, and local structure feature complement each other, which indicates the critical properties of anti-CRISPR proteins. AlphaFold prediction, further motif analysis, and docking experiments further demonstrate that AcrNET can capture the evolutionarily conserved pattern and the interaction between anti-CRISPR and the target implicitly.<br><br>Web server: https://proj.cse.cuhk.edu.hk/aihlab/AcrNET/. Training code and pre-trained model are available at https://github.com/banma12956/AcrNET.<br><br>&#x2022;: Supplementary Information.}, }
@article {pmid37083687, year = {2023}, author = {Figueroa, W and Cazares, A and Cazares, D and Wu, Y and de la Cruz, A and Welch, M and Kameyama, L and Nobrega, FL and Guarneros, G}, title = {Distribution and molecular evolution of the anti-CRISPR family AcrIF7.}, journal = {PLoS biology}, volume = {21}, number = {4}, pages = {e3002072}, doi = {10.1371/journal.pbio.3002072}, pmid = {37083687}, issn = {1545-7885}, abstract = {Anti-clustered regularly interspaced short palindromic repeats (CRISPRs) are proteins capable of blocking CRISPR-Cas systems and typically their genes are located on mobile genetic elements. Since their discovery, numerous anti-CRISPR families have been identified. However, little is known about the distribution and sequence diversity of members within a family, nor how these traits influence the anti-CRISPR's function and evolution. Here, we use AcrIF7 to explore the dissemination and molecular evolution of an anti-CRISPR family. We uncovered 5 subclusters and prevalent anti-CRISPR variants within the group. Remarkably, AcrIF7 homologs display high similarity despite their broad geographical, ecological, and temporal distribution. Although mainly associated with Pseudomonas aeruginosa, AcrIF7 was identified in distinct genetic backgrounds indicating horizontal dissemination, primarily by phages. Using mutagenesis, we recreated variation observed in databases but also extended the sequence diversity of the group. Characterisation of the variants identified residues key for the anti-CRISPR function and other contributing to its mutational tolerance. Moreover, molecular docking revealed that variants with affected function lose key interactions with its CRISPR-Cas target. Analysis of publicly available data and the generated variants suggests that the dominant AcrIF7 variant corresponds to the minimal and optimal anti-CRISPR selected in the family. Our study provides a blueprint to investigate the molecular evolution of anti-CRISPR families.}, }
@article {pmid37083261, year = {2023}, author = {Philippidis, A}, title = {PASTE, Don't Cut: Genome Editing Tool Looks Beyond CRISPR and Prime.}, journal = {Human gene therapy}, volume = {34}, number = {7-8}, pages = {255-258}, doi = {10.1089/hum.2023.29238.bfs}, pmid = {37083261}, issn = {1557-7422}, mesh = {*Gene Editing ; *Clustered Regularly Interspaced Short Palindromic Repeats ; CRISPR-Cas Systems ; Genetic Engineering ; }, }
@article {pmid37083189, year = {2023}, author = {Zhou, M and Li, X and Wen, H and Huang, B and Ren, J and Zhang, J}, title = {The construction of CRISPR/Cas9-mediated FRET 16S rDNA sensor for detection of Mycobacterium tuberculosis.}, journal = {The Analyst}, volume = {}, number = {}, pages = {}, doi = {10.1039/d3an00462g}, pmid = {37083189}, issn = {1364-5528}, abstract = {The simple and efficient detection of nucleic acids is important in the diagnosis of tuberculosis (TB) caused by Mycobacterium tuberculosis (M. tuberculosis). However, base mismatch will lead to false positive and false negative nucleic acid test, which seriously interferes with the accuracy of the final results. Herein, we demonstrated a CRISPR/Cas-9-mediated fluorescent strategy utilizing fluorescence resonance energy transfer (FRET) for the detection of bacteria. High-variable region of M. tuberculosis 16S rDNA fragment was used as the target, and CRISPR/Cas9 was used as the recognition element. The binding of the P1 probe of upconversion nanoparticles (UCNPs) @SiO2-P1 and the P2 probe of Fe3O4@Au-P2 caused the fluorescence quenching of UCNPs. In the presence of the target, the P2 probe hybridized with the target to form double-stranded DNA (dsDNA), which was recognized and cleaved by CRISPR/Cas9, resulting in the breaking of the P1-P2 duplex linkage. UCNPs moved away from Fe3O4@Au under a magnetic field, and the fluorescence signal was restored; bacteria were detected under the excitation of a 980 nm laser source. Using the CRISPR/Cas-9-mediated system, the sensor could distinguish single-base mismatches in 10 bases from the protospacer adjacent motif (PAM) region. The limit of detection (LOD) was 20 CFU mL[-1] and the detection time was 2 h. It developed a new way of accurate nucleic acid detection for disease diagnosis.}, }
@article {pmid37081092, year = {2023}, author = {Leski, TA and Spangler, JR and Wang, Z and Schultzhaus, Z and Taitt, CR and Dean, SN and Stenger, DA}, title = {Machine learning for design of degenerate Cas13a crRNAs using lassa virus as a model of highly variable RNA target.}, journal = {Scientific reports}, volume = {13}, number = {1}, pages = {6506}, pmid = {37081092}, issn = {2045-2322}, mesh = {*RNA/metabolism ; *Lassa virus/genetics ; RNA Processing, Post-Transcriptional ; CRISPR-Cas Systems/genetics ; }, abstract = {The design of minimum CRISPR RNA (crRNA) sets for detection of diverse RNA targets using sequence degeneracy has not been systematically addressed. We tested candidate degenerate Cas13a crRNA sets designed for detection of diverse RNA targets (Lassa virus). A decision tree machine learning (ML) algorithm (RuleFit) was applied to define the top attributes that determine the specificity of degenerate crRNAs to elicit collateral nuclease activity. Although the total number of mismatches (0-4) is important, the specificity depends as well on the spacing of mismatches, and their proximity to the 5' end of the spacer. We developed a predictive algorithm for design of candidate degenerate crRNA sets, allowing improved discrimination between "included" and "excluded" groups of related target sequences. A single degenerate crRNA set adhering to these rules detected representatives of all Lassa lineages. Our general ML approach may be applied to the design of degenerate crRNA sets for any CRISPR/Cas system.}, }
@article {pmid37031406, year = {2023}, author = {Yin, W and Hu, H}, title = {CRISPR/Cas9-Mediated Genome Editing via Homologous Recombination in a Centric Diatom Chaetoceros muelleri.}, journal = {ACS synthetic biology}, volume = {12}, number = {4}, pages = {1287-1296}, doi = {10.1021/acssynbio.3c00051}, pmid = {37031406}, issn = {2161-5063}, mesh = {*Gene Editing ; CRISPR-Cas Systems/genetics ; *Diatoms/genetics ; Chlorophyll A ; Homologous Recombination/genetics ; DNA ; }, abstract = {Chaetoceros, the most abundant genus of marine planktonic diatoms, can be used in mariculture. An effective genetic transformation system with a short transformation period was established in Chaetoceros muelleri by electroporation in our previous study. In this study, a sequence-specific clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 vector applicable for C. muelleri was constructed, and the expressions of sgRNA, resistance gene, and Cas9 gene were driven by the endogenous promoters U6, acetyl-CoA acetyltransferase, and fucoxanthin chlorophyll a/c binding protein, respectively, in the vector. Nitrate reductase (NR) and urease (URE) genes were edited in C. muelleri, and the NR knockout and NR/URE double-knockout lines displayed the strict auxotrophic phenotype. In addition, the DNA double-strand break was repaired by homologous recombination when a donor DNA was introduced. CRISPR/Cas9 technology was successfully applied to C. muelleri with an editing efficiency of up to 86%, providing a molecular tool for the study of basic biology in C. muelleri and its synthetic biology applications.}, }
@article {pmid37024659, year = {2023}, author = {Zhou, J and Liu, G and Zhao, Y and Zhang, R and Tang, X and Li, L and Jia, X and Guo, Y and Wu, Y and Han, Y and Bao, Y and He, Y and Han, Q and Yang, H and Zheng, X and Qi, Y and Zhang, T and Zhang, Y}, title = {An efficient CRISPR-Cas12a promoter editing system for crop improvement.}, journal = {Nature plants}, volume = {9}, number = {4}, pages = {588-604}, pmid = {37024659}, issn = {2055-0278}, mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; Crops, Agricultural/genetics ; Edible Grain ; Promoter Regions, Genetic ; }, abstract = {Promoter editing represents an innovative approach to introduce quantitative trait variation (QTV) in crops. However, an efficient promoter editing system for QTV needs to be established. Here we develop a CRISPR-Cas12a promoter editing (CAPE) system that combines a promoter key-region estimating model and an efficient CRISPR-Cas12a-based multiplexed or singular editing system. CAPE is benchmarked in rice to produce QTV continuums for grain starch content and size by targeting OsGBSS1 and OsGS3, respectively. We then apply CAPE for promoter editing of OsD18, a gene encoding GA3ox in the gibberellin biosynthesis pathway. The resulting lines carry a QTV continuum of semidwarfism without significantly compromising grain measures. Field trials demonstrated that the OsD18 promoter editing lines have the same yield performance and antilodging phenotype as the Green Revolution OsSD1 mutants in different genetic backgrounds. Hence, promoter editing of OsD18 generates a quantitative Green Revolution trait. Together, we demonstrate a CAPE-based promoter editing and tuning pipeline for efficient production of useful QTV continuum in crops.}, }
@article {pmid37017652, year = {2023}, author = {Hao, X and Mu, T and Sharshar, MM and Jia, Y and Zhong, W and Chen, Z and Wen, Q and Yang, M and Wang, C and Xing, J}, title = {CRISPR/Cas12a-Mediated Genome Editing in Thioalkalivibrio versutus.}, journal = {ACS synthetic biology}, volume = {12}, number = {4}, pages = {1204-1215}, doi = {10.1021/acssynbio.2c00676}, pmid = {37017652}, issn = {2161-5063}, mesh = {*Gene Editing/methods ; CRISPR-Cas Systems/genetics ; *Ectothiorhodospiraceae/genetics/metabolism ; Sulfur/metabolism ; }, abstract = {Haloalkaliphilic Thioalkalivibrio versutus, a dominant species for sulfide removal, has attracted increasing attention. However, research on T. versutus is limited by the lack of genetic manipulation tools. In this work, we developed a CRISPR/AsCas12a-mediated system in T. versutus for an efficient and implementable genome editing workflow. Compared to the CRISPR/Cas9-mediated system, the CRISPR/AsCas12a system exhibited enhanced editing efficiency. Additionally, as Cas12a is capable of processing the crRNA maturation independently, the CRISPR/AsCas12a system allowed multiplex gene editing and large-fragment DNA knockout by expressing more than one crRNA under the control of one promoter. Using the CRISPR/AsCas12a system, five key genes of the elemental sulfur oxidation pathway were knocked out. Simultaneous deletion of the rhd and tusA genes disrupted the ability of T. versutus to metabolize elemental sulfur, resulting in a 24.7% increase in elemental sulfur generation and a 15.2% reduction in sulfate production. This genome engineering strategy significantly improved our understanding of sulfur metabolism in Thioalkalivibrio spp.}, }
@article {pmid37014634, year = {2023}, author = {Machens, F and Ran, G and Ruehmkorff, C and Meyer Auf der Heyde, J and Mueller-Roeber, B and Hochrein, L}, title = {PhiReX 2.0: A Programmable and Red Light-Regulated CRISPR-dCas9 System for the Activation of Endogenous Genes in Saccharomyces cerevisiae.}, journal = {ACS synthetic biology}, volume = {12}, number = {4}, pages = {1046-1057}, doi = {10.1021/acssynbio.2c00517}, pmid = {37014634}, issn = {2161-5063}, mesh = {*Saccharomyces cerevisiae/genetics ; Transcriptional Activation/genetics ; *CRISPR-Cas Systems/genetics ; RNA, Guide, Kinetoplastida/genetics ; Transcription Factors/genetics ; }, abstract = {Metabolic engineering approaches do not exclusively require fine-tuning of heterologous genes but oftentimes also modulation or even induction of host gene expression, e.g., in order to rewire metabolic fluxes. Here, we introduce the programmable red light switch PhiReX 2.0, which can rewire metabolic fluxes by targeting endogenous promoter sequences through single-guide RNAs (sgRNAs) and activate gene expression in Saccharomyces cerevisiae upon red light stimulation. The split transcription factor is built from the plant-derived optical dimer PhyB and PIF3, which is fused to a DNA-binding domain based on the catalytically dead Cas9 protein (dCas9) and a transactivation domain. This design combines at least two major advantages: first, the sgRNAs, guiding dCas9 to the promoter of interest, can be exchanged in an efficient and straightforward Golden Gate-based cloning approach, which allows for rational or randomized combination of up to four sgRNAs in a single expression array. Second, target gene expression can be rapidly upregulated by short red light pulses in a light dose-dependent manner and returned to the native expression level by applying far-red light without interfering with the cell culture. Using the native yeast gene CYC1 as an example, we demonstrated that PhiReX 2.0 can upregulate CYC1 gene expression by up to 6-fold in a light intensity-dependent and reversible manner using a single sgRNA.}, }
@article {pmid36973416, year = {2023}, author = {}, title = {A genome-scale CRISPR tool for targeting multiple gene family members at once.}, journal = {Nature plants}, volume = {9}, number = {4}, pages = {511-512}, pmid = {36973416}, issn = {2055-0278}, mesh = {*Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; CRISPR-Cas Systems ; }, }
@article {pmid36973414, year = {2023}, author = {Hu, Y and Patra, P and Pisanty, O and Shafir, A and Belew, ZM and Binenbaum, J and Ben Yaakov, S and Shi, B and Charrier, L and Hyams, G and Zhang, Y and Trabulsky, M and Caldararu, O and Weiss, D and Crocoll, C and Avni, A and Vernoux, T and Geisler, M and Nour-Eldin, HH and Mayrose, I and Shani, E}, title = {Multi-Knock-a multi-targeted genome-scale CRISPR toolbox to overcome functional redundancy in plants.}, journal = {Nature plants}, volume = {9}, number = {4}, pages = {572-587}, pmid = {36973414}, issn = {2055-0278}, mesh = {*Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Arabidopsis/genetics ; Plant Breeding ; Plants/genetics ; Genome, Plant ; CRISPR-Cas Systems ; Plants, Genetically Modified/genetics ; Gene Editing ; }, abstract = {Plant genomes are characterized by large and complex gene families that often result in similar and partially overlapping functions. This genetic redundancy severely hampers current efforts to uncover novel phenotypes, delaying basic genetic research and breeding programmes. Here we describe the development and validation of Multi-Knock, a genome-scale clustered regularly interspaced short palindromic repeat toolbox that overcomes functional redundancy in Arabidopsis by simultaneously targeting multiple gene-family members, thus identifying genetically hidden components. We computationally designed 59,129 optimal single-guide RNAs that each target two to ten genes within a family at once. Furthermore, partitioning the library into ten sublibraries directed towards a different functional group allows flexible and targeted genetic screens. From the 5,635 single-guide RNAs targeting the plant transportome, we generated over 3,500 independent Arabidopsis lines that allowed us to identify and characterize the first known cytokinin tonoplast-localized transporters in plants. With the ability to overcome functional redundancy in plants at the genome-scale level, the developed strategy can be readily deployed by scientists and breeders for basic research and to expedite breeding efforts.}, }
@article {pmid36932144, year = {2023}, author = {}, title = {Removing the toxic enantiomer from crop products with selective gene editing.}, journal = {Nature plants}, volume = {9}, number = {4}, pages = {513-514}, pmid = {36932144}, issn = {2055-0278}, mesh = {*Gene Editing ; *CRISPR-Cas Systems ; Genetic Engineering ; Genome, Plant ; }, }
@article {pmid36914142, year = {2023}, author = {Eslahi, A and Alizadeh, F and Avan, A and Ferns, GA and Moghbeli, M and Reza Abbaszadegan, M and Mojarrad, M}, title = {New advancements in CRISPR based gene therapy of Duchenne muscular dystrophy.}, journal = {Gene}, volume = {867}, number = {}, pages = {147358}, doi = {10.1016/j.gene.2023.147358}, pmid = {36914142}, issn = {1879-0038}, mesh = {Humans ; *Muscular Dystrophy, Duchenne/genetics/therapy ; Dystrophin/genetics ; CRISPR-Cas Systems ; Gene Editing ; Genetic Therapy ; }, abstract = {Duchenne muscular dystrophy (DMD) is caused by the dystrophin gene mutations and is one of the most common and lethal human hereditary disorders. A novel therapeutic approach using CRISPR technology has gained attention in the treatment of DMD. Gene replacement strategies are being proposed as a promising therapeutic option to compensate the loss of function mutations. Although, the large size of the dystrophin gene and the limitations of the existing gene replacement approach, could mean the gene delivery of shortened versions of dystrophin such as midystrophin and microdystrophins. There are also other approaches: including Targeted removal of dystrophin exons to restore the reading-frame; Dual sgRNA-directed DMD exon deletion, CRISPR-SKIP strategy; reframing of dystrophin using Prime Editing technology; exon removal using twin prime technology; TransCRISTI technology to targeted exon integration into dystrophin gene. Here we provide an overview of recent progresses in dystrophin gene editing using updated versions of CRISPR to introduce novel opportunities in DMD gene therapy. Overall, the novel CRISPR based technologies are improving and expanding to allow the application of more precise gene editing for the treatment of DMD.}, }
@article {pmid36893454, year = {2023}, author = {Dallo, T and Krishnakumar, R and Kolker, SD and Ruffing, AM}, title = {High-Density Guide RNA Tiling and Machine Learning for Designing CRISPR Interference in Synechococcus sp. PCC 7002.}, journal = {ACS synthetic biology}, volume = {12}, number = {4}, pages = {1175-1186}, doi = {10.1021/acssynbio.2c00653}, pmid = {36893454}, issn = {2161-5063}, mesh = {*CRISPR-Cas Systems/genetics ; *Synechococcus/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; DNA ; RNA ; }, abstract = {While CRISPRi was previously established in Synechococcus sp. PCC 7002 (hereafter 7002), the design principles for guide RNA (gRNA) effectiveness remain largely unknown. Here, 76 strains of 7002 were constructed with gRNAs targeting three reporter systems to evaluate features that impact gRNA efficiency. Correlation analysis of the data revealed that important features of gRNA design include the position relative to the start codon, GC content, protospacer adjacent motif (PAM) site, minimum free energy, and targeted DNA strand. Unexpectedly, some gRNAs targeting upstream of the promoter region showed small but significant increases in reporter expression, and gRNAs targeting the terminator region showed greater repression than gRNAs targeting the 3' end of the coding sequence. Machine learning algorithms enabled prediction of gRNA effectiveness, with Random Forest having the best performance across all training sets. This study demonstrates that high-density gRNA data and machine learning can improve gRNA design for tuning gene expression in 7002.}, }
@article {pmid36732423, year = {2023}, author = {Kodackattumannil, P and Lekshmi, G and Kottackal, M and Sasi, S and Krishnan, S and Al Senaani, S and Amiri, KMA}, title = {Hidden pleiotropy of agronomic traits uncovered by CRISPR-Cas9 mutagenesis of the tyrosinase CuA-binding domain of the polyphenol oxidase 2 of eggplant.}, journal = {Plant cell reports}, volume = {42}, number = {4}, pages = {825-828}, pmid = {36732423}, issn = {1432-203X}, mesh = {*Monophenol Monooxygenase/genetics ; *Solanum melongena/genetics ; CRISPR-Cas Systems/genetics ; Amino Acid Sequence ; Mutagenesis ; Gene Editing ; }, }
@article {pmid36645771, year = {2023}, author = {Bonsma-Fisher, M and Goyal, S}, title = {Dynamics of immune memory and learning in bacterial communities.}, journal = {eLife}, volume = {12}, number = {}, pages = {}, pmid = {36645771}, issn = {2050-084X}, mesh = {Humans ; *Immunologic Memory ; Evolution, Molecular ; Bacteria/genetics ; *Bacteriophages/genetics ; Models, Theoretical ; CRISPR-Cas Systems ; }, abstract = {From bacteria to humans, adaptive immune systems provide learned memories of past infections. Despite their vast biological differences, adaptive immunity shares features from microbes to vertebrates such as emergent immune diversity, long-term coexistence of hosts and pathogens, and fitness pressures from evolving pathogens and adapting hosts, yet there is no conceptual model that addresses all of these together. To this end, we propose and solve a simple phenomenological model of CRISPR-based adaptive immunity in microbes. We show that in coexisting phage and bacteria populations, immune diversity in both populations is coupled and emerges spontaneously, that bacteria track phage evolution with a context-dependent lag, and that high levels of diversity are paradoxically linked to low overall CRISPR immunity. We define average immunity, an important summary parameter predicted by our model, and use it to perform synthetic time-shift analyses on available experimental data to reveal different modalities of coevolution. Finally, immune cross-reactivity in our model leads to qualitatively different states of evolutionary dynamics, including an influenza-like traveling wave regime that resembles a similar state in models of vertebrate adaptive immunity. Our results show that CRISPR immunity provides a tractable model, both theoretically and experimentally, to understand general features of adaptive immunity.}, }
@article {pmid37080908, year = {2023}, author = {Du, H and Yin, T and Wang, J and Jie, G}, title = {Multifunctional Photoelectrochemical Biosensor Based on ZnIn2S4/ZnS QDs@Au-Ag-Reversed Photocurrent of Cu-Metal-Organic Framework Coupled with CRISPR/Cas-12a-Shearing for Assay of Dual Targets.}, journal = {Analytical chemistry}, volume = {}, number = {}, pages = {}, doi = {10.1021/acs.analchem.3c00846}, pmid = {37080908}, issn = {1520-6882}, abstract = {False positives and negatives in bioanalytical assays remain a persistent problem. Herein, a multifunctional photoelectrochemical (PEC) biosensor based on ZnIn2S4 (ZIS)/ZnS quantum dots (QDs)@Au-Ag-reversed photocurrent of Cu-metal-organic framework (MOF) coupled with CRISPR/Cas-12a-shearing was innovatively developed for assay of dual targets. First, Cu-MOF as a good PEC material shows cathodic photocurrent. Then, numerous ZIS/ZnS QDs were assembled to the Au-Ag nanoparticles (NPs) to prepare a stable and highly amplified signal probe, which can just match the energy level of Cu-MOFs and realized the polarity-reversed photocurrent of Cu-MOF for the first time. As the empty-core nanostructure of Au-Ag NPs has a high specific surface area and low material density, the bimetallic nanocrystal can much increase the reaction rate and improve the redox efficiency. When target CEA-produced cDNA opened the hairpin DNA (HP1 DNA) on the electrode, the ZIS/ZnS QDs@Au-Ag signal probe was conjugated to the electrode via DNA hybridization, achieving a significantly reversed PEC current for CEA detection. Moreover, the specific binding of kanamycin/aptamer generated the acDNA (activator), which can activate the trans-cleavage activity of the CRISPR-CAS12a system on ssDNA, so the signal probe was sheared and caused the obvious decrease of PEC signal for kanamycin detection. The newly developed ZIS/ZnS QDs@Au-Ag NPs displayed excellent PEC properties and reversed photocurrent to MOF and were combined with the unique CRISPR-Cas12a system to achieve sensitive detection of dual targets, which can open a new polarity-reversed PEC sensing platform for rapid and accurate analysis of multiple targets and can effectively avoid false positives results in clinical testing.}, }
@article {pmid37079992, year = {2023}, author = {Han, J and Shin, J and Lee, ES and Cha, BS and Kim, S and Jang, Y and Kim, S and Park, KS}, title = {Cas12a/blocker DNA-based multiplex nucleic acid detection system for diagnosis of high-risk human papillomavirus infection.}, journal = {Biosensors &amp; bioelectronics}, volume = {232}, number = {}, pages = {115323}, doi = {10.1016/j.bios.2023.115323}, pmid = {37079992}, issn = {1873-4235}, abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) proteins are an innovative tool in molecular diagnostics owing to their high specificity and modularity for target nucleic acid sequences. However, the sequence-indiscriminate trans-cleavage activity of the Cas protein renders multiplex detection challenging. In this study, we developed a Cas12a-based multiplex detection system by designing blocker DNA complementary to reporter DNA, which enables the simultaneous detection of two genes with a single Cas protein in a single reaction. As a proof of concept, we chose high-risk human papillomavirus (HPV) 16 and 18 as the model targets and incorporated recombinase polymerase amplification (RPA) and transcription reactions to achieve high accuracy and sensitivity. Using the proposed system, we detected the genes of both HPV 16 and 18 down to 1 aM within 80 min under isothermal conditions. We validated the performance of the system in detecting genomic DNA from various cell lines and clinical samples from cervical cancer patients with high specificity. The proposed system facilitated rapid multiplex detection of high-risk HPVs in a single reaction tube with only Cas12a, thus representing a more user-friendly and economical alternative to previous Cas protein-based multiplex detection assays. The proposed system has considerable potential for point-of-care testing and could be expanded to detect various nucleic acid biomarkers.}, }
@article {pmid37078688, year = {2023}, author = {Huang, X and Zhou, J and Yang, D and Zhang, J and Xia, X and Chen, YE and Xu, J}, title = {Decoding CRISPR-Cas PAM recognition with UniDesign.}, journal = {Briefings in bioinformatics}, volume = {}, number = {}, pages = {}, doi = {10.1093/bib/bbad133}, pmid = {37078688}, issn = {1477-4054}, support = {GM149016/NH/NIH HHS/United States ; }, abstract = {The critical first step in Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated (CRISPR-Cas) protein-mediated gene editing is recognizing a preferred protospacer adjacent motif (PAM) on target DNAs by the protein's PAM-interacting amino acids (PIAAs). Thus, accurate computational modeling of PAM recognition is useful in assisting CRISPR-Cas engineering to relax or tighten PAM requirements for subsequent applications. Here, we describe a universal computational protein design framework (UniDesign) for designing protein-nucleic acid interactions. As a proof of concept, we applied UniDesign to decode the PAM-PIAA interactions for eight Cas9 and two Cas12a proteins. We show that, given native PIAAs, the UniDesign-predicted PAMs are largely identical to the natural PAMs of all Cas proteins. In turn, given natural PAMs, the computationally redesigned PIAA residues largely recapitulated the native PIAAs (74% and 86% in terms of identity and similarity, respectively). These results demonstrate that UniDesign faithfully captures the mutual preference between natural PAMs and native PIAAs, suggesting it is a useful tool for engineering CRISPR-Cas and other nucleic acid-interacting proteins. UniDesign is open-sourced at https://github.com/tommyhuangthu/UniDesign.}, }
@article {pmid37078593, year = {2023}, author = {Walker, MWG and Klompe, SE and Zhang, DJ and Sternberg, SH}, title = {Novel molecular requirements for CRISPR RNA-guided transposition.}, journal = {Nucleic acids research}, volume = {}, number = {}, pages = {}, doi = {10.1093/nar/gkad270}, pmid = {37078593}, issn = {1362-4962}, support = {DP2HG011650/NH/NIH HHS/United States ; }, abstract = {CRISPR-associated transposases (CASTs) direct DNA integration downstream of target sites using the RNA-guided DNA binding activity of nuclease-deficient CRISPR-Cas systems. Transposition relies on several key protein-protein and protein-DNA interactions, but little is known about the explicit sequence requirements governing efficient transposon DNA integration activity. Here, we exploit pooled library screening and high-throughput sequencing to reveal novel sequence determinants during transposition by the Type I-F Vibrio cholerae CAST system (VchCAST). On the donor DNA, large transposon end libraries revealed binding site nucleotide preferences for the TnsB transposase, as well as an additional conserved region that encoded a consensus binding site for integration host factor (IHF). Remarkably, we found that VchCAST requires IHF for efficient transposition, thus revealing a novel cellular factor involved in CRISPR-associated transpososome assembly. On the target DNA, we uncovered preferred sequence motifs at the integration site that explained previously observed heterogeneity with single-base pair resolution. Finally, we exploited our library data to design modified transposon variants that enable in-frame protein tagging. Collectively, our results provide new clues about the assembly and architecture of the paired-end complex formed between TnsB and the transposon DNA, and inform the design of custom payload sequences for genome engineering applications with CAST systems.}, }
@article {pmid37078570, year = {2023}, author = {Xia, B and Viswanatha, R and Hu, Y and Mohr, SE and Perrimon, N}, title = {Pooled genome-wide CRISPR activation screening for rapamycin resistance genes in Drosophila cells.}, journal = {eLife}, volume = {12}, number = {}, pages = {}, pmid = {37078570}, issn = {2050-084X}, support = {GM132087/GM/NIGMS NIH HHS/United States ; CA120964/CA/NCI NIH HHS/United States ; P01CA120964/GF/NIH HHS/United States ; }, mesh = {Animals ; *Drosophila/genetics ; *Sirolimus ; Clustered Regularly Interspaced Short Palindromic Repeats ; CRISPR-Cas Systems ; Genome ; }, abstract = {Loss-of-function and gain-of-function genetic perturbations provide valuable insights into gene function. In Drosophila cells, while genome-wide loss-of-function screens have been extensively used to reveal mechanisms of a variety of biological processes, approaches for performing genome-wide gain-of-function screens are still lacking. Here, we describe a pooled CRISPR activation (CRISPRa) screening platform in Drosophila cells and apply this method to both focused and genome-wide screens to identify rapamycin resistance genes. The screens identified three genes as novel rapamycin resistance genes: a member of the SLC16 family of monocarboxylate transporters (CG8468), a member of the lipocalin protein family (CG5399), and a zinc finger C2H2 transcription factor (CG9932). Mechanistically, we demonstrate that CG5399 overexpression activates the RTK-Akt-mTOR signaling pathway and that activation of insulin receptor (InR) by CG5399 requires cholesterol and clathrin-coated pits at the cell membrane. This study establishes a novel platform for functional genetic studies in Drosophila cells.}, }
@article {pmid37076490, year = {2023}, author = {Kawasaki, S and Ono, H and Hirosawa, M and Kuwabara, T and Sumi, S and Lee, S and Woltjen, K and Saito, H}, title = {Programmable mammalian translational modulators by CRISPR-associated proteins.}, journal = {Nature communications}, volume = {14}, number = {1}, pages = {2243}, pmid = {37076490}, issn = {2041-1723}, mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *CRISPR-Associated Proteins/genetics ; Gene Expression Regulation ; Gene Regulatory Networks ; RNA, Messenger ; Mammals/genetics ; }, abstract = {Translational modulation based on RNA-binding proteins can be used to construct artificial gene circuits, but RNA-binding proteins capable of regulating translation efficiently and orthogonally remain scarce. Here we report CARTRIDGE (Cas-Responsive Translational Regulation Integratable into Diverse Gene control) to repurpose Cas proteins as translational modulators in mammalian cells. We demonstrate that a set of Cas proteins efficiently and orthogonally repress or activate the translation of designed mRNAs that contain a Cas-binding RNA motif in the 5'-UTR. By linking multiple Cas-mediated translational modulators, we designed and built artificial circuits like logic gates, cascades, and half-subtractor circuits. Moreover, we show that various CRISPR-related technologies like anti-CRISPR and split-Cas9 platforms could be similarly repurposed to control translation. Coupling Cas-mediated translational and transcriptional regulation enhanced the complexity of synthetic circuits built by only introducing a few additional elements. Collectively, CARTRIDGE has enormous potential as a versatile molecular toolkit for mammalian synthetic biology.}, }
@article {pmid37013669, year = {2023}, author = {Zhao, NN and Tian, X and Ma, F and Zhang, CY}, title = {CRISPR/Cas12a-enhanced single-molecule counting for sensitive detection of flap endonuclease 1 activity at the single-cell level.}, journal = {Chemical communications (Cambridge, England)}, volume = {59}, number = {33}, pages = {4939-4942}, doi = {10.1039/d3cc00642e}, pmid = {37013669}, issn = {1364-548X}, mesh = {*CRISPR-Cas Systems/genetics ; Flap Endonucleases ; Kinetics ; Biological Assay ; Coloring Agents ; *Biosensing Techniques ; }, abstract = {We develop a new fluorescent biosensor for flap endonuclease 1 (FEN1) assay based on CRISPR/Cas12-enhanced single-molecule counting. This biosensor is simple, selective, and sensitive with a detection limit of 2.325 × 10[-5] U and it is applicable for inhibitor screening, kinetic parameter analysis, and quantifying cellular FEN1 with single-cell sensitivity.}, }
@article {pmid36952025, year = {2023}, author = {Ma, X and Suo, T and Zhao, F and Shang, Z and Chen, Y and Wang, P and Li, B}, title = {Integrating CRISPR/Cas12a with strand displacement amplification for the ultrasensitive aptasensing of cadmium(II).}, journal = {Analytical and bioanalytical chemistry}, volume = {415}, number = {12}, pages = {2281-2289}, pmid = {36952025}, issn = {1618-2650}, mesh = {Humans ; CRISPR-Cas Systems ; Cadmium ; Agriculture ; DNA, Single-Stranded ; *Environmental Pollutants ; Oligonucleotides ; *Biosensing Techniques ; }, abstract = {Cadmium ion (Cd(II)) is a pernicious environmental pollutant that has been shown to contaminate agricultural lands, accumulate through the food chain, and seriously threaten human health. At present, Cd(II) monitoring is dependent on centralized instruments, necessitating the development of rapid and on-site detection platforms. Against this backdrop, the present study reports on the development of a fluorometric aptasensor designed to target Cd(II), which is achieved through the integration of strand displacement amplification (SDA) and CRISPR/Cas12a. In the absence of Cd(II), the aptamer initiates SDA, resulting in the generation of a profusion of ssDNA that activates Cas12a, leading to a substantial increase in fluorescence output. Conversely, the presence of Cd(II) curtails the SDA efficiency, culminating in a significant reduction in fluorescence output. The proposed approach has been demonstrated to enable the selective detection of Cd(II) at concentrations of 60 pM, with the performance of the aptasensor validated in real water and rice samples. The proposed platform based on aptamer-target interaction holds immense promise as a signal-amplified and precise method for the detection of Cd(II) and has the potential to transform current hazard detection practices in food samples.}, }
@article {pmid37077165, year = {2023}, author = {Liu, DN and Wu, HP and Zhou, GH}, title = {Research progress of visual detection in rapid on-site detection of pathogen nucleic acid.}, journal = {Yi chuan = Hereditas}, volume = {45}, number = {4}, pages = {306-323}, doi = {10.16288/j.yczz.22-323}, pmid = {37077165}, issn = {0253-9772}, abstract = {Nucleic acid detection is widely used in pathogen screening and detection due to its high sensitivity and specificity. With the increase of detection requirements and the development of amplification technology, nucleic acid detection methods are gradually developing towards simple, fast and low-cost. Quantitative polymerase chain reaction (qPCR), as the "gold standard" for nucleic acid detection, relies on expensive equipment and professional operators, which is not suitable for rapid on-site detection of pathogens. The visual detection method without relying on excitation light source or complex equipment can present the detection results in a more intuitive and portable way after combining with rapid and efficient amplification technology, which has the potential of point-of-care testing (POCT). This paper focuses on the reported application of amplification technology and CRISPR/Cas technology in visual detection and compares their advantages and disadvantages, so as to provide reference for POCT strategy based on pathogen nucleic acid.}, }
@article {pmid37075815, year = {2023}, author = {Farzanehpour, M and Miri, A and Ghorbani Alvanegh, A and Esmaeili Gouvarchinghaleh, H}, title = {Viral Vectors, Exosomes, and Vexosomes: Potential Armamentarium for Delivering CRISPR/Cas to Cancer Cells.}, journal = {Biochemical pharmacology}, volume = {}, number = {}, pages = {115555}, doi = {10.1016/j.bcp.2023.115555}, pmid = {37075815}, issn = {1873-2968}, abstract = {The underlying cause of cancer is genetic disruption, so gene editing technologies, particularly CRISPR/Cas systems can be used to go against cancer. The field of gene therapy has undergone many transitions over its 40-year history. Despite its many successes, it has also suffered many failures in the battle against malignancies, causing really adverse effects instead of therapeutic outcomes. At the tip of this double-edged sword are viral and non-viral-based vectors, which have profoundly transformed the way scientists and clinicians develop therapeutic platforms. Viruses such as lentivirus, adenovirus, and adeno-associated viruses are the most common viral vectors used for delivering the CRISPR/Cas system into human cells. In addition, among non-viral vectors, exosomes, especially tumor-derived exosomes (TDEs), have proven to be quite effective at delivering this gene editing tool. The combined use of viral vectors and exosomes, called vexosomes, seems to be a solution to overcoming the obstacles of both delivery systems.}, }
@article {pmid37071672, year = {2023}, author = {Bryant, WB and Yang, A and Griffin, SH and Zhang, W and Rafiq, AM and Han, W and Deak, F and Mills, MK and Long, X and Miano, JM}, title = {CRISPR-Cas9 Long-Read Sequencing for Mapping Transgenes in the Mouse Genome.}, journal = {The CRISPR journal}, volume = {6}, number = {2}, pages = {163-175}, doi = {10.1089/crispr.2022.0099}, pmid = {37071672}, issn = {2573-1602}, mesh = {Animals ; Mice ; *Gene Editing ; *CRISPR-Cas Systems/genetics ; Transgenes ; Genome/genetics ; Mice, Transgenic ; }, abstract = {Microinjected transgenes, both large and small, are known to insert randomly into the mouse genome. Traditional methods of mapping a transgene are challenging, thus complicating breeding strategies and accurate interpretation of phenotypes, particularly when a transgene disrupts critical coding or noncoding sequences. As the vast majority of transgenic mouse lines remain unmapped, we developed CRISPR-Cas9 Long-Read Sequencing (CRISPR-LRS) to ascertain transgene integration loci. This novel approach mapped a wide size range of transgenes and uncovered more complex transgene-induced host genome re-arrangements than previously appreciated. CRISPR-LRS offers a facile, informative approach to establish robust breeding practices and will enable researchers to study a gene without confounding genetic issues. Finally, CRISPR-LRS will find utility in rapidly and accurately interrogating gene/genome editing fidelity in experimental and clinical settings.}, }
@article {pmid37071671, year = {2023}, author = {Barrangou, R}, title = {Amplifying CRISPR: Next-Generation Diagnostics.}, journal = {The CRISPR journal}, volume = {6}, number = {2}, pages = {85}, doi = {10.1089/crispr.2023.0004.editorial}, pmid = {37071671}, issn = {2573-1602}, mesh = {*Gene Editing ; *CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; }, }
@article {pmid37071670, year = {2023}, author = {Panda, A and Suvakov, M and Mariani, J and Drucker, KL and Park, Y and Jang, Y and Kollmeyer, TM and Sarkar, G and Bae, T and Kim, JJ and Yoon, WH and Jenkins, RB and Vaccarino, FM and Abyzov, A}, title = {Clonally Selected Lines After CRISPR-Cas Editing Are Not Isogenic.}, journal = {The CRISPR journal}, volume = {6}, number = {2}, pages = {176-182}, doi = {10.1089/crispr.2022.0050}, pmid = {37071670}, issn = {2573-1602}, mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing ; Mutation ; DNA ; }, abstract = {The CRISPR-Cas9 system has enabled researchers to precisely modify/edit the sequence of a genome. A typical editing experiment consists of two steps: (1) editing cultured cells; (2) cell cloning and selection of clones with and without intended edit, presumed to be isogenic. The application of CRISPR-Cas9 system may result in off-target edits, whereas cloning will reveal culture-acquired mutations. We analyzed the extent of the former and the latter by whole genome sequencing in three experiments involving separate genomic loci and conducted by three independent laboratories. In all experiments we hardly found any off-target edits, whereas detecting hundreds to thousands of single nucleotide mutations unique to each clone after relatively short culture of 10-20 passages. Notably, clones also differed in copy number alterations (CNAs) that were several kb to several mb in size and represented the largest source of genomic divergence among clones. We suggest that screening of clones for mutations and CNAs acquired in culture is a necessary step to allow correct interpretation of DNA editing experiments. Furthermore, since culture associated mutations are inevitable, we propose that experiments involving derivation of clonal lines should compare a mix of multiple unedited lines and a mix of multiple edited lines.}, }
@article {pmid37071669, year = {2023}, author = {Cress, B and Barrangou, R}, title = {Special Issue: Manipulating the Microbiome with CRISPR.}, journal = {The CRISPR journal}, volume = {6}, number = {2}, pages = {86}, doi = {10.1089/crispr.2023.0005.cfp}, pmid = {37071669}, issn = {2573-1602}, mesh = {*Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Editing ; CRISPR-Cas Systems/genetics ; *Microbiota/genetics ; }, }
@article {pmid37069118, year = {2023}, author = {Maloshenok, LG and Abushinova, GA and Ryazanova, AY and Bruskin, SA and Zherdeva, VV}, title = {Visualizing the Nucleome Using the CRISPR-Cas9 System: From in vitro to in vivo.}, journal = {Biochemistry. Biokhimiia}, volume = {88}, number = {Suppl 1}, pages = {S123-S149}, pmid = {37069118}, issn = {1608-3040}, mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; Genome ; Genomics ; Microscopy, Fluorescence ; }, abstract = {One of the latest methods in modern molecular biology is labeling genomic loci in living cells using fluorescently labeled Cas protein. The NIH Foundation has made the mapping of the 4D nucleome (the three-dimensional nucleome on a timescale) a priority in the studies aimed to improve our understanding of chromatin organization. Fluorescent methods based on CRISPR-Cas are a significant step forward in visualization of genomic loci in living cells. This approach can be used for studying epigenetics, cell cycle, cellular response to external stimuli, rearrangements during malignant cell transformation, such as chromosomal translocations or damage, as well as for genome editing. In this review, we focused on the application of CRISPR-Cas fluorescence technologies as components of multimodal imaging methods for in vivo mapping of chromosomal loci, in particular, attribution of fluorescence signal to morphological and anatomical structures in a living organism. The review discusses the approaches to the highly sensitive, high-precision labeling of CRISPR-Cas components, delivery of genetically engineered constructs into cells and tissues, and promising methods for molecular imaging.}, }
@article {pmid37062563, year = {2023}, author = {Zhu, R and Jiang, H and Li, C and Li, Y and Peng, M and Wang, J and Wu, Q and Yan, C and Bo, Q and Wang, J and Shen, C and Qin, P}, title = {CRISPR/Cas9-based point-of-care lateral flow biosensor with improved performance for rapid and robust detection of Mycoplasma pneumonia.}, journal = {Analytica chimica acta}, volume = {1257}, number = {}, pages = {341175}, doi = {10.1016/j.aca.2023.341175}, pmid = {37062563}, issn = {1873-4324}, mesh = {Humans ; CRISPR-Cas Systems ; *Pneumonia, Mycoplasma/diagnosis/genetics ; Point-of-Care Systems ; *Biosensing Techniques/methods ; }, abstract = {Screening of acute respiratory infections causes serious challenges in urgent point-of-care scenarios where conventional methods are impractical and alternative techniques suffer from low accuracy, poor robustness, and reliance on sophisticated instruments. As an improvement to this paradigm, we report a point-of-care lateral flow biosensor (LFB) based on the recognition property of clustered regularly interspaced short palindromic repeats (CRISPR)/associated protein 9 (Cas9) and apply it to the detection of Mycoplasma pneumoniae (M. pneumoniae). The designed biosensor employs CRISPR/Cas9 for secondary recognition after preamplification of target gene using specific primer set, avoiding false positives caused by nontarget factors. The high amplification efficiency and low applicable temperatures of recombinase polymerase amplification brings the detection limit of the biosensor to 3 copies even at a preamplification temperature of 25 °C. Its practical application is further demonstrated with 100% accuracy by testing with 43 M. pneumoniae-infected specimens and 80 uninfected specimens. Additionally, the entire detection, including pretreatment, preamplification, CRISPR/Cas9 recognition, and visual analysis, can be completed in 30 min. Featured with the combination of CRISPR/Cas9 and LFB, the biosensor we developed herein ensures excellent convenience, accuracy, and robustness, which endows promising point-of-care screening potential for infectious pathogens.}, }
@article {pmid36944129, year = {2023}, author = {Opstelten, R and Freen-van Heeren, JJ}, title = {CLASH of the Titans: How CAR-T Cells Can Triumph Over Tumors.}, journal = {The CRISPR journal}, volume = {6}, number = {2}, pages = {87-89}, doi = {10.1089/crispr.2023.0003}, pmid = {36944129}, issn = {2573-1602}, mesh = {Humans ; *CRISPR-Cas Systems ; Gene Editing ; *Neoplasms/genetics/therapy ; T-Lymphocytes ; }, }
@article {pmid36944123, year = {2023}, author = {Morales-Moreno, MD and Valdés-Galindo, EG and Reza, MM and Fiordelisio, T and Peon, J and Hernandez-Garcia, A}, title = {Multiplex gRNAs Synergically Enhance Detection of SARS-CoV-2 by CRISPR-Cas12a.}, journal = {The CRISPR journal}, volume = {6}, number = {2}, pages = {116-126}, doi = {10.1089/crispr.2022.0074}, pmid = {36944123}, issn = {2573-1602}, mesh = {Humans ; *SARS-CoV-2/genetics ; *COVID-19/diagnosis ; CRISPR-Cas Systems/genetics ; RNA, Viral/genetics ; Gene Editing ; RNA, Guide, Kinetoplastida/genetics ; }, abstract = {Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) diagnostic methods have a large potential to effectively detect SARS-CoV-2 with sensitivity and specificity nearing 100%, comparable to quantitative polymerase chain reaction. Yet, there is room for improvement. Commonly, one guide CRISPR RNA (gRNA) is used to detect the virus DNA and activate Cas collateral activity, which cleaves a reporter probe. In this study, we demonstrated that using 2-3 gRNAs in parallel can create a synergistic effect, resulting in a 4.5 × faster cleaving rate of the probe and increased sensitivity compared to using individual gRNAs. The synergy is due to the simultaneous activation of CRISPR-Cas12a and the improved performance of each gRNA. This approach was able to detect as few as 10 viral copies of the N-gene of SARS-CoV-2 RNA after a preamplification step using reverse transcription loop-mediated isothermal amplification. The method was able to accurately detect 100% of positive and negative clinical samples in ∼25 min using a fluorescence plate reader and ∼45 min with lateral flow strips.}, }
@article {pmid36912819, year = {2023}, author = {Cullot, G and Amintas, S and Karembé, L and Prouzet-Mauléon, V and Rébillard, J and Boureau, L and Cappellen, D and Bedel, A and Moreau-Gaudry, F and Dulucq, S and Dabernat, S and Turcq, B}, title = {Specific High-Sensitivity Enzymatic Reporter UnLOCKing-Mediated Detection of Oncogenic BCR::ABL1 and EGFR Rearrangements.}, journal = {The CRISPR journal}, volume = {6}, number = {2}, pages = {140-151}, doi = {10.1089/crispr.2022.0070}, pmid = {36912819}, issn = {2573-1602}, mesh = {Humans ; Fusion Proteins, bcr-abl/genetics ; *Carcinoma, Non-Small-Cell Lung/diagnosis/genetics ; *Lung Neoplasms/diagnosis/genetics ; CRISPR-Cas Systems ; Gene Editing ; *Leukemia, Myelogenous, Chronic, BCR-ABL Positive/diagnosis/genetics ; ErbB Receptors/genetics ; }, abstract = {Advances in molecular medicine have placed nucleic acid detection methods at the center of an increasing number of clinical applications. Polymerase chain reaction (PCR)-based diagnostics have been widely adopted for their versatility, specificity, and sensitivity. However, recently reported clustered regularly interspaced short palindromic repeats-based methods have demonstrated equivalent to superior performance, with increased portability and reduced processing time and cost. In this study, we applied Specific High-Sensitivity Enzymatic Reporter UnLOCKing (SHERLOCK) technology to the detection of oncogenic rearrangements. We implemented SHERLOCK for the detection of BCR::ABL1 mRNA, a hallmark of chronic myeloid leukemia (CML), and EGFR DNA oncogenic alleles, frequently detected in glioblastoma and non-small cell lung cancer (NSCLC). SHERLOCK enabled rapid, sensitive, and variant-specific detection of BCR::ABL1 and EGFR alterations. Compared with the gold-standard PCR-based methods currently used in clinic, SHERLOCK achieved equivalent to greater sensitivity, suggesting it could be a new tool in CML and NSCLC, to detect low level of molecular residual disease.}, }
@article {pmid36683240, year = {2023}, author = {Lv, Y and Sun, Y and Zhou, Y and Khan, IM and Niazi, S and Yue, L and Zhang, Y and Wang, Z}, title = {Cascade DNA Circuits Mediated CRISPR-Cas12a Fluorescent Aptasensor based on Multifunctional Fe3 O4 @hollow-TiO2 @MoS2 Nanochains for Tetracycline Determination.}, journal = {Small (Weinheim an der Bergstrasse, Germany)}, volume = {19}, number = {16}, pages = {e2206105}, doi = {10.1002/smll.202206105}, pmid = {36683240}, issn = {1613-6829}, mesh = {*CRISPR-Cas Systems/genetics ; Molybdenum ; Tetracycline ; Anti-Bacterial Agents ; DNA ; Oligonucleotides ; Coloring Agents ; *Biosensing Techniques ; }, abstract = {Herein, for the first time, the CRISPR-Cas12a system is combined with aptamer, cascaded dynamic DNA network circuits, and Fe3 O4 @hollow-TiO2 @MoS2 nanochains (Fe3 O4 @h-TiO2 @MoS2 NCs) to construct an efficient sensing platform for tetracycline (TC) analysis. In this strategy, specific recognition of the target is transduced and amplified into H1-H2 duplexes containing the specific sequence of Cas12a-crRNA through an aptamer recognition module and the dual amplification dynamic DNA network. Subsequently, the obtained activated Cas12a protein non-specifically cleaves the adjacent reporter gene ssDNA-FAM to dissociate the FAM molecule from the quencher Fe3 O4 @h-TiO2 @MoS2 NCs, resulting in the recovery of the fluorescence signal and further signal amplification. Particularly, the synthesized multifunctional Fe3 O4 @h-TiO2 @MoS2 NCs composites also exhibit superb magnetic separability and photocatalytic degradation ability. Under optimal conditions, the aptasensor displays a distinct linear relationship with the logarithm of TC concentration, and the limit of detection is as low as 0.384 pg mL[-1] . Furthermore, the results of spiked recovery confirm the viability of the proposed aptasensor for TC quantification in real samples. This study extends the application of the CRISPR-Cas12a system in the field of analytical sensing and contributes new insights into the exploration of reliable tools for monitoring and treating hazards in food and environment.}, }
@article {pmid36637878, year = {2023}, author = {Kohabir, KAV and Nooi, LO and Brink, A and Brakenhoff, RH and Sistermans, EA and Wolthuis, RMF}, title = {In Vitro CRISPR-Cas12a-Based Detection of Cancer-Associated TP53 Hotspot Mutations Beyond the crRNA Seed Region.}, journal = {The CRISPR journal}, volume = {6}, number = {2}, pages = {127-139}, doi = {10.1089/crispr.2022.0077}, pmid = {36637878}, issn = {2573-1602}, mesh = {Humans ; CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems ; Gene Editing ; Bacterial Proteins/genetics ; *CRISPR-Associated Proteins/genetics ; DNA/genetics ; Mutation ; Endonucleases/genetics ; *Neoplasms/diagnosis/genetics ; Tumor Suppressor Protein p53/genetics ; }, abstract = {Cost-effective and time-efficient detection of oncogenic mutations supports improved presymptomatic cancer diagnostics and post-treatment disease monitoring. Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12a is an RNA-guided endonuclease that, upon protospacer adjacent motif (PAM)-dependent recognition of target DNA in cis, exhibits indiscriminate ssDNase activity in trans, which can be harnessed for diagnostics. TP53, one of the most frequently mutated tumor suppressor genes in cancer, displays recurring point mutations at so-called "hotspots." In this study, we optimized Cas12a-based assay conditions for in vitro detection of six TP53 hotspot mutations at the codon for p.R273, located outside the Cas12a seed region, and evaluated the specificities of four commercial Cas12a variants. We found that nonengineered LbCas12a significantly outperformed the other tested nucleases specifically in distinguishing mutant p.R273 codons in synthetic DNA, mock cell-free DNA, and tissue biopsies, despite the suboptimal PAM-distal positioning of the corresponding mutations. Future clinical Cas12a-based applications may include point-of-care tumor analysis, cost-effective mutation screening, and improved monitoring of individual cancer patients.}, }
@article {pmid36367987, year = {2023}, author = {Patchsung, M and Homchan, A and Aphicho, K and Suraritdechachai, S and Wanitchanon, T and Pattama, A and Sappakhaw, K and Meesawat, P and Wongsatit, T and Athipanyasilp, A and Jantarug, K and Athipanyasilp, N and Buahom, J and Visanpattanasin, S and Niljianskul, N and Chaiyen, P and Tinikul, R and Wichukchinda, N and Mahasirimongkol, S and Sirijatuphat, R and Angkasekwinai, N and Crone, MA and Freemont, PS and Joung, J and Ladha, A and Abudayyeh, O and Gootenberg, J and Zhang, F and Chewapreecha, C and Chanarat, S and Horthongkham, N and Pakotiprapha, D and Uttamapinant, C}, title = {A Multiplexed Cas13-Based Assay with Point-of-Care Attributes for Simultaneous COVID-19 Diagnosis and Variant Surveillance.}, journal = {The CRISPR journal}, volume = {6}, number = {2}, pages = {99-115}, pmid = {36367987}, issn = {2573-1602}, support = {216457/WT_/Wellcome Trust/United Kingdom ; BB/M025632/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; }, mesh = {Humans ; *SARS-CoV-2/genetics ; *COVID-19/diagnosis ; COVID-19 Testing ; Pandemics ; Point-of-Care Systems ; CRISPR-Cas Systems/genetics ; Gene Editing ; }, abstract = {Point-of-care (POC) nucleic acid detection technologies are poised to aid gold-standard technologies in controlling the COVID-19 pandemic, yet shortcomings in the capability to perform critically needed complex detection-such as multiplexed detection for viral variant surveillance-may limit their widespread adoption. Herein, we developed a robust multiplexed clustered regularly interspaced short palindromic repeats (CRISPR)-based detection using LwaCas13a and PsmCas13b to simultaneously diagnose severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and pinpoint the causative SARS-CoV-2 variant of concern (VOC)-including globally dominant VOCs Delta (B.1.617.2) and Omicron (B.1.1.529)-all the while maintaining high levels of accuracy upon the detection of multiple SARS-CoV-2 gene targets. The platform has several attributes suitable for POC use: premixed, freeze-dried reagents for easy use and storage; convenient direct-to-eye or smartphone-based readouts; and a one-pot variant of the multiplexed detection. To reduce reliance on proprietary reagents and enable sustainable use of such a technology in low- and middle-income countries, we locally produced and formulated our own recombinase polymerase amplification reaction and demonstrated its equivalent efficiency to commercial counterparts. Our tool-CRISPR-based detection for simultaneous COVID-19 diagnosis and variant surveillance that can be locally manufactured-may enable sustainable use of CRISPR diagnostics technologies for COVID-19 and other diseases in POC settings.}, }
@article {pmid36258038, year = {2023}, author = {Mondal, G and VanLith, CJ and Nicolas, CT and Thompson, WS and Cao, WS and Hillin, L and Haugo, BJ and Brien, DRO and Kocher, JP and Kaiser, RA and Lillegard, JB}, title = {Activation of homology-directed DNA repair plays key role in CRISPR-mediated genome correction.}, journal = {Gene therapy}, volume = {30}, number = {3-4}, pages = {386-397}, pmid = {36258038}, issn = {1476-5462}, mesh = {Animals ; Mice ; *CRISPR-Cas Systems ; *Gene Editing ; Recombinational DNA Repair ; DNA Breaks, Double-Stranded ; DNA ; DNA Repair ; }, abstract = {Gene editing for the cure of inborn errors of metabolism (IEMs) has been limited by inefficiency of adult hepatocyte targeting. Here, we demonstrate that in utero CRISPR/Cas9-mediated gene editing in a mouse model of hereditary tyrosinemia type 1 provides stable cure of the disease. Following this, we performed an extensive gene expression analysis to explore the inherent characteristics of fetal/neonatal hepatocytes that make them more susceptible to efficient gene editing than adult hepatocytes. We showed that fetal and neonatal livers are comprised of proliferative hepatocytes with abundant expression of genes involved in homology-directed repair (HDR) of DNA double-strand breaks (DSBs), key for efficient gene editing by CRISPR/Cas9. We demonstrated the same is true of hepatocytes after undergoing a regenerative stimulus (partial hepatectomy), where post-hepatectomy cells show a higher efficiency of HDR and correction. Specifically, we demonstrated that HDR-related genome correction is most effective in the replicative phase, or S-phase, of an actively proliferating cell. In conclusion, this study shows that taking advantage of or triggering cell proliferation, specifically DNA replication in S-phase, may serve as an important tool to improve efficiency of CRISPR/Cas9-mediated genome editing in the liver and provide a curative therapy for IEMs in both children and adults.}, }
@article {pmid37073287, year = {2021}, author = {Lu, J and Fang, W and Huang, J and Li, S}, title = {The application of genome editing technology in fish.}, journal = {Marine life science &amp; technology}, volume = {3}, number = {3}, pages = {326-346}, pmid = {37073287}, issn = {2662-1746}, abstract = {The advent and development of genome editing technology has opened up the possibility of directly targeting and modifying genomic sequences in the field of life sciences with rapid developments occurring in the last decade. As a powerful tool to decipher genome data at the molecular biology level, genome editing technology has made important contributions to elucidating many biological problems. Currently, the three most widely used genome editing technologies include: zinc finger nucleases (ZFN), transcription activator like effector nucleases (TALEN), and clustered regularly interspaced short palindromic repeats (CRISPR). Researchers are still striving to create simpler, more efficient, and accurate techniques, such as engineered base editors and new CRISPR/Cas systems, to improve editing efficiency and reduce off-target rate, as well as a near-PAMless SpCas9 variants to expand the scope of genome editing. As one of the important animal protein sources, fish has significant economic value in aquaculture. In addition, fish is indispensable for research as it serves as the evolutionary link between invertebrates and higher vertebrates. Consequently, genome editing technologies were applied extensively in various fish species for basic functional studies as well as applied research in aquaculture. In this review, we focus on the application of genome editing technologies in fish species detailing growth, gender, and pigmentation traits. In addition, we have focused on the construction of a zebrafish (Danio rerio) disease model and high-throughput screening of functional genes. Finally, we provide some of the future perspectives of this technology.}, }
@article {pmid37077216, year = {2021}, author = {Symeonidi, E and Regalado, J and Schwab, R and Weigel, D}, title = {CRISPR-finder: A high throughput and cost-effective method to identify successfully edited Arabidopsis thaliana individuals.}, journal = {Quantitative plant biology}, volume = {2}, number = {}, pages = {e1}, pmid = {37077216}, issn = {2632-8828}, abstract = {Genome editing with the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR associated protein) system allows mutagenesis of a targeted region of the genome using a Cas endonuclease and an artificial guide RNA. Both because of variable efficiency with which such mutations arise and because the repair process produces a spectrum of mutations, one needs to ascertain the genome sequence at the targeted locus for many individuals that have been subjected to mutagenesis. We provide a complete protocol for the generation of amplicons up until the identification of the exact mutations in the targeted region. CRISPR-finder can be used to process thousands of individuals in a single sequencing run. We successfully identified an ISOCHORISMATE SYNTHASE 1 mutant line in which the production of salicylic acid was impaired compared to the wild type, as expected. These features establish CRISPR-finder as a high-throughput, cost-effective and efficient genotyping method of individuals whose genomes have been targeted using the CRISPR/Cas9 system.}, }
@article {pmid37059915, year = {2023}, author = {Lear, SK and Lopez, SC and González-Delgado, A and Bhattarai-Kline, S and Shipman, SL}, title = {Temporally resolved transcriptional recording in E. coli DNA using a Retro-Cascorder.}, journal = {Nature protocols}, volume = {}, number = {}, pages = {}, pmid = {37059915}, issn = {1750-2799}, abstract = {Biological signals occur over time in living cells. Yet most current approaches to interrogate biology, particularly gene expression, use destructive techniques that quantify signals only at a single point in time. A recent technological advance, termed the Retro-Cascorder, overcomes this limitation by molecularly logging a record of gene expression events in a temporally organized genomic ledger. The Retro-Cascorder works by converting a transcriptional event into a DNA barcode using a retron reverse transcriptase and then storing that event in a unidirectionally expanding clustered regularly interspaced short palindromic repeats (CRISPR) array via acquisition by CRISPR-Cas integrases. This CRISPR array-based ledger of gene expression can be retrieved at a later point in time by sequencing. Here we describe an implementation of the Retro-Cascorder in which the relative timing of transcriptional events from multiple promoters of interest is recorded chronologically in Escherichia coli populations over multiple days. We detail the molecular components required for this technology, provide a step-by-step guide to generate the recording and retrieve the data by Illumina sequencing, and give instructions for how to use custom software to infer the relative transcriptional timing from the sequencing data. The example recording is generated in 2 d, preparation of sequencing libraries and sequencing can be accomplished in 2-3 d, and analysis of data takes up to several hours. This protocol can be implemented by someone familiar with basic bacterial culture, molecular biology and bioinformatics. Analysis can be minimally run on a personal computer.}, }
@article {pmid37059525, year = {2023}, author = {Schilling, C and Klau, LJ and Aachmann, FL and Rühmann, B and Schmid, J and Sieber, V}, title = {CRISPR-Cas9 driven structural elucidation of the heteroexopolysaccharides from Paenibacillus polymyxa DSM 365.}, journal = {Carbohydrate polymers}, volume = {312}, number = {}, pages = {120763}, doi = {10.1016/j.carbpol.2023.120763}, pmid = {37059525}, issn = {1879-1344}, mesh = {Humans ; Carbohydrate Sequence ; *Paenibacillus polymyxa/genetics ; CRISPR-Cas Systems ; Polysaccharides/chemistry ; Magnetic Resonance Spectroscopy ; }, abstract = {Paenibacillus polymyxa is a Gram-positive soil bacterium known for producing a wide range of exopolysaccharides. However, due to the biopolymer's complexity, structural elucidation has so far been inconclusive. Combinatorial knock-outs of glycosyltransferases were generated in order to separate distinct polysaccharides produced by P. polymyxa. Using a complementary analytical approach consisting of carbohydrate fingerprints, sequence analysis, methylation analysis as well as NMR spectroscopy, the structure of the repeating units of two additional heteroexopolysaccharides termed paenan I and paenan III were elucidated. Results for paenan I identified a trisaccharide backbone consisting of 1➔4-β-d-Glc, 1➔4-β-d-Man and a 1,3,4-branching β-d-Gal residue with a sidechain comprising of a terminal β-d-Gal[3,4-Pyr] and 1➔3-β-d-Glc. For paenan III, results indicated a backbone consisting of 1➔3-β-d-Glc, 1,3,4-linked α-d-Man and 1,3,4-linked α-d-GlcA. NMR analysis indicated monomeric β-d-Glc and α-d-Man sidechains for the branching Man and GlcA residues respectively.}, }
@article {pmid36424489, year = {2023}, author = {Yarnall, MTN and Ioannidi, EI and Schmitt-Ulms, C and Krajeski, RN and Lim, J and Villiger, L and Zhou, W and Jiang, K and Garushyants, SK and Roberts, N and Zhang, L and Vakulskas, CA and Walker, JA and Kadina, AP and Zepeda, AE and Holden, K and Ma, H and Xie, J and Gao, G and Foquet, L and Bial, G and Donnelly, SK and Miyata, Y and Radiloff, DR and Henderson, JM and Ujita, A and Abudayyeh, OO and Gootenberg, JS}, title = {Drag-and-drop genome insertion of large sequences without double-strand DNA cleavage using CRISPR-directed integrases.}, journal = {Nature biotechnology}, volume = {41}, number = {4}, pages = {500-512}, pmid = {36424489}, issn = {1546-1696}, mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Integrases ; DNA Cleavage ; Gene Editing ; DNA/genetics ; DNA End-Joining Repair/genetics ; }, abstract = {Programmable genome integration of large, diverse DNA cargo without DNA repair of exposed DNA double-strand breaks remains an unsolved challenge in genome editing. We present programmable addition via site-specific targeting elements (PASTE), which uses a CRISPR-Cas9 nickase fused to both a reverse transcriptase and serine integrase for targeted genomic recruitment and integration of desired payloads. We demonstrate integration of sequences as large as ~36 kilobases at multiple genomic loci across three human cell lines, primary T cells and non-dividing primary human hepatocytes. To augment PASTE, we discovered 25,614 serine integrases and cognate attachment sites from metagenomes and engineered orthologs with higher activity and shorter recognition sequences for efficient programmable integration. PASTE has editing efficiencies similar to or exceeding those of homology-directed repair and non-homologous end joining-based methods, with activity in non-dividing cells and in vivo with fewer detectable off-target events. PASTE expands the capabilities of genome editing by allowing large, multiplexed gene insertion without reliance on DNA repair pathways.}, }
@article {pmid36008610, year = {2023}, author = {Shy, BR and Vykunta, VS and Ha, A and Talbot, A and Roth, TL and Nguyen, DN and Pfeifer, WG and Chen, YY and Blaeschke, F and Shifrut, E and Vedova, S and Mamedov, MR and Chung, JJ and Li, H and Yu, R and Wu, D and Wolf, J and Martin, TG and Castro, CE and Ye, L and Esensten, JH and Eyquem, J and Marson, A}, title = {High-yield genome engineering in primary cells using a hybrid ssDNA repair template and small-molecule cocktails.}, journal = {Nature biotechnology}, volume = {41}, number = {4}, pages = {521-531}, pmid = {36008610}, issn = {1546-1696}, support = {K08 AI153767/AI/NIAID NIH HHS/United States ; L30 AI140341/AI/NIAID NIH HHS/United States ; L40 AI140341/AI/NIAID NIH HHS/United States ; P01 AI138962/AI/NIAID NIH HHS/United States ; }, mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *DNA, Single-Stranded/genetics ; Genome ; Recombinational DNA Repair ; Mutation ; DNA ; Gene Editing ; DNA End-Joining Repair ; }, abstract = {Enhancing CRISPR-mediated site-specific transgene insertion efficiency by homology-directed repair (HDR) using high concentrations of double-stranded DNA (dsDNA) with Cas9 target sequences (CTSs) can be toxic to primary cells. Here, we develop single-stranded DNA (ssDNA) HDR templates (HDRTs) incorporating CTSs with reduced toxicity that boost knock-in efficiency and yield by an average of around two- to threefold relative to dsDNA CTSs. Using small-molecule combinations that enhance HDR, we could further increase knock-in efficiencies by an additional roughly two- to threefold on average. Our method works across a variety of target loci, knock-in constructs and primary human cell types, reaching HDR efficiencies of >80-90%. We demonstrate application of this approach for both pathogenic gene variant modeling and gene-replacement strategies for IL2RA and CTLA4 mutations associated with Mendelian disorders. Finally, we develop a good manufacturing practice (GMP)-compatible process for nonviral chimeric antigen receptor-T cell manufacturing, with knock-in efficiencies (46-62%) and yields (>1.5 × 10[9] modified cells) exceeding those of conventional approaches.}, }
@article {pmid37059363, year = {2023}, author = {Kattner, AA}, title = {When it doesn't run in the blood(vessels) - events involved in vascular disorders.}, journal = {Biomedical journal}, volume = {}, number = {}, pages = {}, doi = {10.1016/j.bj.2023.03.004}, pmid = {37059363}, issn = {2320-2890}, abstract = {In the current issue of the Biomedical Journal the underlying pathology of hemodynamic compromise in acute small subcortical infarction are elucidated. A follow-up study in patients with childhood Kawasaki disease is presented, as well as an insight into the gradually decreasing antigen expression in cases of acute myeloid leukemia. Furthermore this issue provides an exciting update concerning COVID-19 and the use of CRISPR-Cas, a review about computational approaches in the research of kidney stone formation, factors connected to central precocious puberty, and why a rock star of paleogenetics recently received a Nobel Prize. Additionally, this issue contains an article proposing the repurposing of the lung cancer drug Capmatinib, a study of how the gut microbiome develops in neonates, an impulse about the role of the transmembrane protein TMED3 in esophageal carcinoma, and the revelation about how competing endogenous RNA influences ischemic stroke. Lastly, genetic reasons for male infertility are discussed, as well as the relation between non-alcoholic fatty liver disease and chronic kidney disease.}, }
@article {pmid37058530, year = {2023}, author = {Schelling, MA and Nguyen, GT and Sashital, DG}, title = {CRISPR-Cas effector specificity and cleavage site determine phage escape outcomes.}, journal = {PLoS biology}, volume = {21}, number = {4}, pages = {e3002065}, doi = {10.1371/journal.pbio.3002065}, pmid = {37058530}, issn = {1545-7885}, abstract = {CRISPR-mediated interference relies on complementarity between a guiding CRISPR RNA (crRNA) and target nucleic acids to provide defense against bacteriophage. Phages escape CRISPR-based immunity mainly through mutations in the protospacer adjacent motif (PAM) and seed regions. However, previous specificity studies of Cas effectors, including the class 2 endonuclease Cas12a, have revealed a high degree of tolerance of single mismatches. The effect of this mismatch tolerance has not been extensively studied in the context of phage defense. Here, we tested defense against lambda phage provided by Cas12a-crRNAs containing preexisting mismatches against the genomic targets in phage DNA. We find that most preexisting crRNA mismatches lead to phage escape, regardless of whether the mismatches ablate Cas12a cleavage in vitro. We used high-throughput sequencing to examine the target regions of phage genomes following CRISPR challenge. Mismatches at all locations in the target accelerated emergence of mutant phage, including mismatches that greatly slowed cleavage in vitro. Unexpectedly, our results reveal that a preexisting mismatch in the PAM-distal region results in selection of mutations in the PAM-distal region of the target. In vitro cleavage and phage competition assays show that dual PAM-distal mismatches are significantly more deleterious than combinations of seed and PAM-distal mismatches, resulting in this selection. However, similar experiments with Cas9 did not result in emergence of PAM-distal mismatches, suggesting that cut-site location and subsequent DNA repair may influence the location of escape mutations within target regions. Expression of multiple mismatched crRNAs prevented new mutations from arising in multiple targeted locations, allowing Cas12a mismatch tolerance to provide stronger and longer-term protection. These results demonstrate that Cas effector mismatch tolerance, existing target mismatches, and cleavage site strongly influence phage evolution.}, }
@article {pmid37052300, year = {2023}, author = {McBride, TM and Cameron, SC and Fineran, PC and Fagerlund, RD}, title = {The biology and type I/III hybrid nature of type I-D CRISPR-Cas systems.}, journal = {The Biochemical journal}, volume = {480}, number = {7}, pages = {471-488}, doi = {10.1042/BCJ20220073}, pmid = {37052300}, issn = {1470-8728}, mesh = {*CRISPR-Cas Systems ; *CRISPR-Associated Proteins/genetics ; Biology ; }, abstract = {Prokaryotes have adaptive defence mechanisms that protect them from mobile genetic elements and viral infection. One defence mechanism is called CRISPR-Cas (clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins). There are six different types of CRISPR-Cas systems and multiple subtypes that vary in composition and mode of action. Type I and III CRISPR-Cas systems utilise multi-protein complexes, which differ in structure, nucleic acid binding and cleaving preference. The type I-D system is a chimera of type I and III systems. Recently, there has been a burst of research on the type I-D CRISPR-Cas system. Here, we review the mechanism, evolution and biotechnological applications of the type I-D CRISPR-Cas system.}, }
@article {pmid36628413, year = {2023}, author = {Kan, J and Cai, Y and Cheng, C and Chen, S and Jiang, C and He, Z and Yang, P}, title = {CRISPR/Cas9-guided knockout of eIF4E improves Wheat yellow mosaic virus resistance without yield penalty.}, journal = {Plant biotechnology journal}, volume = {21}, number = {5}, pages = {893-895}, pmid = {36628413}, issn = {1467-7652}, mesh = {CRISPR-Cas Systems/genetics ; Triticum/genetics/metabolism ; Eukaryotic Initiation Factor-4E/genetics/metabolism ; *Potyviridae ; *Mosaic Viruses/metabolism ; *Hordeum/metabolism ; }, }
@article {pmid36591868, year = {2023}, author = {Li, Y and Wu, P and Zhu, M and Liang, M and Zhang, L and Zong, Y and Wan, M}, title = {High-Performance Delivery of a CRISPR Interference System via Lipid-Polymer Hybrid Nanoparticles Combined with Ultrasound-Mediated Microbubble Destruction for Tumor-Specific Gene Repression.}, journal = {Advanced healthcare materials}, volume = {12}, number = {10}, pages = {e2203082}, doi = {10.1002/adhm.202203082}, pmid = {36591868}, issn = {2192-2659}, mesh = {Humans ; CRISPR-Cas Systems ; Polymers ; Clustered Regularly Interspaced Short Palindromic Repeats ; Microbubbles ; *Neoplasms/genetics ; *Nanoparticles ; Lipids ; *MicroRNAs ; }, abstract = {The dCas9-based CRISPR interference (CRISPRi) system efficiently silences genes without causing detectable off-target activity, thus showing great potential for the treatment of cancer at the transcriptional level. However, due to the large size of the commonly used CRISPRi system, effective delivery of the system has been a challenge that hinders its application in the clinic. Herein, a combination of pH-responsive lipid-polymer hybrid nanoparticles (PLPNs) and ultrasound-mediated microbubble destruction (UMMD) is used for the delivery of the CRISPRi system. The core-shell structure of PLPNs can effectively be loaded with the CRISPRi plasmid, and increases the time spent in the circulating in vivo, and "actively target" cancer cells. Moreover, the combination of PLPNs with UMMD achieves a higher cellular uptake of the CRISPRi plasmid in vitro and retention in vivo. Furthermore, when PLPNs loaded with a CRISPRi plasmid that targets microRNA-10b (miR-10b) are used in combination with UMMD, it results in the effective repression of miR-10b in breast cancer, simultaneous disturbance of multiple cell migration and invasion-related signaling pathways, and a significant inhibition of lung metastasis. Thus, the established system presents a versatile, highly efficient, and safe strategy for delivery of the CRISPRi system both in vitro and in vivo.}, }
@article {pmid37052221, year = {2023}, author = {Wang, D and Wang, X and Ye, F and Zou, J and Qu, J and Jiang, X}, title = {An Integrated Amplification-Free Digital CRISPR/Cas-Assisted Assay for Single Molecule Detection of RNA.}, journal = {ACS nano}, volume = {}, number = {}, pages = {}, doi = {10.1021/acsnano.2c10143}, pmid = {37052221}, issn = {1936-086X}, abstract = {Conventional nucleic acid detection technologies usually rely on amplification to improve sensitivity, which has drawbacks, such as amplification bias, complicated operation, high requirements for complex instruments, and aerosol pollution. To address these concerns, we developed an integrated assay for the enrichment and single molecule digital detection of nucleic acid based on a CRISPR/Cas13a and microwell array. In our design, magnetic beads capture and concentrate the target from a large volume of sample, which is 100 times larger than reported earlier. The target-induced CRISPR/Cas13a cutting reaction was then dispersed and limited to a million individual femtoliter-sized microwells, thereby enhancing the local signal intensity to achieve single-molecule detection. The limit of this assay for amplification-free detection of SARS-CoV-2 is 2 aM. The implementation of this study will establish a "sample-in-answer-out" single-RNA detection technology without amplification and improve the sensitivity and specificity while shortening the detection time. This research has broad prospects in clinical application.}, }
@article {pmid36988873, year = {2023}, author = {Sinclair, F and Begum, AA and Dai, CC and Toth, I and Moyle, PM}, title = {Recent advances in the delivery and applications of nonviral CRISPR/Cas9 gene editing.}, journal = {Drug delivery and translational research}, volume = {13}, number = {5}, pages = {1500-1519}, pmid = {36988873}, issn = {2190-3948}, mesh = {*Gene Editing ; *CRISPR-Cas Systems ; Genetic Therapy ; Gene Transfer Techniques ; Genetic Vectors ; }, abstract = {The CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 genome editing system has been a major technological breakthrough that has brought revolutionary changes to genome editing for therapeutic and diagnostic purposes and precision medicine. With the advent of the CRISPR/Cas9 system, one of the critical limiting factors has been the safe and efficient delivery of this system to cells or tissues of interest. Several approaches have been investigated to find delivery systems that can attain tissue-targeted delivery, lowering the chances of off-target editing. While viral vectors have shown promise for in vitro, in vivo and ex vivo delivery of CRISPR/Cas9, their further clinical applications have been restricted due to shortcomings including limited cargo packaging capacity, difficulties with large-scale production, immunogenicity and insertional mutagenesis. Rapid progress in nonviral delivery vectors, including the use of lipid, polymer, peptides, and inorganic nanoparticle-based delivery systems, has established nonviral delivery approaches as a viable alternative to viral vectors. This review will introduce the molecular mechanisms of the CRISPR/Cas9 gene editing system, current strategies for delivering CRISPR/Cas9-based tools, an overview of strategies for overcoming off-target genome editing, and approaches for improving genome targeting and tissue targeting. We will also highlight current developments and recent clinical trials for the delivery of CRISPR/Cas9. Finally, future directions for overcoming the limitations and adaptation of this technology for clinical trials will be discussed.}, }
@article {pmid36898908, year = {2023}, author = {Awan, MJA and Mahmood, MA and Naqvi, RZ and Mansoor, S}, title = {PASTE: a high-throughput method for large DNA insertions.}, journal = {Trends in plant science}, volume = {28}, number = {5}, pages = {509-511}, doi = {10.1016/j.tplants.2023.02.013}, pmid = {36898908}, issn = {1878-4372}, mesh = {*CRISPR-Cas Systems/genetics ; *DNA Breaks, Double-Stranded ; Gene Editing/methods ; DNA ; }, abstract = {Prime editing (PE) enables precise genome editing at targeted locus without inducing double-stranded breaks (DSBs). Despite its precision, PE lacks the tendency to integrate large DNA fragments into the genome. Recently, Yarnall et al. reported clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 and an integrase-based system that conducts targeted integration of large DNA sequences (~36 kb) into the genome more efficiently.}, }
@article {pmid36806179, year = {2023}, author = {Gasser, T}, title = {[Introduction to Gene Therapy].}, journal = {Fortschritte der Neurologie-Psychiatrie}, volume = {91}, number = {4}, pages = {129-134}, doi = {10.1055/a-2002-5176}, pmid = {36806179}, issn = {1439-3522}, mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Genetic Therapy ; RNA ; }, abstract = {After many years of research and numerous setbacks, there are now undeniable success stories of gene therapies, namely the direct modification of genetic information on the DNA or RNA level. Both "ex vivo" strategies, i. e. the genetic manipulation of patient cells in a dish, as well as "in vivo" approaches are being used successfully. In addition to the supplementation of defective genes, the use of the CRISPR-Cas9 system to alter nuclear DNA sequences and the sequence-specific interference with the transcriptional process on the RNA level can be designated as gene therapies in a broad sense.}, }
@article {pmid35993441, year = {2023}, author = {Guo, W and Wang, H and Kumar Tharkeshwar, A and Couthouis, J and Braems, E and Masrori, P and Van Schoor, E and Fan, Y and Ahuja, K and Moisse, M and Jacquemyn, M and Furtado Madeiro da Costa, R and Gajjar, M and Balusu, S and Tricot, T and Fumagalli, L and Hersmus, N and Janky, R and Impens, F and Vanden Berghe, P and Ho, R and Thal, DR and Vandenberghe, R and Hegde, ML and Chandran, S and De Strooper, B and Daelemans, D and Van Damme, P and Van Den Bosch, L and Verfaillie, C}, title = {CRISPR/Cas9 screen in human iPSC-derived cortical neurons identifies NEK6 as a novel disease modifier of C9orf72 poly(PR) toxicity.}, journal = {Alzheimer's &amp; dementia : the journal of the Alzheimer's Association}, volume = {19}, number = {4}, pages = {1245-1259}, pmid = {35993441}, issn = {1552-5279}, support = {NC/N001419/1/NC3RS_/National Centre for the Replacement, Refinement and Reduction of Animals in Research/United Kingdom ; R01 NS088645/NS/NINDS NIH HHS/United States ; RF1 NS112719/NS/NINDS NIH HHS/United States ; R01 NS094535/NS/NINDS NIH HHS/United States ; R03 AG064266/AG/NIA NIH HHS/United States ; MC_EX_MR/N50192X/1/MRC_/Medical Research Council/United Kingdom ; MR/N013255/1/MRC_/Medical Research Council/United Kingdom ; /NS/NINDS NIH HHS/United States ; }, mesh = {Animals ; Humans ; *Amyotrophic Lateral Sclerosis/genetics ; *Frontotemporal Dementia/genetics ; *Induced Pluripotent Stem Cells/metabolism ; C9orf72 Protein/genetics ; Tumor Suppressor Protein p53/genetics/metabolism ; CRISPR-Cas Systems ; Zebrafish/genetics/metabolism ; Neurons/metabolism ; DNA Repeat Expansion/genetics ; NIMA-Related Kinases/genetics/metabolism ; }, abstract = {INTRODUCTION: The most common genetic cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) are hexanucleotide repeats in chromosome 9 open reading frame 72 (C9orf72). These repeats produce dipeptide repeat proteins with poly(PR) being the most toxic one.<br><br>METHODS: We performed a kinome-wide CRISPR/Cas9 knock-out screen in human induced pluripotent stem cell (iPSC) -derived cortical neurons to identify modifiers of poly(PR) toxicity, and validated the role of candidate modifiers using in vitro, in vivo, and ex-vivo studies.<br><br>RESULTS: Knock-down of NIMA-related kinase 6 (NEK6) prevented neuronal toxicity caused by poly(PR). Knock-down of nek6 also ameliorated the poly(PR)-induced axonopathy in zebrafish and NEK6 was aberrantly expressed in C9orf72 patients. Suppression of NEK6 expression and NEK6 activity inhibition rescued axonal transport defects in cortical neurons from C9orf72 patient iPSCs, at least partially by reversing p53-related DNA damage.<br><br>DISCUSSION: We identified NEK6, which regulates poly(PR)-mediated p53-related DNA damage, as a novel therapeutic target for C9orf72 FTD/ALS.}, }
@article {pmid37051232, year = {2023}, author = {Liu, X and Li, G and Liu, Y and Zhou, F and Huang, X and Li, K}, title = {Advances in CRISPR/Cas gene therapy for inborn errors of immunity.}, journal = {Frontiers in immunology}, volume = {14}, number = {}, pages = {1111777}, pmid = {37051232}, issn = {1664-3224}, mesh = {Humans ; *CRISPR-Cas Systems ; *Gene Editing ; Genetic Therapy ; Mutation ; Technology ; }, abstract = {Inborn errors of immunity (IEIs) are a group of inherited disorders caused by mutations in the protein-coding genes involved in innate and/or adaptive immunity. Hematopoietic stem cell transplantation (HSCT) is a mainstay definitive therapy for many severe IEIs. However, the lack of HLA-matched donors increases the risk of developing severe immunological complications. Gene therapy provides long-term clinical benefits and could be an attractive therapeutic strategy for IEIs. In this review, we describe the development and evolution of clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated proteins (Cas) gene-editing systems, including double-strand break (DSB)-based gene editing and DSB-free base editing or prime editing systems. Here, we discuss the advances in and issues associated with CRISPR/Cas gene editing tools and their potential as therapeutic alternatives for IEIs. We also highlight the progress of preclinical studies for the treatment of human genetic diseases, including IEIs, using CRISR/Cas and ongoing clinical trials based on this versatile technology.}, }
@article {pmid37050170, year = {2023}, author = {Tyagi, A and Ali, S and Park, S and Bae, H}, title = {Exploring the Potential of Multiomics and Other Integrative Approaches for Improving Waterlogging Tolerance in Plants.}, journal = {Plants (Basel, Switzerland)}, volume = {12}, number = {7}, pages = {}, pmid = {37050170}, issn = {2223-7747}, abstract = {Soil flooding has emerged as a serious threat to modern agriculture due to the rapid global warming and climate change, resulting in catastrophic crop damage and yield losses. The most detrimental effects of waterlogging in plants are hypoxia, decreased nutrient uptake, photosynthesis inhibition, energy crisis, and microbiome alterations, all of which result in plant death. Although significant advancement has been made in mitigating waterlogging stress, it remains largely enigmatic how plants perceive flood signals and translate them for their adaptive responses at a molecular level. With the advent of multiomics, there has been significant progress in understanding and decoding the intricacy of how plants respond to different stressors which have paved the way towards the development of climate-resistant smart crops. In this review, we have provided the overview of the effect of waterlogging in plants, signaling (calcium, reactive oxygen species, nitric oxide, hormones), and adaptive responses. Secondly, we discussed an insight into past, present, and future prospects of waterlogging tolerance focusing on conventional breeding, transgenic, multiomics, and gene-editing approaches. In addition, we have also highlighted the importance of panomics for developing waterlogging-tolerant cultivars. Furthermore, we have discussed the role of high-throughput phenotyping in the screening of complex waterlogging-tolerant traits. Finally, we addressed the current challenges and future perspectives of waterlogging signal perception and transduction in plants, which warrants future investigation.}, }
@article {pmid37047677, year = {2023}, author = {Anuchina, AA and Zaynitdinova, MI and Demchenko, AG and Evtushenko, NA and Lavrov, AV and Smirnikhina, SA}, title = {Bridging Gaps in HDR Improvement: The Role of MAD2L2, SCAI, and SCR7.}, journal = {International journal of molecular sciences}, volume = {24}, number = {7}, pages = {}, pmid = {37047677}, issn = {1422-0067}, mesh = {Humans ; *CRISPR-Cas Systems ; HEK293 Cells ; *Recombinational DNA Repair ; Gene Editing/methods ; DNA End-Joining Repair ; Ligases/genetics ; Mad2 Proteins/genetics ; }, abstract = {This study aimed to enhance homology-directed repair (HDR) efficiency in CRISPR/Cas-mediated genome editing by targeting three key factors regulating the balance between HDR and non-homologous end joining (NHEJ): MAD2L2, SCAI, and Ligase IV. In order to achieve this, a cellular model using mutated eGFP was designed to monitor HDR events. Results showed that MAD2L2 knockdown and SCR7 treatment significantly improved HDR efficiency during Cas9-mediated HDR repair of the mutated eGFP gene in the HEK293T cell line. Fusion protein Cas9-SCAI did not improve HDR. This study is the first to demonstrate that MAD2L2 knockdown during CRISPR-mediated gene editing in HEK293T cells can increase precise correction by up to 10.2 times. The study also confirmed a moderate but consistent effect of SCR7, an inhibitor of Ligase IV, which increased HDR by 1.7 times. These findings provide valuable insights into improving HDR-based genome editing efficiency.}, }
@article {pmid37047489, year = {2023}, author = {Yıldırım, K and Kavas, M and Küçük, &#x130;S and Seçgin, Z and Saraç, &#xc7;G}, title = {Development of Highly Efficient Resistance to Beet Curly Top Iran Virus (Becurtovirus) in Sugar Beet (B. vulgaris) via CRISPR/Cas9 System.}, journal = {International journal of molecular sciences}, volume = {24}, number = {7}, pages = {}, pmid = {37047489}, issn = {1422-0067}, mesh = {*CRISPR-Cas Systems ; *Beta vulgaris/genetics ; Iran ; Gene Editing/methods ; Vegetables ; Sugars ; }, abstract = {Beet Curly Top Iran Virus (BCTIV, Becurtovirus) is a dominant and widespread pathogen responsible for great damage and yield reduction in sugar beet production in the Mediterranean and Middle East. CRISPR-based gene editing is a versatile tool that has been successfully used in plants to improve resistance against many viral pathogens. In this study, the efficiency of gRNA/Cas9 constructs targeting the expressed genes of BCTIV was assessed in sugar beet leaves by their transient expression. Almost all positive control sugar beets revealed systemic infection and severe disease symptoms (90%), with a great biomass reduction (68%) after BCTIV agroinoculation. On the other hand, sugar beets co-agronioculated with BCTIV and gRNA/Cas9 indicated much lower systemic infection (10-55%), disease symptoms and biomass reduction (13-45%). Viral inactivation was also verified by RCA and qPCR assays for gRNA/Cas9 treated sugar beets. PCR-RE digestion and sequencing assays confirmed the gRNA/Cas9-mediated INDEL mutations at the target sites of the BCTIV genome and represented high efficiencies (53-88%), especially for those targeting BCTIV's movement gene and its overlapping region between capsid and ssDNA regulator genes. A multiplex CRISPR approach was also tested. The most effective four gRNAs targeting all the genes of BCTIV were cloned into a Cas9-containing vector and agroinoculated into virus-infected sugar beet leaves. The results of this multiplex CRISPR system revealed almost complete viral resistance with inhibition of systemic infection and mutant escape. This is the first report of CRSIPR-mediated broad-spectrum resistance against Becurtovirus in sugar beet.}, }
@article {pmid37043430, year = {2023}, author = {Porto, EM and Komor, AC}, title = {In the business of base editors: Evolution from bench to bedside.}, journal = {PLoS biology}, volume = {21}, number = {4}, pages = {e3002071}, pmid = {37043430}, issn = {1545-7885}, mesh = {Humans ; Adolescent ; *CRISPR-Cas Systems/genetics ; *Gene Editing ; Genetic Engineering ; Genetic Therapy ; }, abstract = {With the advent of recombinant DNA technology in the 1970s, the idea of using gene therapies to treat human genetic diseases captured the interest and imagination of scientists around the world. Years later, enabled largely by the development of CRISPR-based genome editing tools, the field has exploded, with academic labs, startup biotechnology companies, and large pharmaceutical corporations working in concert to develop life-changing therapeutics. In this Essay, we highlight base editing technologies and their development from bench to bedside. Base editing, first reported in 2016, is capable of installing C•G to T•A and A•T to G•C point mutations, while largely circumventing some of the pitfalls of traditional CRISPR/Cas9 gene editing. Despite their youth, these technologies have been widely used by both academic labs and therapeutics-based companies. Here, we provide an overview of the mechanics of base editing and its use in clinical trials.}, }
@article {pmid37020015, year = {2023}, author = {Sasnauskas, G and Tamulaitiene, G and Druteika, G and Carabias, A and Silanskas, A and Kazlauskas, D and Venclovas, &#x10c; and Montoya, G and Karvelis, T and Siksnys, V}, title = {TnpB structure reveals minimal functional core of Cas12 nuclease family.}, journal = {Nature}, volume = {616}, number = {7956}, pages = {384-389}, pmid = {37020015}, issn = {1476-4687}, mesh = {*Endonucleases/metabolism ; *DNA Transposable Elements ; Gene Editing ; RNA ; CRISPR-Cas Systems/genetics ; }, abstract = {The widespread TnpB proteins of IS200/IS605 transposon family have recently emerged as the smallest RNA-guided nucleases capable of targeted genome editing in eukaryotic cells[1,2]. Bioinformatic analysis identified TnpB proteins as the likely predecessors of Cas12 nucleases[3-5], which along with Cas9 are widely used for targeted genome manipulation. Whereas Cas12 family nucleases are well characterized both biochemically and structurally[6], the molecular mechanism of TnpB remains unknown. Here we present the cryogenic-electron microscopy structures of the Deinococcus radiodurans TnpB-reRNA (right-end transposon element-derived RNA) complex in DNA-bound and -free forms. The structures reveal the basic architecture of TnpB nuclease and the molecular mechanism for DNA target recognition and cleavage that is supported by biochemical experiments. Collectively, these results demonstrate that TnpB represents the minimal structural and functional core of the Cas12 protein family and provide a framework for developing TnpB-based genome editing tools.}, }
@article {pmid37047235, year = {2023}, author = {Naeem, M and Alkhnbashi, OS}, title = {Current Bioinformatics Tools to Optimize CRISPR/Cas9 Experiments to Reduce Off-Target Effects.}, journal = {International journal of molecular sciences}, volume = {24}, number = {7}, pages = {}, doi = {10.3390/ijms24076261}, pmid = {37047235}, issn = {1422-0067}, abstract = {The CRISPR-Cas system has evolved into a cutting-edge technology that has transformed the field of biological sciences through precise genetic manipulation. CRISPR/Cas9 nuclease is evolving into a revolutionizing method to edit any gene of any species with desirable outcomes. The swift advancement of CRISPR-Cas technology is reflected in an ever-expanding ecosystem of bioinformatics tools designed to make CRISPR/Cas9 experiments easier. To assist researchers with efficient guide RNA designs with fewer off-target effects, nuclease target site selection, and experimental validation, bioinformaticians have built and developed a comprehensive set of tools. In this article, we will review the various computational tools available for the assessment of off-target effects, as well as the quantification of nuclease activity and specificity, including web-based search tools and experimental methods, and we will describe how these tools can be optimized for gene knock-out (KO) and gene knock-in (KI) for model organisms. We also discuss future directions in precision genome editing and its applications, as well as challenges in target selection, particularly in predicting off-target effects.}, }
@article {pmid37047101, year = {2023}, author = {Tenea, GN}, title = {Metabiotics Signature through Genome Sequencing and In Vitro Inhibitory Assessment of a Novel Lactococcus lactis Strain UTNCys6-1 Isolated from Amazonian Camu-Camu Fruits.}, journal = {International journal of molecular sciences}, volume = {24}, number = {7}, pages = {}, doi = {10.3390/ijms24076127}, pmid = {37047101}, issn = {1422-0067}, abstract = {Metabiotics are the structural components of probiotic bacteria, functional metabolites, and/or signaling molecules with numerous beneficial properties. A novel Lactococcus lactis strain, UTNCys6-1, was isolated from wild Amazonian camu-camu fruits (Myrciaria dubia), and various functional metabolites with antibacterial capacity were found. The genome size is 2,226,248 base pairs, and it contains 2248 genes, 2191 protein-coding genes (CDSs), 50 tRNAs, 6 rRNAs, 1 16S rRNA, 1 23S rRNA, and 1 tmRNA. The average GC content is 34.88%. In total, 2148 proteins have been mapped to the EggNOG database. The specific annotation consisted of four incomplete prophage regions, one CRISPR-Cas array, six genomic islands (GIs), four insertion sequences (ISs), and four regions of interest (AOI regions) spanning three classes of bacteriocins (enterolysin_A, nisin_Z, and sactipeptides). Based on pangenome analysis, there were 6932 gene clusters, of which 751 (core genes) were commonly observed within the 11 lactococcal strains. Among them, 3883 were sample-specific genes (cloud genes) and 2298 were shell genes, indicating high genetic diversity. A sucrose transporter of the SemiSWEET family (PTS system: phosphoenolpyruvate-dependent transport system) was detected in the genome of UTNCys6-1 but not the other 11 lactococcal strains. In addition, the metabolic profile, antimicrobial susceptibility, and inhibitory activity of both protein-peptide extract (PPE) and exopolysaccharides (EPSs) against several foodborne pathogens were assessed in vitro. Furthermore, UTNCys6-1 was predicted to be a non-human pathogen that was unable to tolerate all tested antibiotics except gentamicin; metabolized several substrates; and lacks virulence factors (VFs), genes related to the production of biogenic amines, and acquired antibiotic resistance genes (ARGs). Overall, this study highlighted the potential of this strain for producing bioactive metabolites (PPE and EPSs) for agri-food and pharmaceutical industry use.}, }
@article {pmid37045931, year = {2023}, author = {Wang, R and Shu, X and Zhao, H and Xue, Q and Liu, C and Wu, A and Cheng, F and Wang, L and Zhang, Y and Feng, J and Wu, N and Li, M}, title = {Associate toxin-antitoxin with CRISPR-Cas to kill multidrug-resistant pathogens.}, journal = {Nature communications}, volume = {14}, number = {1}, pages = {2078}, pmid = {37045931}, issn = {2041-1723}, abstract = {CreTA, CRISPR-regulated toxin-antitoxin (TA), safeguards CRISPR-Cas immune systems by inducing cell dormancy/death upon their inactivation. Here, we characterize a bacterial CreTA associating with the I-F CRISPR-Cas in Acinetobacter. CreT is a distinct bactericidal small RNA likely targeting several essential RNA molecules that are required to initiate protein synthesis. CreA guides the CRISPR effector to transcriptionally repress CreT. We further demonstrate a proof-of-concept antimicrobial strategy named ATTACK, which AssociaTes TA and CRISPR-Cas to Kill multidrug resistant (MDR) pathogens. In this design, CRISPR-Cas is programed to target antibiotic resistance gene(s) to selectively kill MDR pathogens or cure their resistance, and when CRISPR-Cas is inactivated or suppressed by unwanted genetic or non-genetic events/factors, CreTA triggers cell death as the last resort. Our data highlight the diversity of RNA toxins coevolving with CRISPR-Cas, and illuminate a combined strategy of CRISPR and TA antimicrobials to 'ATTACK' MDR pathogens.}, }
@article {pmid37043060, year = {2023}, author = {Achudhan, AB and Kannan, P and Saleena, LM}, title = {CRISPR detection in metagenome-assembled genomes (MAGs) of coal mine.}, journal = {Functional &amp; integrative genomics}, volume = {23}, number = {2}, pages = {122}, pmid = {37043060}, issn = {1438-7948}, abstract = {Bacterial and archaeal CRISPR-Cas systems provide adaptive immune protection against foreign mobile genetic elements. When viruses infect bacteria, a small portion of the viral DNA is inserted into the bacterial DNA in a specific pattern to produce segments known as CRISPR arrays. Metagenome assembled genomes (MAGs) were used in our study to identify the CRISPR sequence for determining the interacted phage. Metagenomic data from a coal mine was used to perform a computational study. From raw reads, 206151 contigs were assembled. Then contigs were clustered into 150 Metagenome assembled genomes from which 78 non-redundant MAGs were selected. Using the CHECKM standard, seven MAGs were found to have >80 completeness and <20 contaminations. Those MAGs were analyzed for the presence of CRISPR elements. Out of seven MAGs, four MAGs have the CRISPR elements and are searched against the VIROblast database. CRISPR arrays have 4, 1, 3, and 7 spacer sequences in the MAGs of Burkholderia, Acinetobacter, Oxalobacteraceae, and Burkholderia multivorans respectively. The uncultured Caudovirales phage genomic regions were present in the genomes of Burkholderia, Oxalobacteriaceae, and Burkholderia multivorans. This study follows the unconventional metagenomics workflow to provide a better understanding of bacteria and phage interactions.}, }
@article {pmid37042651, year = {2023}, author = {Buyukyoruk, M and Henriques, WS and Wiedenheft, B}, title = {Clarifying CRISPR: Why Repeats Identified in the Human Genome Should Not Be Considered CRISPRs.}, journal = {The CRISPR journal}, volume = {}, number = {}, pages = {}, doi = {10.1089/crispr.2022.0106}, pmid = {37042651}, issn = {2573-1602}, abstract = {Clustered regularly interspaced short palindromic repeats (CRISPRs) and their associated genes (cas) are essential components of adaptive immune systems that protect bacteria and archaea from viral infection. CRISPR-Cas systems are found in about 40% of bacterial and 85% of archaeal genomes, but not in eukaryotic genomes. Recently, an article published in Communications Biology reported the identification of 12,572 putative CRISPRs in the human genome, which they call "hCRISPR." In this study, we attempt to reproduce this analysis and show that repetitive elements identified as putative CRISPR loci in the human genome contain neither the repeat-spacer-repeat architecture nor the cas genes characteristic of functional CRISPR systems.}, }
@article {pmid37041450, year = {2023}, author = {Li, L and Li, S and Gu, D and Xu, Y and Wang, J}, title = {CRISPR-Cas12-Based Diagnostic Applications in Infectious and Zoonotic Diseases.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2621}, number = {}, pages = {267-278}, pmid = {37041450}, issn = {1940-6029}, mesh = {Animals ; CRISPR-Cas Systems ; Sensitivity and Specificity ; Zoonoses ; *Communicable Diseases/genetics ; RNA ; *Nucleic Acids/genetics ; }, abstract = {Rapid detection of infectious and zoonotic diseases is very important for pathogen identification and infection control. Molecular diagnostic assays are well-known for high accuracy and sensitivity; however, conventional methods such as real-time PCR may require professional instruments and operations, preventing their wide applications in scenarios including animal quarantine. The recently developed CRISPR diagnostic (CRISPR-Dx) methods, employing the trans-cleavage activities of either Cas12 (e.g., HOLMES) or Cas13 (e.g., SHERLOCK), have shown great potential in rapid and convenient nucleic acid detection. Guided by specially designed CRISPR RNA (crRNA), Cas12 binds target DNA sequences and trans-cleaves ssDNA reporters, generating detectable signals, while Cas13 recognizes target ssRNA and trans-cleaves ssRNA reporters. To achieve high detection sensitivity, both HOLMES and SHERLOCK systems can be combined with pre-amplification procedures including both PCR and isothermal amplifications. Here, we present the employment of the HOLMESv2 method for convenient detection of the infectious and zoonotic diseases. Specifically, target nucleic acid is first amplified by LAMP or RT-LAMP, and the products are then detected by the thermophilic Cas12b. In addition, Cas12b reaction can be combined with LAMP amplification to achieve one-pot reaction systems. In this chapter, we provide a step-by-step description of the HOLMESv2-mediated rapid and sensitive detection of Japanese encephalitis virus (JEV), an RNA pathogen as an example.}, }
@article {pmid37041195, year = {2023}, author = {Kong, X and Zhang, H and Li, G and Wang, Z and Kong, X and Wang, L and Xue, M and Zhang, W and Wang, Y and Lin, J and Zhou, J and Shen, X and Wei, Y and Zhong, N and Bai, W and Yuan, Y and Shi, L and Zhou, Y and Yang, H}, title = {Engineered CRISPR-OsCas12f1 and RhCas12f1 with robust activities and expanded target range for genome editing.}, journal = {Nature communications}, volume = {14}, number = {1}, pages = {2046}, pmid = {37041195}, issn = {2041-1723}, mesh = {Animals ; *Gene Editing ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; CRISPR-Associated Protein 9/metabolism ; Dependovirus/genetics ; Mammals/genetics ; }, abstract = {The type V-F CRISPR-Cas12f system is a strong candidate for therapeutic applications due to the compact size of the Cas12f proteins. In this work, we identify six uncharacterized Cas12f1 proteins with nuclease activity in mammalian cells from assembled bacterial genomes. Among them, OsCas12f1 (433 aa) from Oscillibacter sp. and RhCas12f1 (415 aa) from Ruminiclostridium herbifermentans, which respectively target 5' T-rich Protospacer Adjacent Motifs (PAMs) and 5' C-rich PAMs, show the highest editing activity. Through protein and sgRNA engineering, we generate enhanced OsCas12f1 (enOsCas12f1) and enRhCas12f1 variants, with 5'-TTN and 5'-CCD (D = not C) PAMs respectively, exhibiting much higher editing efficiency and broader PAMs, compared with the engineered variant Un1Cas12f1 (Un1Cas12f1_ge4.1). Furthermore, by fusing the destabilized domain with enOsCas12f1, we generate inducible-enOsCas12f1 and demonstate its activity in vivo by single adeno-associated virus delivery. Finally, dead enOsCas12f1-based epigenetic editing and gene activation can also be achieved in mammalian cells. This study thus provides compact gene editing tools for basic research with remarkable promise for therapeutic applications.}, }
@article {pmid37036877, year = {2023}, author = {Bai, Y and He, Y and Shen, CZ and Li, K and Li, DL and He, ZQ}, title = {CRISPR/Cas9-Mediated genomic knock out of tyrosine hydroxylase and yellow genes in cricket Gryllus bimaculatus.}, journal = {PloS one}, volume = {18}, number = {4}, pages = {e0284124}, pmid = {37036877}, issn = {1932-6203}, mesh = {Animals ; *Tyrosine 3-Monooxygenase/genetics/metabolism ; *Melanins/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Mutation ; Genomics ; }, abstract = {Gryllus bimaculatus is an emerging model organism in various fields of biology such as behavior, neurology, physiology and genetics. Recently, application of reverse genetics provides an opportunity of understanding the functional genomics and manipulating gene regulation networks with specific physiological response in G. bimaculatus. By using CRISPR/Cas9 system in G. bimaculatus, we present an efficient knockdown of Tyrosine hydroxylase (TH) and yellow-y, which are involved in insect melanin and catecholamine-biosynthesis pathway. As an enzyme catalyzing the conversion of tyrosine to 3,4-dihydroxyphenylalanine, TH confines the first step reaction in the pathway. Yellow protein (dopachrome conversion enzyme, DCE) is also involved in the melanin biosynthetic pathway. The regulation system and molecular mechanism of melanin biogenesis in the pigmentation and their physiological functions in G. bimaculatus hasn't been well defined by far for lacking of in vivo models. Deletion and insertion of nucleotides in target sites of both TH and Yellow are detected in both F0 individuals and the inheritable F1 progenies. We confirm that TH and yellow-y are down-regulated in mutants by quantitative real-time PCR analysis. Compared with the control group, mutations of TH and yellow-y genes result in defects in pigmentation. Most F0 nymphs with mutations of TH gene die by the first instar, and the only adult had significant defects in the wings and legs. However, we could not get any homozygotes of TH mutants for all the F2 die by the first instar. Therefore, TH gene is very important for the growth and development of G. bimaculatus. When the yellow-y gene is knocked out, 71.43% of G. bimaculatus are light brown, with a slight mosaic on the abdomen. The yellow-y gene can be inherited stably through hybridization experiment with no obvious phenotype except lighter cuticular color. The present loss of function study indicates the essential roles of TH and yellow in pigmentation, and TH possesses profound and extensive effects of dopamine synthesis in embryonic development in G. bimaculatus.}, }
@article {pmid37036720, year = {2022}, author = {Testa, G and Mainardi, M and Vannini, E and Pancrazi, L and Cattaneo, A and Costa, M}, title = {Disentangling the signaling complexity of nerve growth factor receptors by CRISPR/Cas9.}, journal = {FASEB journal : official publication of the Federation of American Societies for Experimental Biology}, volume = {36}, number = {11}, pages = {e22498}, doi = {10.1096/fj.202101760RR}, pmid = {37036720}, issn = {1530-6860}, mesh = {Rats ; Animals ; *Receptor, trkA/genetics/metabolism ; *Receptor, Nerve Growth Factor/genetics ; Nerve Growth Factor/genetics/metabolism ; CRISPR-Cas Systems ; Receptors, Nerve Growth Factor/genetics/metabolism ; }, abstract = {The binding of nerve growth factor (NGF) to the tropomyosin-related kinase A (TrkA) and p75[NTR] receptors activates a large variety of pathways regulating critical processes as diverse as proliferation, differentiation, membrane potential, synaptic plasticity, and pain. To ascertain the details of TrkA-p75[NTR] interaction and cooperation, a plethora of experiments, mostly based on receptor overexpression or downregulation, have been performed. Among the heterogeneous cellular systems used for studying NGF signaling, the PC12 pheochromocytoma-derived cell line is a widely used model. By means of CRISPR/Cas9 genome editing, we created PC12 cells lacking TrkA, p75[NTR] , or both. We found that TrkA-null cells become unresponsive to NGF. Conversely, the absence of p75[NTR] enhances the phosphorylation of TrkA and its effectors. Using a patch-clamp, we demonstrated that the individual activation of TrkA and p75[NTR] by NGF results in antagonizing effects on the membrane potential. These newly developed PC12 cell lines can be used to investigate the specific roles of TrkA and p75[NTR] in a genetically defined cellular model, thus providing a useful platform for future studies and further gene editing.}, }
@article {pmid37036212, year = {2023}, author = {Tsuji, T and Zhang, Y and Tseng, YH}, title = {Generation of Brown Fat-Specific Knockout Mice Using a Combined Cre-LoxP, CRISPR-Cas9, and Adeno-Associated Virus Single-Guide RNA System.}, journal = {Journal of visualized experiments : JoVE}, volume = {}, number = {193}, pages = {}, doi = {10.3791/65083}, pmid = {37036212}, issn = {1940-087X}, mesh = {Mice ; Animals ; Mice, Knockout ; *CRISPR-Cas Systems ; *RNA, Guide, CRISPR-Cas Systems ; Adipose Tissue, Brown ; Dependovirus/genetics ; }, abstract = {Brown adipose tissue (BAT) is an adipose depot specialized in energy dissipation that can also serve as an endocrine organ via the secretion of bioactive molecules. The creation of BAT-specific knockout mice is one of the most popular approaches for understanding the contribution of a gene of interest to BAT-mediated energy regulation. The conventional gene targeting strategy utilizing the Cre-LoxP system has been the principal approach to generate tissue-specific knockout mice. However, this approach is time-consuming and tedious. Here, we describe a protocol for the rapid and efficient knockout of a gene of interest in BAT using a combined Cre-LoxP, CRISPR-Cas9, and adeno-associated virus (AAV) single-guide RNA (sgRNA) system. The interscapular BAT is located in the deep layer between the muscles. Thus, the BAT must be exposed in order to inject the AAV precisely and directly into the BAT within the visual field. Appropriate surgical handling is crucial to prevent damage to the sympathetic nerves and vessels, such as the Sultzer's vein that connects to the BAT. To minimize tissue damage, there is a critical need to understand the three-dimensional anatomical location of the BAT and the surgical skills required in the technical steps. This protocol highlights the key technical procedures, including the design of sgRNAs targeting the gene of interest, the preparation of AAV-sgRNA particles, and the surgery for the direct microinjection of AAV into both BAT lobes for generating BAT-specific knockout mice, which can be broadly applied to study the biological functions of genes in BAT.}, }
@article {pmid37035707, year = {2023}, author = {Sun, R and Zhao, Y and Wang, W and Liu, JG and Chen, C}, title = {Nonspecific interactions between Cas12a and dsDNA located downstream of the PAM mediate target search and assist AsCas12a for DNA cleavage.}, journal = {Chemical science}, volume = {14}, number = {14}, pages = {3839-3851}, pmid = {37035707}, issn = {2041-6520}, abstract = {Cas12a is one of the most commonly used Cas proteins for genome editing and gene regulation. The first key step for Cas12a to fulfill its function is to search for its target among numerous nonspecific and off-target sites. Cas12a utilizes one-dimensional diffusion along the contour of dsDNA to efficiently search for its target. However, due to a lack of structural information of the transient diffusing complex, the residues mediating the one-dimensional diffusion of Cas12a are unknown. Here, combining single-molecule and ensemble assays, we found that nonspecific interactions between Cas12a and dsDNA at the PAM downstream cause asymmetric target search regions of Cas12a flanking the PAM site, which guided us to identify a positive-charge-enriched alpha helix in the REC2 domain serving as a conserved element to facilitate one-dimensional diffusion-driven target search of AsCas12a, LbCas12a and FnCas12a. In addition, this alpha helix assists the target cleavage process of AsCas12a via stabilizing the cleavage states. Thus, neutralizing positive charges within this helix not only significantly slows target search but also enhances the specificity of AsCas12a both in vitro and in living cells. Similar behaviors are detected when residues mediating diffusion of SpCas9 are mutated. Thus, engineering residues mediating diffusion on dsDNA is a new avenue to optimize and enrich the versatile CRISPR-Cas toolbox.}, }
@article {pmid37035482, year = {2023}, author = {Pougnet, R and Derbez, B and Troadec, MB}, title = {Mapping the 'Ethical' Controversy of Human Heritable Genome Editing: a Multidisciplinary Approach.}, journal = {Asian bioethics review}, volume = {15}, number = {2}, pages = {189-204}, pmid = {37035482}, issn = {1793-9453}, abstract = {UNLABELLED: Genome editing, for instance by CRISPR-Cas, is a major advancement of the last 10 years in medicine but questions ethically our practices. In particular, human embryo heritable genome editing is a source of great controversy. We explored how this ethical question was debated in the literature from PubMed database, in a period of 4 years (2016-2020) around the announcement of the 'CRISPR babies' Chinese experiment in November 2018. We evaluated the weight of the arguments for and against this topic, through an analysis of reviews published on this question. The most important arguments come from the technical perspective: safety issues and benefits, putative long-term effects on the future generations and the need to assess this aspect. Next, foreseeable clinical benefits and the alternatives to these methods are discussed. The number of people that would benefit from such techniques is also considered. However, social and anthropological issues are addressed in a more disparate way. Parenthood and desire for children are sometimes overlooked. Few authors mention social justice, stigmatisation and equality of access. Consent and information are more clearly addressed, as well as the question of the relationship between generations. Finally, the effects on the nature of humankind or human species are far from being consensual; the risks of enhancement, eugenics and transhumanism are raised. We conclude that the risks associated with the immaturity of the technique were at the forefront of the ethical debate on human embryo heritable genome editing. Their consequences were seen as more immediate and easier to handle than those of sociological or anthropological projections, which are more speculative in nature.<br><br>SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s41649-022-00234-1.}, }
@article {pmid37034382, year = {2022}, author = {Hasanzadeh, A and Hamblin, MR and Kiani, J and Noori, H and Hardie, JM and Karimi, M and Shafiee, H}, title = {Could artificial intelligence revolutionize the development of nanovectors for gene therapy and mRNA vaccines?.}, journal = {Nano today}, volume = {47}, number = {}, pages = {}, pmid = {37034382}, issn = {1748-0132}, support = {R01 AI138800/AI/NIAID NIH HHS/United States ; R01 EB033866/EB/NIBIB NIH HHS/United States ; R33 AI140489/AI/NIAID NIH HHS/United States ; R61 AI140489/AI/NIAID NIH HHS/United States ; }, abstract = {Gene therapy enables the introduction of nucleic acids like DNA and RNA into host cells, and is expected to revolutionize the treatment of a wide range of diseases. This growth has been further accelerated by the discovery of CRISPR/Cas technology, which allows accurate genomic editing in a broad range of cells and organisms in vitro and in vivo. Despite many advances in gene delivery and the development of various viral and non-viral gene delivery vectors, the lack of highly efficient non-viral systems with low cellular toxicity remains a challenge. The application of cutting-edge technologies such as artificial intelligence (AI) has great potential to find new paradigms to solve this issue. Herein, we review AI and its major subfields including machine learning (ML), neural networks (NNs), expert systems, deep learning (DL), computer vision and robotics. We discuss the potential of AI-based models and algorithms in the design of targeted gene delivery vehicles capable of crossing extracellular and intracellular barriers by viral mimicry strategies. We finally discuss the role of AI in improving the function of CRISPR/Cas systems, developing novel nanobots, and mRNA vaccine carriers.}, }
@article {pmid37033919, year = {2023}, author = {Braun, T and Pruene, A and Darguzyte, M and Vom Stein, AF and Nguyen, PH and Wagner, DL and Kath, J and Roig-Merino, A and Heuser, M and Riehm, LL and Schneider, A and Awerkiew, S and Talbot, SR and Bleich, A and Figueiredo, C and Bornhäuser, M and Stripecke, R}, title = {Non-viral TRAC-knocked-in CD19[KI]CAR-T and gp350[KI]CAR-T cells tested against Burkitt lymphomas with type 1 or 2 EBV infection: In vivo cellular dynamics and potency.}, journal = {Frontiers in immunology}, volume = {14}, number = {}, pages = {1086433}, pmid = {37033919}, issn = {1664-3224}, mesh = {Humans ; Mice ; Animals ; *Epstein-Barr Virus Infections ; *Burkitt Lymphoma/therapy ; Herpesvirus 4, Human ; *Receptors, Chimeric Antigen ; Hepatitis A Virus Cellular Receptor 2 ; Programmed Cell Death 1 Receptor ; Prospective Studies ; Receptors, Antigen, T-Cell, alpha-beta ; }, abstract = {INTRODUCTION: The ubiquitous Epstein-Barr virus (EBV) is an oncogenic herpes virus associated with several human malignancies. EBV is an immune-evasive pathogen that promotes CD8[+] T cell exhaustion and dysregulates CD4[+] T cell functions. Burkitt lymphoma (BL) is frequently associated with EBV infections. Since BL relapses after conventional therapies are difficult to treat, we evaluated prospective off-the-shelf edited CAR-T cell therapies targeting CD19 or the EBV gp350 cell surface antigen.<br><br>METHODS: We used CRISPR/Cas9 gene editing methods to knock in (KI) the CD19CAR.CD28z or gp350CAR.CD28z into the T cell receptor (TCR) alpha chain (TRAC) locus.<br><br>RESULTS: Applying upscaled methods with the ExPERT ATx[&#xae;] MaxCyte system, KI efficacy was ~20% of the total ~2 &#xd7; 10[8] TCR-knocked-out (KO) generated cells. [KO]TCR[KI]CAR-T cells were co-cultured in vitro with the gp350[+]CD19[+] BL cell lines Daudi (infected with type 1 EBV) or with Jiyoye (harboring a lytic type 2 EBV). Both types of CAR-T cells showed cytotoxic effects against the BL lines in vitro. CD8[+ KI]CAR-T cells showed higher persistency than CD4[+ KI]CAR-T cells after in vitro co-culture with BL and upregulation of the activation/exhaustion markers PD-1, LAG-3, and TIM-3. Two preclinical in vivo xenograft models were set up with Nod.Rag.Gamma mice injected intravenously (i.v.) with 2 &#xd7; 10[5] Daudi/fLuc-GFP or with Jiyoye/fLuc-GFP cells. Compared with the non-treated controls, mice challenged with BL and treated with CD19[KI]CAR-T cells showed delayed lymphoma dissemination with lower EBV DNA load. Notably, for the Jiyoye/fLuc-GFP model, almost exclusively CD4[+] CD19[KI]CAR-T cells were detectable at the endpoint analyses in the bone marrow, with increased frequencies of regulatory T cells (Tregs) and TIM-3[+]CD4[+] T cells. Administration of gp350[KI]CAR-T cells to mice after Jiyoye/GFP-fLuc challenge did not inhibit BL growth in vivo but reduced the EBV DNA load in the bone marrow and promoted gp350 antigen escape. CD8[+]PD-1[+]LAG-3[+] gp350[KI]CAR-T cells were predominant in the bone marrow.<br><br>DISCUSSION: The two types of [KO]TCR[KI]CAR-T cells showed different therapeutic effects and in vivo dynamics. These findings reflect the complexities of the immune escape mechanisms of EBV, which may interfere with the CAR-T cell property and potency and should be taken into account for future clinical translation.}, }
@article {pmid37032710, year = {2023}, author = {Illa-Berenguer, E and LaFayette, PR and Parrott, WA}, title = {Editing efficiencies with Cas9 orthologs, Cas12a endonucleases, and temperature in rice.}, journal = {Frontiers in genome editing}, volume = {5}, number = {}, pages = {1074641}, pmid = {37032710}, issn = {2673-3439}, abstract = {The advent of CRISPR-Cas technology has made it the genome editing tool of choice in all kingdoms of life, including plants, which can have large, highly duplicated genomes. As a result, finding adequate target sequences that meet the specificities of a given Cas nuclease on any gene of interest remains challenging in many cases. To assess target site flexibility, we tested five different Cas9/Cas12a endonucleases (SpCas9, SaCas9, St1Cas9, Mb3Cas12a, and AsCas12a) in embryogenic rice calli from Taipei 309 at 37°C (optimal temperature for most Cas9/Cas12a proteins) and 27°C (optimal temperature for tissue culture) and measured their editing rates under regular tissue culture conditions using Illumina sequencing. StCas9 and AsCas12 were not functional as tested, regardless of the temperature used. SpCas9 was the most efficient endonuclease at either temperature, regardless of whether monoallelic or biallelic edits were considered. Mb3Cas12a at 37°C was the next most efficient endonuclease. Monoallelic edits prevailed for both SaCas9 and Mb3Cas12a at 27°C, but biallelic edits prevailed at 37°C. Overall, the use of other Cas9 orthologs, the use of Cas12a endonucleases, and the optimal temperature can expand the range of targetable sequences.}, }
@article {pmid36876986, year = {2023}, author = {Inotsume, M and Chiba, T and Matsushima, T and Kurimoto, R and Nakajima, M and Kato, T and Shishido, K and Liu, L and Kawakami, K and Asahara, H}, title = {One-step generation of mice with gene editing by Tol2 transposon-dependent gRNA delivery.}, journal = {FEBS letters}, volume = {597}, number = {7}, pages = {975-984}, doi = {10.1002/1873-3468.14605}, pmid = {36876986}, issn = {1873-3468}, mesh = {Mice ; Animals ; *Gene Editing ; *CRISPR-Cas Systems ; Plasmids ; Mice, Knockout ; Transposases/genetics ; }, abstract = {Conditional knockout mice are valuable tools for examining the functions of targeted genes in a time- and space-specific manner. Here, we generated gene-edited mice by using the Tol2 transposon to introduce guide RNA (gRNA) into fertilized eggs obtained by crossing LSL (loxP-stop-loxP)-CRISPR-associated 9 (Cas9) mice, which express Cas9 in a Cre-dependent manner, with CAG-CreER mice. Transposase mRNA and plasmid DNA, which contained a gRNA sequence for the gene encoding tyrosinase flanked by the transposase recognition sequence, were injected together into fertilized eggs. As a result, the transcribed gRNA cleaved the target genome in a Cas9-dependent manner. Using this method, it is possible to generate conditional genome-edited mice more easily in a shorter period of time.}, }
@article {pmid36864759, year = {2023}, author = {Feng, YL and Liu, SC and Chen, RD and Sun, XN and Xiao, JJ and Xiang, JF and Xie, AY}, title = {Proximal binding of dCas9 at a DNA double strand break stimulates homology-directed repair as a local inhibitor of classical non-homologous end joining.}, journal = {Nucleic acids research}, volume = {51}, number = {6}, pages = {2740-2758}, pmid = {36864759}, issn = {1362-4962}, mesh = {Animals ; *DNA Breaks, Double-Stranded ; *CRISPR-Cas Systems ; DNA End-Joining Repair ; Recombinational DNA Repair ; Gene Editing/methods ; DNA/genetics ; DNA Repair ; Mammals/genetics ; }, abstract = {In CRISPR/Cas9 genome editing, the tight and persistent target binding of Cas9 provides an opportunity for efficient genetic and epigenetic modification on genome. In particular, technologies based on catalytically dead Cas9 (dCas9) have been developed to enable genomic regulation and live imaging in a site-specific manner. While post-cleavage target residence of CRISPR/Cas9 could alter the pathway choice in repair of Cas9-induced DNA double strand breaks (DSBs), it is possible that dCas9 residing adjacent to a break may also determine the repair pathway for this DSB, providing an opportunity to control genome editing. Here, we found that loading dCas9 onto a DSB-adjacent site stimulated homology-directed repair (HDR) of this DSB by locally blocking recruitment of classical non-homologous end-joining (c-NHEJ) factors and suppressing c-NHEJ in mammalian cells. We further repurposed dCas9 proximal binding to increase HDR-mediated CRISPR genome editing by up to 4-fold while avoiding exacerbation of off-target effects. This dCas9-based local inhibitor provided a novel strategy of c-NHEJ inhibition in CRISPR genome editing in place of small molecule c-NHEJ inhibitors, which are often used to increase HDR-mediated genome editing but undesirably exacerbate off-target effects.}, }
@article {pmid36705519, year = {2023}, author = {Warsinger-Pepe, N and Chang, C and Desroberts, CR and Akbari, OS}, title = {Polycomb response elements reduce leaky expression of Cas9 under temperature-inducible Hsp70Bb promoter in Drosophila melanogaster.}, journal = {G3 (Bethesda, Md.)}, volume = {13}, number = {4}, pages = {}, pmid = {36705519}, issn = {2160-1836}, mesh = {Animals ; *Drosophila melanogaster/genetics ; Temperature ; CRISPR-Cas Systems ; *Drosophila Proteins/genetics ; Heat-Shock Proteins/genetics ; Response Elements ; Hot Temperature ; }, abstract = {Heat-shock-inducible expression of genes through the use of heat-inducible promoters is commonly used in research despite leaky expression of downstream genes of interest without targeted induction (i.e. heat shock). The development of non-leaky inducible expression systems is of broad interest for both basic and applied studies, to precisely control gene expression. Here we characterize the use of Polycomb response elements and the inducible Heat-shock protein 70Bb promoter, previously described as a non-leaky inducible system, to regulate Cas9 endonuclease levels and function in Drosophila melanogaster after varying both heat-shock durations and rearing temperatures. We show that Polycomb response elements can significantly reduce expression of Cas9 under Heat-shock protein 70Bb promoter control using a range of conditions, corroborating previously published results. We further demonstrate that this low transcript level of heat-induced Cas9 is sufficient to induce mutant mosaic phenotypes. Incomplete suppression of an inducible Cas9 system by Polycomb response elements with no heat-shock suggests that further regulatory elements are required to precisely control Cas9 expression and abundance.}, }
@article {pmid37032058, year = {2023}, author = {Wang, X and Jin, W and Yang, Y and Ma, H and Liu, H and Lei, J and Wu, Y and Zhang, L}, title = {CRISPR/Cas12a-mediated Enzymatic recombinase amplification for rapid visual quantitative authentication of halal food.}, journal = {Analytica chimica acta}, volume = {1255}, number = {}, pages = {341144}, doi = {10.1016/j.aca.2023.341144}, pmid = {37032058}, issn = {1873-4324}, mesh = {Animals ; Swine ; *CRISPR-Cas Systems ; *Drug Contamination ; Fluorescence ; Food Safety ; Recombinases/genetics ; Nucleic Acid Amplification Techniques ; }, abstract = {Economically motivated adulteration (EMA) has become a concern in food safety. We propose a CRISPR/Cas12a Mediated Enzymatic Recombinase Amplification detection system (CAMERA) that integrates Enzymatic Recombinase Amplification (ERA) and Cas12a cleavage to detect halal food adulteration. We designed and screened crRNA targeting CLEC, a porcine-specific nuclear single-copy gene, and optimized the reagent concentrations and incubation times for the ERA and Cas12a cleavage steps. CAMERA was highly specific for pork ingredients detection. The DNA concentration and fluorescence signal intensity relationship was linear at DNA concentrations of 20-0.032 ng/μL. CAMERA detected as few as two CLEC copies and quantified samples with porcine DNA content as low as 5% within 25 min. The system could be operated in a miniaturized working mode that requires no technical expertise or professional equipment, making CAMERA a valuable tool in resource-limited areas for the qualitative and quantitative detection of pork ingredients in halal food.}, }
@article {pmid37029174, year = {2023}, author = {Feng, Z and Kong, D and Jin, W and He, K and Zhao, J and Liu, B and Xu, H and Yu, X and Feng, S}, title = {Rapid detection of isocitrate dehydrogenase 1 mutation status in glioma based on Crispr-Cas12a.}, journal = {Scientific reports}, volume = {13}, number = {1}, pages = {5748}, pmid = {37029174}, issn = {2045-2322}, mesh = {Adult ; Humans ; Isocitrate Dehydrogenase/genetics/metabolism ; *Brain Neoplasms/genetics/diagnosis ; CRISPR-Cas Systems/genetics ; *Glioma/diagnosis/genetics ; Mutation ; }, abstract = {The aim is to use Crispr-Cas12a for the rapid detection of the single nucleotide polymorphism (SNP) of isocitrate dehydrogenase 1 (IDH1)-R132H locus and explore the effectiveness and consistency of this method with direct sequencing method for detecting IDH1-R132H of glioma tissue samples. 58 previous frozen tissue and 46 recent fresh tissue samples of adult diffuse glioma were selected to detect IDH1-R132H using Crispr-Cas12a. The results of immunohistochemistry (IHC) and direct sequencing methods were analyzed. We calculated the efficiency index of Crispr-Cas12a and IHC, and analyzed the consistency among Crispr-Cas12a, IHC and direct sequencing method using paired Chi-sequare test and Kappa identity test. We accomplished the rapid detection of IDH1-R132H in 60 min using Crispr-Cas12a. Regarding direct sequencing method as the gold standard, the sensitivity, specificity and consistency rate of Crispr-Cas12a was 91.4%, 95.7% and 93.1% in the frozen sample group, while 96.1%, 89.7% and 92.0% in the fresh sample group, respectively. Kappa test showed good consistency between the two methods (k = 0.858). Crispr-Cas12a can quickly and accurately detect IDH1-R132H and has good stability. It is a promising method to detect IDH1 mutation status intraoperatively.}, }
@article {pmid36917981, year = {2023}, author = {Wu, Q and Wu, J and Karim, K and Chen, X and Wang, T and Iwama, S and Carobbio, S and Keen, P and Vidal-Puig, A and Kotter, MR and Bassett, A}, title = {Massively parallel characterization of CRISPR activator efficacy in human induced pluripotent stem cells and neurons.}, journal = {Molecular cell}, volume = {83}, number = {7}, pages = {1125-1139.e8}, doi = {10.1016/j.molcel.2023.02.011}, pmid = {36917981}, issn = {1097-4164}, mesh = {Humans ; *Induced Pluripotent Stem Cells ; Clustered Regularly Interspaced Short Palindromic Repeats ; CRISPR-Cas Systems ; Neurons ; Transcriptional Activation ; Chromatin/genetics ; }, abstract = {CRISPR activation (CRISPRa) is an important tool to perturb transcription, but its effectiveness varies between target genes. We employ human pluripotent stem cells with thousands of randomly integrated barcoded reporters to assess epigenetic features that influence CRISPRa efficacy. Basal expression levels are influenced by genomic context and dramatically change during differentiation to neurons. Gene activation by dCas9-VPR is successful in most genomic contexts, including developmentally repressed regions, and activation level is anti-correlated with basal gene expression, whereas dCas9-p300 is ineffective in stem cells. Certain chromatin states, such as bivalent chromatin, are particularly sensitive to dCas9-VPR, whereas constitutive heterochromatin is less responsive. We validate these rules at endogenous genes and show that activation of certain genes elicits a change in the stem cell transcriptome, sometimes showing features of differentiated cells. Our data provide rules to predict CRISPRa outcome and highlight its utility to screen for factors driving stem cell differentiation.}, }
@article {pmid36804635, year = {2023}, author = {Yan, AL and Du, SW and Palczewski, K}, title = {Genome editing, a superior therapy for inherited retinal diseases.}, journal = {Vision research}, volume = {206}, number = {}, pages = {108192}, doi = {10.1016/j.visres.2023.108192}, pmid = {36804635}, issn = {1878-5646}, mesh = {Humans ; *Gene Editing/methods ; CRISPR-Cas Systems/genetics ; Genetic Therapy/methods ; *Retinal Diseases/genetics/therapy ; Retina ; }, abstract = {Gene augmentation and genome editing are promising strategies for the treatment of monogenic inherited retinal diseases. Although gene augmentation treatments are commercially available for inherited retinal diseases, there are many shortcomings that need to be addressed, like progressive retinal degeneration and diminishing efficacy over time. Innovative CRISPR-Cas9-based genome editing technologies have broadened the proportion of treatable genetic disorders and can greatly improve or complement treatment outcomes from gene augmentation. Progress in this relatively new field involves the development of therapeutics including gene disruption, ablate-and-replace strategies, and precision gene correction techniques, such as base editing and prime editing. By making direct edits to endogenous DNA, genome editing theoretically guarantees permanent gene correction and long-lasting treatment effects. Improvements to delivery modalities aimed at limiting persistent gene editor activity have displayed an improved safety profile and minimal off-target editing. Continued progress to advance precise gene correction and associated delivery strategies will establish genome editing as the preferred treatment for genetic retinal disorders. This commentary describes the applications, strengths, and drawbacks of conventional gene augmentation approaches, recent advances in precise genome editing in the retina, and promising preclinical strategies to facilitate the use of robust genome editing therapies in human patients.}, }
@article {pmid36006888, year = {2023}, author = {Wan, Y and Jiang, Z}, title = {TransCrispr: Transformer Based Hybrid Model for Predicting CRISPR/Cas9 Single Guide RNA Cleavage Efficiency.}, journal = {IEEE/ACM transactions on computational biology and bioinformatics}, volume = {20}, number = {2}, pages = {1518-1528}, doi = {10.1109/TCBB.2022.3201631}, pmid = {36006888}, issn = {1557-9964}, mesh = {*CRISPR-Cas Systems/genetics ; *RNA, Guide, CRISPR-Cas Systems ; RNA, Guide, Kinetoplastida/genetics ; Gene Editing/methods ; Neural Networks, Computer ; }, abstract = {CRISPR/Cas9 is a widely used genome editing tool for site-directed modification of deoxyribonucleic acid (DNA) nucleotide sequences. However, how to accurately predict and evaluate the on- and off-target effects of single guide RNA (sgRNA) is one of the key problems for CRISPR/Cas9 system. Using computational methods to obtain high cell-specific sensitivity and specificity is a prerequisite for the optimal design of sgRNAs. Inspired by the work of predecessors, we found that sgRNA on-target knockout efficacy was not only related to the original sequence but also affected by important biological features. Hence, we introduce a novel approach called TransCrispr, which integrates Transformer and convolutional neural network (CNN) architecture to predict sgRNA knockout efficacy. Firstly, we encode the sequence data and send the transformed sgRNA sequence, positional information, and biological features into the network as input. Then, the convolutional neural network will automatically learn an appropriate feature representation for the sgRNA sequence and combine it with the positional information for self-attention learning of the Transformer. Finally, a regression score is generated by predicting biological features. Experiments on seven public datasets illustrate that TransCrispr outperforms state-of-the-art methods in terms of prediction accuracy and generalization ability.}, }
@article {pmid37031646, year = {2023}, author = {Mukherjee, S and Perveen, S and Negi, A and Sharma, R}, title = {Evolution of tuberculosis diagnostics: From molecular strategies to nanodiagnostics.}, journal = {Tuberculosis (Edinburgh, Scotland)}, volume = {140}, number = {}, pages = {102340}, pmid = {37031646}, issn = {1873-281X}, abstract = {Tuberculosis has remained a global concern for public health affecting the lives of people for ages. Approximately 10 million people are affected by the disease and 1.5 million succumb to the disease worldwide annually. The COVID-19 pandemic has highlighted the role of early diagnosis to win the battle against such infectious diseases. Thus, advancement in the diagnostic approaches to provide early detection forms the foundation to eradicate and manage contagious diseases like tuberculosis. The conventional diagnostic strategies include microscopic examination, chest X-ray and tuberculin skin test. The limitations associated with sensitivity and specificity of these tests demands for exploring new techniques like probe-based assays, CRISPR-Cas and microRNA detection. The aim of the current review is to envisage the correlation between both the conventional and the newer approaches to enhance the specificity and sensitivity. A significant emphasis has been placed upon nanodiagnostic approaches manipulating quantum dots, magnetic nanoparticles, and biosensors for accurate diagnosis of latent, active and drug-resistant TB. Additionally, we would like to ponder upon a reliable method that is cost-effective, reproducible, require minimal infrastructure and provide point-of-care to the patients.}, }
@article {pmid37030198, year = {2023}, author = {Mingarro, G and Del Olmo, ML}, title = {Corrigendum to "Improvements in the genetic editing technologies: CRISPR-Cas and beyond" [Gene 852 (2023) 147064].}, journal = {Gene}, volume = {870}, number = {}, pages = {147404}, doi = {10.1016/j.gene.2023.147404}, pmid = {37030198}, issn = {1879-0038}, }
@article {pmid37029313, year = {2023}, author = {Hassan, S and Ganai, BA}, title = {Deciphering the recent trends in pesticide bioremediation using genome editing and multi-omics approaches: a review.}, journal = {World journal of microbiology &amp; biotechnology}, volume = {39}, number = {6}, pages = {151}, pmid = {37029313}, issn = {1573-0972}, abstract = {Pesticide pollution in recent times has emerged as a grave environmental problem contaminating both aquatic and terrestrial ecosystems owing to their widespread use. Bioremediation using gene editing and system biology could be developed as an eco-friendly and proficient tool to remediate pesticide-contaminated sites due to its advantages and greater public acceptance over the physical and chemical methods. However, it is indispensable to understand the different aspects associated with microbial metabolism and their physiology for efficient pesticide remediation. Therefore, this review paper analyses the different gene editing tools and multi-omics methods in microbes to produce relevant evidence regarding genes, proteins and metabolites associated with pesticide remediation and the approaches to contend against pesticide-induced stress. We systematically discussed and analyzed the recent reports (2015-2022) on multi-omics methods for pesticide degradation to elucidate the mechanisms and the recent advances associated with the behaviour of microbes under diverse environmental conditions. This study envisages that CRISPR-Cas, ZFN and TALEN as gene editing tools utilizing Pseudomonas, Escherichia coli and Achromobacter sp. can be employed for remediation of chlorpyrifos, parathion-methyl, carbaryl, triphenyltin and triazophos by creating gRNA for expressing specific genes for the bioremediation. Similarly, systems biology accompanying multi-omics tactics revealed that microbial strains from Paenibacillus, Pseudomonas putida, Burkholderia cenocepacia, Rhodococcus sp. and Pencillium oxalicum are capable of degrading deltamethrin, p-nitrophenol, chlorimuron-ethyl and nicosulfuron. This review lends notable insights into the research gaps and provides potential solutions for pesticide remediation by using different microbe-assisted technologies. The inferences drawn from the current study will help researchers, ecologists, and decision-makers gain comprehensive knowledge of value and application of systems biology and gene editing in bioremediation assessments.}, }
@article {pmid37028423, year = {2023}, author = {Usluer, S and Hallast, P and Crepaldi, L and Zhou, Y and Urgo, K and Dincer, C and Su, J and Noell, G and Alasoo, K and El Garwany, O and Gerety, SS and Newman, B and Dovey, OM and Parts, L}, title = {Optimized whole-genome CRISPR interference screens identify ARID1A-dependent growth regulators in human induced pluripotent stem cells.}, journal = {Stem cell reports}, volume = {}, number = {}, pages = {}, doi = {10.1016/j.stemcr.2023.03.008}, pmid = {37028423}, issn = {2213-6711}, abstract = {Perturbing expression is a powerful way to understand the role of individual genes, but can be challenging in important models. CRISPR-Cas screens in human induced pluripotent stem cells (iPSCs) are of limited efficiency due to DNA break-induced stress, while the less stressful silencing with an inactive Cas9 has been considered less effective so far. Here, we developed the dCas9-KRAB-MeCP2 fusion protein for screening in iPSCs from multiple donors. We found silencing in a 200 bp window around the transcription start site in polyclonal pools to be as effective as using wild-type Cas9 for identifying essential genes, but with much reduced cell numbers. Whole-genome screens to identify ARID1A-dependent dosage sensitivity revealed the PSMB2 gene, and enrichment of proteasome genes among the hits. This selective dependency was replicated with a proteasome inhibitor, indicating a targetable drug-gene interaction. Many more plausible targets in challenging cell models can be efficiently identified with our approach.}, }
@article {pmid37028002, year = {2023}, author = {Zhu, Y and Wu, J and Zhou, Q}, title = {Functional DNA sensors integrated with nucleic acid signal amplification strategies for non-nucleic acid targets detection.}, journal = {Biosensors &amp; bioelectronics}, volume = {230}, number = {}, pages = {115282}, doi = {10.1016/j.bios.2023.115282}, pmid = {37028002}, issn = {1873-4235}, abstract = {In addition to carrying and transmitting genetic material, some DNA molecules have specific binding ability or catalytic function. DNA with this special function is collectively referred to as functional DNA (fDNA), such as aptamer, DNAzyme and so on. fDNA has the advantages of simple synthetic process, low cost and low toxicity. It also has high chemical stability, recognition specificity and biocompatibility. In recent years, fDNA biosensors have been widely investigated as signal recognition elements and signal transduction elements for the detection of non-nucleic acid targets. However, the main problem of fDNA sensors is their limited sensitivity to trace targets, especially when the affinity of fDNA to the targets is low. To further improve the sensitivity, various nucleic acid signal amplification strategies (NASAS) are explored to improve the limit of detection of fDNA. In this review, we will introduce four NASAS (hybridization chain reaction, entropy-driven catalysis, rolling circle amplification, CRISPR/Cas system) and the corresponding design principles. The principle and application of these fDNA sensors integrated with signal amplification strategies for detection of non-nucleic acid targets are summarized. Finally, the main challenges and application prospects of NASAS integrated fDNA biosensing system are discussed.}, }
@article {pmid37025992, year = {2023}, author = {Bandara, G and Falduto, GH and Luker, A and Bai, Y and Pfeiffer, A and Lack, J and Metcalfe, DD and Olivera, A}, title = {CRISPR/Cas9-engineering of HMC-1.2 cells renders a human mast cell line with a single D816V-KIT mutation: An improved preclinical model for research on mastocytosis.}, journal = {Frontiers in immunology}, volume = {14}, number = {}, pages = {1078958}, pmid = {37025992}, issn = {1664-3224}, mesh = {Humans ; Animals ; Mice ; *Mastocytosis, Systemic/drug therapy/genetics/pathology ; CRISPR-Cas Systems ; Proto-Oncogene Proteins c-kit/genetics/metabolism ; *Mastocytosis/genetics ; Mutation ; Cell Line ; }, abstract = {The HMC-1.2 human mast cell (huMC) line is often employed in the study of attributes of neoplastic huMCs as found in patients with mastocytosis and their sensitivity to interventional drugs in vitro and in vivo. HMC-1.2 cells express constitutively active KIT, an essential growth factor receptor for huMC survival and function, due to the presence of two oncogenic mutations (D816V and V560G). However, systemic mastocytosis is commonly associated with a single D816V-KIT mutation. The functional consequences of the coexisting KIT mutations in HMC-1.2 cells are unknown. We used CRISPR/Cas9-engineering to reverse the V560G mutation in HMC-1.2 cells, resulting in a subline (HMC-1.3) with a single mono-allelic D816V-KIT variant. Transcriptome analyses predicted reduced activity in pathways involved in survival, cell-to-cell adhesion, and neoplasia in HMC-1.3 compared to HMC-1.2 cells, with differences in expression of molecular components and cell surface markers. Consistently, subcutaneous inoculation of HMC-1.3 into mice produced significantly smaller tumors than HMC-1.2 cells, and in colony assays, HMC-1.3 formed less numerous and smaller colonies than HMC-1.2 cells. However, in liquid culture conditions, the growth of HMC-1.2 and HMC-1.3 cells was comparable. Phosphorylation levels of ERK1/2, AKT and STAT5, representing pathways associated with constitutive oncogenic KIT signaling, were also similar between HMC-1.2 and HMC-1.3 cells. Despite these similarities in liquid culture, survival of HMC-1.3 cells was diminished in response to various pharmacological inhibitors, including tyrosine kinase inhibitors used clinically for treatment of advanced systemic mastocytosis, and JAK2 and BCL2 inhibitors, making HMC-1.3 more susceptible to these drugs than HMC-1.2 cells. Our study thus reveals that the additional V560G-KIT oncogenic variant in HMC-1.2 cells modifies transcriptional programs induced by D816V-KIT, confers a survival advantage, alters sensitivity to interventional drugs, and increases the tumorigenicity, suggesting that engineered huMCs with a single D816V-KIT variant may represent an improved preclinical model for mastocytosis.}, }
@article {pmid37021953, year = {2023}, author = {Wu, SZ and Ryken, SE and Bezanilla, M}, title = {CRISPR-Cas9 Genome Editing in the Moss Physcomitrium (Formerly Physcomitrella) patens.}, journal = {Current protocols}, volume = {3}, number = {4}, pages = {e725}, doi = {10.1002/cpz1.725}, pmid = {37021953}, issn = {2691-1299}, mesh = {*Gene Editing/methods ; CRISPR-Cas Systems ; *Bryopsida/genetics ; Homologous Recombination ; Plasmids ; }, abstract = {Until recently, precise genome editing has been limited to a few organisms. The ability of Cas9 to generate double stranded DNA breaks at specific genomic sites has greatly expanded molecular toolkits in many organisms and cell types. Before CRISPR-Cas9 mediated genome editing, P. patens was unique among plants in its ability to integrate DNA via homologous recombination. However, selection for homologous recombination events was required to obtain edited plants, limiting the types of editing that were possible. Now with CRISPR-Cas9, molecular manipulations in P. patens have greatly expanded. This protocol describes a method to generate a variety of different genome edits. The protocol describes a streamlined method to generate the Cas9/sgRNA expression constructs, design homology templates, transform, and quickly genotype plants. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Constructing the Cas9/sgRNA transient expression vector Alternate Protocol 1: Shortcut to generating single and pooled Cas9/sgRNA expression vectors Basic Protocol 2: Designing the oligonucleotide-based homology-directed repair (HDR) template Alternate Protocol 2: Designing the plasmid-based HDR template Basic Protocol 3: Inducing genome editing by transforming CRISPR vector into P. patens protoplasts Basic Protocol 4: Identifying edited plants.}, }
@article {pmid36793210, year = {2023}, author = {Cromer, MK and Majeti, KR and Rettig, GR and Murugan, K and Kurgan, GL and Bode, NM and Hampton, JP and Vakulskas, CA and Behlke, MA and Porteus, MH}, title = {Comparative analysis of CRISPR off-target discovery tools following ex vivo editing of CD34[+] hematopoietic stem and progenitor cells.}, journal = {Molecular therapy : the journal of the American Society of Gene Therapy}, volume = {31}, number = {4}, pages = {1074-1087}, doi = {10.1016/j.ymthe.2023.02.011}, pmid = {36793210}, issn = {1525-0024}, mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; CRISPR-Associated Protein 9/genetics ; RNA, Guide, Kinetoplastida/genetics ; Hematopoietic Stem Cells/metabolism ; }, abstract = {While a number of methods exist to investigate CRISPR off-target (OT) editing, few have been compared head-to-head in primary cells after clinically relevant editing processes. Therefore, we compared in silico tools (COSMID, CCTop, and Cas-OFFinder) and empirical methods (CHANGE-Seq, CIRCLE-Seq, DISCOVER-Seq, GUIDE-Seq, and SITE-Seq) after ex vivo hematopoietic stem and progenitor cell (HSPC) editing. We performed editing using 11 different gRNAs complexed with Cas9 protein (high-fidelity [HiFi] or wild-type versions), then performed targeted next-generation sequencing of nominated OT sites identified by in silico and empirical methods. We identified an average of less than one OT site per guide RNA (gRNA) and all OT sites generated using HiFi Cas9 and a 20-nt gRNA were identified by all OT detection methods with the exception of SITE-seq. This resulted in high sensitivity for the majority of OT nomination tools and COSMID, DISCOVER-Seq, and GUIDE-Seq attained the highest positive predictive value (PPV). We found that empirical methods did not identify OT sites that were not also identified by bioinformatic methods. This study supports that refined bioinformatic algorithms could be developed that maintain both high sensitivity and PPV, thereby enabling more efficient identification of potential OT sites without compromising a thorough examination for any given gRNA.}, }
@article {pmid36793209, year = {2023}, author = {Yu, SY and Carlaw, T and Thomson, T and Birkenshaw, A and Basha, G and Kurek, D and Huang, C and Kulkarni, J and Zhang, LH and Ross, CJD}, title = {A luciferase reporter mouse model to optimize in vivo gene editing validated by lipid nanoparticle delivery of adenine base editors.}, journal = {Molecular therapy : the journal of the American Society of Gene Therapy}, volume = {31}, number = {4}, pages = {1159-1166}, doi = {10.1016/j.ymthe.2023.02.009}, pmid = {36793209}, issn = {1525-0024}, mesh = {Mice ; Animals ; *Gene Editing/methods ; *CRISPR-Cas Systems ; Adenine ; Disease Models, Animal ; Luciferases/genetics ; }, abstract = {The rapid development of CRISPR genome editing technology has provided the potential to treat genetic diseases effectively and precisely. However, efficient and safe delivery of genome editors to affected tissues remains a challenge. Here, we developed luminescent ABE (LumA), a luciferase reporter mouse model containing the R387X mutation (c.A1159T) in the luciferase gene located in the Rosa26 locus of the mouse genome. This mutation eliminates luciferase activity but can be restored upon A-to-G correction by SpCas9 adenine base editors (ABEs). The LumA mouse model was validated through intravenous injection of two FDA-approved lipid nanoparticle (LNP) formulations consisting of either MC3 or ALC-0315 ionizable cationic lipids, encapsulated with ABE mRNA and LucR387X-specific guide RNA (gRNA). Whole-body bioluminescence live imaging showed consistent restoration of luminescence lasting up to 4 months in treated mice. Compared with mice carrying the wild-type luciferase gene, the ALC-0315 and MC3 LNP groups showed 83.5% ± 17.5% and 8.4% ± 4.3% restoration of luciferase activity in the liver, respectively, as measured by tissue luciferase assays. These results demonstrated successful development of a luciferase reporter mouse model that can be used to evaluate the efficacy and safety of different genome editors, LNP formulations, and tissue-specific delivery systems for optimizing genome editing therapeutics.}, }
@article {pmid36755493, year = {2023}, author = {Tiukacheva, EA and Ulianov, SV and Karpukhina, A and Razin, SV and Vassetzky, Y}, title = {3D genome alterations and editing in pathology.}, journal = {Molecular therapy : the journal of the American Society of Gene Therapy}, volume = {31}, number = {4}, pages = {924-933}, doi = {10.1016/j.ymthe.2023.02.005}, pmid = {36755493}, issn = {1525-0024}, mesh = {Humans ; *Gene Editing ; Genome, Human ; Cell Nucleus ; *Neoplasms/genetics/therapy ; CRISPR-Cas Systems ; }, abstract = {The human genome is folded into a multi-level 3D structure that controls many nuclear functions including gene expression. Recently, alterations in 3D genome organization were associated with several genetic diseases and cancer. As a consequence, experimental approaches are now being developed to modify the global 3D genome organization and that of specific loci. Here, we discuss emerging experimental approaches of 3D genome editing that may prove useful in biomedicine.}, }
@article {pmid36733252, year = {2023}, author = {Li, B and Zhao, D and Li, Y and Yang, Y and Zhu, X and Li, J and Bi, C and Zhang, X}, title = {Obtaining the best igRNAs for bystander-less correction of all ABE-reversible pathogenic SNVs using high-throughput screening.}, journal = {Molecular therapy : the journal of the American Society of Gene Therapy}, volume = {31}, number = {4}, pages = {1167-1176}, doi = {10.1016/j.ymthe.2023.01.028}, pmid = {36733252}, issn = {1525-0024}, mesh = {*Gene Editing ; *Adenine Nucleotides ; High-Throughput Screening Assays ; CRISPR-Cas Systems ; }, abstract = {Imperfect -gRNA (igRNA) provides a simple strategy for single-base editing of a base editor. However, a significant number of igRNAs need to be generated and tested for each target locus to achieve efficient single-base reversion of pathogenic single nucleotide variations (SNVs), which hinders the direct application of this technology. To provide ready-to-use igRNAs for single-base and bystander-less correction of all the adenine base editor (ABE)-reversible pathogenic SNVs, we employed a high-throughput method to edit all 5,253 known ABE-reversible pathogenic SNVs, each with multiple systematically designed igRNAs, and two libraries of 96,000 igRNAs were tested. A total of 1,988 SNV loci could be single-base reversed by igRNA with a >30% efficiency. Among these 1,988 loci, 378 SNV loci exhibited an efficiency of more than 90%. At the same time, the bystander editing efficiency of 76.62% of the SNV loci was reduced to 0%, while remaining below 1% for another 18.93% of the loci. These ready-to-use igRNAs provided the best solutions for a substantial portion of the 4,657 pathogenic/likely pathogenic SNVs. In this work, we overcame one of the most significant obstacles of base editors and provide a ready-to-use platform for the genetic treatment of diseases caused by ABE-reversible SNVs.}, }
@article {pmid36733251, year = {2023}, author = {Gao, S and Wang, Y and Qi, T and Wei, J and Hu, Z and Liu, J and Sun, S and Liu, H and Wang, Y}, title = {Genome editing with natural and engineered CjCas9 orthologs.}, journal = {Molecular therapy : the journal of the American Society of Gene Therapy}, volume = {31}, number = {4}, pages = {1177-1187}, doi = {10.1016/j.ymthe.2023.01.029}, pmid = {36733251}, issn = {1525-0024}, mesh = {Animals ; Swine ; *Gene Editing ; *CRISPR-Cas Systems ; Mutation ; DNA/genetics ; Genome ; Mammals ; }, abstract = {CjCas9 is one of the smallest CRISPR-associated (Cas9) nucleases for mammalian genome editing. However, it requires a long N4RYAC (R = A or G; Y = C or T) protospacer-adjacent motif (PAM), limiting its DNA targeting scope. In this study, we investigated the PAMs of three CjCas9 orthologs, including Hsp1Cas9, Hsp2Cas9, and CcuCas9, by performing a GFP-activation assay. Interestingly, Hsp1Cas9 and CcuCas9 recognized unique N4RAA and N4CNA PAMs, respectively. We further generated an Hsp1Cas9-Hsp2Cas9 chimeric Cas9 (Hsp1-Hsp2Cas9), which recognized a simple N4CY PAM. Genome-wide off-target analysis revealed that Hsp1-Hsp2Cas9 has very few off-targets compared to SpCas9. By analyzing the crystal structure of CjCas9, we identified eight mutations that can improve the specificity and generate a high-fidelity Hsp1-Hsp2Cas9-Y. Hsp1-Hsp2Cas9-Y enables the knockout of B4GALNT2 and CMAH in porcine fetal fibroblasts (PFFs). Moreover, we developed a high-fidelity Hsp1-Hsp2Cas9-KY which displayed undetectable off-targets revealed by GUIDE-seq at four tested loci. These natural and engineered Cas9 nucleases enabled efficient genome editing in multiple mammalian cells, expanding the DNA targeting scope.}, }
@article {pmid37024653, year = {2023}, author = {Zou, RS and Liu, Y and Gaido, OER and Konig, MF and Mog, BJ and Shen, LL and Aviles-Vazquez, F and Marin-Gonzalez, A and Ha, T}, title = {Improving the sensitivity of in vivo CRISPR off-target detection with DISCOVER-Seq.}, journal = {Nature methods}, volume = {}, number = {}, pages = {}, pmid = {37024653}, issn = {1548-7105}, abstract = {Discovery of off-target CRISPR-Cas activity in patient-derived cells and animal models is crucial for genome editing applications, but currently exhibits low sensitivity. We demonstrate that inhibition of DNA-dependent protein kinase catalytic subunit accumulates the repair protein MRE11 at CRISPR-Cas-targeted sites, enabling high-sensitivity mapping of off-target sites to positions of MRE11 binding using chromatin immunoprecipitation followed by sequencing. This technique, termed DISCOVER-Seq+, discovered up to fivefold more CRISPR off-target sites in immortalized cell lines, primary human cells and mice compared with previous methods. We demonstrate applicability to ex vivo knock-in of a cancer-directed transgenic T cell receptor in primary human T cells and in vivo adenovirus knock-out of cardiovascular risk gene PCSK9 in mice. Thus, DISCOVER-Seq+ is, to our knowledge, the most sensitive method to-date for discovering off-target genome editing in vivo.}, }
@article {pmid37024652, year = {2023}, author = {Jiménez, C and Crosetto, N}, title = {Discovering CRISPR-Cas off-target breaks.}, journal = {Nature methods}, volume = {}, number = {}, pages = {}, pmid = {37024652}, issn = {1548-7105}, }
@article {pmid37022538, year = {2023}, author = {Sufyan, M and Daraz, U and Hyder, S and Zulfiqar, U and Iqbal, R and Eldin, SM and Rafiq, F and Mahmood, N and Shahzad, K and Uzair, M and Fiaz, S and Ali, I}, title = {An overview of genome engineering in plants, including its scope, technologies, progress and grand challenges.}, journal = {Functional &amp; integrative genomics}, volume = {23}, number = {2}, pages = {119}, pmid = {37022538}, issn = {1438-7948}, abstract = {Genome editing is a useful, adaptable, and favored technique for both functional genomics and crop enhancement. Over the years, rapidly evolving genome editing technologies, including clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas), transcription activator-like effector nucleases (TALENs), and zinc finger nucleases (ZFNs), have shown broad application prospects in gene function research and improvement of critical agronomic traits in many crops. These technologies have also opened up opportunities for plant breeding. These techniques provide excellent chances for the quick modification of crops and the advancement of plant science in the future. The current review describes various genome editing techniques and how they function, particularly CRISPR/Cas9 systems, which can contribute significantly to the most accurate characterization of genomic rearrangement and plant gene functions as well as the enhancement of critical traits in field crops. To accelerate the use of gene-editing technologies for crop enhancement, the speed editing strategy of gene-family members was designed. As it permits genome editing in numerous biological systems, the CRISPR technology provides a valuable edge in this regard that particularly captures the attention of scientists.}, }
@article {pmid37022199, year = {2023}, author = {Liu, ZL and Hu, EZ and Niu, DK}, title = {Investigating the Relationship between CRISPR-Cas Content and Growth Rate in Bacteria.}, journal = {Microbiology spectrum}, volume = {}, number = {}, pages = {e0340922}, doi = {10.1128/spectrum.03409-22}, pmid = {37022199}, issn = {2165-0497}, abstract = {CRISPR-Cas systems provide adaptive immunity for prokaryotic cells by recognizing and eliminating the recurrent genetic invaders whose sequences had been captured in a prior infection and stored in the CRISPR arrays as spacers. However, the biological/environmental factors determining the efficiency of this immune system have yet to be fully characterized. Recent studies in cultured bacteria showed that slowing the growth rate of bacterial cells could promote their acquisition of novel spacers. This study examined the relationship between the CRISPR-Cas content and the minimal doubling time across the bacteria and the archaea domains. Every completely sequenced genome could be used to predict a minimal doubling time. With a large data set of 4,142 bacterial samples, we found that the predicted minimal doubling times are positively correlated with spacer number and other parameters of the CRISPR-Cas systems, like array number, Cas gene cluster number, and Cas gene number. Different data sets gave different results. Weak results were obtained in analyzing bacterial empirical minimal doubling times and the archaea domain. Still, the conclusion of more spacers in slowly grown prokaryotes was supported. In addition, we found that the minimal doubling times are negatively correlated with the occurrence of prophages, and the spacer numbers per array are negatively associated with the number of prophages. These observations support the existence of an evolutionary trade-off between bacterial growth and adaptive defense against virulent phages. IMPORTANCE Accumulating evidence indicates that slowing the growth of cultured bacteria could stimulate their CRISPR spacer acquisition. We observed a positive correlation between CRISPR-Cas content and cell cycle duration across the bacteria domain. This observation extends the physiological conclusion to an evolutionary one. In addition, the correlation provides evidence supporting the existence of a trade-off between bacterial growth/reproduction and antiviral resistance.}, }
@article {pmid35640601, year = {2022}, author = {Nguyen, T and Ramachandran, H and Martins, S and Krutmann, J and Rossi, A}, title = {Identification of genome edited cells using CRISPRnano.}, journal = {Nucleic acids research}, volume = {50}, number = {W1}, pages = {W199-W203}, pmid = {35640601}, issn = {1362-4962}, mesh = {*Genome/genetics ; *High-Throughput Nucleotide Sequencing/methods ; *Nanopores ; *Sequence Analysis, DNA/methods ; *Software ; Whole Genome Sequencing ; *Gene Editing/methods ; *CRISPR-Cas Systems ; Workflow ; Mutagenesis, Site-Directed ; Internet ; Computers ; Data Visualization ; Time Factors ; Cell Line ; }, abstract = {Genome engineering-induced cleavage sites can be resolved by non-homologous end joining (NHEJ) or homology-directed repair (HDR). Identifying genetically modified clones at the target locus remains an intensive and laborious task. Different workflows and software that rely on deep sequencing data have been developed to detect and quantify targeted mutagenesis. Nevertheless, these pipelines require high-quality reads generated by Next Generation Sequencing (NGS) platforms. Here, we have developed a robust, versatile, and easy-to-use computational webserver named CRISPRnano (www.CRISPRnano.de) that enables the analysis of low-quality reads generated by affordable and portable sequencers including Oxford Nanopore Technologies (ONT) devices. CRISPRnano allows fast and accurate identification, quantification, and visualization of genetically modified cell lines, it is compatible with NGS and ONT sequencing reads, and it can be used without an internet connection.}, }
@article {pmid37020597, year = {2023}, author = {Meng, H and Nan, M and Li, Y and Ding, Y and Yin, Y and Zhang, M}, title = {Application of CRISPR-Cas9 gene editing technology in basic research, diagnosis and treatment of colon cancer.}, journal = {Frontiers in endocrinology}, volume = {14}, number = {}, pages = {1148412}, pmid = {37020597}, issn = {1664-2392}, mesh = {Humans ; *Gene Editing ; CRISPR-Cas Systems ; Gene Expression Regulation ; *Colonic Neoplasms ; Technology ; }, abstract = {Colon cancer is the fourth leading cause of cancer death worldwide, and its progression is accompanied by a complex array of genetic variations. CRISPR/Cas9 can identify new drug-resistant or sensitive mutations in colon cancer, and can use gene editing technology to develop new therapeutic targets and provide personalized treatments, thereby significantly improving the treatment of colon cancer patients. CRISPR/Cas9 systems are driving advances in biotechnology. RNA-directed Cas enzymes have accelerated the pace of basic research and led to clinical breakthroughs. This article reviews the rapid development of CRISPR/Cas in colon cancer, from gene editing to transcription regulation, gene knockout, genome-wide CRISPR tools, therapeutic targets, stem cell genomics, immunotherapy, metabolism-related genes and inflammatory bowel disease. In addition, the limitations and future development of CRISPR/Cas9 in colon cancer studies are reviewed. In conclusion, this article reviews the application of CRISPR-Cas9 gene editing technology in basic research, diagnosis and treatment of colon cancer.}, }
@article {pmid37020072, year = {2023}, author = {Liu, Z and Quan, L and Ma, F and Yang, M and Jiang, X and Chen, X}, title = {Determination of adenosine by CRISPR-Cas12a system based on duplexed aptamer and molecular beacon reporter linked to gold nanoparticles.}, journal = {Mikrochimica acta}, volume = {190}, number = {5}, pages = {173}, pmid = {37020072}, issn = {1436-5073}, mesh = {Adenosine ; Gold ; CRISPR-Cas Systems ; *Metal Nanoparticles ; *Aptamers, Nucleotide ; DNA, Single-Stranded ; }, abstract = {Adenosine as a potential tumor marker is of great value for clinical disease diagnosis. Since the CRISPR-cas12a system is only capable of recognizing nucleic acid targets we expanded the CRISPR-cas12a system to determine small molecules by designing a duplexed aptamer (DA) converting g-RNA recognition of adenosine to recognition of aptamer complementary DNA strands (ACD). To further improve the sensitivity of determination, we designed a molecule beacon (MB)/gold nanoparticle (AuNP)-based reporter, which has higher sensitivity than traditional ssDNA reporter. In addition, the AuNP-based reporter enables more efficient and fast determination. The determination of adenosine under 488-nm excitation can be realized within 7 min, which is more than 4 times faster than traditional ssDNA reporter. The linear determination range of the assay to adenosine was 0.5-100 μM with the determination limit of 15.67 nM. The assay was applied to recovery determination of adenosine in serum samples with satisfactory results. The recoveries were between 91 and 106% and the RSD values of different concertation were below 4.8%. This sensitive, highly selective, and stable sensing system is expected to play a role in the clinical determination of adenosine and other biomolecules.}, }
@article {pmid37018323, year = {2023}, author = {Bridgeland, A and Biswas, S and Tsakirpaloglou, N and Thomson, MJ and Septiningsih, EM}, title = {Optimization of gene editing in cowpea through protoplast transformation and agroinfiltration by targeting the phytoene desaturase gene.}, journal = {PloS one}, volume = {18}, number = {4}, pages = {e0283837}, pmid = {37018323}, issn = {1932-6203}, mesh = {*Gene Editing/methods ; *Vigna/genetics ; CRISPR-Cas Systems ; Protoplasts/metabolism ; }, abstract = {Cowpea (Vigna unguiculata) is a legume staple widely grown across Sub-Saharan Africa and other tropical and sub-tropical regions. Considering projected climate change and global population increases, cowpea's adaptation to hot climates, resistance to drought, and nitrogen-fixing capabilities make it an especially attractive crop for facing future challenges. Despite these beneficial traits, efficient varietal improvement is challenging in cowpea due to its recalcitrance to transformation and long regeneration times. Transient gene expression assays can provide solutions to alleviate these issues as they allow researchers to test gene editing constructs before investing in the time and resource- intensive process of transformation. In this study, we developed an improved cowpea protoplast isolation protocol, a transient protoplast assay, and an agroinfiltration assay to be used for initial testing and validation of gene editing constructs and for gene expression studies. To test these protocols, we assessed the efficacy of a CRISPR-Cas9 construct containing four multiplexed single-guide RNA (sgRNA) sequences using polyethylene glycol (PEG)-mediated transformation and agroinfiltration with phytoene desaturase (PDS) as the target gene. Sanger sequencing of DNA from transformed protoplasts and agroinfiltrated cowpea leaves revealed several large deletions in the target sequences. The protoplast system and agroinfiltration protocol developed in this study provide versatile tools to test gene editing components before initiating plant transformation, thus improving the chance of using active sgRNAs and attaining the desired edits and target phenotype.}, }
@article {pmid36964239, year = {2023}, author = {}, title = {Gene editing holds promise for babies with deadly immune disease.}, journal = {Nature}, volume = {616}, number = {7955}, pages = {11}, pmid = {36964239}, issn = {1476-4687}, mesh = {*Gene Editing ; *CRISPR-Cas Systems ; Genetic Therapy ; }, }
@article {pmid37020030, year = {2023}, author = {Nakagawa, R and Hirano, H and Omura, SN and Nety, S and Kannan, S and Altae-Tran, H and Yao, X and Sakaguchi, Y and Ohira, T and Wu, WY and Nakayama, H and Shuto, Y and Tanaka, T and Sano, FK and Kusakizako, T and Kise, Y and Itoh, Y and Dohmae, N and van der Oost, J and Suzuki, T and Zhang, F and Nureki, O}, title = {Cryo-EM structure of the transposon-associated TnpB enzyme.}, journal = {Nature}, volume = {}, number = {}, pages = {}, pmid = {37020030}, issn = {1476-4687}, abstract = {The class 2 type V CRISPR effector Cas12 is thought to have evolved from the IS200/IS605 superfamily of transposon-associated TnpB proteins[1]. Recent studies have identified TnpB proteins as miniature RNA-guided DNA endonucleases[2,3]. TnpB associates with a single, long RNA (ωRNA) and cleaves double-stranded DNA targets complementary to the ωRNA guide. However, the RNA-guided DNA cleavage mechanism of TnpB and its evolutionary relationship with Cas12 enzymes remain unknown. Here we report the cryo-electron microscopy (cryo-EM) structure of Deinococcus radiodurans ISDra2 TnpB in complex with its cognate ωRNA and target DNA. In the structure, the ωRNA adopts an unexpected architecture and forms a pseudoknot, which is conserved among all guide RNAs of Cas12 enzymes. Furthermore, the structure, along with our functional analysis, reveals how the compact TnpB recognizes the ωRNA and cleaves target DNA complementary to the guide. A structural comparison of TnpB with Cas12 enzymes suggests that CRISPR-Cas12 effectors acquired an ability to recognize the protospacer-adjacent motif-distal end of the guide RNA-target DNA heteroduplex, by either asymmetric dimer formation or diverse REC2 insertions, enabling engagement in CRISPR-Cas adaptive immunity. Collectively, our findings provide mechanistic insights into TnpB function and advance our understanding of the evolution from transposon-encoded TnpB proteins to CRISPR-Cas12 effectors.}, }
@article {pmid37018843, year = {2023}, author = {Rallapalli, KL and Komor, AC}, title = {The Design and Application of DNA-Editing Enzymes as Base Editors.}, journal = {Annual review of biochemistry}, volume = {}, number = {}, pages = {}, doi = {10.1146/annurev-biochem-052521-013938}, pmid = {37018843}, issn = {1545-4509}, abstract = {DNA-editing enzymes perform chemical reactions on DNA nucleobases. These reactions can change the genetic identity of the modified base or modulate gene expression. Interest in DNA-editing enzymes has burgeoned in recent years due to the advent of clustered regularly interspaced short palindromic repeat-associated (CRISPR-Cas) systems, which can be used to direct their DNA-editing activity to specific genomic loci of interest. In this review, we showcase DNA-editing enzymes that have been repurposed or redesigned and developed into programmable base editors. These include deaminases, glycosylases, methyltransferases, and demethylases. We highlight the astounding degree to which these enzymes have been redesigned, evolved, and refined and present these collective engineering efforts as a paragon for future efforts to repurpose and engineer other families of enzymes. Collectively, base editors derived from these DNA-editing enzymes facilitate programmable point mutation introduction and gene expression modulation by targeted chemical modification of nucleobases. Expected final online publication date for the Annual Review of Biochemistry, Volume 92 is June 2023. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.}, }
@article {pmid37018798, year = {2023}, author = {Tian, T and Zhou, X}, title = {CRISPR-Based Biosensing Strategies: Technical Development and Application Prospects.}, journal = {Annual review of analytical chemistry (Palo Alto, Calif.)}, volume = {}, number = {}, pages = {}, doi = {10.1146/annurev-anchem-090822-014725}, pmid = {37018798}, issn = {1936-1335}, abstract = {Biosensing based on CRISPR-Cas systems is a young but rapidly evolving technology. The unprecedented properties of the CRISPR-Cas system provide an innovative tool for developing new-generation biosensing strategies. To date, a series of nucleic acid and non-nucleic acid detection methods have been developed based on the CRISPR platform. In this review, we first introduce the core biochemical properties underpinning the development of CRISPR bioassays, such as diverse reaction temperatures, programmability in design, high reaction efficiency, and recognition specificity, and highlight recent efforts to improve these parameters. We then introduce the technical developments, including how to improve sensitivity and quantification capabilities, develop multiplex assays, achieve convenient one-pot assays, create advanced sensors, and extend the applications of detection. Finally, we analyze obstacles to the commercial application of CRISPR detection technology and explore development opportunities and directions. Expected final online publication date for the Annual Review of Analytical Chemistry, Volume 16 is June 2023. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.}, }
@article {pmid37018035, year = {2023}, author = {Muzahid, NH and Hussain, MH and Huët, MAL and Dwiyanto, J and Su, TT and Reidpath, D and Mustapha, F and Ayub, Q and Tan, HS and Rahman, S}, title = {Molecular characterization and comparative genomic analysis of Acinetobacter baumannii isolated from the community and the hospital: an epidemiological study in Segamat, Malaysia.}, journal = {Microbial genomics}, volume = {9}, number = {4}, pages = {}, doi = {10.1099/mgen.0.000977}, pmid = {37018035}, issn = {2057-5858}, abstract = {Acinetobacter baumannii is a common cause of multidrug-resistant (MDR) nosocomial infections around the world. However, little is known about the persistence and dynamics of A. baumannii in a healthy community. This study investigated the role of the community as a prospective reservoir for A. baumannii and explored possible links between hospital and community isolates. A total of 12 independent A. baumannii strains were isolated from human faecal samples from the community in Segamat, Malaysia, in 2018 and 2019. Another 15 were obtained in 2020 from patients at the co-located tertiary public hospital. The antimicrobial resistance profile and biofilm formation ability were analysed, and the relatedness of community and hospital isolates was determined using whole-genome sequencing (WGS). Antibiotic profile analysis revealed that 12 out of 15 hospital isolates were MDR, but none of the community isolates were MDR. However, phylogenetic analysis based on single-nucleotide polymorphisms (SNPs) and a pangenome analysis of core genes showed clustering between four community and two hospital strains. Such clustering of strains from two different settings based on their genomes suggests that these strains could persist in both. WGS revealed 41 potential resistance genes on average in the hospital strains, but fewer (n=32) were detected in the community strains. In contrast, 68 virulence genes were commonly seen in strains from both sources. This study highlights the possible transmission threat to public health posed by virulent A. baumannii present in the gut of asymptomatic individuals in the community.}, }
@article {pmid37010073, year = {2023}, author = {Liang, J and Tan, Y}, title = {Highly efficient CRISPR-mediated base editing for the gut Bacteroides spp. with pnCasBS-CBE.}, journal = {Biotechnology journal}, volume = {}, number = {}, pages = {e2200504}, doi = {10.1002/biot.202200504}, pmid = {37010073}, issn = {1860-7314}, abstract = {Bacteroidales are the most abundant order of bacteria in the healthy human gut and have the potential as a therapeutic agent. We constructed a pnCasBS-CBE system for base editing in the Bacteroides thetaiotaomicron to expand their genetic toolkit, which is able to efficiently convert a C:G to a T:A in the genome. As a functional proof-of-concept, we used the pnCasBS-CBE system to successfully introduce nonsynonymous mutation and stop codons to the genes involved in carbohydrate metabolism. The system also allowed for multiplexed gene editing with a single plasmid, enabling efficient editing of up to four genes in a single experiment. Furthermore, the pnCasBS-CBE editing system was validated and successfully applied in four other non-model gut Bacteroides species for genome editing. An unbiased genome-wide SNPs analysis indicated that the pnCasBS-CBE system showed high fidelity and applicability. Thus, this study provides a powerful CRISPR-mediated genome editing toolbox for functional genomic analysis in Bacteroidales. This article is protected by copyright. All rights reserved.}, }
@article {pmid36993517, year = {2023}, author = {Lampe, GD and King, RT and Halpin-Healy, TS and Klompe, SE and Hogan, MI and Vo, PLH and Tang, S and Chavez, A and Sternberg, SH}, title = {Targeted DNA integration in human cells without double-strand breaks using CRISPR RNA-guided transposases.}, journal = {bioRxiv : the preprint server for biology}, volume = {}, number = {}, pages = {}, pmid = {36993517}, abstract = {Traditional genome-editing reagents such as CRISPR-Cas9 achieve targeted DNA modification by introducing double-strand breaks (DSBs), thereby stimulating localized DNA repair by endogenous cellular repair factors. While highly effective at generating heterogenous knockout mutations, this approach suffers from undesirable byproducts and an inability to control product purity. Here we develop a system in human cells for programmable, DSB-free DNA integration using Type I CRISPR-associated transposons (CASTs). To adapt our previously described CAST systems, we optimized DNA targeting by the QCascade complex through a comprehensive assessment of protein design, and we developed potent transcriptional activators by exploiting the multi-valent recruitment of the AAA+ ATPase, TnsC, to genomic sites targeted by QCascade. After initial detection of plasmid-based transposition, we screened 15 homologous CAST systems from a wide range of bacterial hosts, identified a CAST homolog from Pseudoalteromonas that exhibited improved activity, and increased integration efficiencies through parameter optimization. We further discovered that bacterial ClpX enhances genomic integration by multiple orders of magnitude, and we propose that this critical accessory factor functions to drive active disassembly of the post-transposition CAST complex, akin to its demonstrated role in Mu transposition. Our work highlights the ability to functionally reconstitute complex, multi-component machineries in human cells, and establishes a strong foundation to realize the full potential of CRISPR-associated transposons for human genome engineering.}, }
@article {pmid36737854, year = {2023}, author = {Lei, H and Zeng, T and Ye, X and Fan, R and Xiong, W and Tian, T and Zhou, X}, title = {Chemical Control of CRISPR Gene Editing via Conditional Diacylation Crosslinking of Guide RNAs.}, journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)}, volume = {10}, number = {10}, pages = {e2206433}, pmid = {36737854}, issn = {2198-3844}, mesh = {*Gene Editing ; *CRISPR-Cas Systems/genetics ; RNA, Guide, Kinetoplastida/genetics ; }, abstract = {Conditional control of RNA structure and function has emerged as an effective toolkit. Here, a strategy based on a one-step introduction of diacylation linkers and azide groups on the 2'-OH of RNA is advance. Selected from eight phosphine reagents, it is found that 2-(diphenylphosphino)ethylamine has excellent performance in reducing azides via a Staudinger reduction to obtain the original RNA. It is demonstrated that the enzymatic activities of Cas13 and Cas9 can be regulated by chemically modified guide RNAs, and further achieved ligand-induced gene editing in living cells by a controllable CRISPR/Cas9 system.}, }
@article {pmid36378073, year = {2023}, author = {Kobayashi, R and Kawabata-Iwakawa, R and Sugiyama, M and Oyama, T and Ohtsuka, M and Horii, T and Morita, S and Nishiyama, M and Hatada, I}, title = {Multiplexed genome editing by in vivo electroporation of Cas9 ribonucleoproteins effectively induces endometrial carcinoma in mice.}, journal = {International journal of cancer}, volume = {152}, number = {11}, pages = {2331-2337}, doi = {10.1002/ijc.34342}, pmid = {36378073}, issn = {1097-0215}, mesh = {Mice ; Female ; Humans ; Animals ; *Gene Editing/methods ; CRISPR-Cas Systems ; Ribonucleoproteins/genetics ; Electroporation/methods ; *Endometrial Neoplasms/genetics ; }, abstract = {Synergistic effects among multiple gene mutations are involved in cancer development and progression. However, developing genetically modified mouse models to analyze various combinations of mutations is extremely labor-intensive and time-consuming. To address these problems, we developed a novel method for in vivo multiplexed genome editing of the murine uterus to model human endometrial carcinoma (EMC). To do this, we injected a CRISPR-Cas9 ribonucleoprotein complex into the uterine cavity of adult female mice, followed by electroporation. Evaluation of reporter mice demonstrated that genome editing occurred specifically in uterine epithelial cells, which are the origin of EMCs. Simultaneous targeting of Pten/Trp53/Lkb1, or targeting of Pten/Lkb1 along with the Ctnnb1ΔEx3 mutation, resulted in efficient generation of invasive tumors in wild-type females within 3 months. This novel method will enable rapid and easy validation of many combinations of gene mutations that lead to endometrial carcinogenesis.}, }
@article {pmid37009498, year = {2023}, author = {Pan, Y and Xia, H and He, Y and Zeng, S and Shen, Z and Huang, W}, title = {The progress of molecules and strategies for the treatment of HBV infection.}, journal = {Frontiers in cellular and infection microbiology}, volume = {13}, number = {}, pages = {1128807}, pmid = {37009498}, issn = {2235-2988}, mesh = {Humans ; *Hepatitis B virus/physiology ; Virus Replication ; *Hepatitis B/drug therapy ; Interferon-alpha/pharmacology ; Antiviral Agents/pharmacology/therapeutic use/metabolism ; DNA, Circular/metabolism/pharmacology/therapeutic use ; DNA, Viral/genetics ; }, abstract = {Hepatitis B virus infections have always been associated with high levels of mortality. In 2019, hepatitis B virus (HBV)-related diseases resulted in approximately 555,000 deaths globally. In view of its high lethality, the treatment of HBV infections has always presented a huge challenge. The World Health Organization (WHO) came up with ambitious targets for the elimination of hepatitis B as a major public health threat by 2030. To accomplish this goal, one of the WHO's strategies is to develop curative treatments for HBV infections. Current treatments in a clinical setting included 1 year of pegylated interferon alpha (PEG-IFNα) and long-term nucleoside analogues (NAs). Although both treatments have demonstrated outstanding antiviral effects, it has been difficult to develop a cure for HBV. The reason for this is that covalently closed circular DNA (cccDNA), integrated HBV DNA, the high viral burden, and the impaired host immune responses all hinder the development of a cure for HBV. To overcome these problems, there are clinical trials on a number of antiviral molecules being carried out, all -showing promising results so far. In this review, we summarize the functions and mechanisms of action of various synthetic molecules, natural products, traditional Chinese herbal medicines, as clustered regularly interspaced short palindromic repeats and their associated proteins (CRISPR/Cas)-based systems, zinc finger nucleases (ZFNs), and transcription activator-like effector nucleases (TALENs), all of which could destroy the stability of the HBV life cycle. In addition, we discuss the functions of immune modulators, which can enhance or activate the host immune system, as well some representative natural products with anti-HBV effects.}, }
@article {pmid37007603, year = {2022}, author = {Gomez, MA and Berkoff, KC and Gill, BK and Iavarone, AT and Lieberman, SE and Ma, JM and Schultink, A and Karavolias, NG and Wyman, SK and Chauhan, RD and Taylor, NJ and Staskawicz, BJ and Cho, MJ and Rokhsar, DS and Lyons, JB}, title = {CRISPR-Cas9-mediated knockout of CYP79D1 and CYP79D2 in cassava attenuates toxic cyanogen production.}, journal = {Frontiers in plant science}, volume = {13}, number = {}, pages = {1079254}, pmid = {37007603}, issn = {1664-462X}, abstract = {Cassava (Manihot esculenta) is a starchy root crop that supports over a billion people in tropical and subtropical regions of the world. This staple, however, produces the neurotoxin cyanide and requires processing for safe consumption. Excessive consumption of insufficiently processed cassava, in combination with protein-poor diets, can have neurodegenerative impacts. This problem is further exacerbated by drought conditions which increase this toxin in the plant. To reduce cyanide levels in cassava, we used CRISPR-mediated mutagenesis to disrupt the cytochrome P450 genes CYP79D1 and CYP79D2 whose protein products catalyze the first step in cyanogenic glucoside biosynthesis. Knockout of both genes eliminated cyanide in leaves and storage roots of cassava accession 60444; the West African, farmer-preferred cultivar TME 419; and the improved variety TMS 91/02324. Although knockout of CYP79D2 alone resulted in significant reduction of cyanide, mutagenesis of CYP79D1 did not, indicating these paralogs have diverged in their function. The congruence of results across accessions indicates that our approach could readily be extended to other preferred or improved cultivars. This work demonstrates cassava genome editing for enhanced food safety and reduced processing burden, against the backdrop of a changing climate.}, }
@article {pmid37005025, year = {2023}, author = {Yi, M and Gong, Y and Zhan, Q and Dai, Y and Yang, T and Cheng, X and Ding, S and Gu, B and Cheng, W and Zhang, D}, title = {A one-pot CRISPR-Cas12a-based toolbox enables determination of terminal deoxynucleotidyl transferase activity for acute leukemia screening.}, journal = {Analytica chimica acta}, volume = {1254}, number = {}, pages = {341115}, doi = {10.1016/j.aca.2023.341115}, pmid = {37005025}, issn = {1873-4324}, mesh = {Humans ; CRISPR-Cas Systems ; DNA Nucleotidylexotransferase ; Biological Assay ; *Biomedical Research ; DNA, Single-Stranded/genetics ; *Precursor Cell Lymphoblastic Leukemia-Lymphoma/diagnosis/genetics ; *Biosensing Techniques ; }, abstract = {An isothermal, one-pot toolbox (called OPT-Cas) based on CRISPR-Cas12a collateral cleavage capability is proposed for highly sensitive and selective determination of terminal deoxynucleotidyl transferase (TdT) activity. Oligonucleotide primers with 3'-hydroxyl (OH) terminal were randomly introduced for TdT-induced elongation. In the presence of TdT, dTTP nucleotides polymerized at the 3' terminals of the primers to generate abundant polyT-tails, which function as triggers for the synchronous activation of Cas12a proteins. Finally, the activated Cas12a trans-cleaved FAM and BHQ1 dual-labeled single-stranded DNA (ssDNA-FQ) reporters, producing significantly amplified fluorescence signals. This one-pot assay, that is primer, crRNA, Cas12a protein and ssDNA-FQ reporter are all in one tube, allows simple but high-sensitive quantification of TdT activity with a low detection limit of 6.16 × 10[-5] U μL[-1] in the concentration scope from 1 × 10[-4] U μL[-1] to 1 × 10[-1] U μL[-1], and achieves extraordinary selectivity with other interfering proteins. Furthermore, the OPT-Cas was successfully used to detect TdT in complex matrices and accurate determination of TdT activity in acute lymphoblastic leukemia cells, which might be a reliable technique platform for the diagnosis of TdT-related diseases and biomedical research applications.}, }
@article {pmid37004306, year = {2023}, author = {Deng, X and Yuan, J and Chen, L and Chen, H and Wei, C and Nielsen, PH and Wuertz, S and Qiu, G}, title = {CRISPR-Cas phage defense systems and prophages in Candidatus Accumulibacter.}, journal = {Water research}, volume = {235}, number = {}, pages = {119906}, doi = {10.1016/j.watres.2023.119906}, pmid = {37004306}, issn = {1879-2448}, abstract = {Candidatus Accumulibacter plays a major role in enhanced biological phosphorus removal (EBPR) from wastewater. Although bacteriophages have been shown to represent fatal threats to Ca. Accumulibacter organisms and thus interfere with the stability of the EBPR process, little is known about the ability of different Ca. Accumulibacter strains to resist phage infections. We conducted a systematic analysis of the occurrence and characteristics of clustered regularly interspaced short palindromic repeats and associated proteins (CRISPR-Cas) systems and prophages in Ca. Accumulibacter lineage members (43 in total, including 10 newly recovered genomes). Results indicate that 28 Ca. Accumulibacter genomes encode CRISPR-Cas systems. They were likely acquired via horizontal gene transfer, conveying a distinct adaptivity to phage predation to different Ca. Accumulibacter members. Major differences in the number of spacers show the unique phage resistance of these members. A comparison of the spacers in closely related Ca. Accumulibacter members from distinct geographical locations indicates that habitat isolation may have resulted in the acquisition of resistance to different phages by different Ca. Accumulibacter. Long-term operation of three laboratory-scale EBPR bioreactors revealed high relative abundances of Ca. Accumulibacter with CRISPSR-Cas systems. Their specific resistance to phages in these reactors was indicated by spacer analysis. Metatranscriptomic analyses showed the activation of the CRISPR-Cas system under both anaerobic and aerobic conditions. Additionally, 133 prophage regions were identified in 43 Ca. Accumulibacter genomes. Twenty-seven of them (in 19 genomes) were potentially active. Major differences in the occurrence of CRISPR-Cas systems and prophages in Ca. Accumulibacter will lead to distinct responses to phage predation. This study represents the first systematic analysis of CRISPR-Cas systems and prophages in the Ca. Accumulibacter lineage, providing new perspectives on the potential impacts of phages on Ca. Accumulibacter and EBPR systems.}, }
@article {pmid36958604, year = {2023}, author = {Bondy-Denomy, J and Maxwell, KL and Davidson, AR}, title = {Ten Years of Anti-CRISPR Research.}, journal = {Journal of molecular biology}, volume = {435}, number = {7}, pages = {168058}, doi = {10.1016/j.jmb.2023.168058}, pmid = {36958604}, issn = {1089-8638}, mesh = {*CRISPR-Cas Systems/genetics ; *Bacteriophages ; }, }
@article {pmid36933297, year = {2023}, author = {Yi, JY and Kim, M and Ahn, JH and Kim, BG and Son, J and Sung, C}, title = {CRISPR/deadCas9-based high-throughput gene doping analysis (HiGDA): A proof of concept for exogenous human erythropoietin gene doping detection.}, journal = {Talanta}, volume = {258}, number = {}, pages = {124455}, doi = {10.1016/j.talanta.2023.124455}, pmid = {36933297}, issn = {1873-3573}, mesh = {Humans ; *CRISPR-Cas Systems ; *Erythropoietin/genetics ; }, abstract = {A genetic approach targeted toward improving athletic performance is called gene doping and is prohibited by the World Anti-Doping Agency. Currently, the clustered regularly interspaced short palindromic repeats-associated protein (Cas)-related assays have been utilized to detect genetic deficiencies or mutations. Among the Cas proteins, deadCas9 (dCas9), a nuclease-deficient mutant of Cas9, acts as a DNA binding protein with a target-specific single guide RNA. On the basis of the principles, we developed a dCas9-based high-throughput gene doping analysis for exogenous gene detection. The assay comprises two distinctive dCas9s, a magnetic bead immobilized capture dCas9 for exogenous gene isolation and a biotinylated dCas9 with streptavidin-polyHRP that enables rapid signal amplification. For efficient biotin labeling via maleimide-thiol chemistry, two cysteine residues of dCas9 were structurally validated, and the Cys574 residue was identified as an essential labeling site. As a result, we succeeded in detecting the target gene in a concentration as low as 12.3 fM (7.41 × 10[5] copies) and up to 10 nM (6.07 × 10[11] copies) in a whole blood sample within 1 h with HiGDA. Assuming an exogenous gene transfer scenario, we added a direct blood amplification step to establish a rapid analytical procedure while detecting target genes with high sensitivity. Finally, we detected the exogenous human erythropoietin gene at concentrations as low as 2.5 copies within 90 min in 5 μL of the blood sample. Herein, we propose that HiGDA is a very fast, highly sensitive, and practical detection method for actual doping field in the future.}, }
@article {pmid36928506, year = {2023}, author = {Trivedi, V and Ramesh, A and Wheeldon, I}, title = {Analyzing CRISPR screens in non-conventional microbes.}, journal = {Journal of industrial microbiology &amp; biotechnology}, volume = {50}, number = {1}, pages = {}, doi = {10.1093/jimb/kuad006}, pmid = {36928506}, issn = {1476-5535}, mesh = {Animals ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Genomics ; Genome ; Computational Biology ; Mammals/genetics ; }, abstract = {UNLABELLED: The multifaceted nature of CRISPR screens has propelled advancements in the field of functional genomics. Pooled CRISPR screens involve creating programmed genetic perturbations across multiple genomic sites in a pool of host cells subjected to a challenge, empowering researchers to identify genetic causes of desirable phenotypes. These genome-wide screens have been widely used in mammalian cells to discover biological mechanisms of diseases and drive the development of targeted drugs and therapeutics. Their use in non-model organisms, especially in microbes to improve bioprocessing-relevant phenotypes, has been limited. Further compounding this issue is the lack of bioinformatic algorithms for analyzing microbial screening data with high accuracy. Here, we describe the general approach and underlying principles for conducting pooled CRISPR knockout screens in non-conventional yeasts and performing downstream analysis of the screening data, while also reviewing state-of-the-art algorithms for identification of CRISPR screening outcomes. Application of pooled CRISPR screens to non-model yeasts holds considerable potential to uncover novel metabolic engineering targets and improve industrial bioproduction.<br><br>ONE-SENTENCE SUMMARY: This mini-review describes experimental and computational approaches for functional genomic screening using CRISPR technologies in non-conventional microbes.}, }
@article {pmid36924897, year = {2023}, author = {Qiao, L and Gao, M and Yi, X and Peng, H and Zhang, R and Yao, W and Sun, G and He, X}, title = {Biomimetic gene editing system for precise tumor cell reprogramming and augmented tumor therapy.}, journal = {Journal of controlled release : official journal of the Controlled Release Society}, volume = {356}, number = {}, pages = {663-677}, doi = {10.1016/j.jconrel.2023.03.020}, pmid = {36924897}, issn = {1873-4995}, mesh = {Humans ; *Gene Editing ; CRISPR-Cas Systems/genetics ; Biomimetics ; Cellular Reprogramming ; *Neoplasms/genetics/therapy/metabolism ; }, abstract = {The abnormal level of hypoxia-inducible factor-1 alpha (HIF-1α) is closely related to cancer metastasis and treatment resistance. CRISPR-Cas9-based gene editing technology has sparked profound hope to solve this issue by precise gene disruption, although the in vivo application remains hindered by the lack of a safe and efficient delivery strategy. Herein, we developed a cell membrane biomimetic core-shell system for light-controllable, precise gene editing. The inner core of the system comprises protamine for CRISPR-Cas9/sgRNA plasmid (pCas9) loading and calcium ions for efficient pCas9 transfection. The shell of the system is camouflaged by a cell membrane and modified with AS1411 aptamers for tumor targeting and photosensitizers to induce lysosomal escape and pCas9 release through reactive oxygen species production, thereby producing light-controllable enhanced gene editing. Neoplastic H1299 cells were reprogrammed using the biomimetic gene editing system upon laser irradiation with reduced VEGF and Vimentin expression, leading to enhanced antimetastatic effects. Genetic disruption of HIF-1α augmented the in vivo chemotherapeutic efficacy of paclitaxel. Our approach of using a membrane-camouflaged system combined with light augmentation provides a potential solution for the in vivo delivery of CRISPR-Cas9 as well as a feasible strategy for cancer therapy.}, }
@article {pmid36899271, year = {2023}, author = {Kingwell, K}, title = {Testing the genome-editing toolkit in cardiomyopathy.}, journal = {Nature reviews. Drug discovery}, volume = {22}, number = {4}, pages = {270}, pmid = {36899271}, issn = {1474-1784}, mesh = {Humans ; *Gene Editing ; CRISPR-Cas Systems/genetics ; *Cardiomyopathies/genetics ; }, }
@article {pmid36881874, year = {2023}, author = {Dahlvang, JD and Dick, JK and Sangala, JA and Kennedy, PR and Pomeroy, EJ and Snyder, KM and Moushon, JM and Thefaine, CE and Wu, J and Hamilton, SE and Felices, M and Miller, JS and Walcheck, B and Webber, BR and Moriarity, BS and Hart, GT}, title = {Ablation of SYK Kinase from Expanded Primary Human NK Cells via CRISPR/Cas9 Enhances Cytotoxicity and Cytokine Production.}, journal = {Journal of immunology (Baltimore, Md. : 1950)}, volume = {210}, number = {8}, pages = {1108-1122}, pmid = {36881874}, issn = {1550-6606}, support = {R01 AI143828/AI/NIAID NIH HHS/United States ; R01 AI146031/AI/NIAID NIH HHS/United States ; R21 AI149659/AI/NIAID NIH HHS/United States ; }, mesh = {Humans ; Syk Kinase/genetics ; *Cytomegalovirus ; CRISPR-Cas Systems ; Killer Cells, Natural ; *Cytomegalovirus Infections ; Cytokines ; Antibody-Dependent Cell Cytotoxicity ; }, abstract = {CMV infection alters NK cell phenotype and function toward a more memory-like immune state. These cells, termed adaptive NK cells, typically express CD57 and NKG2C but lack expression of the FcRγ-chain (gene: FCER1G, FcRγ), PLZF, and SYK. Functionally, adaptive NK cells display enhanced Ab-dependent cellular cytotoxicity (ADCC) and cytokine production. However, the mechanism behind this enhanced function is unknown. To understand what drives enhanced ADCC and cytokine production in adaptive NK cells, we optimized a CRISPR/Cas9 system to ablate genes from primary human NK cells. We ablated genes that encode molecules in the ADCC pathway, such as FcRγ, CD3ζ, SYK, SHP-1, ZAP70, and the transcription factor PLZF, and tested subsequent ADCC and cytokine production. We found that ablating the FcRγ-chain caused a modest increase in TNF-α production. Ablation of PLZF did not enhance ADCC or cytokine production. Importantly, SYK kinase ablation significantly enhanced cytotoxicity, cytokine production, and target cell conjugation, whereas ZAP70 kinase ablation diminished function. Ablating the phosphatase SHP-1 enhanced cytotoxicity but reduced cytokine production. These results indicate that the enhanced cytotoxicity and cytokine production of CMV-induced adaptive NK cells is more likely due to the loss of SYK than the lack of FcRγ or PLZF. We found the lack of SYK expression could improve target cell conjugation through enhanced CD2 expression or limit SHP-1-mediated inhibition of CD16A signaling, leading to enhanced cytotoxicity and cytokine production.}, }
@article {pmid36871676, year = {2023}, author = {Boubakri, H}, title = {Recent progress in CRISPR/Cas9-based genome editing for enhancing plant disease resistance.}, journal = {Gene}, volume = {866}, number = {}, pages = {147334}, doi = {10.1016/j.gene.2023.147334}, pmid = {36871676}, issn = {1879-0038}, mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; Transcription Activator-Like Effector Nucleases/genetics ; Disease Resistance/genetics ; Plants/genetics ; Genome, Plant ; }, abstract = {Nowadays, agricultural production is strongly affected by both climate change and pathogen attacks which seriously threaten global food security. For a long time, researchers have been waiting for a tool allowing DNA/RNA manipulation to tailor genes and their expression. Some earlier genetic manipulation methods such as meganucleases (MNs), zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) allowed site directed modification but their successful rate was limited due to lack of flexibility when targeting a 'site-specific nucleic acid'. The discovery of clustered regularly interspaced short palindrome repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system has revolutionized genome editing domain in different living organisms during the past 9 years. Based on RNA-guided DNA/RNA recognition, CRISPR/Cas9 optimizations have offered an unrecorded scientific opportunity to engineer plants resistant to diverse pathogens. In this report, we describe the main characteristics of the primary reported-genome editing tools ((MNs, ZFNs, TALENs) and evaluate the different CRISPR/Cas9 methods and achievements in developing crop plants resistant to viruses, fungi and bacteria.}, }
@article {pmid36868446, year = {2023}, author = {Abe, T and Kaneko, M and Kiyonari, H}, title = {A reverse genetic approach in geckos with the CRISPR/Cas9 system by oocyte microinjection.}, journal = {Developmental biology}, volume = {497}, number = {}, pages = {26-32}, doi = {10.1016/j.ydbio.2023.02.005}, pmid = {36868446}, issn = {1095-564X}, mesh = {Animals ; Mice ; *CRISPR-Cas Systems/genetics ; *Lizards/genetics ; Microinjections ; Reverse Genetics ; Gene Editing/methods ; Oocytes ; }, abstract = {Reptiles are important model organisms in developmental and evolutionary biology, but are used less widely than other amniotes such as mouse and chicken. One of the main reasons for this is that has proven difficult to conduct CRISPR/Cas9-mediated genome editing in many reptile species despite the widespread use of this technology in other taxa. Certain features of reptile reproductive systems make it difficult to access one-cell or early-stage zygotes, which represents a key impediment to gene editing techniques. Recently, Rasys and colleagues reported a genome editing method using oocyte microinjection that allowed them to produce genome-edited Anolis lizards. This method opened a new avenue to reverse genetics studies in reptiles. In the present article, we report the development of a related method for genome editing in the Madagascar ground gecko (Paroedura picta), a well-established experimental model, and describe the generation of Tyr and Fgf10 gene-knockout geckos in the F0 generation.}, }
@article {pmid36736009, year = {2023}, author = {Han, J and Li, X and Li, W and Yang, Q and Li, Z and Cheng, Z and Lv, L and Zhang, L and Han, D}, title = {Isolation and preliminary functional analysis of FvICE1, involved in cold and drought tolerance in Fragaria vesca through overexpression and CRISPR/Cas9 technologies.}, journal = {Plant physiology and biochemistry : PPB}, volume = {196}, number = {}, pages = {270-280}, doi = {10.1016/j.plaphy.2023.01.048}, pmid = {36736009}, issn = {1873-2690}, mesh = {*Fragaria/metabolism ; Stress, Physiological ; Drought Resistance ; CRISPR-Cas Systems/genetics ; Gene Expression Regulation, Plant ; Plant Proteins/genetics/metabolism ; Droughts ; Plants, Genetically Modified/metabolism ; }, abstract = {Cold and drought stresses are serious problems of strawberry cultivation in temperate and subtropical regions. In the molecular regulation system of cold and drought stresses, ICE transcription factors (TFs) are crucial. In this research, the FvICE1 was isolated from Fragaria vesca 'Hawaii 4', a bioinformatics analysis was conducted, overexpression vector and CRISPR/cas9 vector were constructed. The results showed that FvICE1 was a member of the bHLH TF family, with a length of 1608 bp, encoding 535 amino acids, and its molecular formula was C2504H3987N745O811S22. By observing the fusion protein 35S-FvICE1-GFP, it was found that FvICE1 was a nuclear protein. The qRT-PCR results demonstrated that FvICE1 was significantly upregulated in different tissues of Fragaria vesca after cold, drought, salt and heat treatments. The wild type (WT) strawberry was selected as the control group, FvICE1-overexpression strawberries showed high tolerance to cold and drought treatments at the phenotypic and physiological levels. On the contrary, fvice1 mutant strawberries obtained by CRISPR/cas9 editing technology had lower tolerance to cold and drought treatments. Moreover, the expression of FvCBF1, FvCBF2, FvCBF3, FvCOR413, FvRD22 and FvKIN1 was positively regulated in the FvICE1-overexpression strawberries and inhibited in fvice1 mutant strawberries. Overall, the current results suggested that FvICE1 functioned as a positively regulator of cold and drought resistances.}, }
@article {pmid36478145, year = {2023}, author = {Ge, X and Xu, J and Yang, Z and Yang, X and Wang, Y and Chen, Y and Wang, P and Li, F}, title = {Efficient genotype-independent cotton genetic transformation and genome editing.}, journal = {Journal of integrative plant biology}, volume = {65}, number = {4}, pages = {907-917}, doi = {10.1111/jipb.13427}, pmid = {36478145}, issn = {1744-7909}, mesh = {*Gene Editing/methods ; *Gossypium/genetics ; CRISPR-Cas Systems/genetics ; Plants, Genetically Modified/genetics ; Genotype ; Transformation, Genetic ; }, abstract = {Cotton (Gossypium spp.) is one of the most important fiber crops worldwide. In the last two decades, transgenesis and genome editing have played important roles in cotton improvement. However, genotype dependence is one of the key bottlenecks in generating transgenic and gene-edited cotton plants through either particle bombardment or Agrobacterium-mediated transformation. Here, we developed a shoot apical meristem (SAM) cell-mediated transformation system (SAMT) that allowed the transformation of recalcitrant cotton genotypes including widely grown upland cotton (Gossypium hirsutum), Sea island cotton (Gossypium barbadense), and Asiatic cotton (Gossypium arboreum). Through SAMT, we successfully introduced two foreign genes, GFP and RUBY, into SAM cells of some recalcitrant cotton genotypes. Within 2-3 months, transgenic adventitious shoots generated from the axillary meristem zone could be recovered and grown into whole cotton plants. The GFP fluorescent signal and betalain accumulation could be observed in various tissues in GFP- and RUBY-positive plants, as well as in their progenies, indicating that the transgenes were stably integrated into the genome and transmitted to the next generation. Furthermore, using SAMT, we successfully generated edited cotton plants with inheritable targeted mutagenesis in the GhPGF and GhRCD1 genes through CRISPR/Cas9-mediated genome editing. In summary, the established SAMT transformation system here in this study bypasses the embryogenesis process during tissue culture in a conventional transformation procedure and significantly accelerates the generation of transgenic and gene-edited plants for genetic improvement of recalcitrant cotton varieties.}, }
@article {pmid36460630, year = {2023}, author = {Wang, C and Li, Y and Wang, N and Yu, Q and Li, Y and Gao, J and Zhou, X and Ma, N}, title = {An efficient CRISPR/Cas9 platform for targeted genome editing in rose (Rosa hybrida).}, journal = {Journal of integrative plant biology}, volume = {65}, number = {4}, pages = {895-899}, doi = {10.1111/jipb.13421}, pmid = {36460630}, issn = {1744-7909}, mesh = {*Gene Editing/methods ; CRISPR-Cas Systems/genetics ; *Rosa/genetics ; Plants/genetics ; }, abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-related nuclease 9 (Cas9) system enables precise, simple editing of genes in many animals and plants. However, this system has not been applied to rose (Rosa hybrida) due to the genomic complexity and lack of an efficient transformation technology for this plant. Here, we established a platform for screening single-guide RNAs (sgRNAs) with high editing efficiency for CRISPR/Cas9-mediated gene editing in rose using suspension cells. We used the Arabidopsis thaliana U6-29 promoter, which showed high activity for driving sgRNA expression, to modify the CRISPR/Cas9 system. We used our highly efficient optimized CRISPR/Cas9 system to successfully edit RhEIN2, encoding an indispensable component of the ethylene signaling pathway, resulting in ethylene insensitivity in rose. Our optimized CRISPR/Cas9 system provides a powerful toolbox for functional genomics, molecular breeding, and synthetic biology in rose.}, }
@article {pmid36096547, year = {2023}, author = {Ling, J and Jenny, LA and Zhou, A and Tsang, SH}, title = {Therapeutic Gene Editing in Inherited Retinal Disorders.}, journal = {Cold Spring Harbor perspectives in medicine}, volume = {13}, number = {4}, pages = {}, pmid = {36096547}, issn = {2157-1422}, mesh = {Humans ; *Gene Editing ; CRISPR-Cas Systems ; *Retinal Diseases/genetics/therapy ; }, abstract = {Since the development of CRISPR/Cas9 gene editing in 2012, therapeutic editing research has produced several phase 1-2a trials. Here we provide an overview of the mechanisms and applications of various gene-editing technologies including adeno-associated virus vectors, lentiviruses, CRISPR/Cas9 systems, base and prime editing, antisense oligonucleotides, short-hairpin RNAs, Cas13, and adenosine deaminase acting on RNA for the treatment of various inherited retinal diseases (IRDs). We outline the various stages of clinical trials using these technologies and the impacts they have made in advancing the practice of medicine.}, }
@article {pmid35670672, year = {2022}, author = {Konstantakos, V and Nentidis, A and Krithara, A and Paliouras, G}, title = {CRISPRedict: a CRISPR-Cas9 web tool for interpretable efficiency predictions.}, journal = {Nucleic acids research}, volume = {50}, number = {W1}, pages = {W191-W198}, pmid = {35670672}, issn = {1362-4962}, mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; RNA, Guide, Kinetoplastida/genetics ; }, abstract = {The development of the CRISPR-Cas9 technology has provided a simple yet powerful system for genome editing. Current gRNA design tools serve as an important platform for the efficient application of the CRISPR systems. However, most of the existing tools are black-box models that suffer from limitations, such as variable performance and unclear mechanism of decision making. Here, we introduce CRISPRedict, an interpretable gRNA efficiency prediction model for CRISPR-Cas9 gene editing. Its strength lies in the fact that it can accurately predict efficient guide RNAs-with equivalent performance to state-of-the-art tools-while being a simple linear model. Implemented as a user-friendly web server, CRISPRedict offers (i) quick and accurate predictions across various experimental conditions (e.g. U6/T7 transcription); (ii) regression and classification models for scoring gRNAs and (iii) multiple visualizations to explain the obtained results. Given its performance, interpretability, and versatility, we expect that it will assist researchers in the gRNA design process and facilitate genome editing research. CRISPRedict is available for use at http://www.crispredict.org/.}, }
@article {pmid37004283, year = {2023}, author = {Zhang, Y and Chen, Y and Zhang, Q and Liu, Y and Zhang, X}, title = {An aM-level sensitive cascade CRISPR-Dx system (ASCas) for rapid detection of RNA without pre-amplification.}, journal = {Biosensors &amp; bioelectronics}, volume = {230}, number = {}, pages = {115248}, doi = {10.1016/j.bios.2023.115248}, pmid = {37004283}, issn = {1873-4235}, abstract = {The CRISPR/Cas system is known as one of the directions of the next generation of mainstream molecular diagnostic technology. However, most current CRISPR/Cas molecular diagnostics still rely on the pre-amplification of nucleic acid due to the limited sensitivity of CRISPR/Cas alone, which has no significant advantage over commercial Taqman-PCR and TwistAmp® Exo kits. Herein, we report an aM-level sensitive cascade CRISPR-Dx system (ASCas) that eliminates nucleic acid pre-amplification, thus avoiding aerosol contamination and greatly reducing the testing environment and personnel skill requirements for molecular diagnostics. Most importantly, the Cas13a nucleases with high sensitivity and trans-cleavage efficiency can rapidly cleaved RNA bubbles on the hybridized cascade probe at low concentration target RNA detection, which results in the destruction of the cascade probe and releases a large amount of trigger DNA for further signal amplification of secondary Cas12a reactions. Therefore, the ASCas system achieves amplification-free, ultra-sensitivity (1 aM), and ultra-fast (20 min) RNA detection. In addition, the ASCas system replaces the complicated screening process of primers and probes with the programmed Cas13a-crRNA design so that a suitable detection system can be constructed more quickly and straightforwardly for the mutation-prone SARS-CoV-2 virus.}, }
@article {pmid37002219, year = {2023}, author = {Thakku, SG and Lirette, J and Murugesan, K and Chen, J and Theron, G and Banaei, N and Blainey, PC and Gomez, J and Wong, SY and Hung, DT}, title = {Genome-wide tiled detection of circulating Mycobacterium tuberculosis cell-free DNA using Cas13.}, journal = {Nature communications}, volume = {14}, number = {1}, pages = {1803}, pmid = {37002219}, issn = {2041-1723}, mesh = {Cell-Free System ; DNA, Bacterial/analysis/genetics ; *Mycobacterium tuberculosis/genetics ; Genome, Bacterial ; CRISPR-Cas Systems ; Humans ; Tuberculosis/diagnosis/microbiology ; }, abstract = {Detection of microbial cell-free DNA (cfDNA) circulating in the bloodstream has emerged as a promising new approach for diagnosing infection. Microbial diagnostics based on cfDNA require assays that can detect rare and highly fragmented pathogen nucleic acids. We now report WATSON (Whole-genome Assay using Tiled Surveillance Of Nucleic acids), a method to detect low amounts of pathogen cfDNA that couples pooled amplification of genomic targets tiled across the genome with pooled CRISPR/Cas13-based detection of these targets. We demonstrate that this strategy of tiling improves cfDNA detection compared to amplification and detection of a single targeted locus. WATSON can detect cfDNA from Mycobacterium tuberculosis in plasma of patients with active pulmonary tuberculosis, a disease that urgently needs accurate, minimally-invasive, field-deployable diagnostics. We thus demonstrate the potential for translating WATSON to a lateral flow platform. WATSON demonstrates the ability to capitalize on the strengths of targeting microbial cfDNA to address the need for point-of-care diagnostic tests for infectious diseases.}, }
@article {pmid37001495, year = {2023}, author = {Malech, HL and Notarangelo, LD}, title = {Gene therapy for inborn errors of immunity: Base editing comes into play.}, journal = {Cell}, volume = {186}, number = {7}, pages = {1302-1304}, doi = {10.1016/j.cell.2023.03.001}, pmid = {37001495}, issn = {1097-4172}, mesh = {Humans ; Adenine ; *CRISPR-Cas Systems/genetics ; *Gene Editing ; Genetic Therapy ; Mutation ; Genetic Diseases, Inborn/genetics/therapy ; *Immune System Diseases/genetics/therapy ; }, abstract = {CRISPR-Cas9-based base editing allows precise base editing to achieve conversion of adenosine to guanine or cytosine to thymidine. In this issue of Cell, McAuley et al. use adenine base editing to correct a single base-pair mutation causing human CD3δ deficiency, demonstrating superior efficiency of genetic correction with reduced undesired genetic alterations compared with standard CRISPR-Cas9 editing.}, }
@article {pmid37000655, year = {2023}, author = {Kulishova, LM and Vokhtantsev, IP and Kim, DV and Zharkov, DO}, title = {[Mechanisms of the Specificity of the CRISPR/Cas9 System in Genome Editing].}, journal = {Molekuliarnaia biologiia}, volume = {57}, number = {2}, pages = {269-284}, pmid = {37000655}, issn = {0026-8984}, mesh = {Humans ; *Gene Editing ; *CRISPR-Cas Systems/genetics ; Biotechnology ; RNA, Guide, Kinetoplastida/genetics ; Genome ; }, abstract = {The CRISPR/Cas9 system, which was discovered recently, utilizes nucleases targeted by sequence complementarity and is originally intended to protect bacteria from foreign genetic elements. The system provided a convenient tool for manipulating the genomes of living cells. The CRISPR/Cas9 genomic editing technology moved beyond the laboratory and already found application in biotechnology and agriculture. However, off-target activity of the CRISPR/Cas9 system can cause oncogenic mutations and thus limits its use for genome editing in human cells for medical purposes. Many studies are therefore aimed at developing variants of the CRISPR/Cas9 system with improved accuracy. The review considers the mechanisms of precise and erroneous actions of Cas9 RNA-guided nuclease, natural and artificial variants of RNA-targeted nucleases, possibilities to modulate their specificity through guide RNA modifications, and other approaches to increasing the accuracy of the CRISPR/Cas9 system in genome editing.}, }
@article {pmid36958206, year = {2023}, author = {Wang, Y and Chen, H and Gao, H and Wei, H and Wang, Y and Mu, K and Liu, L and Dai, E and Rong, Z and Wang, S}, title = {CESSAT: A chemical additive-enhanced single-step accurate CRISPR/Cas13 testing system for field-deployable ultrasensitive detection and genotyping of SARS-CoV-2 variants of concern.}, journal = {Biosensors &amp; bioelectronics}, volume = {229}, number = {}, pages = {115238}, pmid = {36958206}, issn = {1873-4235}, mesh = {Humans ; *COVID-19/diagnosis ; CRISPR-Cas Systems/genetics ; Genotype ; SARS-CoV-2/genetics ; *Biosensing Techniques ; RNA, Viral/genetics ; }, abstract = {The continued emergence of SARS-CoV-2 variants of concern (VOCs) has raised great challenges for epidemic prevention and control. A rapid, sensitive, and on-site SARS-CoV-2 genotyping technique is urgently needed for individual diagnosis and routine surveillance. Here, a field-deployable ultrasensitive CRISPR-based diagnostics system, called Chemical additive-Enhanced Single-Step Accurate CRISPR/Cas13 Testing system (CESSAT), for simultaneous screening of SARS-CoV-2 and its five VOCs (Alpha, Beta, Gamma, Delta, and Omicron) within 40 min was reported. In this system, a single-step reverse transcription recombinase polymerase amplification-CRISPR/Cas13a assay was incorporated with optimized extraction-free viral lysis and reagent lyophilization, which could eliminate complicated sample processing steps and rigorous reagent storage conditions. Remarkably, 10% glycine as a chemical additive could improve the assay sensitivity by 10 times, making the limit of detection as low as 1 copy/μL (5 copies/reaction). A compact optic fiber-integrated smartphone-based device was developed for sample lysis, assay incubation, fluorescence imaging, and result interpretation. CESSAT could specifically differentiate the synthetic pseudovirus of SARS-CoV-2 and its five VOCs. The genotyping results for 40 clinical samples were in 100% concordance with standard method. We believe this simple but efficient enhancement strategy can be widely incorporated with existing Cas13a-based assays, thus leading a substantial progress in the development and application of rapid, ultrasensitive, and accurate nucleic acid analysis technology.}, }
@article {pmid37003351, year = {2023}, author = {Huang, L and Wang, D and Chen, H and Hu, J and Dai, X and Liu, C and Li, A and Shen, X and Qi, C and Sun, H and Zhang, D and Chen, T and Jiang, Y}, title = {CRISPR-detector: fast and accurate detection, visualization, and annotation of genome-wide mutations induced by genome editing events.}, journal = {Journal of genetics and genomics = Yi chuan xue bao}, volume = {}, number = {}, pages = {}, doi = {10.1016/j.jgg.2023.03.010}, pmid = {37003351}, issn = {1673-8527}, abstract = {The leading-edge CRISPR/Cas technology is revolutionizing biotechnologies through genome editing. To track on/off-target events with emerging new editing techniques, improved bioinformatic tools are indispensable. Existing tools suffer from limitations in speed and scalability, especially with whole-genome sequencing (WGS) data analysis. To address these limitations, we have developed a comprehensive tool called CRISPR-detector, a web-based and locally deployable pipeline for genome editing sequence analysis. The core analysis module of CRISPR-detector is based on the Sentieon TNscope pipeline, with additional novel annotation and visualization modules designed to fit CRISPR applications. Co-analysis of the treated and control samples is performed to remove background variants existing prior to genome editing. CRISPR-detector offers optimized scalability, enabling WGS data analysis beyond Browser Extensible Data (BED) file-defined regions, with improved accuracy due to haplotype-based variant calling to handle sequencing errors. In addition, the tool also provides integrated structural variation (SV) calling and included functional and clinical annotation of editing-induced mutations appreciated by users. These advantages facilitate rapid and efficient detection of mutations induced by genome editing events, especially for datasets generated from WGS. The web-based version of CRISPR-detector is available at https://db.cngb.org/crispr-detector, and the locally deployable version is available at https://github.com/hlcas/CRISPR-detector.}, }
@article {pmid37002157, year = {2023}, author = {Zhao, Z and Shang, P and Mohanraju, P and Geijsen, N}, title = {Prime editing: advances and therapeutic applications.}, journal = {Trends in biotechnology}, volume = {}, number = {}, pages = {}, doi = {10.1016/j.tibtech.2023.03.004}, pmid = {37002157}, issn = {1879-3096}, abstract = {Clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR-Cas)-mediated genome editing has revolutionized biomedical research and will likely change the therapeutic and diagnostic landscape. However, CRISPR-Cas9, which edits DNA by activating DNA double-strand break (DSB) repair pathways, is not always sufficient for gene therapy applications where precise mutation repair is required. Prime editing, the latest revolution in genome-editing technologies, can achieve any possible base substitution, insertion, or deletion without the requirement for DSBs. However, prime editing is still in its infancy, and further development is needed to improve editing efficiency and delivery strategies for therapeutic applications. We summarize latest developments in the optimization of prime editor (PE) variants with improved editing efficiency and precision. Moreover, we highlight some potential therapeutic applications.}, }
@article {pmid37001687, year = {2023}, author = {Djermoun, S and Reuter, A and Derollez, E and Lesterlin, C and Bigot, S}, title = {Reprogramming Targeted-Antibacterial-Plasmids (TAPs) to achieve broad-host range antibacterial activity.}, journal = {Plasmid}, volume = {126}, number = {}, pages = {102680}, doi = {10.1016/j.plasmid.2023.102680}, pmid = {37001687}, issn = {1095-9890}, abstract = {The emergence and spread of antimicrobial resistance results in antibiotic inefficiency against multidrug resistant bacterial strains. Alternative treatment to antibiotics must be investigated to fight bacterial infections and limit this global public health problem. We recently developed an innovative strategy based on mobilizable Targeted-Antibacterial-Plasmids (TAPs) that deliver CRISPR/Cas systems with strain-specific antibacterial activity, using the F plasmid conjugation machinery for transfer into the targeted strains. These TAPs were shown to specifically kill a variety of Enterobacteriaceae strains, including E. coli K12 and the pathogen strains EPEC, Enterobacter cloacae and Citrobacter rodentium. Here, we extend the host-range of TAPs using the RP4 plasmid conjugation system for their mobilization, thus allowing the targeting of E. coli but also phylogenetically distant species, including Salmonella enterica Thyphimurium, Klebsiella pneumoniae, Vibrio cholerae, and Pseudomonas aeruginosa. This work demonstrates the versatility of the TAP strategy and represents a significant step toward the development of non-antibiotic strain-specific antimicrobial treatments.}, }
@article {pmid36997536, year = {2023}, author = {Yan, N and Feng, H and Sun, Y and Xin, Y and Zhang, H and Lu, H and Zheng, J and He, C and Zuo, Z and Yuan, T and Li, N and Xie, L and Wei, W and Sun, Y and Zuo, E}, title = {Cytosine base editors induce off-target mutations and adverse phenotypic effects in transgenic mice.}, journal = {Nature communications}, volume = {14}, number = {1}, pages = {1784}, pmid = {36997536}, issn = {2041-1723}, mesh = {Animals ; Mice ; Mice, Transgenic ; *Cytosine/metabolism ; Mutation ; *Gene Editing/methods ; RNA/genetics ; CRISPR-Cas Systems ; }, abstract = {Base editors have been reported to induce off-target mutations in cultured cells, mouse embryos and rice, but their long-term effects in vivo remain unknown. Here, we develop a Systematic evaluation Approach For gene Editing tools by Transgenic mIce (SAFETI), and evaluate the off-target effects of BE3, high fidelity version of CBE (YE1-BE3-FNLS) and ABE (ABE7.10[F148A]) in ~400 transgenic mice over 15 months. Whole-genome sequence analysis reveals BE3 expression generated de novo mutations in the offspring of transgenic mice. RNA-seq analysis reveals both BE3 and YE1-BE3-FNLS induce transcriptome-wide SNVs, and the numbers of RNA SNVs are positively correlated with CBE expression levels across various tissues. By contrast, ABE7.10[F148A] shows no detectable off-target DNA or RNA SNVs. Notably, we observe abnormal phenotypes including obesity and developmental delay in mice with permanent genomic BE3 overexpression during long-time monitoring, elucidating a potentially overlooked aspect of side effects of BE3 in vivo.}, }
@article {pmid36997524, year = {2023}, author = {Lee, J and Lim, K and Kim, A and Mok, YG and Chung, E and Cho, SI and Lee, JM and Kim, JS}, title = {Prime editing with genuine Cas9 nickases minimizes unwanted indels.}, journal = {Nature communications}, volume = {14}, number = {1}, pages = {1786}, pmid = {36997524}, issn = {2041-1723}, mesh = {*CRISPR-Cas Systems/genetics ; *Deoxyribonuclease I/metabolism ; Mutation ; INDEL Mutation ; DNA ; }, abstract = {Unlike CRISPR-Cas9 nucleases, which yield DNA double-strand breaks (DSBs), Cas9 nickases (nCas9s), which are created by replacing key catalytic amino-acid residues in one of the two nuclease domains of S. pyogenesis Cas9 (SpCas9), produce nicks or single-strand breaks. Two SpCas9 variants, namely, nCas9 (D10A) and nCas9 (H840A), which cleave target (guide RNA-pairing) and non-target DNA strands, respectively, are widely used for various purposes, including paired nicking, homology-directed repair, base editing, and prime editing. In an effort to define the off-target nicks caused by these nickases, we perform Digenome-seq, a method based on whole genome sequencing of genomic DNA treated with a nuclease or nickase of interest, and find that nCas9 (H840A) but not nCas9 (D10A) can cleave both strands, producing unwanted DSBs, albeit less efficiently than wild-type Cas9. To inactivate the HNH nuclease domain further, we incorporate additional mutations into nCas9 (H840A). Double-mutant nCas9 (H840A + N863A) does not exhibit the DSB-inducing behavior in vitro and, either alone or in fusion with the M-MLV reverse transcriptase (prime editor, PE2 or PE3), induces a lower frequency of unwanted indels, compared to nCas9 (H840A), caused by error-prone repair of DSBs. When incorporated into prime editor and used with engineered pegRNAs (ePE3), we find that the nCas9 variant (H840A + N854A) dramatically increases the frequency of correct edits, but not unwanted indels, yielding the highest purity of editing outcomes compared to nCas9 (H840A).}, }
@article {pmid36995680, year = {2023}, author = {Preisinger, D and Winogrodzki, T and Klinger, B and Schnieke, A and Rieblinger, B}, title = {Genome Editing in Pigs.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2631}, number = {}, pages = {393-417}, pmid = {36995680}, issn = {1940-6029}, mesh = {Swine/genetics ; Animals ; Female ; *Gene Editing ; *Genetic Engineering/methods ; Nuclear Transfer Techniques ; Oocytes ; Zygote ; CRISPR-Cas Systems ; Animals, Genetically Modified/genetics ; }, abstract = {The generation of genetically engineered (GE) pigs for disease modeling and xenotransplantation has been massively facilitated by the discovery of the CRISPR/Cas9 system. For livestock, genome editing is a powerful tool when used in combination with either somatic cell nuclear transfer (SCNT) or microinjection (MI) into fertilized oocytes. To generate either knockout or knock-in animals using SCNT, genome editing is carried out in vitro. This has the advantage that fully characterized cells are being employed to generate cloned pigs, predetermining their genetic makeups. However, this technique is labor-intensive and, hence, SCNT is better suited for more challenging projects such as the generation of multi-knockout- and knock-in pigs. Alternatively, CRISPR/Cas9 is introduced directly into fertilized zygotes via microinjection to produce knockout pigs more rapidly. Finally, the embryos are each transferred into recipient sows to deliver GE piglets.Both techniques, SCNT and MI, are technically challenging and therefore require skilled expertise, especially when applied for porcine embryos. Here, we present a detailed laboratory protocol for the generation of knockout and knock-in porcine somatic donor cells for SCNT and knockout pigs via microinjection. We describe the state-of-the-art method for isolation, cultivation, and manipulation of porcine somatic cells, which can then be used for SCNT. Moreover, we describe the isolation and maturation of porcine oocytes, their manipulation by microinjection, and the embryo transfer into surrogate sows.}, }
@article {pmid36995678, year = {2023}, author = {Daniel, JG and Yu, X and Ferguson, AC and Shavit, JA}, title = {CRISPR/Cas9-Mediated Genome Editing in Zebrafish.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2631}, number = {}, pages = {371-380}, pmid = {36995678}, issn = {1940-6029}, mesh = {Animals ; *Gene Editing ; *CRISPR-Cas Systems/genetics ; Zebrafish/genetics ; Genome ; }, abstract = {The CRISPR/Cas9 system is a powerful tool for genome editing in zebrafish. This workflow takes advantage of the genetic tractability of zebrafish and will allow users to edit genomic sites and produce mutant lines using selective breeding. Established lines may then be employed by researchers for downstream genetic and phenotypic analyses.}, }
@article {pmid36995675, year = {2023}, author = {Sato, M and Nakamura, A and Sekiguchi, M and Matsuwaki, T and Miura, H and Gurumurthy, CB and Kakuta, S and Ohtsuka, M}, title = {Improved Genome Editing via Oviductal Nucleic Acids Delivery (i-GONAD): Protocol Steps and Additional Notes.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2631}, number = {}, pages = {325-340}, pmid = {36995675}, issn = {1940-6029}, mesh = {Humans ; Pregnancy ; Female ; Mice ; Animals ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Fallopian Tubes ; Oviducts ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Electroporation/methods ; Gonads ; }, abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR) technology has made it possible to produce genome-edited (GE) animals more easily and rapidly than before. In most cases, GE mice are produced by microinjection (MI) or by in vitro electroporation (EP) of CRISPR reagents into fertilized eggs (zygotes). Both of these approaches require ex vivo handling of isolated embryos and their subsequent transfer into another set of mice (called recipient or pseudopregnant mice). Such experiments are performed by highly skilled technicians (especially for MI). We recently developed a novel genome editing method, called "GONAD (Genome-editing via Oviductal Nucleic Acids Delivery)," which can completely eliminate the ex vivo handling of embryos. We also made improvements to the GONAD method, termed "improved-GONAD (i-GONAD)." The i-GONAD method involves injection of CRISPR reagents into the oviduct of an anesthetized pregnant female using a mouthpiece-controlled glass micropipette under a dissecting microscope, followed by EP of the entire oviduct allowing the CRISPR reagents to enter into the zygotes present inside the oviduct, in situ. After the i-GONAD procedure, the mouse recovered from anesthesia is allowed to continue the pregnancy to full term to deliver its pups. The i-GONAD method does not require pseudopregnant female animals for embryo transfer, unlike the methods relying on ex vivo handling of zygotes. Therefore, the i-GONAD method can reduce the number of animals used, compared to the traditional methods. In this chapter, we describe some newer technical tips about the i-GONAD method. Additionally, even though the detailed protocols of GONAD and i-GONAD have been published elsewhere (Gurumurthy et al., Curr Protoc Hum Genet 88:15.8.1-15.8.12, 2016 Nat Protoc 14:2452-2482, 2019), we provide all the protocol steps of i-GONAD in this chapter so that the reader can find most of the information, needed for performing i-GONAD experiments, in one place.}, }
@article {pmid36995673, year = {2023}, author = {Schaeffer, L and Lindner, L and Pavlovic, G and Hérault, Y and Birling, MC}, title = {CRISMERE Chromosome Engineering in Mouse and Rat.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2631}, number = {}, pages = {277-297}, pmid = {36995673}, issn = {1940-6029}, mesh = {Mice ; Rats ; Animals ; *DNA Copy Number Variations ; *Genome ; Genomics ; Mutation ; Chromosomes ; CRISPR-Cas Systems/genetics ; Genetic Engineering ; }, abstract = {CRISPR/Cas9 technology is a versatile tool for engineering biology that has dramatically transformed our ability to manipulate genomes. In this protocol, we use its capacity to generate two double-strand breaks simultaneously, at precise positions in the genome, to generate mouse or rat lines with deletion, inversion, and duplication of a specific genomic segment. The technic is called CRISMERE for CRISpr-MEdiated REarrangement. This protocol describes the different steps to generate and validate the different chromosomal rearrangements that can be obtained with the technology. These new genetic configurations can be useful to model rare diseases with copy number variation, understand the genomic organization, or provide genetic tools (like balancer chromosome) to keep lethal mutations.}, }
@article {pmid36995672, year = {2023}, author = {Davis, DJ and Men, H and Bryda, EC}, title = {Electroporation-Mediated CRISPR/Cas9 Genome Editing in Rat Zygotes.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2631}, number = {}, pages = {267-276}, pmid = {36995672}, issn = {1940-6029}, mesh = {Rats ; Animals ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Zygote ; Electroporation/methods ; Electroporation Therapies ; }, abstract = {Genetic engineering in the rat has been revolutionized by the development of CRISPR-based genome editing tools. Conventional methods for inserting genome editing elements such as CRISPR/Cas9 reagents into rat zygotes include cytoplasmic or pronuclear microinjections. These techniques are labor-intensive, require specialized micromanipulator equipment, and are technically challenging. Here, we describe a simple and effective method for zygote electroporation in which CRISPR/Cas9 reagents are introduced into rat zygotes via pores produced by precise electrical pulses applied to the cells. Zygote electroporation allows for high-throughput efficient genome editing in rat embryos.}, }
@article {pmid36995671, year = {2023}, author = {Garza, S and Paik, R}, title = {CRISPR/Cas9 Endonuclease-Mediated Mouse Genome Editing of One-Cell and/or Two-Cell Embryos by Electroporation, and the Use of Rad51 to Enhance Knock-In Allele Homozygosity via Interhomolog Repair Mechanism.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2631}, number = {}, pages = {253-266}, pmid = {36995671}, issn = {1940-6029}, mesh = {Animals ; Mice ; *Gene Editing/methods ; *CRISPR-Associated Protein 9/genetics ; CRISPR-Cas Systems/genetics ; Alleles ; Electroporation/methods ; DNA ; Gene Knock-In Techniques ; }, abstract = {Electroporation of mouse embryos with CRISPR/Cas9 endonuclease tool is a facile and efficient method to edit endogenous genome sequences for generating genetically engineered mouse models (GEMMs). Common genome engineering projects, such as knock-out (KO), conditional knock-out (cKO), point mutation, and small foreign DNA (<1 Kb) knock-in (KI) alleles, can be effectively accomplished with a simple electroporation procedure. The use of electroporation in sequential gene editing at the one-cell (0.7 days post-coitum (dpc)) and at two-cell (1.5 dpc) embryonic stages provides a fast and compelling protocol to safely introduce multiple gene modifications on the same chromosome by limiting chromosomal fractures. In addition, the co-electroporation of the ribonucleoprotein (RNP) complex and single-stranded oligodeoxynucleotide (ssODN) donor DNA with the strand exchange protein Rad51 can significantly increase the number of homozygous founders. Here we describe a comprehensive guideline for mouse embryo electroporation to generate GEMMs and the implementation of Rad51 in RNP/ssODN complex EP medium protocol.}, }
@article {pmid36995669, year = {2023}, author = {Wefers, B and Wurst, W and Kühn, R}, title = {Gene Editing in Mouse Zygotes Using the CRISPR/Cas9 System.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2631}, number = {}, pages = {207-230}, pmid = {36995669}, issn = {1940-6029}, mesh = {Animals ; Mice ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Zygote/metabolism ; Gene Targeting/methods ; Mice, Knockout ; RNA, Guide, Kinetoplastida/genetics ; }, abstract = {Engineering of the mouse germline is a key technology in biomedical research for studying the function of genes in health and disease. Since the first knockout mouse was described in 1989, gene targeting was based on recombination of vector encoded sequences in mouse embryonic stem cell lines and their introduction into preimplantation embryos to obtain germline chimeric mice. This approach has been replaced in 2013 by the application of the RNA-guided CRISPR/Cas9 nuclease system, which is introduced into zygotes and directly creates targeted modifications in the mouse genome. Upon the introduction of Cas9 nuclease and guide RNAs into one-cell embryos, sequence-specific double-strand breaks are created that are highly recombinogenic and processed by DNA repair enzymes. Gene editing commonly refers to the diversity of DSB repair products that include imprecise deletions or precise sequence modifications copied from repair template molecules. Since gene editing can now be easily applied directly in mouse zygotes, it has rapidly become the standard procedure for generating genetically engineered mice. This article covers the design of guide RNAs, knockout and knockin alleles, options for donor delivery, preparation of reagents, microinjection or electroporation of zygotes, and the genotyping of pups derived from gene editing projects.}, }
@article {pmid36995667, year = {2023}, author = {Narina, S and Connelly, JP and Pruett-Miller, SM}, title = {High-Throughput Analysis of CRISPR-Cas9 Editing Outcomes in Cell and Animal Models Using CRIS.py.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2631}, number = {}, pages = {155-182}, pmid = {36995667}, issn = {1940-6029}, mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Mutation ; Models, Animal ; }, abstract = {Genome editing using the CRISPR-Cas9 platform creates precise modifications in cells and whole organisms. Although knockout (KO) mutations can occur at high frequencies, determining the editing rates in a pool of cells or selecting clones that contain only KO alleles can be a challenge. User-defined knock-in (KI) modifications are achieved at much lower rates, making the identification of correctly modified clones even more challenging. The high-throughput format of targeted next-generation sequencing (NGS) provides a platform allowing sequence information to be gathered from a one to thousands of samples. However, it also poses a challenge in terms of analyzing the large amount of data that is generated. In this chapter, we present and discuss CRIS.py, a simple and highly versatile Python-based program for analyzing NGS data for genome-editing outcomes. CRIS.py can be used to analyze sequencing results for any kind of modification or multiplex modifications specified by the user. Moreover, CRIS.py runs on all fastq files found in a directory, thereby concurrently analyzing all uniquely indexed samples. CRIS.py results are consolidated into two summary files, which allows users to sort and filter results and quickly identify the clones (or animals) of greatest interest.}, }
@article {pmid36995665, year = {2023}, author = {Mackenzie, M and Fower, A and Allan, AJ and Codner, GF and Bunton-Stasyshyn, RK and Teboul, L}, title = {Genotyping Genome-Edited Founders and Subsequent Generation.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2631}, number = {}, pages = {103-134}, pmid = {36995665}, issn = {1940-6029}, mesh = {Animals ; *CRISPR-Cas Systems ; *Gene Editing ; Genotype ; Mutation ; Genome ; }, abstract = {Targeted nucleases allow the production of many types of genetic mutations directly in the early embryo. However, the outcome of their activity is a repair event of unpredictable nature, and the founder animals that are produced are generally of a mosaic nature. Here, we present the molecular assays and genotyping strategies that will support the screening of the first generation for potential founders and the validation of positive animals in the subsequent generation, according to the type of mutation generated.}, }
@article {pmid36995664, year = {2023}, author = {Lintott, LG and Nutter, LMJ}, title = {Genetic and Molecular Quality Control of Genetically Engineered Mice.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2631}, number = {}, pages = {53-101}, pmid = {36995664}, issn = {1940-6029}, mesh = {Mice ; Animals ; Humans ; *Gene Editing/methods ; *Genetic Engineering ; Embryonic Stem Cells ; Transgenes ; Quality Control ; CRISPR-Cas Systems ; Mammals/genetics ; }, abstract = {Genetically engineered mice are used as avatars to understand mammalian gene function and develop therapies for human disease. During genetic modification, unintended changes can occur, and these changes may result in misassigned gene-phenotype relationships leading to incorrect or incomplete experimental interpretations. The types of unintended changes that may occur depend on the allele type being made and the genetic engineering approach used. Here we broadly categorize allele types as deletions, insertions, base changes, and transgenes derived from engineered embryonic stem (ES) cells or edited mouse embryos. However, the methods we describe can be adapted to other allele types and engineering strategies. We describe the sources and consequ ences of common unintended changes and best practices for detecting both intended and unintended changes by screening and genetic and molecular quality control (QC) of chimeras, founders, and their progeny. Employing these practices, along with careful allele design and good colony management, will increase the chance that investigations using genetically engineered mice will produce high-quality reproducible results, to enable a robust understanding of gene function, human disease etiology, and therapeutic development.}, }
@article {pmid36995636, year = {2023}, author = {González, MN and Massa, GA and Andersson, M and Storani, L and Olsson, N and Décima Oneto, CA and Hofvander, P and Feingold, SE}, title = {CRISPR/Cas9 Technology for Potato Functional Genomics and Breeding.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2653}, number = {}, pages = {333-361}, pmid = {36995636}, issn = {1940-6029}, mesh = {*CRISPR-Cas Systems/genetics ; *Solanum tuberosum/genetics ; Plant Breeding ; Gene Editing/methods ; Genomics ; }, abstract = {Cultivated potato (Solanum tuberosum L.) is one of the most important staple food crops worldwide. Its tetraploid and highly heterozygous nature poses a great challenge to its basic research and trait improvement through traditional mutagenesis and/or crossbreeding. The establishment of the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) as a gene editing tool has allowed the alteration of specific gene sequences and their concomitant gene function, providing powerful technology for potato gene functional analysis and improvement of elite cultivars. This technology relies on a short RNA molecule called single guide RNA (sgRNA) that directs the Cas9 nuclease to induce a site-specific double-stranded break (DSB). Further, repair of the DSB by the error-prone non-homologous end joining (NHEJ) mechanism leads to the introduction of targeted mutations, which can be used to produce the loss of function of specific gene(s). In this chapter, we describe experimental procedures to apply the CRISPR/Cas9 technology for potato genome editing. First, we provide strategies for target selection and sgRNA design and describe a Golden Gate-based cloning system to obtain a sgRNA/Cas9-encoding binary vector. We also describe an optimized protocol for ribonucleoprotein (RNP) complex assembly. The binary vector can be used for both Agrobacterium-mediated transformation and transient expression in potato protoplasts, while the RNP complexes are intended to obtain edited potato lines through protoplast transfection and plant regeneration. Finally, we describe procedures to identify the gene-edited potato lines. The methods described here are suitable for potato gene functional analysis and breeding.}, }
@article {pmid36995634, year = {2023}, author = {Wu, FH and Hsu, CT and Lin, CS}, title = {Targeted Insertion in Nicotiana benthamiana Genomes via Protoplast Regeneration.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2653}, number = {}, pages = {297-315}, pmid = {36995634}, issn = {1940-6029}, mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Tobacco/genetics ; Protoplasts ; Oligodeoxyribonucleotides ; }, abstract = {Insertion of a specific sequence in a targeted region for precise editing is still a major challenge in plants. Current protocols rely on inefficient homology-directed repair or non-homologous end-joining with modified double-stranded oligodeoxyribonucleotides (dsODNs) as donors. We developed a simple protocol that eliminates the need for expensive equipment, chemicals, modifications of donor DNA, and complicated vector construction. The protocol uses polyethylene glycol (PEG)-calcium to deliver low-cost, unmodified single-stranded oligodeoxyribonucleotides (ssODNs) and CRISPR/Cas9 ribonucleoprotein (RNP) complexes into Nicotiana benthamiana protoplasts. Regenerated plants were obtained from edited protoplasts with an editing frequency of up to 50% at the target locus. The inserted sequence was inherited to the next generation; this method thus opens the possibility for the future exploration of genomes by targeted insertion in plants.}, }
@article {pmid36995633, year = {2023}, author = {Jiang, Q and Yang, Q and Harwood, W and Tang, H and Wei, Y and Zheng, Y}, title = {A CRISPR/Cas9 Protocol for Target Gene Editing in Barley.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2653}, number = {}, pages = {287-296}, pmid = {36995633}, issn = {1940-6029}, mesh = {*Gene Editing/methods ; CRISPR-Cas Systems/genetics ; *Hordeum/genetics ; CRISPR-Associated Protein 9/genetics ; Genome ; }, abstract = {Previous studies of gene function rely on the existing natural genetic variation or on induction of mutations by physical or chemical mutagenesis. The availability of alleles in nature, and random mutagenesis induced by physical or chemical means, limits the depth of research. The CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) system provides the means to rapidly modify genomes in a precise and predictable way, making it possible to modulate gene expression and modify the epigenome. Barley is the most appropriate model species for functional genomic analysis of common wheat. Therefore, the genome editing system of barley is very important for the study of wheat gene function. Here we detail a protocol for barley gene editing. The effectiveness of this method has been confirmed in our previous published studies.}, }
@article {pmid36995632, year = {2023}, author = {Quach, T and Nguyen, H and Meyer, O and Sato, SJ and Clemente, TE and Guo, M}, title = {Introduction of Genome Editing Reagents and Genotyping of Derived Edited Alleles in Soybean (Glycine max (L.) Merr.).}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2653}, number = {}, pages = {273-285}, pmid = {36995632}, issn = {1940-6029}, mesh = {*Gene Editing/methods ; *Soybeans/genetics ; CRISPR-Cas Systems/genetics ; Alleles ; Genotype ; }, abstract = {Cas9-based genome editing is a powerful genetic tool for loci specifically targeted for genome modification. This chapter describes up-to-date protocols using Cas9-based genome editing technology, including vector construction with GoldenBraid assembly, Agrobacterium-mediated soybean transformation, and identification of editing in the genome.}, }
@article {pmid36995630, year = {2023}, author = {Lawrenson, T and Atkinson, N and Forner, M and Harwood, W}, title = {Highly Efficient Gene Knockout in Medicago truncatula Genotype R108 Using CRISPR-Cas9 System and an Optimized Agrobacterium Transformation Method.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2653}, number = {}, pages = {221-252}, pmid = {36995630}, issn = {1940-6029}, mesh = {*Agrobacterium/genetics ; CRISPR-Cas Systems/genetics ; *Medicago truncatula/genetics ; Gene Knockout Techniques ; Genotype ; }, abstract = {Medicago truncatula is the model plant species for studying symbioses with nitrogen-fixing rhizobia and arbuscular mycorrhizae, where edited mutants are invaluable for elucidating the contributions of known genes in these processes. Streptococcus pyogenes Cas9 (SpCas9)-based genome editing is a facile means of achieving loss of function, including where multiple gene knockouts are desired in a single generation. We describe how the user can customize our vector to target single or multiple genes, then how the vector is used to make M. truncatula transgenic plants containing target site mutations. Finally, obtaining transgene-free homozygous mutants is covered.}, }
@article {pmid36995629, year = {2023}, author = {Dhokane, D and Kancharla, N and Savarimuthu, A and Bhadra, B and Bandyopadhyay, A and Dasgupta, S}, title = {Genome Editing in Chlamydomonas reinhardtii Using Cas9-gRNA Ribonucleoprotein Complex: A Step-by-Step Guide.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2653}, number = {}, pages = {207-217}, pmid = {36995629}, issn = {1940-6029}, mesh = {*Gene Editing ; CRISPR-Cas Systems/genetics ; *Chlamydomonas reinhardtii/genetics/metabolism ; RNA, Guide, Kinetoplastida/genetics ; Ribonucleoproteins/genetics/metabolism ; }, abstract = {Genome editing technologies have provided opportunities to manipulate literally any genomic location, opening new avenues for reverse genetics-based improvements. Among them, CRISPR/Cas9 is the most versatile tool for genome editing applications in prokaryotes and eukaryotes. Here, we provide a guide to successfully carry out high-efficiency genome editing in Chlamydomonas reinhardtii using preassembled CRISPR/Cas9-gRNA ribonucleoprotein (RNP) complexes.}, }
@article {pmid36995627, year = {2023}, author = {Becker, M and Hensel, G}, title = {Ribonucleoprotein (RNP)-Mediated Targeted Mutagenesis in Barley (Hordeum vulgare L.).}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2653}, number = {}, pages = {187-197}, pmid = {36995627}, issn = {1940-6029}, mesh = {*Hordeum/genetics ; Ribonucleoproteins/genetics ; Genetic Engineering/methods ; Mutagenesis ; Edible Grain/genetics ; CRISPR-Cas Systems ; Genome, Plant ; }, abstract = {The crop species barley is a genetic model for the small grain temperate cereals. Thanks to the availability of whole genome sequence and the development of customizable endonucleases, site-directed genome modification has recently revolutionized genetic engineering. Several platforms have been established in plants, with the most flexible one offered by the clustered regularly interspaced short palindromic repeats (CRISPR) technology. In this protocol, commercially available synthetic guide RNAs (gRNAs), Cas enzymes, or custom-generated reagents are used for targeted mutagenesis in barley. The protocol has been successfully used with immature embryo explants to generate site-specific mutations in regenerants. As the double-strand break-inducing reagents are customizable and can be efficiently delivered, pre-assembled ribonucleoprotein (RNP) complexes allow efficient generation of genome-modified plants.}, }
@article {pmid36995625, year = {2023}, author = {Fang, H and Culver, JN and Niedz, RP and Qi, Y}, title = {Delivery of CRISPR-Cas12a Ribonucleoprotein Complex for Genome Editing in an Embryogenic Citrus Cell Line.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2653}, number = {}, pages = {153-171}, pmid = {36995625}, issn = {1940-6029}, mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Plants/genetics ; Ribonucleoproteins/genetics/metabolism ; Cell Line ; }, abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR) technology is a powerful genome editing tool. Recently developed CRISPR-Cas12a system confers several advantages over CRISPR-Cas9, making it ideal for use in plant genome editing and crop improvement. While traditional transformation methods based on plasmid delivery pose concerns associated with transgene integration and off-target effects, CRISPR-Cas12a delivered as ribonucleoproteins (RNPs) can effectively alleviate these potential issues. Here we present a detailed protocol for LbCas12a-mediated genome editing using RNP delivery in Citrus protoplasts. This protocol provides a comprehensive guideline for RNP component preparation, RNP complex assembly and delivery, and editing efficiency assessment.}, }
@article {pmid36995623, year = {2023}, author = {Yuan, G and Tuskan, GA and Yang, X}, title = {Use of Fluorescent Protein Reporters for Assessing and Detecting Genome Editing Reagents and Transgene Expression in Plants.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2653}, number = {}, pages = {115-127}, pmid = {36995623}, issn = {1940-6029}, mesh = {*Gene Editing ; Plants, Genetically Modified/genetics ; *CRISPR-Cas Systems ; Indicators and Reagents ; Transgenes ; Genome, Plant/genetics ; }, abstract = {Fluorescent protein reporters have been widely used for monitoring the expression of target genes in various engineered organisms. Although a wide range of analytical approaches (e.g., genotyping PCR, digital PCR, DNA sequencing) have been utilized to detect and identify genome editing reagents and transgene expression in genetically modified plants, these methods are usually limited to use in the late stages of plant transformation and can only be used invasively. Here we describe GFP- and eYGFPuv-based strategies and methods for assessing and detecting genome editing reagents and transgene expression in plants, including protoplast transformation, leaf infiltration, and stable transformation. These methods and strategies enable easy, noninvasive screening of genome editing and transgenic events in plants.}, }
@article {pmid36995622, year = {2023}, author = {Xiong, J and Wang, C and Wang, K}, title = {Construction of CRISPR/Cas9 Multiplex Genome Editing System in Rice.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2653}, number = {}, pages = {107-114}, pmid = {36995622}, issn = {1940-6029}, mesh = {*Gene Editing/methods ; CRISPR-Cas Systems/genetics ; *Oryza/genetics ; Mutation ; }, abstract = {Multiplex genome editing (MGE) technologies constitute essential tools for rapid genome modification of multiple targets in one gene or multiple genes simultaneously. However, the vector construction process is complicated, and the number of mutation targets is constrained using the conventional binary vectors. Here, we describe a simple CRISPR/Cas9 MGE system based on classical isocaudomer technique in rice, which is comprised of only two simple vectors, and can theoretically be used to edit an unlimited number of genes simultaneously.}, }
@article {pmid36995619, year = {2023}, author = {Li, G and Sretenovic, S and Coleman, G and Qi, Y}, title = {Base Editing in Poplar Through an Agrobacterium-Mediated Transformation Method.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2653}, number = {}, pages = {53-71}, pmid = {36995619}, issn = {1940-6029}, mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Agrobacterium/genetics ; Promoter Regions, Genetic ; }, abstract = {CRISPR-Cas9 systems have revolutionized genome editing in plants and facilitated gene knockout and functional genomic studies in woody plants, like poplar. However, in tree species, previous studies have only focused on targeting indel mutations through CRISPR-based nonhomologous end joining (NHEJ) pathway. Cytosine base editors (CBEs) and adenine base editors (ABEs) enable C-to-T and A-to-G base changes, respectively. These base editors can introduce premature stop codons and amino acid changes, alter RNA splicing sites, and edit cis-regulatory elements of promoters. Base editing systems have only been recently established in trees. In this chapter, we describe a detailed, robust, and thoroughly tested protocol for preparing T-DNA vectors with two highly efficient CBEs, PmCDA1-BE3 and A3A/Y130F-BE3, and the highly efficient ABE8e as well as delivering the T-DNA through an improved protocol for Agrobacterium-mediated transformation in poplar. This chapter will provide promising application potential for precise base editing in poplar and other trees.}, }
@article {pmid36995618, year = {2023}, author = {Liang, D and Liu, Y and Li, C and Wen, Q and Xu, J and Geng, L and Liu, C and Jin, H and Gao, Y and Zhong, H and Dawson, J and Tian, B and Barco, B and Su, X and Dong, S and Li, C and Elumalai, S and Que, Q and Jepson, I and Shi, L}, title = {CRISPR/LbCas12a-Mediated Genome Editing in Soybean.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2653}, number = {}, pages = {39-52}, pmid = {36995618}, issn = {1940-6029}, mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Soybeans/genetics/metabolism ; Endonucleases/genetics ; Plants, Genetically Modified/genetics/metabolism ; Genome, Plant/genetics ; }, abstract = {Currently methods for generating soybean edited lines are time-consuming, inefficient, and limited to certain genotypes. Here we describe a fast and highly efficient genome editing method based on CRISPR-Cas12a nuclease system in soybean. The method uses Agrobacterium-mediated transformation to deliver editing constructs and uses aadA or ALS genes as selectable marker. It only takes about 45 days to obtain greenhouse-ready edited plants at higher than 30% transformation efficiency and 50% editing rate. The method is applicable to other selectable markers including EPSPS and has low transgene chimera rate. The method is also genotype-flexible and has been applied to genome editing of several elite soybean varieties.}, }
@article {pmid36995616, year = {2023}, author = {Sretenovic, S and Tang, X and Ren, Q and Zhang, Y and Qi, Y}, title = {PAM-Less CRISPR-SpRY Genome Editing in Plants.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2653}, number = {}, pages = {3-19}, pmid = {36995616}, issn = {1940-6029}, mesh = {*Gene Editing/methods ; CRISPR-Cas Systems/genetics ; CRISPR-Associated Protein 9/genetics ; Genome, Plant ; *Oryza/genetics/metabolism ; }, abstract = {Engineered SpCas9 variant, SpRY, has been demonstrated to facilitate protospacer adjacent motif (PAM) unrestricted targeting of genomic DNA in various biological systems. Here we describe fast, efficient, and robust preparation of SpRY-derived genome and base editors that can be easily adapted to target various DNA sequences in plants due to modular Gateway assembly. Presented are detailed protocols for preparing T-DNA vectors for genome and base editors and for assessing genome editing efficiency through transient expression of these reagents in rice protoplasts.}, }
@article {pmid36995603, year = {2023}, author = {He, L and He, Z and Li, Y and Sun, H and Wang, H}, title = {In Vivo Investigation of Gene Function in Muscle Stem Cells by CRISPR/Cas9-Mediated Genome Editing.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2640}, number = {}, pages = {287-311}, pmid = {36995603}, issn = {1940-6029}, mesh = {Mice ; Animals ; *Gene Editing/methods ; CRISPR-Cas Systems/genetics ; *Satellite Cells, Skeletal Muscle ; Muscles ; }, abstract = {Skeletal muscle satellite cells (SCs) are adult stem cells responsible for muscle development and injury-induced muscle regeneration. Functional elucidation of intrinsic regulatory factors governing SC activity is constrained partially by the technological limitations in editing SCs in vivo. Although the power of CRISPR/Cas9 in genome manipulation has been widely documented, its application in endogenous SCs remains largely untested. Our recent study generates a muscle-specific genome editing system leveraging the Cre-dependent Cas9 knockin mice and AAV9-mediated sgRNAs delivery, which allows gene disruption in SCs in vivo. Here, we illustrate the step-by-step procedure for achieving efficient editing using the above system.}, }
@article {pmid36995602, year = {2023}, author = {Nakamura, K and Tanaka, T and Yamanouchi, K}, title = {In Vivo Modeling of Skeletal Muscle Diseases Using the CRISPR/Cas9 System in Rats.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2640}, number = {}, pages = {277-285}, pmid = {36995602}, issn = {1940-6029}, mesh = {Animals ; Rats ; Mice ; Humans ; *Dystrophin/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Muscle, Skeletal/metabolism ; Genetic Therapy/methods ; *Muscular Dystrophy, Duchenne/genetics/therapy ; Disease Models, Animal ; }, abstract = {The CRISPR/Cas9 system is a powerful gene editing tool that can be used to modify a target gene in almost all species. It unlocks the possibility of generating knockout or knock-in genes in laboratory animals other than mice. The Dystrophin gene is implicated in human Duchenne muscular dystrophy; however, Dystrophin gene mutant mice do not show severe muscle degenerating phenotypes when compared to humans. On the other hand, Dystrophin gene mutant rats made with the CRISPR/Cas9 system show more severe phenotypes than those seen in mice. The phenotypes seen in dystrophin mutant rats are more representative of the features of human DMD. This implies that rats are better models of human skeletal muscle diseases than mice. In this chapter, we present a detailed protocol for the generation of gene-modified rats by microinjection into embryos using the CRISPR/Cas9 system.}, }
@article {pmid36997438, year = {2023}, author = {Awan, MJA and Aslam, MQ and Naqvi, RZ and Amin, I and Mansoor, S}, title = {A graft that crafts nontransgenic and genome-edited plants.}, journal = {Trends in plant science}, volume = {}, number = {}, pages = {}, doi = {10.1016/j.tplants.2023.03.020}, pmid = {36997438}, issn = {1878-4372}, abstract = {Grafting in plants facilitates the transmission of biomolecules across the union formation. Recently, Yang et al. demonstrated that inter- and intraspecific grafting in plants can be exploited for trafficking tRNA-tagged mobile reagents of the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system from the transgenic rootstock to wild-type scion for genetic improvement in plants through targeted mutagenesis.}, }
@article {pmid36995631, year = {2023}, author = {Lawrenson, T and Youles, M and Chhetry, M and Clarke, M and Harwood, W and Hundleby, P}, title = {Efficient Targeted Mutagenesis in Brassica Crops Using CRISPR/Cas Systems.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2653}, number = {}, pages = {253-271}, pmid = {36995631}, issn = {1940-6029}, abstract = {CRISPR/Cas has been established for targeted mutagenesis in many plant species since 2013, including Brassica napus and Brassica oleracea. Since that time, improvements have been made in terms of efficiency and choice of CRISPR systems. This protocol encompasses improved Cas9 efficiency and an alternative Cas12a system, allowing more challenging and diverse editing outcomes to be achieved.}, }
@article {pmid36995628, year = {2023}, author = {Becker, M and Hensel, G}, title = {Ribonucleoprotein (RNP)-Mediated Allele Replacement in Barley (Hordeum vulgare L.) Leaves.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2653}, number = {}, pages = {199-205}, pmid = {36995628}, issn = {1940-6029}, abstract = {Varietal differences within a species with agronomic importance are often based on minor changes in the genomic sequence. For example, fungus-resistant and fungus-susceptible wheat varieties may vary in only one amino acid. The situation is similar with the reporter genes Gfp and Yfp where two base pairs cause a shift in the emission spectrum from green to yellow. Methods of targeted double-strand break induction now allow this exchange precisely with the simultaneous transfer of the desired repair template. However, these changes rarely lead to a selective advantage that can be used in generating such mutant plants. The protocol presented here allows a corresponding allele replacement at the cellular level using ribonucleoprotein complexes in combination with an appropriate repair template. The efficiencies achieved are comparable to other methods with direct DNA transfer or integration of the corresponding building blocks in the host genome. They are in the range of 35 percent, considering one allele in a diploid organism as barley and using Cas9 RNP complexes.}, }
@article {pmid36995626, year = {2023}, author = {Ma, X and Li, X and Li, Z}, title = {Transgene-Free Genome Editing in Nicotiana benthamiana with CRISPR/Cas9 Delivered by a Rhabdovirus Vector.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2653}, number = {}, pages = {173-185}, pmid = {36995626}, issn = {1940-6029}, abstract = {The clustered regularly interspersed short palindromic repeats (CRISPR)/Cas systems have become the most widely adopted genome editing platform owing to their unprecedented simplicity, efficiency, and versatility. Typically, the genome editing enzyme is expressed in plant cells from an integrated transgene delivered by either Agrobacterium-mediated or biolistic transformation. Recently, plant virus vectors have emerged as promising tools for the in planta delivery of CRISPR/Cas reagent. Here, we provide a protocol for CRISPR/Cas9-mediated genome editing in the model tobacco plant Nicotiana benthamiana using a recombinant negative-stranded RNA rhabdovirus vector. The method is based on infection of N. benthamiana with a Sonchus yellow net virus (SYNV)-based vector that carries the Cas9 and guide RNA expression cassettes to target specific genome loci for mutagenesis. With this method, mutant plants free of foreign DNA can be obtained within 4-5 months.}, }
@article {pmid36995621, year = {2023}, author = {Wu, Y and Zhang, T}, title = {Designing Guide-RNA for Generating Premature Stop Codons for Gene Knockout Using CRISPR-BETS.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2653}, number = {}, pages = {95-105}, pmid = {36995621}, issn = {1940-6029}, abstract = {Cytosine base editors (CBEs) accurately modify target sites by mediating a C to T change (or a G to A change on the opposite strand). This allows us to install premature stop codons for gene knockout. However, highly specific sgRNAs (single-guide RNAs) are necessary for the CRISPR-Cas nuclease to work efficiently. In this study, we introduce a method of designing highly specific gRNA to generate premature stop codons and knock out a gene using CRISPR-BETS software.}, }
@article {pmid36945419, year = {2023}, author = {Koseki, S and Hong, L and Yudistyra, V and Stan, T and Tysinger, E and Silverstein, R and Kramme, C and Amrani, N and Savic, N and Pacesa, M and Rodriguez, TS and Ponnapati, M and Jacobson, J and Church, G and Truant, R and Jinek, M and Kleinstiver, B and Sontheimer, E and Chatterjee, P}, title = {PAM-Flexible Genome Editing with an Engineered Chimeric Cas9.}, journal = {Research square}, volume = {}, number = {}, pages = {}, pmid = {36945419}, abstract = {CRISPR enzymes require a defined protospacer adjacent motif (PAM) flanking a guide RNA-programmed target site, limiting their sequence accessibility for robust genome editing applications. In this study, we recombine the PAM-interacting domain of SpRY, a broad-targeting Cas9 possessing an NRN > NYN PAM preference, with the N-terminus of Sc++, a Cas9 with simultaneously broad, efficient, and accurate NNG editing capabilities, to generate a chimeric enzyme with highly flexible PAM preference: SpRYc. We demonstrate that SpRYc leverages properties of both enzymes to specifically edit diverse NNN PAMs and disease-related loci for potential therapeutic applications. In total, the unique approaches to generate SpRYc, coupled with its robust flexibility, highlight the power of integrative protein design for Cas9 engineering and motivate downstream editing applications that require precise genomic positioning.}, }
@article {pmid36912653, year = {2023}, author = {Li, J and Wang, X and Zou, J and Yang, K and Wang, X and Wang, Y and Zhang, H and Huang, H and Su, X and Yao, B and Luo, H and Qin, X}, title = {Identification and Characterization of the Determinants of Copper Resistance in the Acidophilic Fungus Acidomyces richmondensis MEY-1 Using the CRISPR/Cas9 System.}, journal = {Applied and environmental microbiology}, volume = {89}, number = {3}, pages = {e0210722}, pmid = {36912653}, issn = {1098-5336}, mesh = {Copper/pharmacology/metabolism ; CRISPR-Cas Systems ; Gene Editing ; *Ascomycota/genetics/metabolism ; Transcription Factors/genetics/metabolism ; *P-type ATPases/genetics ; }, abstract = {Copper (Cu) homeostasis has not been well documented in filamentous fungi, especially extremophiles. One of the main obstacles impeding their characterization is the lack of a powerful genome-editing tool. In this study, we applied a CRISPR/Cas9 system for efficient targeted gene disruption in the acidophilic fungus Acidomyces richmondensis MEY-1, formerly known as Bispora sp. strain MEY-1. Using this system, we investigated the basis of Cu tolerance in strain MEY-1. This strain has extremely high Cu tolerance among filamentous fungi, and the transcription factor ArAceA (A. richmondensis AceA) has been shown to be involved in this process. The ArAceA deletion mutant (ΔArAceA) exhibits specific growth defects at Cu concentrations of ≥10 mM and is transcriptionally more sensitive to Cu than the wild-type strain. In addition, the putative metallothionein ArCrdA was involved in Cu tolerance only under high Cu concentrations. MEY-1 has no Aspergillus nidulans CrpA homologs, which are targets of AceA-like transcription factors and play a role in Cu tolerance. Instead, we identified the Cu-transporting P-type ATPase ArYgA, homologous to A. nidulans YgA, which was involved in pigmentation rather than Cu tolerance. When the ΔArYgA mutant was grown on medium supplemented with Cu ions, the black color was completely restored. The lack of CrpA homologs in A. richmondensis MEY-1 and its high tolerance to Cu suggest that a novel Cu detoxification mechanism differing from the AceA-CrpA axis exists. IMPORTANCE Filamentous fungi are widely distributed worldwide and play an important ecological role as decomposers. However, the mechanisms of their adaptability to various environments are not fully understood. Various extremely acidophilic filamentous fungi have been isolated from acidic mine drainage (AMD) with extremely low pH and high heavy metal and sulfate concentrations, including A. richmondensis. The lack of genetic engineering tools, particularly genome-editing tools, hinders the study of these acidophilic and heavy metal-resistant fungi at the molecular level. Here, we first applied a CRISPR/Cas9-mediated gene-editing system to A. richmondensis MEY-1. Using this system, we identified and characterized the determinants of Cu resistance in A. richmondensis MEY-1. The conserved roles of the Cu-binding transcription factor ArAceA in Cu tolerance and the Cu-transporting P-type ATPase ArYgA in the Cu-dependent production of pigment were confirmed. Our findings provide insights into the molecular basis of Cu tolerance in the acidophilic fungus A. richmondensis MEY-1. Furthermore, the CRISPR/Cas9 system used here would be a powerful tool for studies of the mechanisms of adaptability of acidophilic fungi to extreme environments.}, }
@article {pmid36890230, year = {2023}, author = {Puig-Saus, C and Sennino, B and Peng, S and Wang, CL and Pan, Z and Yuen, B and Purandare, B and An, D and Quach, BB and Nguyen, D and Xia, H and Jilani, S and Shao, K and McHugh, C and Greer, J and Peabody, P and Nayak, S and Hoover, J and Said, S and Jacoby, K and Dalmas, O and Foy, SP and Conroy, A and Yi, MC and Shieh, C and Lu, W and Heeringa, K and Ma, Y and Chizari, S and Pilling, MJ and Ting, M and Tunuguntla, R and Sandoval, S and Moot, R and Hunter, T and Zhao, S and Saco, JD and Perez-Garcilazo, I and Medina, E and Vega-Crespo, A and Baselga-Carretero, I and Abril-Rodriguez, G and Cherry, G and Wong, DJ and Hundal, J and Chmielowski, B and Speiser, DE and Bethune, MT and Bao, XR and Gros, A and Griffith, OL and Griffith, M and Heath, JR and Franzusoff, A and Mandl, SJ and Ribas, A}, title = {Neoantigen-targeted CD8[+] T cell responses with PD-1 blockade therapy.}, journal = {Nature}, volume = {615}, number = {7953}, pages = {697-704}, pmid = {36890230}, issn = {1476-4687}, mesh = {Humans ; *Antigens, Neoplasm/immunology ; *CD8-Positive T-Lymphocytes/immunology/metabolism ; *Immunotherapy ; *Melanoma/drug therapy/genetics/immunology/pathology ; Receptors, Antigen, T-Cell/immunology/metabolism ; *Immune Checkpoint Inhibitors/pharmacology/therapeutic use ; HLA Antigens/immunology ; Neoplasm Metastasis ; Precision Medicine ; Gene Editing ; CRISPR-Cas Systems ; Mutation ; }, abstract = {Neoantigens are peptides derived from non-synonymous mutations presented by human leukocyte antigens (HLAs), which are recognized by antitumour T cells[1-14]. The large HLA allele diversity and limiting clinical samples have restricted the study of the landscape of neoantigen-targeted T cell responses in patients over their treatment course. Here we applied recently developed technologies[15-17] to capture neoantigen-specific T cells from blood and tumours from patients with metastatic melanoma with or without response to anti-programmed death receptor 1 (PD-1) immunotherapy. We generated personalized libraries of neoantigen-HLA capture reagents to single-cell isolate the T cells and clone their T cell receptors (neoTCRs). Multiple T cells with different neoTCR sequences (T cell clonotypes) recognized a limited number of mutations in samples from seven patients with long-lasting clinical responses. These neoTCR clonotypes were recurrently detected over time in the blood and tumour. Samples from four patients with no response to anti-PD-1 also demonstrated neoantigen-specific T cell responses in the blood and tumour to a restricted number of mutations with lower TCR polyclonality and were not recurrently detected in sequential samples. Reconstitution of the neoTCRs in donor T cells using non-viral CRISPR-Cas9 gene editing demonstrated specific recognition and cytotoxicity to patient-matched melanoma cell lines. Thus, effective anti-PD-1 immunotherapy is associated with the presence of polyclonal CD8[+] T cells in the tumour and blood specific for a limited number of immunodominant mutations, which are recurrently recognized over time.}, }
@article {pmid36759244, year = {2023}, author = {Ben Yacoub, T and Wohlschlegel, J and Sahel, JA and Zeitz, C and Audo, I}, title = {[CRISPR/Cas9: From research to therapeutic application].}, journal = {Journal francais d'ophtalmologie}, volume = {46}, number = {4}, pages = {398-407}, doi = {10.1016/j.jfo.2022.10.008}, pmid = {36759244}, issn = {1773-0597}, mesh = {Humans ; *CRISPR-Cas Systems ; Gene Editing/methods ; *Neoplasms ; }, abstract = {For several decades, genome engineering has raised interest among many researchers and physicians in the study of genetic disorders and their treatments. Compared to its predecessors, zinc-finger nucleases (ZFN) and transcription activator-like effectors (TALEN), clustered regularly interspaced short palindromic repeats (CRISPR/Cas9) is currently the most efficient molecular tool for genome editing. This system, originally identified as a bacterial adaptive immune system, is capable of cutting and modifying any gene of a large number of living organisms. Numerous trials using this technology are being developed to provide effective treatment for several diseases, such as cancer, cardiovascular and ophthalmic disorders. In research, this technology is increasingly used for genetic disease modelling, providing meaningful models of relevant studies as well as a better understanding of underlying pathological mechanisms. Many molecular tools are now available to put this technique into practice in laboratories, and despite the technical and ethical issues raised by manipulation of the genome, CRIPSR/Cas9 offers a new breath of hope for therapeutic research around the world.}, }
@article {pmid36356599, year = {2023}, author = {Foy, SP and Jacoby, K and Bota, DA and Hunter, T and Pan, Z and Stawiski, E and Ma, Y and Lu, W and Peng, S and Wang, CL and Yuen, B and Dalmas, O and Heeringa, K and Sennino, B and Conroy, A and Bethune, MT and Mende, I and White, W and Kukreja, M and Gunturu, S and Humphrey, E and Hussaini, A and An, D and Litterman, AJ and Quach, BB and Ng, AHC and Lu, Y and Smith, C and Campbell, KM and Anaya, D and Skrdlant, L and Huang, EY and Mendoza, V and Mathur, J and Dengler, L and Purandare, B and Moot, R and Yi, MC and Funke, R and Sibley, A and Stallings-Schmitt, T and Oh, DY and Chmielowski, B and Abedi, M and Yuan, Y and Sosman, JA and Lee, SM and Schoenfeld, AJ and Baltimore, D and Heath, JR and Franzusoff, A and Ribas, A and Rao, AV and Mandl, SJ}, title = {Non-viral precision T cell receptor replacement for personalized cell therapy.}, journal = {Nature}, volume = {615}, number = {7953}, pages = {687-696}, pmid = {36356599}, issn = {1476-4687}, support = {K08 AI139375/AI/NIAID NIH HHS/United States ; }, mesh = {Humans ; Antigens, Neoplasm/genetics/immunology ; Biopsy ; *Cell- and Tissue-Based Therapy/adverse effects/methods ; Cytokine Release Syndrome/complications ; Disease Progression ; Encephalitis/complications ; *Gene Editing ; Gene Knock-In Techniques ; Gene Knockout Techniques ; Genes, T-Cell Receptor alpha ; Genes, T-Cell Receptor beta ; Mutation ; *Neoplasms/complications/genetics/immunology/therapy ; Patient Safety ; *Precision Medicine/adverse effects/methods ; *Receptors, Antigen, T-Cell/genetics/immunology/metabolism ; *T-Lymphocytes/immunology/metabolism ; *Transgenes/genetics ; HLA Antigens/immunology ; CRISPR-Cas Systems ; }, abstract = {T cell receptors (TCRs) enable T cells to specifically recognize mutations in cancer cells[1-3]. Here we developed a clinical-grade approach based on CRISPR-Cas9 non-viral precision genome-editing to simultaneously knockout the two endogenous TCR genes TRAC (which encodes TCRα) and TRBC (which encodes TCRβ). We also inserted into the TRAC locus two chains of a neoantigen-specific TCR (neoTCR) isolated from circulating T cells of patients. The neoTCRs were isolated using a personalized library of soluble predicted neoantigen-HLA capture reagents. Sixteen patients with different refractory solid cancers received up to three distinct neoTCR transgenic cell products. Each product expressed a patient-specific neoTCR and was administered in a cell-dose-escalation, first-in-human phase I clinical trial (NCT03970382). One patient had grade 1 cytokine release syndrome and one patient had grade 3 encephalitis. All participants had the expected side effects from the lymphodepleting chemotherapy. Five patients had stable disease and the other eleven had disease progression as the best response on the therapy. neoTCR transgenic T cells were detected in tumour biopsy samples after infusion at frequencies higher than the native TCRs before infusion. This study demonstrates the feasibility of isolating and cloning multiple TCRs that recognize mutational neoantigens. Moreover, simultaneous knockout of the endogenous TCR and knock-in of neoTCRs using single-step, non-viral precision genome-editing are achieved. The manufacture of neoTCR engineered T cells at clinical grade, the safety of infusing up to three gene-edited neoTCR T cell products and the ability of the transgenic T cells to traffic to the tumours of patients are also demonstrated.}, }
@article {pmid36995617, year = {2023}, author = {Wada, N and Osakabe, K and Osakabe, Y}, title = {Type I-D CRISPR System-Mediated Genome Editing in Plants.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2653}, number = {}, pages = {21-38}, pmid = {36995617}, issn = {1940-6029}, abstract = {Genome editing has revolutionized plant research and plant breeding by enabling precise genome manipulation. In particular, the application of type II CRISPR-Cas9 systems to genome editing has proved an important milestone, accelerating genetic engineering and the analysis of gene function. On the other hand, the potential of other types of CRISPR-Cas systems, especially many of the most abundant type I CRISPR-Cas systems, remains unexplored. We recently developed a novel genome editing tool, TiD, based on the type I-D CRISPR-Cas system. In this chapter, we describe a protocol for genome editing of plant cells using TiD. This protocol allows the application of TiD to induce short insertion and deletions (indels) or long-range deletions at target sites with high specificity in tomato cells.}, }
@article {pmid36995210, year = {2023}, author = {Mahler, M and Malone, LM and van den Berg, DF and Smith, LM and Brouns, SJJ and Fineran, PC}, title = {An OmpW-dependent T4-like phage infects Serratia sp. ATCC 39006.}, journal = {Microbial genomics}, volume = {9}, number = {3}, pages = {}, doi = {10.1099/mgen.0.000968}, pmid = {36995210}, issn = {2057-5858}, abstract = {Serratia sp. ATCC 39006 is a Gram-negative bacterium that has been used to study the function of phage defences, such as CRISPR-Cas, and phage counter-defence mechanisms. To expand our phage collection to study the phage-host interaction with Serratia sp. ATCC 39006, we isolated the T4-like myovirus LC53 in Ōtepoti Dunedin, Aotearoa New Zealand. Morphological, phenotypic and genomic characterization revealed that LC53 is virulent and similar to other Serratia, Erwinia and Kosakonia phages belonging to the genus Winklervirus. Using a transposon mutant library, we identified the host ompW gene as essential for phage infection, suggesting that it encodes the phage receptor. The genome of LC53 encodes all the characteristic T4-like core proteins involved in phage DNA replication and generation of viral particles. Furthermore, our bioinformatic analysis suggests that the transcriptional organization of LC53 is similar to that of Escherichia coli phage T4. Importantly, LC53 encodes 18 tRNAs, which likely compensate for differences in GC content between phage and host genomes. Overall, this study describes a newly isolated phage infecting Serratia sp. ATCC 39006 that expands the diversity of phages available to study phage-host interactions.}, }
@article {pmid36993865, year = {2023}, author = {Ahmad, M}, title = {Plant breeding advancements with "CRISPR-Cas" genome editing technologies will assist future food security.}, journal = {Frontiers in plant science}, volume = {14}, number = {}, pages = {1133036}, pmid = {36993865}, issn = {1664-462X}, abstract = {Genome editing techniques are being used to modify plant breeding, which might increase food production sustainably by 2050. A product made feasible by genome editing is becoming better known, because of looser regulation and widespread acceptance. The world's population and food supply would never have increased proportionally under current farming practices. The development of plants and food production has been greatly impacted by global warming and climate change. Therefore, minimizing these effects is crucial for agricultural production that is sustainable. Crops are becoming more resilient to abiotic stress because of sophisticated agricultural practices and a better understanding of the abiotic stress response mechanism. Both conventional and molecular breeding techniques have been used to create viable crop types both processes are time-consuming. Recently, plant breeders have shown an interest in genome editing approaches for genetic manipulation that use clustered regularly interspaced short palindromic repeats (CRISPR/Cas9). To ensure the security of the food supply in the future, plant kinds with desired traits must be developed. A completely new era in plant breeding has begun because of the revolution in genome editing techniques based on the CRISPR/CRISPR-associated nuclease (Cas9) systems. All plants may effectively target a particular gene or group of loci using Cas9 and single-guide RNA (sgRNA). CRISPR/Cas9 can thereby save time and labor compared to conventional breeding methods. An easy, quick, and efficient method for directly altering the genetic sequences in cells is with the CRISPR and Cas9 systems. The CRISPR-Cas9 system, which was developed from components of the earliest known bacterial immune system, allows for targeted gene breakage and gene editing in a variety of cells/RNA sequences to guide endonuclease cleavage specificity in the CRISPR-Cas9 system. Editing can be directed to practically any genomic site by altering the guide RNA (gRNA) sequence and delivering it to a target cell along with the Cas9 endonuclease. We summarize recent CRISPR/Cas9 plant research findings, investigate potential applications in plant breeding, and make predictions about likely future breakthroughs and approaches to food security through 2050.}, }
@article {pmid36993599, year = {2023}, author = {Meers, C and Le, H and Pesari, SR and Hoffmann, FT and Walker, MWG and Gezelle, J and Sternberg, SH}, title = {Transposon-encoded nucleases use guide RNAs to selfishly bias their inheritance.}, journal = {bioRxiv : the preprint server for biology}, volume = {}, number = {}, pages = {}, doi = {10.1101/2023.03.14.532601}, pmid = {36993599}, abstract = {Insertion sequences (IS) are compact and pervasive transposable elements found in bacteria, which encode only the genes necessary for their mobilization and maintenance. IS 200 /IS 605 elements undergo 'peel-and-paste' transposition catalyzed by a TnpA transposase, but intriguingly, they also encode diverse, TnpB- and IscB-family proteins that are evolutionarily related to the CRISPR-associated effectors Cas12 and Cas9, respectively. Recent studies demonstrated that TnpB-family enzymes function as RNA-guided DNA endonucleases, but the broader biological role of this activity has remained enigmatic. Here we show that TnpB/IscB are essential to prevent permanent transposon loss as a consequence of the TnpA transposition mechanism. We selected a family of related IS elements from Geobacillus stearothermophilus that encode diverse TnpB/IscB orthologs, and showed that a single TnpA transposase was active for transposon excision. The donor joints formed upon religation of IS-flanking sequences were efficiently targeted for cleavage by RNA-guided TnpB/IscB nucleases, and co-expression of TnpB together with TnpA led to significantly greater transposon retention, relative to conditions in which TnpA was expressed alone. Remarkably, TnpA and TnpB/IscB recognize the same AT-rich transposon-adjacent motif (TAM) during transposon excision and RNA-guided DNA cleavage, respectively, revealing a striking convergence in the evolution of DNA sequence specificity between collaborating transposase and nuclease proteins. Collectively, our study reveals that RNA-guided DNA cleavage is a primal biochemical activity that arose to bias the selfish inheritance and spread of transposable elements, which was later co-opted during the evolution of CRISPR-Cas adaptive immunity for antiviral defense.}, }
@article {pmid36992681, year = {2023}, author = {Sukegawa, S and Nureki, O and Toki, S and Saika, H}, title = {Genome editing in rice mediated by miniature size Cas nuclease SpCas12f.}, journal = {Frontiers in genome editing}, volume = {5}, number = {}, pages = {1138843}, pmid = {36992681}, issn = {2673-3439}, abstract = {Cas9 derived from Streptococcus pyogenes (SpCas9) is used widely in genome editing using the CRISPR-Cas system due to its high activity, but is a relatively large molecule (1,368 amino acid (a.a.) residues). Recently, targeted mutagenesis in human cells and maize using Cas12f derived from Syntrophomonas palmitatica (SpCas12f)-a very small Cas of 497 a.a, which is a more suitable size for virus vectors-was reported. However, there are no reports of genome editing using SpCas12f in crops other than maize. In this study, we applied SpCas12f to genome editing in rice-one of the most important staple crops in the world. An expression vector encoding rice codon-optimized SpCas12f and sgRNA for OsTubulin as a target was introduced into rice calli by Agrobacterium-mediated transformation. Molecular analysis of SpCas12f-transformed calli showed that mutations were introduced successfully into the target region. Detailed analysis by amplicon sequencing revealed estimated mutation frequencies (a ratio of the number of mutated calli to that of SpCas12f-transformed calli) of 28.8% and 55.6% in two targets. Most mutation patterns were deletions, but base substitutions and insertions were also confirmed at low frequency. Moreover, off-target mutations by SpCas12f were not found. Furthermore, mutant plants were regenerated successfully from the mutated calli. It was confirmed that the mutations in the regenerated plants were inherited to the next-generation. In the previous report in maize, mutations were introduced by treatment with heat shock at 45°C for 4 h per day for 3 days; no mutations were introduced under normal growth conditions at 28°C. Surprisingly, however, mutations can be introduced without heat-shock treatment in rice. This might be due to the culture conditions, with relatively higher temperature (30°C or higher) and constant light during callus proliferation. Taken together, we demonstrated that SpCas12f can be used to achieve targeted mutagenesis in rice. SpCas12f is thus a useful tool for genome editing in rice and is suitable for virus vector-mediated genome editing due to its very small size.}, }
@article {pmid36992399, year = {2023}, author = {Mazurov, D and Ramadan, L and Kruglova, N}, title = {Packaging and Uncoating of CRISPR/Cas Ribonucleoproteins for Efficient Gene Editing with Viral and Non-Viral Extracellular Nanoparticles.}, journal = {Viruses}, volume = {15}, number = {3}, pages = {}, doi = {10.3390/v15030690}, pmid = {36992399}, issn = {1999-4915}, abstract = {Rapid progress in gene editing based on clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas) has revolutionized functional genomic studies and genetic disease correction. While numerous gene editing applications have been easily adapted by experimental science, the clinical utility of CRISPR/Cas remains very limited due to difficulty in delivery to primary cells and possible off-target effects. The use of CRISPR in the form of a ribonucleoprotein (RNP) complex substantially reduces the time of DNA exposure to the effector nuclease and minimizes its off-target activity. The traditional electroporation and lipofection methods lack the cell-type specificity of RNP delivery, can be toxic for cells, and are less efficient when compared to nanoparticle transporters. This review focuses on CRISPR/Cas RNP packaging and delivery using retro/lentiviral particles and exosomes. First, we briefly describe the natural stages of viral and exosomal particle formation, release and entry into the target cells. This helps us understand the mechanisms of CRISPR/Cas RNP packaging and uncoating utilized by the current delivery systems, which we discuss afterward. Much attention is given to the exosomes released during viral particle production that can be passively loaded with RNPs as well as the mechanisms necessary for particle fusion, RNP release, and transportation inside the target cells. Collectively, together with specific packaging mechanisms, all these factors can substantially influence the editing efficiency of the system. Finally, we discuss ways to improve CRISPR/Cas RNP delivery using extracellular nanoparticles.}, }
@article {pmid36983530, year = {2023}, author = {Wang, D and Jin, S and Lu, Q and Chen, Y}, title = {Advances and Challenges in CRISPR/Cas-Based Fungal Genome Engineering for Secondary Metabolite Production: A Review.}, journal = {Journal of fungi (Basel, Switzerland)}, volume = {9}, number = {3}, pages = {}, pmid = {36983530}, issn = {2309-608X}, abstract = {Fungi represent an important source of bioactive secondary metabolites (SMs), which have wide applications in many fields, including medicine, agriculture, human health, and many other industries. The genes involved in SM biosynthesis are usually clustered adjacent to each other into a region known as a biosynthetic gene cluster (BGC). The recent advent of a diversity of genetic and genomic technologies has facilitated the identification of many cryptic or uncharacterized BGCs and their associated SMs. However, there are still many challenges that hamper the broader exploration of industrially important secondary metabolites. The recent advanced CRISPR/Cas system has revolutionized fungal genetic engineering and enabled the discovery of novel bioactive compounds. In this review, we firstly introduce fungal BGCs and their relationships with associated SMs, followed by a brief summary of the conventional strategies for fungal genetic engineering. Next, we introduce a range of state-of-the-art CRISPR/Cas-based tools that have been developed and review recent applications of these methods in fungi for research on the biosynthesis of SMs. Finally, the challenges and limitations of these CRISPR/Cas-based systems are discussed and directions for future research are proposed in order to expand their applications and improve efficiency for fungal genetic engineering.}, }
@article {pmid36983076, year = {2023}, author = {Zhang, Y and Geng, Y and Li, S and Shi, T and Ma, X and Hua, R and Fang, L}, title = {Efficient Knocking Out of the Organophosphorus Insecticides Degradation Gene opdB in Cupriavidus nantongensis X1[T] via CRISPR/Cas9 with Red System.}, journal = {International journal of molecular sciences}, volume = {24}, number = {6}, pages = {}, pmid = {36983076}, issn = {1422-0067}, mesh = {*Insecticides/metabolism ; CRISPR-Cas Systems/genetics ; Organophosphorus Compounds/metabolism ; *Cupriavidus/genetics/metabolism ; Gene Editing/methods ; }, abstract = {Cupriavidus nantongensis X1[T] is a type strain of the genus Cupriavidus, that can degrade eight kinds of organophosphorus insecticides (OPs). Conventional genetic manipulations in Cupriavidus species are time-consuming, difficult, and hard to control. The clustered regularly interspaced short palindromic repeat (CRISPR)/associated protein 9 (Cas9) system has emerged as a powerful tool for genome editing applied in prokaryotes and eukaryotes due to its simplicity, efficiency, and accuracy. Here, we combined CRISPR/Cas9 with the Red system to perform seamless genetic manipulation in the X1[T] strain. Two plasmids, pACasN and pDCRH were constructed. The pACasN plasmid contained Cas9 nuclease and Red recombinase, and the pDCRH plasmid contained the dual single-guide RNA (sgRNA) of organophosphorus hydrolase (OpdB) in the X1[T] strain. For gene editing, two plasmids were transferred to the X1[T] strain and a mutant strain in which genetic recombination had taken place, resulting in the targeted deletion of opdB. The incidence of homologous recombination was over 30%. Biodegradation experiments suggested that the opdB gene was responsible for the catabolism of organophosphorus insecticides. This study was the first to use the CRISPR/Cas9 system for gene targeting in the genus Cupriavidus, and it furthered our understanding of the process of degradation of organophosphorus insecticides in the X1[T] strain.}, }
@article {pmid36982987, year = {2023}, author = {Perdigão, PRL and Ollington, B and Sai, H and Leung, A and Sacristan-Reviriego, A and van der Spuy, J}, title = {Retinal Organoids from an AIPL1 CRISPR/Cas9 Knockout Cell Line Successfully Recapitulate the Molecular Features of LCA4 Disease.}, journal = {International journal of molecular sciences}, volume = {24}, number = {6}, pages = {}, pmid = {36982987}, issn = {1422-0067}, support = {MR/P02582X/1/MRC_/Medical Research Council/United Kingdom ; }, mesh = {Child, Preschool ; Humans ; *Leber Congenital Amaurosis/pathology ; Carrier Proteins/metabolism ; Adaptor Proteins, Signal Transducing/metabolism ; CRISPR-Cas Systems/genetics ; Cell Line ; Organoids/metabolism ; Eye Proteins/genetics/metabolism ; }, abstract = {Aryl hydrocarbon receptor-interacting protein-like 1 (AIPL1) is expressed in photoreceptors where it facilitates the assembly of phosphodiesterase 6 (PDE6) which hydrolyses cGMP within the phototransduction cascade. Genetic variations in AIPL1 cause type 4 Leber congenital amaurosis (LCA4), which presents as rapid loss of vision in early childhood. Limited in vitro LCA4 models are available, and these rely on patient-derived cells harbouring patient-specific AIPL1 mutations. While valuable, the use and scalability of individual patient-derived LCA4 models may be limited by ethical considerations, access to patient samples and prohibitive costs. To model the functional consequences of patient-independent AIPL1 mutations, CRISPR/Cas9 was implemented to produce an isogenic induced pluripotent stem cell line harbouring a frameshift mutation in the first exon of AIPL1. Retinal organoids were generated using these cells, which retained AIPL1 gene transcription, but AIPL1 protein was undetectable. AIPL1 knockout resulted in a decrease in rod photoreceptor-specific PDE6α and β, and increased cGMP levels, suggesting downstream dysregulation of the phototransduction cascade. The retinal model described here provides a novel platform to assess functional consequences of AIPL1 silencing and measure the rescue of molecular features by potential therapeutic approaches targeting mutation-independent pathogenesis.}, }
@article {pmid36982961, year = {2023}, author = {Zhang, K and Zhang, W and Qin, M and Li, Y and Wang, H}, title = {Characterization and Application of the Sugar Transporter Zmo0293 from Zymomonas mobilis.}, journal = {International journal of molecular sciences}, volume = {24}, number = {6}, pages = {}, pmid = {36982961}, issn = {1422-0067}, mesh = {*Zymomonas/genetics/metabolism ; Glucose/metabolism ; Escherichia coli/genetics/metabolism ; Biological Transport ; Ethanol/metabolism ; }, abstract = {Zymomonas mobilis is a natural ethanologen with many desirable characteristics, which makes it an ideal industrial microbial biocatalyst for the commercial production of desirable bioproducts. Sugar transporters are responsible for the import of substrate sugars and the conversion of ethanol and other products. Glucose-facilitated diffusion protein Glf is responsible for facilitating the diffusion of glucose uptake in Z. mobilis. However, another sugar transporter-encoded gene, ZMO0293, is poorly characterized. We employed gene deletion and heterologous expression mediated by the CRISPR/Cas method to investigate the role of ZMO0293. The results showed that deletion of the ZMO0293 gene slowed growth and reduced ethanol production and the activities of key enzymes involved in glucose metabolism in the presence of high concentrations of glucose. Moreover, ZMO0293 deletion caused different transcriptional changes in some genes of the Entner Doudoroff (ED) pathway in the ZM4-ΔZM0293 strain but not in ZM4 cells. The integrated expression of ZMO0293 restored the growth of the glucose uptake-defective strain Escherichia coli BL21(DE3)-ΔptsG. This study reveals the function of the ZMO0293 gene in Z. mobilis in response to high concentrations of glucose and provides a new biological part for synthetic biology.}, }
@article {pmid36982733, year = {2023}, author = {Moniruzzaman, M and Darwish, AG and Ismail, A and El-Kereamy, A and Tsolova, V and El-Sharkawy, I}, title = {Seedlessness Trait and Genome Editing-A Review.}, journal = {International journal of molecular sciences}, volume = {24}, number = {6}, pages = {}, pmid = {36982733}, issn = {1422-0067}, mesh = {*Gene Editing ; *Vitis/genetics ; Plant Breeding ; Seeds/genetics ; Fruit/genetics ; CRISPR-Cas Systems/genetics ; }, abstract = {Parthenocarpy and stenospermocarpy are the two mechanisms underlying the seedless fruit set program. Seedless fruit occurs naturally and can be produced using hormone application, crossbreeding, or ploidy breeding. However, the two types of breeding are time-consuming and sometimes ineffective due to interspecies hybridization barriers or the absence of appropriate parental genotypes to use in the breeding process. The genetic engineering approach provides a better prospect, which can be explored based on an understanding of the genetic causes underlying the seedlessness trait. For instance, CRISPR/Cas is a comprehensive and precise technology. The prerequisite for using the strategy to induce seedlessness is identifying the crucial master gene or transcription factor liable for seed formation/development. In this review, we primarily explored the seedlessness mechanisms and identified the potential candidate genes underlying seed development. We also discussed the CRISPR/Cas-mediated genome editing approaches and their improvements.}, }
@article {pmid36982690, year = {2023}, author = {Belalov, IS and Sokolov, AA and Letarov, AV}, title = {Diversity-Generating Retroelements in Prokaryotic Immunity.}, journal = {International journal of molecular sciences}, volume = {24}, number = {6}, pages = {}, pmid = {36982690}, issn = {1422-0067}, mesh = {*Bacteria/genetics ; *Retroelements/genetics ; Prokaryotic Cells ; Proteins/genetics ; CRISPR-Cas Systems ; }, abstract = {Adaptive immunity systems found in different organisms fall into two major types. Prokaryotes possess CRISPR-Cas systems that recognize former invaders using memorized (captured) pieces of their DNA as pathogen signatures. Mammals possess a vast repertoire of antibodies and T-cell receptor variants generated in advance. In this second type of adaptive immunity, a pathogen presentation to the immune system specifically activates the cells that express matching antibodies or receptors. These cells proliferate to fight the infection and form the immune memory. The principle of preemptive production of diverse defense proteins for future use can hypothetically take place in microbes too. We propose a hypothesis that prokaryotes employ diversity-generating retroelements to prepare defense proteins against yet-unknown invaders. In this study, we test this hypothesis with the methods of bioinformatics and identify several candidate defense systems based on diversity-generating retroelements.}, }
@article {pmid36982409, year = {2023}, author = {Kim, MS and Ko, SR and Jung, YJ and Kang, KK and Lee, YJ and Cho, YG}, title = {Knockout Mutants of OsPUB7 Generated Using CRISPR/Cas9 Revealed Abiotic Stress Tolerance in Rice.}, journal = {International journal of molecular sciences}, volume = {24}, number = {6}, pages = {}, pmid = {36982409}, issn = {1422-0067}, mesh = {*Oryza/metabolism ; CRISPR-Cas Systems ; Plants, Genetically Modified/genetics/metabolism ; Plant Breeding ; Stress, Physiological/genetics ; Plant Proteins/metabolism ; Gene Expression Regulation, Plant ; Droughts ; }, abstract = {Plants produce and accumulate stress-resistant substances when exposed to abiotic stress, which involves a protein conversion mechanism that breaks down stress-damaged proteins and supplies usable amino acids. Eukaryotic protein turnover is mostly driven by the ubiquitination pathway. Among the three enzymes required for protein degradation, E3 ubiquitin ligase plays a pivotal role in most cells, as it determines the specificity of ubiquitination and selects target proteins for degradation. In this study, to investigate the function of OsPUB7 (Plant U-box gene in Oryza sativa), we constructed a CRISPR/Cas9 vector, generated OsPUB7 gene-edited individuals, and evaluated resistance to abiotic stress using gene-edited lines. A stress-tolerant phenotype was observed as a result of drought and salinity stress treatment in the T2OsPUB7 gene-edited null lines (PUB7-GE) lacking the T-DNA. In addition, although PUB7-GE did not show any significant change in mRNA expression analysis, it showed lower ion leakage and higher proline content than the wild type (WT). Protein-protein interaction analysis revealed that the expression of the genes (OsPUB23, OsPUB24, OsPUB66, and OsPUB67) known to be involved in stress increased in PUB7-GE and this, by forming a 1-node network with OsPUB66 and OsPUB7, acted as a negative regulator of drought and salinity stress. This result provides evidence that OsPUB7 will be a useful target for both breeding and future research on drought tolerance/abiotic stress in rice.}, }
@article {pmid36982266, year = {2023}, author = {Mohan, K and Dubey, SK and Jung, K and Dubey, R and Wang, QJ and Prajapati, S and Roney, J and Abney, J and Kleinman, ME}, title = {Long-Term Evaluation of Retinal Morphology and Function in Rosa26-Cas9 Knock-In Mice.}, journal = {International journal of molecular sciences}, volume = {24}, number = {6}, pages = {}, pmid = {36982266}, issn = {1422-0067}, support = {R01EY028206/NH/NIH HHS/United States ; K08EY021757/NH/NIH HHS/United States ; }, mesh = {Mice ; Animals ; *CRISPR-Cas Systems ; *Retina/metabolism ; Gene Editing/methods ; Electroretinography ; Genetic Vectors ; }, abstract = {The CRISPR/Cas9 system is a robust, efficient, and cost-effective gene editing tool widely adopted in translational studies of ocular diseases. However, in vivo CRISPR-based editing in animal models poses challenges such as the efficient delivery of the CRISPR components in viral vectors with limited packaging capacity and a Cas9-associated immune response. Using a germline Cas9-expressing mouse model would help to overcome these limitations. Here, we evaluated the long-term effects of SpCas9 expression on retinal morphology and function using Rosa26-Cas9 knock-in mice. We observed abundant SpCas9 expression in the RPE and retina of Rosa26-Cas9 mice using the real-time polymerase chain reaction (RT-PCR), Western blotting, and immunostaining. SD-OCT imaging and histological analysis of the RPE, retinal layers, and vasculature showed no apparent structural abnormalities in adult and aged Cas9 mice. Full-field electroretinogram of adult and aged Cas9 mice showed no long-term functional changes in the retinal tissues because of constitutive Cas9 expression. The current study showed that both the retina and RPE maintain their phenotypic and functional features in Cas9 knock-in mice, establishing this as an ideal animal model for developing therapeutics for retinal diseases.}, }
@article {pmid36982223, year = {2023}, author = {Dyrkheeva, NS and Malakhova, AA and Zakharenko, AL and Okorokova, LS and Shtokalo, DN and Pavlova, SV and Medvedev, SP and Zakian, SM and Nushtaeva, AA and Tupikin, AE and Kabilov, MR and Khodyreva, SN and Luzina, OA and Salakhutdinov, NF and Lavrik, OI}, title = {Transcriptomic Analysis of CRISPR/Cas9-Mediated PARP1-Knockout Cells under the Influence of Topotecan and TDP1 Inhibitor.}, journal = {International journal of molecular sciences}, volume = {24}, number = {6}, pages = {}, pmid = {36982223}, issn = {1422-0067}, mesh = {CRISPR-Cas Systems ; DNA ; DNA Repair ; DNA Topoisomerases, Type I/genetics/metabolism ; Esterases/metabolism ; *Phosphoric Diester Hydrolases/metabolism ; Proteasome Endopeptidase Complex/metabolism ; *Topotecan/pharmacology ; Transcriptome ; Poly (ADP-Ribose) Polymerase-1/metabolism ; }, abstract = {Topoisomerase 1 (TOP1) is an enzyme that regulates DNA topology and is essential for replication, recombination, and other processes. The normal TOP1 catalytic cycle involves the formation of a short-lived covalent complex with the 3' end of DNA (TOP1 cleavage complex, TOP1cc), which can be stabilized, resulting in cell death. This fact substantiates the effectiveness of anticancer drugs-TOP1 poisons, such as topotecan, that block the relegation of DNA and fix TOP1cc. Tyrosyl-DNA phosphodiesterase 1 (TDP1) is able to eliminate TOP1cc. Thus, TDP1 interferes with the action of topotecan. Poly(ADP-ribose) polymerase 1 (PARP1) is a key regulator of many processes in the cell, such as maintaining the integrity of the genome, regulation of the cell cycle, cell death, and others. PARP1 also controls the repair of TOP1cc. We performed a transcriptomic analysis of wild type and PARP1 knockout HEK293A cells treated with topotecan and TDP1 inhibitor OL9-119 alone and in combination. The largest number of differentially expressed genes (DEGs, about 4000 both up- and down-regulated genes) was found in knockout cells. Topotecan and OL9-119 treatment elicited significantly fewer DEGs in WT cells and negligible DEGs in PARP1-KO cells. A significant part of the changes caused by PARP1-KO affected the synthesis and processing of proteins. Differences under the action of treatment with TOP1 or TDP1 inhibitors alone were found in the signaling pathways for the development of cancer, DNA repair, and the proteasome. The drug combination resulted in DEGs in the ribosome, proteasome, spliceosome, and oxidative phosphorylation pathways.}, }
@article {pmid36981028, year = {2023}, author = {Mizushima, S and Sasanami, T and Ono, T and Kuroiwa, A}, title = {Current Approaches to and the Application of Intracytoplasmic Sperm Injection (ICSI) for Avian Genome Editing.}, journal = {Genes}, volume = {14}, number = {3}, pages = {}, pmid = {36981028}, issn = {2073-4425}, mesh = {Animals ; Male ; Humans ; *Gene Editing ; *Sperm Injections, Intracytoplasmic ; CRISPR-Cas Systems/genetics ; Semen ; Chickens/genetics ; }, abstract = {Poultry are one of the most valuable resources for human society. They are also recognized as a powerful experimental animal for basic research on embryogenesis. Demands for the supply of low-allergen eggs and bioreactors have increased with the development of programmable genome editing technology. The CRISPR/Cas9 system has recently been used to produce transgenic animals and various animals in the agricultural industry and has also been successfully adopted for the modification of chicken and quail genomes. In this review, we describe the successful establishment of genome-edited lines combined with germline chimera production systems mediated by primordial germ cells and by viral infection in poultry. The avian intracytoplasmic sperm injection (ICSI) system that we previously established and recent advances in ICSI for genome editing are also summarized.}, }
@article {pmid36980270, year = {2023}, author = {Martínez-Fernández, C and Jha, S and Aliagas, E and Holmberg, CI and Nadal, E and Cerón, J}, title = {BAP1 Malignant Pleural Mesothelioma Mutations in Caenorhabditis elegans Reveal Synthetic Lethality between ubh-4/BAP1 and the Proteasome Subunit rpn-9/PSMD13.}, journal = {Cells}, volume = {12}, number = {6}, pages = {}, pmid = {36980270}, issn = {2073-4409}, mesh = {Animals ; Humans ; *Mesothelioma, Malignant ; Caenorhabditis elegans/genetics/metabolism ; *Mesothelioma/genetics/pathology ; Proteasome Endopeptidase Complex/genetics ; Synthetic Lethal Mutations ; Mutation/genetics ; Ubiquitin Thiolesterase/genetics/metabolism ; Tumor Suppressor Proteins/genetics ; }, abstract = {The deubiquitinase BAP1 (BRCA1-associated protein 1) is associated with BAP1 tumor predisposition syndrome (TPDS). BAP1 is a tumor suppressor gene whose alterations in cancer are commonly caused by gene mutations leading to protein loss of function. By CRISPR-Cas, we have generated mutations in ubh-4, the BAP1 ortholog in Caenorhabditis elegans, to model the functional impact of BAP1 mutations. We have found that a mimicked BAP1 cancer missense mutation (UBH-4 A87D; BAP1 A95D) resembles the phenotypes of ubh-4 deletion mutants. Despite ubh-4 being ubiquitously expressed, the gene is not essential for viability and its deletion causes only mild phenotypes without affecting 20S proteasome levels. Such viability facilitated an RNAi screen for ubh-4 genetic interactors that identified rpn-9, the ortholog of human PSMD13, a gene encoding subunit of the regulatory particle of the 26S proteasome. ubh-4[A87D], similarly to ubh-4 deletion, cause a synthetic genetic interaction with rpn-9 inactivation affecting body size, lifespan, and the development of germ cells. Finally, we show how ubh-4 inactivation sensitizes animals to the chemotherapeutic agent Bortezomib, which is a proteasome inhibitor. Thus, we have established a model to study BAP1 cancer-related mutations in C. elegans, and our data points toward vulnerabilities that should be studied to explore therapeutic opportunities within the complexity of BAP1 tumors.}, }
@article {pmid36977923, year = {2023}, author = {Shi, P and Murphy, MR and Aparicio, AO and Kesner, JS and Fang, Z and Chen, Z and Trehan, A and Guo, Y and Wu, X}, title = {Collateral activity of the CRISPR/RfxCas13d system in human cells.}, journal = {Communications biology}, volume = {6}, number = {1}, pages = {334}, pmid = {36977923}, issn = {2399-3642}, mesh = {Humans ; *CRISPR-Cas Systems ; *RNA/genetics ; Transcriptome ; Bacteria/genetics ; }, abstract = {CRISPR/Cas13 systems are increasingly used for programmable targeting of RNAs. While Cas13 nucleases are capable of degrading both target RNAs and bystander RNAs in vitro and in bacteria, initial studies fail to detect collateral degradation of non-target RNAs in eukaryotic cells. Here we show that RfxCas13d, also known as CasRx, a widely used Cas13 system, can cause collateral transcriptome destruction when targeting abundant reporter RNA and endogenous RNAs, resulting in proliferation defect in target cells. While these results call for caution of using RfxCas13d for targeted RNA knockdown, we demonstrated that the collateral activity can be harnessed for selective depletion of a specific cell population defined by a marker RNA in an in vitro setting.}, }
@article {pmid36978193, year = {2023}, author = {Ordon, J and Kiel, N and Becker, D and Kretschmer, C and Schulze-Lefert, P and Stuttmann, J}, title = {Targeted gene deletion with SpCas9 and multiple guide RNAs in Arabidopsis thaliana: four are better than two.}, journal = {Plant methods}, volume = {19}, number = {1}, pages = {30}, pmid = {36978193}, issn = {1746-4811}, abstract = {BACKGROUND: In plant genome editing, RNA-guided nucleases such as Cas9 from Streptococcus pyogenes (SpCas9) predominantly induce small insertions or deletions at target sites. This can be used for inactivation of protein-coding genes by frame shift mutations. However, in some cases, it may be advantageous to delete larger chromosomal segments. This is achieved by simultaneously inducing double strand breaks upstream and downstream of the segment to be deleted. Experimental approaches for the deletion of larger chromosomal segments have not been systematically evaluated.<br><br>RESULTS: We designed three pairs of guide RNAs for deletion of a&#x2009;~&#x2009;2.2&#xa0;kb chromosomal segment containing the Arabidopsis WRKY30 locus. We tested how the combination of guide RNA pairs and co-expression of the exonuclease TREX2 affect the frequency of wrky30 deletions in editing experiments. Our data demonstrate that compared to one pair of guide RNAs, two pairs increase the frequency of chromosomal deletions. The exonuclease TREX2 enhanced mutation frequency at individual target sites and shifted the mutation profile towards larger deletions. However, TREX2 did not elevate the frequency of chromosomal segment deletions.<br><br>CONCLUSIONS: Multiplex editing with at least two pairs of guide RNAs (four guide RNAs in total) elevates the frequency of chromosomal segment deletions at least at the AtWRKY30 locus, and thus simplifies the selection of corresponding mutants. Co-expression of the TREX2 exonuclease can be used as a general strategy to increase editing efficiency in Arabidopsis without obvious negative effects.}, }
@article {pmid36977792, year = {2023}, author = {Kolvenbach, CM and Dworschak, GC and Rieke, JM and Woolf, AS and Reutter, H and Odermatt, B and Hilger, AC}, title = {Modelling human lower urinary tract malformations in zebrafish.}, journal = {Molecular and cellular pediatrics}, volume = {10}, number = {1}, pages = {2}, pmid = {36977792}, issn = {2194-7791}, abstract = {Advances in molecular biology are improving our understanding of the genetic causes underlying human congenital lower urinary tract (i.e., bladder and urethral) malformations. This has recently led to the identification of the first disease-causing variants in the gene BNC2 for isolated lower urinary tract anatomical obstruction (LUTO), and of WNT3 and SLC20A1 as genes implicated in the pathogenesis of the group of conditions called bladder-exstrophy-epispadias complex (BEEC). Implicating candidate genes from human genetic data requires evidence of their influence on lower urinary tract development and evidence of the found genetic variants' pathogenicity. The zebrafish (Danio rerio) has many advantages for use as a vertebrate model organism for the lower urinary tract. Rapid reproduction with numerous offspring, comparable anatomical kidney and lower urinary tract homology, and easy genetic manipulability by Morpholino®-based knockdown or CRISPR/Cas editing are among its advantages. In addition, established marker staining for well-known molecules involved in urinary tract development using whole-mount in situ hybridization (WISH) and the usage of transgenic lines expressing fluorescent protein under a tissue-specific promoter allow easy visualization of phenotypic abnormalities of genetically modified zebrafish. Assays to examine the functionality of the excretory organs can also be modeled in vivo with the zebrafish. The approach of using these multiple techniques in zebrafish not only enables rapid and efficient investigation of candidate genes for lower urinary tract malformations derived from human data, but also cautiously allows transferability of causality from a non-mammalian vertebrate to humans.}, }
@article {pmid36976005, year = {2023}, author = {Wang, C and Sun, Z and Hu, Y and Li, D and Guo, Q and Wang, M}, title = {A Novel Anti-CRISPR AcrIE9.2 Is Associated with Dissemination of blaKPC Plasmids in Klebsiella pneumoniae Sequence Type 15.}, journal = {Antimicrobial agents and chemotherapy}, volume = {}, number = {}, pages = {e0154722}, doi = {10.1128/aac.01547-22}, pmid = {36976005}, issn = {1098-6596}, abstract = {Sequence type (ST) 15 has become an emerging clone of carbapenem-resistant Klebsiella pneumoniae in which type I-E* CRISPR-Cas usually exists, indicating that the CRISPR-Cas system may not be able to block the transfer of blaKPC plasmids. The purpose of this study was to explore the mechanisms underlying dissemination of blaKPC plasmids in K. pneumoniae ST15. The type I-E* CRISPR-Cas system was present in 98.0% of 612 nonduplicate K. pneumoniae ST15 strains (88 clinical isolates and 524 from the NCBI database). Twelve ST15 clinical isolates were completely sequenced, and self-targeted protospacers were found on blaKPC plasmids flanked by a protospacer adjacent motif (PAM) of AAT in 11 isolates. The type I-E* CRISPR-Cas system was cloned from a clinical isolate and expressed in Escherichia coli BL21(DE3). In BL21(DE3) harboring the CRISPR system, the transformation efficiency of protospacer-bearing plasmids with a PAM of AAT was reduced by 96.2% compared to the empty vector, indicating that the type I-E* CRISPR-Cas system impeded blaKPC plasmid transfer. BLAST for known anti-CRISPR (Acr) amino acid sequences uncovered a novel AcrIE9-like protein with 40.5% to 44.6% sequence identity with AcrIE9 designated AcrIE9.2, which was present in 90.1% (146 of 162) of ST15 strains carrying both blaKPC and the CRISPR-Cas system. When AcrIE9.2 was cloned and expressed in a ST15 clinical isolate, the conjugation frequency of a CRISPR-targeted blaKPC plasmid was increased from 3.96 × 10[-6] to 2.01 × 10[-4] compared to the AcrIE9.2 absent strain. In conclusion, AcrIE9.2 may be associated with the dissemination of blaKPC in ST15 by repressing CRISPR-Cas activity.}, }
@article {pmid36975789, year = {2023}, author = {Li, X and Bao, N and Yan, Z and Yuan, XZ and Wang, SG and Xia, PF}, title = {Degradation of Antibiotic Resistance Genes by VADER with CRISPR-Cas Immunity.}, journal = {Applied and environmental microbiology}, volume = {}, number = {}, pages = {e0005323}, doi = {10.1128/aem.00053-23}, pmid = {36975789}, issn = {1098-5336}, abstract = {The evolution and dissemination of antibiotic resistance genes (ARGs) are prompting severe health and environmental issues. While environmental processes, e.g., biological wastewater treatment, are key barriers to prevent the spread of ARGs, they are often sources of ARGs at the same time, requiring upgraded biotechnology. Here, we present VADER, a synthetic biology system for the degradation of ARGs based on CRISPR-Cas immunity, an archaeal and bacterial immune system for eliminating invading foreign DNAs, to be implemented for wastewater treatment processes. Navigated by programmable guide RNAs, VADER targets and degrades ARGs depending on their DNA sequences, and by employing an artificial conjugation machinery, IncP, it can be delivered via conjugation. The system was evaluated by degrading plasmid-borne ARGs in Escherichia coli and further demonstrated via the elimination of ARGs on the environmentally relevant RP4 plasmid in Pseudomonas aeruginosa. Next, a prototype conjugation reactor at a 10-mL scale was devised, and 100% of the target ARG was eliminated in the transconjugants receiving VADER, giving a proof of principle for the implementation of VADER in bioprocesses. By generating a nexus of synthetic biology and environmental biotechnology, we believe that our work is not only an enterprise for tackling ARG problems but also a potential solution for managing undesired genetic materials in general in the future. IMPORTANCE Antibiotic resistance has been causing severe health problems and has led to millions of deaths in recent years. Environmental processes, especially those of the wastewater treatment sector, are an important barrier to the spread of antibiotic resistance from the pharmaceutical industry, hospitals, or civil sewage. However, they have been identified as a nonnegligible source of antibiotic resistance at the same time, as antibiotic resistance with its main cause, antibiotic resistance genes (ARGs), may accumulate in biological treatment units. Here, we transplanted the CRISPR-Cas system, an immune system via programmable DNA cleavage, to tackle the antibiotic resistance problem raised in wastewater treatment processes, and we propose a new sector specialized in ARG removal with a conjugation reactor to implement the CRISPR-Cas system. Our study provides a new angle for resolving public health issues via the implementation of synthetic biology in environmental contexts at the process level.}, }
@article {pmid36974713, year = {2023}, author = {Song, D and Wang, F and Ju, Y and He, Q and Sun, T and Deng, W and Ding, R and Zhang, C and Xu, Q and Qi, C and Bao, J}, title = {Application and development of noninvasive biomarkers for colorectal cancer screening: a systematic review.}, journal = {International journal of surgery (London, England)}, volume = {}, number = {}, pages = {}, doi = {10.1097/JS9.0000000000000260}, pmid = {36974713}, issn = {1743-9159}, abstract = {BACKGROUND: Colorectal cancer (CRC) is the second most common cause of cancer-related death (9.4% of the 9.9 million cancer deaths). However, CRC develops slowly, and early detection and intervention can effectively improve the survival rate and quality of life. Although colonoscopy can detect and diagnose CRC, it is unsuitable for CRC screening in average-risk populations. Some commercial kits based on DNA mutation or methylation are approved for screening, but the low sensitivity for advanced adenoma or early-stage CRC would limit the applications.<br><br>MAIN RESULTS: Recently, researchers have focused on developing noninvasive or minimally invasive, easily accessible biomarkers with higher sensitivity and accuracy for CRC screening. Numerous reports describe advances in biomarkers, including DNA mutations and methylation, mRNA and miRNA, gut microbes, and metabolites, as well as low-throughput multiomics panels. In small cohorts, the specificity and sensitivity improved when fecal immunochemical testing combined with other biomarkers; further verification in large cohorts is expected. In addition, the continuous improvement of laboratory technology has also improved the sensitivity of detection technology, such as PCR, and the application of CRISPR/Cas technology. Besides, artificial intelligence has extensively promoted the mining of biomarkers. Machine learning was performed to construct a diagnosis model for CRC screening based on the cfDNA fragment features from whole-genome sequencing data. In another study, multiomics markers, including cfDNA, epigenetic, and protein signals, were also discovered by machine learning. Finally, advancements in sensor technology promote the applicability of volatile organic compounds in CRC early detection.<br><br>CONCLUSION: Here, the authors review advances in early detection and screening of CRC based on different biomarker types. Most studies reported optimistic findings based on preliminary research, and prospective clinical studies are ongoing. These promising biomarkers are expected to more accurately identify early-stage patients with CRC and be applied in the future.}, }
@article {pmid36974013, year = {2023}, author = {Huang, W and Wei, H and Wang, C and Wang, J and Chen, L and Chen, W and Liu, Y and Zheng, Y and Lin, M}, title = {[Establishment and preliminary evaluation of a fluorescent recombinase-aided amplification/CRISPR-Cas12a system for rapid detection of Plasmodium falciparum].}, journal = {Zhongguo xue xi chong bing fang zhi za zhi = Chinese journal of schistosomiasis control}, volume = {35}, number = {1}, pages = {38-43}, doi = {10.16250/j.32.1374.2022240}, pmid = {36974013}, issn = {1005-6661}, mesh = {Humans ; Plasmodium falciparum/genetics ; Sensitivity and Specificity ; Nucleic Acid Amplification Techniques/methods ; Recombinases/metabolism ; CRISPR-Cas Systems ; *Malaria, Falciparum/genetics ; *Malaria/diagnosis ; DNA Primers ; *Malaria, Vivax ; RNA, Ribosomal, 18S/genetics ; }, abstract = {OBJECTIVE: To establish a fluorescent assay for rapid detection of Plasmodium falciparum based on recombinaseaided amplification (RAA) and CRISPR-Cas12a system,and to preliminarily evaluate the diagnostic efficiency of this system.<br><br>METHODS: The 18S ribosomal RNA (rRNA) gene of P. falciparum was selected as the target sequence, and three pairs of RAA primers and CRISPR-derived RNA (crRNA) were designed and synthesized. The optimal combination of RAA primers and crRNA was screened and the reaction conditions of the system were optimized to create a fluorescent RAA/CRISPR-Cas12a system. The plasmid containing 18S rRNA gene of the P. falciparum strain 3D7 was generated, and diluted into concentrations of 1 000, 100, 10, 1 copy/&#x3bc;L for the fluorescent RAA/CRISPR-Cas12a assay, and its sensitivity was evaluated. The genomic DNA from P. vivax, P. malariae, P. ovum, hepatitis B virus, human immunodeficiency virus and Treponema pallidum was employed as templates for the fluorescent RAA/CRISPR-Cas12a assay, and its specificity was evaluated. Fifty malaria clinical samples were subjected to the fluorescent RAA/CRISPR-Cas12a assay and nested PCR assay, and the consistency between two assays was compared. In addition, P. falciparum strain 3D7 was cultured in vitro. Then, the culture was diluted into blood samples with parasite densities of 1 000, 500, 200, 50, 10 parasites/&#x3bc;L with healthy volunteers' O-positive red blood cells for the RAA/CRISPR-Cas12a assay, and the detection efficiency was tested.<br><br>RESULTS: The Pf-F3/Pf-R3/crRNA2 combination, 2.5 &#x3bc;L as the addition amount of B buffer, 40 min as the RAA reaction time, 37 &#xb0;C as the reaction temperature of the CRISPR-Cas12a system were employed to establish the fluorescent RAA/CRISPR-Cas12a system. Such a system was effective to detect the plasmid containing 18S rRNA gene of the P. falciparum strain 3D7 at a concentration of 1 copy/&#x3bc;L, and presented fluorescent signals for detection of P. falciparum, but failed to detect P. ovum, P. malariae, P. vivax, T. pallidum, hepatitis B virus or human immunodeficiency virus. The fluorescent RAA/CRISPR-Cas12a system and nested PCR assay showed completely consistent results for detection of 50 malaria clinical samples (kappa = 1.0, P < 0.001). Following 6-day in vitro culture of the P. falciparum strain 3D7, 10 mL cultures were generated and the fluorescent RAA/CRISPR-Cas12a system showed the minimal detection limit of 50 parasites/&#x3bc;L.<br><br>CONCLUSIONS: The fluorescent RAA/CRISPR-Cas12a system is rapid, sensitive and specific for detection of P. falciparum, which shows promising value for rapid detection and risk monitoring of P. falciparum.}, }
@article {pmid36971438, year = {2023}, author = {Sconocchia, T and Foßelteder, J and Köhnke, T and Majeti, R and Reinisch, A}, title = {Engineering Oncogenic Heterozygous Gain-of-Function Mutations in Human Hematopoietic Stem and Progenitor Cells.}, journal = {Journal of visualized experiments : JoVE}, volume = {}, number = {193}, pages = {}, doi = {10.3791/64558}, pmid = {36971438}, issn = {1940-087X}, mesh = {Humans ; *Gain of Function Mutation ; *Gene Editing/methods ; Hematopoietic Stem Cells ; Mutation ; CRISPR-Cas Systems ; }, abstract = {Throughout their lifetime, hematopoietic stem and progenitor cells (HSPCs) acquire somatic mutations. Some of these mutations alter HSPC functional properties such as proliferation and differentiation, thereby promoting the development of hematologic malignancies. Efficient and precise genetic manipulation of HSPCs is required to model, characterize, and better understand the functional consequences of recurrent somatic mutations. Mutations can have a deleterious effect on a gene and result in loss-of-function (LOF) or, in stark contrast, may enhance function or even lead to novel characteristics of a particular gene, termed gain-of-function (GOF). In contrast to LOF mutations, GOF mutations almost exclusively occur in a heterozygous fashion. Current genome-editing protocols do not allow for the selective targeting of individual alleles, hampering the ability to model heterozygous GOF mutations. Here, we provide a detailed protocol on how to engineer heterozygous GOF hotspot mutations in human HSPCs by combining CRISPR/Cas9-mediated homology-directed repair and recombinant AAV6 technology for efficient DNA donor template transfer. Importantly, this strategy makes use of a dual fluorescent reporter system to allow for the tracking and purification of successfully heterozygously edited HSPCs. This strategy can be employed to precisely investigate how GOF mutations affect HSPC function and their progression toward hematological malignancies.}, }
@article {pmid36746911, year = {2023}, author = {Abdulmalek, HW and Yazgan-Karataş, A}, title = {Improvement of Bacilysin Production in Bacillus subtilis by CRISPR/Cas9-Mediated Editing of the 5'-Untranslated Region of the bac Operon.}, journal = {Journal of microbiology and biotechnology}, volume = {33}, number = {3}, pages = {410-418}, doi = {10.4014/jmb.2209.09035}, pmid = {36746911}, issn = {1738-8872}, mesh = {*Bacillus subtilis/metabolism ; 5' Untranslated Regions ; *CRISPR-Cas Systems ; Operon ; Dipeptides/genetics ; }, abstract = {Bacilysin is a dipeptide antibiotic composed of L-alanine and L-anticapsin produced by certain strains of Bacillus subtilis. Bacilysin is gaining increasing attention in industrial agriculture and pharmaceutical industries due to its potent antagonistic effects on various bacterial, fungal, and algal pathogens. However, its use in industrial applications is hindered by its low production in the native producer. The biosynthesis of bacilysin is mainly based on the bacABCDEF operon. Examination of the sequence surrounding the upstream of the bac operon did not reveal a clear, strong ribosome binding site (RBS). Therefore, in this study, we aimed to investigate the impact of RBS as a potential route to improve bacilysin production. For this, the 5' untranslated region (5'UTR) of the bac operon was edited using the CRISPR/Cas9 approach by introducing a strong ribosome binding sequence carrying the canonical Shine-Dalgarno sequence (TAAGGAGG) with an 8 nt spacing from the AUG start codon. Strong RBS substitution resulted in a 2.87-fold increase in bacilysin production without affecting growth. Strong RBS substitution also improved the mRNA stability of the bac operon. All these data revealed that extensive RBS engineering is a promising key option for enhancing bacilysin production in its native producers.}, }
@article {pmid36701041, year = {2023}, author = {Mathew, SM}, title = {Strategies for generation of mice via CRISPR/HDR-mediated knock-in.}, journal = {Molecular biology reports}, volume = {50}, number = {4}, pages = {3189-3204}, pmid = {36701041}, issn = {1573-4978}, mesh = {Animals ; Mice ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Recombinational DNA Repair ; Genetic Therapy ; DNA/genetics ; }, abstract = {CRISPR/Cas9 framework is generally used to generate genetically modified mouse models. The clustered regularly interspaced short palindromic repeat gene editing technique, can efficiently generate knock-outs using the non-homologous end-joining repair pathway. Small knock-ins also work precisely using a repair template with help of homology-directed-repair (HDR) mechanism. However, when the fragment size is larger than 4-5 kb, the knock-in tends to be error prone and the efficiency decreases. Certain types of modifications, in particular insertions of very large DNA fragments (10-100 kb) or entire gene replacements, are still difficult. The HDR process needs further streamlining and improvement. Here in this review, we describe methods to enhance the efficiency of the knock-in through checking each step from the guide design to the microinjection and choice of the oocyte donors. This helps in understanding the parameters that can be modified to get improved knock-in efficiency via CRISPR targeting.}, }
@article {pmid36478032, year = {2023}, author = {Beracochea, V and Stritzler, M and Radonic, L and Bottero, E and Jozefkowicz, C and Darqui, F and Ayub, N and Bilbao, ML and Soto, G}, title = {CRISPR/Cas9-mediated knockout of SPL13 radically increases lettuce yield.}, journal = {Plant cell reports}, volume = {42}, number = {3}, pages = {645-647}, pmid = {36478032}, issn = {1432-203X}, mesh = {*CRISPR-Cas Systems/genetics ; *Lettuce/genetics ; Gene Knockout Techniques ; Mutation ; Gene Editing ; }, }
@article {pmid36974023, year = {2023}, author = {Li, M and Qin, Z and Yin, K and Zheng, B}, title = {[Application of CRISPR/Cas systems in the nucleic acid detection of pathogens: a review].}, journal = {Zhongguo xue xi chong bing fang zhi za zhi = Chinese journal of schistosomiasis control}, volume = {35}, number = {1}, pages = {98-103}, doi = {10.16250/j.32.1374.2022227}, pmid = {36974023}, issn = {1005-6661}, abstract = {Rapid, sensitive and specific detection tools are critical for the prevention and control of infectious diseases. The in vitro nucleic acid amplification assays, including polymerase chain reaction and isothermal amplification technology, have been widely used for the detection of pathogens. Recently, nucleic acid detection-based on clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas) have been developed, which are rapid, highly sensitive, highly specific, and portable. This review describes the classification and principle of CRISPR/Cas systems and their applications in pathogen detection, and discusses the prospects of CRISPR/Cas systems.}, }
@article {pmid36970694, year = {2023}, author = {Hu, Y and Jiang, J and Wang, D and Guo, Q and Wang, M}, title = {Coexistence of bla KPC-IncFII plasmids and type I-E[*] CRISPR-Cas systems in ST15 Klebsiella pneumoniae.}, journal = {Frontiers in microbiology}, volume = {14}, number = {}, pages = {1125531}, pmid = {36970694}, issn = {1664-302X}, abstract = {The CRISPR-Cas system in Klebsiella pneumoniae can prevent the entry of bla KPC-IncF plasmids. However, some clinical isolates bear the KPC-2 plasmids despite carrying the CRISPR-Cas system. The purpose of this study was to characterize the molecular features of these isolates. A total of 697 clinical K. pneumoniae isolates were collected from 11 hospitals in China, and tested for the presence of CRISPR-Cas systems using polymerase chain reaction. Overall, 164 (23.5%) of 697 K. pneumoniae isolates had type I-E[*] (15.9%) or type I-E (7.7%) CRISPR-Cas systems. The most prevalent sequence type among isolates carrying type I-E[*] CRISPR was ST23 (45.9%), followed by ST15 (18.9%). Isolates with CRISPR-Cas system were more susceptible to ten antimicrobials tested, including carbapenems, compared with the CRISPR-negative isolates. However, there were still 21 CRISPR-Cas-carrying isolates that showed resistance to carbapenems, and these isolates were subjected to whole-genome sequencing. Thirteen of these 21 isolates carried bla KPC-2-bearing plasmids, of which nine had a new plasmid type, IncFIIK34, and two had IncFII(PHN7A8) plasmids. In addition, 12 of these 13 isolates belonged to ST15, while only eight (5.6%, 8/143) isolates belonged to ST15 in carbapenem-susceptible K. pneumoniae carrying CRISPR-Cas systems. In conclusion, we found that bla KPC-2-bearing IncFII plasmids could co-exist with the type I-E[*] CRISPR-Cas systems in ST15 K. pneumoniae.}, }
@article {pmid36969374, year = {2023}, author = {Papaioannou, NY and Patsali, P and Naiisseh, B and Papasavva, PL and Koniali, L and Kurita, R and Nakamura, Y and Christou, S and Sitarou, M and Mussolino, C and Cathomen, T and Kleanthous, M and Lederer, CW}, title = {High-efficiency editing in hematopoietic stem cells and the HUDEP-2 cell line based on in vitro mRNA synthesis.}, journal = {Frontiers in genome editing}, volume = {5}, number = {}, pages = {1141618}, pmid = {36969374}, issn = {2673-3439}, abstract = {Introduction: Genome editing tools, such as CRISPR/Cas, TALE nucleases and, more recently, double-strand-break-independent editors, have been successfully used for gene therapy and reverse genetics. Among various challenges in the field, tolerable and efficient delivery of editors to target cells and sites, as well as independence from commercially available tools for flexibility and fast adoption of new editing technology are the most pressing. For many hematopoietic research applications, primary CD34[+] cells and the human umbilical cord-derived progenitor erythroid 2 (HUDEP-2) cell line are highly informative substrates and readily accessible for in vitro manipulation. Moreover, ex vivo editing of CD34[+] cells has immediate therapeutic relevance. Both cell types are sensitive to standard transfection procedures and reagents, such as lipofection with plasmid DNA, calling for more suitable methodology in order to achieve high efficiency and tolerability of editing with editors of choice. These challenges can be addressed by RNA delivery, either as a mixture of guide RNA and mRNA for CRISRP/Cas-based systems or as a mixture of mRNAs for TALENs. Compared to ribonucleoproteins or proteins, RNA as vector creates flexibility by removing dependence on commercial availability or laborious in-house preparations of novel editor proteins. Compared to DNA, RNA is less toxic and by obviating nuclear transcription and export of mRNA offers faster kinetics and higher editing efficiencies. Methods: Here, we detail an in vitro transcription protocol based on plasmid DNA templates with the addition of Anti-Reverse Cap Analog (ARCA) using T7 RNA polymerase, and poly (A) tailing using poly (A) polymerase, combined with nucleofection of HUDEP-2 and patient-derived CD34[+] cells. Our protocol for RNA-based delivery employs widely available reagents and equipment and can easily be adopted for universal in vitro delivery of genome editing tools. Results and Discussion: Drawing on a common use case, we employ the protocol to target a β-globin mutation and to reactivate γ-globin expression as two potential therapies for β-hemoglobinopathies, followed by erythroid differentiation and functional analyses. Our protocol allows high editing efficiencies and unimpaired cell viability and differentiation, with scalability, suitability for functional assessment of editing outcomes and high flexibility in the application to different editors.}, }
@article {pmid36968399, year = {2023}, author = {Kumari, A and Sharma, D and Sharma, P and ,  and Wang, C and Verma, V and Patil, A and Imran, M and Singh, MP and Kumar, K and Paritosh, K and Caragea, D and Kapoor, S and Chandel, G and Grover, A and Jagadish, SVK and Katiyar-Agarwal, S and Agarwal, M}, title = {Meta-QTL and haplo-pheno analysis reveal superior haplotype combinations associated with low grain chalkiness under high temperature in rice.}, journal = {Frontiers in plant science}, volume = {14}, number = {}, pages = {1133115}, pmid = {36968399}, issn = {1664-462X}, abstract = {Chalk, an undesirable grain quality trait in rice, is primarily formed due to high temperatures during the grain-filling process. Owing to the disordered starch granule structure, air spaces and low amylose content, chalky grains are easily breakable during milling thereby lowering head rice recovery and its market price. Availability of multiple QTLs associated with grain chalkiness and associated attributes, provided us an opportunity to perform a meta-analysis and identify candidate genes and their alleles contributing to enhanced grain quality. From the 403 previously reported QTLs, 64 Meta-QTLs encompassing 5262 non-redundant genes were identified. MQTL analysis reduced the genetic and physical intervals and nearly 73% meta-QTLs were narrower than 5cM and 2Mb, revealing the hotspot genomic regions. By investigating expression profiles of 5262 genes in previously published datasets, 49 candidate genes were shortlisted on the basis of their differential regulation in at least two of the datasets. We identified non-synonymous allelic variations and haplotypes in 39 candidate genes across the 3K rice genome panel. Further, we phenotyped a subset panel of 60 rice accessions by exposing them to high temperature stress under natural field conditions over two Rabi cropping seasons. Haplo-pheno analysis uncovered haplotype combinations of two starch synthesis genes, GBSSI and SSIIa, significantly contributing towards the formation of grain chalk in rice. We, therefore, report not only markers and pre-breeding material, but also propose superior haplotype combinations which can be introduced using either marker-assisted breeding or CRISPR-Cas based prime editing to generate elite rice varieties with low grain chalkiness and high HRY traits.}, }
@article {pmid36967582, year = {2023}, author = {Nieves-Cordones, M and Amo, J and Hurtado-Navarro, L and Martínez-Martínez, A and Martínez, V and Rubio, F}, title = {Inhibition of SlSKOR by SlCIPK23-SlCBL1/9 uncovers CIPK-CBL-target network rewiring in land plants.}, journal = {The New phytologist}, volume = {}, number = {}, pages = {}, doi = {10.1111/nph.18910}, pmid = {36967582}, issn = {1469-8137}, abstract = {Transport of K[+] to the xylem is a key process in the mineral nutrition of the shoots. Although CIPK-CBL complexes have been widely shown to regulate K[+] uptake transport systems, no information is available about the xylem ones. Here, we studied the physiological roles of the voltage-gated K[+] channel SlSKOR and its regulation by the SlCIPK23-SlCBL1/9 complexes in tomato plants. We phenotyped gene-edited slskor and slcipk23 tomato knock-out mutants and carried out two-electrode voltage-clamp (TEVC) and BiFC assays in Xenopus oocytes as key approaches. SlSKOR was preferentially expressed in the root stele and was important not only for K[+] transport to shoots but also, indirectly, for that of Ca[2+] , Mg[2+] , Na[+] , NO3 [-] and Cl[-] . Surprisingly, the SlCIPK23-SlCBL1/9 complexes turned out to be negative regulators of SlSKOR. Inhibition of SlSKOR by SlCIPK23-SlCBL1/9 was observed in Xenopus oocytes and tomato plants. Regulation of SKOR-like channels by CIPK23-CBL1 complexes was also present in Medicago, grapevine and lettuce but not in Arabidopsis and saltwater cress. Our results provide a molecular framework for coordinating root K[+] uptake and its translocation to the shoot by SlCIPK23-SlCBL1/9 in tomato plants. Moreover, they evidenced that CIPK-CBL-target networks have evolved differently in land plants.}, }
@article {pmid36967147, year = {2023}, author = {Hosoba, K and Morita, T and Zhang, Y and Kishi, H and Yamamoto, T and Miyamoto, T}, title = {High-efficient CRISPR/Cas9-mediated gene targeting to establish cell models of ciliopathies.}, journal = {Methods in cell biology}, volume = {175}, number = {}, pages = {85-95}, doi = {10.1016/bs.mcb.2022.07.020}, pmid = {36967147}, issn = {0091-679X}, mesh = {Animals ; Humans ; *CRISPR-Cas Systems/genetics ; Gene Editing/methods ; Gene Targeting/methods ; Recombinational DNA Repair ; *Ciliopathies/genetics ; Mammals/genetics ; }, abstract = {Primary cilia are antenna-like structures developed on the cell surface of mammalian cells during the quiescent G0 phase. Primary cilia in mammalian cells receive extracellular signals for early development and cell tissue homeostasis. Ciliopathies characterized with congenital anomalies such as cerebellar hypoplasia, polycystic kidney and polydactyly are caused by germline mutations of ciliary structure- and function-related genes. Gene knock-out techniques in ciliated cultured cells with the uniformed genetic background are useful to evaluate the pathophysiological roles of ciliopathy-related gene products. Genome editing technology has been applied into the gene knock-out in many types of cultured cell lines. However, the frequency of genome editing varies according to cell species and cycle because of dependency on error-free homology-directed repair (HDR) activity. The human telomerase reverse transcriptase-immortalized retinal pigmented epithelial cell line (hTERT-RPE1) is well known for its suitability in cilia research. However, the efficacy of the HDR-mediated knock-out clone isolation was low. Here, we introduce the clustered regularly interspaced short palindromic repeats-obligate ligation-gated recombination (CRISPR-ObLiGaRe) system, which is a nonhomologous end-joining (NHEJ)-mediated gene targeting method, to generate the knock-out clones effectively even in the lower-HDR activity cell lines including hTERT-RPE1 cell. This CRISPR-ObLiGaRe system is a powerful tool for establishing ciliopathy model cell libraries and identifying each gene function in cilia-related phenotypes.}, }
@article {pmid36967127, year = {2023}, author = {June, CH}, title = {The Unlikely Development of CAR T Cells: a Brief History and Prospects for the Future.}, journal = {The Keio journal of medicine}, volume = {72}, number = {1}, pages = {26}, doi = {10.2302/kjm.ABSTRACT_72_1-1}, pmid = {36967127}, issn = {1880-1293}, mesh = {Humans ; *CRISPR-Cas Systems ; Gene Editing/methods ; Immunotherapy, Adoptive ; *Neoplasms/genetics/therapy ; T-Lymphocytes ; }, abstract = {The quest to cure cancer has been one of the Holy Grails of medicine. The discovery I am going to share with you, CAR T cells, is a promising new form of therapy of cancer that offers the prospect of curing cancer using the immune system. CAR T cells are the first form of synthetic biology to enter the practice of medicine. The notion of using the immune system to fight cancer is an old idea. Over a century ago, bacteria were ground up and injected into patients with late-stage cancers, and occasionally the cancer would disappear. However, we now have precise tools for genetic editing and gene insertion like CRISPR/Cas9 to rewrite the DNA code, offering the possibility to improve the immune system over what has evolved in a Darwinian fashion. In 2017 for the first time, CAR T cells were approved for the treatment of cancer. Today they are used worldwide in more than 15,000 patients and they offer the promise to move beyond cancer to other fields of medicine such as autoimmune disease and heart disease. Here I will discuss the promises and challenges faced by the evolving CAR T cell industry.}, }
@article {pmid36964659, year = {2023}, author = {Bendixen, L and Jensen, TI and Bak, RO}, title = {CRISPR/Cas-mediated transcriptional modulation: The therapeutic promises of CRISPRa and CRISPRi.}, journal = {Molecular therapy : the journal of the American Society of Gene Therapy}, volume = {}, number = {}, pages = {}, doi = {10.1016/j.ymthe.2023.03.024}, pmid = {36964659}, issn = {1525-0024}, abstract = {The CRISPR/Cas system is commonly known for its ability to cleave DNA in a programmable manner, which has democratized gene editing and facilitated recent breakthroughs in gene therapy. However, newer iterations of the technology using nuclease-disabled Cas enzymes have spurred a variety of different types of genetic engineering platforms such as transcriptional modulation using the CRISPR activation (CRISPRa) and CRISPR interference (CRISPRi) systems. This review introduces the creation of these programmable transcriptional modulators, various methods of delivery utilized for these systems, and recent technological developments. CRISPRa and CRISPRi have also been implemented in genetic screens for interrogating gene function and discovering genes involved in various biological pathways. We describe recent compelling examples of how these tools have become powerful means to unravel genetic networks and uncovering important information about devastating diseases. Finally, we provide an overview of preclinical studies in which transcriptional modulation has been used therapeutically, and we discuss potential future directions of these novel modalities.}, }
@article {pmid36924688, year = {2023}, author = {Li, L and Tian, H and Wang, G and Ren, S and Ma, T and Wang, Y and Ge, S and Zhang, Y and Yu, J}, title = {Ready-to-use interactive dual-readout differential lateral flow biosensor for two genotypes of human papillomavirus.}, journal = {Biosensors &amp; bioelectronics}, volume = {228}, number = {}, pages = {115224}, doi = {10.1016/j.bios.2023.115224}, pmid = {36924688}, issn = {1873-4235}, mesh = {Humans ; Human Papillomavirus Viruses ; *Papillomavirus Infections/diagnosis ; Gold/chemistry ; *Metal Nanoparticles/chemistry ; *Biosensing Techniques/methods ; Papillomaviridae/genetics ; Genotype ; CRISPR-Cas Systems ; }, abstract = {Ready-to-use in vitro diagnosis of multiple genotypes is vital for the prevention and treatment of cervical cancer. Herein, a paper-film-based interactive dual readout differential lateral flow biosensor is proposed to simultaneously assay two high-risk types of human papillomavirus (HPV) within the body-fluid. The CuCo2S4/ZnIn2S4 heterostructure is fabricated on the paper-film compound chip with high thermostability, and surface sulfur vacancy is introduced by mild annealing treatment to endow unexceptionable photoexcitation activity, such structure can be served as an initial energy harvester and converter. With the assistance of differential channels, the dual-target-propelled self-assembly of annular DNA and the cleavage activity of CRISPR-Cas12a are stepwise activated by sequential solution transfer. Accordingly, the input and release of polydopamine-coated gold nanoparticles with photothermal/photoelectric characteristic were implemented. The fabricated biosensor not only realized intelligent thermal-response without large instruments, but also actuated dynamic interfacial charge separation and transfer kinetics to further transmit photoelectric-signal, resulting in desirable interactive dual-signal with low limit-of-detection (0.21 pM for HPV-18 and 42.92 pM for HPV-16). Thanks to the sophisticated design of differential lateral flow paper-film compound chip and interactive dual-signal amplification strategy, sensitive detection of two HPV genotypes is realized, which provides a promising candidate for home medical intelligent diagnosis.}, }
@article {pmid36878066, year = {2023}, author = {Zhu, Z and Guo, Y and Wang, C and Yang, Z and Li, R and Zeng, Z and Li, H and Zhang, D and Yang, L}, title = {An ultra-sensitive one-pot RNA-templated DNA ligation rolling circle amplification-assisted CRISPR/Cas12a detector assay for rapid detection of SARS-CoV-2.}, journal = {Biosensors &amp; bioelectronics}, volume = {228}, number = {}, pages = {115179}, pmid = {36878066}, issn = {1873-4235}, mesh = {Humans ; SARS-CoV-2/genetics ; *COVID-19/diagnosis/genetics ; CRISPR-Cas Systems/genetics ; *Biosensing Techniques/methods ; Nucleic Acid Amplification Techniques/methods ; DNA ; RNA ; }, abstract = {Rapid, sensitive, and one-pot diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) plays an extremely important role in point-of-care testing (POCT). Herein, we report an ultra-sensitive and rapid one-pot enzyme-catalyzed rolling circle amplification-assisted CRISPR/FnCas12a assay, termed OPERATOR. OPERATOR employs a single well-designed single-strand padlock DNA, containing a protospacer adjacent motif (PAM) site and a sequence complementary to the target RNA which procedure converts and amplifies genomic RNA to DNA by RNA-templated DNA ligation and multiply-primed rolling circle amplification (MRCA). The MRCA amplicon of single-stranded DNA is cleaved by the FnCas12a/crRNA complex and detected via a fluorescence reader or lateral flow strip. OPERATOR presents outstanding advantages including ultra-sensitivity (1.625 copies per reaction), high specificity (100%), rapid reaction speed (∼30 min), easy operation, low cost, and on-spot visualization. Furthermore, we established a POCT platform by combining OPERATOR with rapid RNA release and a lateral flow strip without professional equipment. The high performance of OPERATOR in SARS-CoV-2 tests was confirmed using both reference materials and clinical samples, and the results suggest that is readily adaptable for point-of-care testing of other RNA viruses.}, }
@article {pmid36812945, year = {2023}, author = {Boontawon, T and Nakazawa, T and Choi, YJ and Ro, HS and Oh, M and Kawauchi, M and Sakamoto, M and Honda, Y}, title = {Double-gene targeting with preassembled Cas9 ribonucleoprotein for safe genome editing in the edible mushroom Pleurotus ostreatus.}, journal = {FEMS microbiology letters}, volume = {370}, number = {}, pages = {}, doi = {10.1093/femsle/fnad015}, pmid = {36812945}, issn = {1574-6968}, mesh = {Gene Editing/methods ; *Pleurotus/genetics/metabolism ; *Agaricales/genetics ; CRISPR-Cas Systems ; Ribonucleoproteins/genetics/metabolism ; Gene Targeting ; }, abstract = {CRISPR/Cas9 has potential for efficient molecular breeding. Recently, a foreign-DNA-free gene-targeting technology was established by introducing a preassembled Cas9 ribonucleoprotein (RNP) complex into the oyster mushroom Pleurotus ostreatus. However, the target gene was restricted to such a gene like pyrG, since screening of a genome-edited strain was indispensable and could be performed via examination of 5-fluoroorotic acid (5-FOA) resistance caused by the disruption of the target gene. In this study, we simultaneously introduced the Cas9 RNP complex targeting fcy1, a mutation that conferred P. ostreatus resistance to 5-fluorocytosine (5-FC), together with that targeting pyrG. A total of 76 5-FOA resistant strains were isolated during the first screening. Subsequently, a 5-FC resistance examination was conducted, and three strains exhibited resistance. Genomic PCR experiments followed by DNA sequencing revealed that mutations were successfully introduced into fcy1 and pyrG in the three strains. The results indicated that double gene-edited mutants could be obtained in one experiment employing 5-FOA resistance screening for strains with Cas9 RNP incorporation. This work may pave the way for safe CRISPR/Cas9 technology to isolate mutant strains in any gene of interest without an ectopic marker gene.}, }
@article {pmid36682390, year = {2023}, author = {Sheikh Beig Goharrizi, MA and Ghodsi, S and Memarjafari, MR}, title = {Implications of CRISPR-Cas9 Genome Editing Methods in Atherosclerotic Cardiovascular Diseases.}, journal = {Current problems in cardiology}, volume = {48}, number = {5}, pages = {101603}, doi = {10.1016/j.cpcardiol.2023.101603}, pmid = {36682390}, issn = {1535-6280}, mesh = {Humans ; Gene Editing/methods ; CRISPR-Cas Systems ; *Cardiovascular Diseases/genetics/therapy ; CRISPR-Associated Protein 9/genetics/metabolism ; *Atherosclerosis/genetics/therapy ; }, abstract = {Today, new methods have been developed to treat or modify the natural course of cardiovascular diseases (CVDs), including atherosclerosis, by the clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9 (CRISPR-Cas9) system. Genome-editing tools are CRISPR-related palindromic short iteration systems such as CRISPR-Cas9, a valuable technology for achieving somatic and germinal genomic manipulation in model cells and organisms for various applications, including the creation of deletion alleles. Mutations in genomic deoxyribonucleic acid and new genes' placement have emerged. Based on World Health Organization fact sheets, 17.9 million people die from CVDs each year, an estimated 32% of all deaths worldwide. 85% of all CVD deaths are due to acute coronary events and strokes. This review discusses the applications of CRISPR-Cas9 technology throughout atherosclerotic disease research and the prospects for future in vivo genome editing therapies. We also describe several limitations that must be considered to achieve the full scientific and therapeutic potential of cardiovascular genome editing in the treatment of atherosclerosis.}, }
@article {pmid36055523, year = {2023}, author = {Liu, S and Duan, X and Peng, F and Wang, Y and Liu, Y and Wan, X and Zhang, J and Li, X and Sun, X}, title = {A tunable genome editing system of the prime editor mediated by dihydrofolate reductase.}, journal = {Journal of genetics and genomics = Yi chuan xue bao}, volume = {50}, number = {3}, pages = {204-207}, doi = {10.1016/j.jgg.2022.08.004}, pmid = {36055523}, issn = {1673-8527}, mesh = {*Gene Editing ; *Tetrahydrofolate Dehydrogenase/genetics/metabolism ; CRISPR-Cas Systems/genetics ; }, }
@article {pmid36963196, year = {2023}, author = {Ma, L and Zhang, W and Yin, L and Li, Y and Zhuang, J and Shen, L and Man, S}, title = {A SERS-signalled, CRISPR/Cas-powered bioassay for amplification-free and anti-interference detection of SARS-CoV-2 in foods and environmental samples using a single tube-in-tube vessel.}, journal = {Journal of hazardous materials}, volume = {452}, number = {}, pages = {131195}, pmid = {36963196}, issn = {1873-3336}, abstract = {The pandemic of COVID-19 creates an imperative need for sensitive and portable detection of SARS-CoV-2. We devised a SERS-read, CRISPR/Cas-powered nanobioassay, termed as OVER-SARS-CoV-2 (One-Vessel Enhanced RNA test on SARS-CoV-2), which enabled supersensitive, ultrafast, accurate and portable detection of SARS-CoV-2 in a single vessel in an amplification-free and anti-interference manner. The SERS nanoprobes were constructed by conjugating gold nanoparticles with Raman reporting molecular and single-stranded DNA (ssDNA) probes, whose aggregation-to-dispersion changes can be finely tuned by target-activated Cas12a though trans-cleavage of linker ssDNA. As such, the nucleic acid signals could be dexterously converted and amplified to SERS signals. By customizing an ingenious vessel, the steps of RNA reverse transcription, Cas12a trans-cleavage and SERS nanoprobes crosslinking can be integrated into a single and disposal vessel. It was proved that our proposed nanobioassay was able to detect SARS-CoV-2 as low as 200 copies/mL without any pre-amplification within 45 min. In addition, the proposed nanobioassay was confirmed by clinical swab samples and challenged for SARS-CoV-2 detection in simulated complex environmental and food samples. This work enriches the arsenal of CRISPR-based diagnostics (CRISPR-Dx) and provides a novel and robust platform for SARS-CoV-2 decentralized detection, which can be put into practice in the near future.}, }
@article {pmid36961900, year = {2023}, author = {Rubio, A and Sprang, M and Garzón, A and Moreno-Rodriguez, A and Pachón-Ibáñez, ME and Pachón, J and Andrade-Navarro, MA and Pérez-Pulido, AJ}, title = {Analysis of bacterial pangenomes reduces CRISPR dark matter and reveals strong association between membranome and CRISPR-Cas systems.}, journal = {Science advances}, volume = {9}, number = {12}, pages = {eadd8911}, pmid = {36961900}, issn = {2375-2548}, abstract = {CRISPR-Cas systems are prokaryotic acquired immunity mechanisms, which are found in 40% of bacterial genomes. They prevent viral infections through small DNA fragments called spacers. However, the vast majority of these spacers have not yet been associated with the virus they recognize, and it has been named CRISPR dark matter. By analyzing the spacers of tens of thousands of genomes from six bacterial species, we have been able to reduce the CRISPR dark matter from 80% to as low as 15% in some of the species. In addition, we have observed that, when a genome presents CRISPR-Cas systems, this is accompanied by particular sets of membrane proteins. Our results suggest that when bacteria present membrane proteins that make it compete better in its environment and these proteins are, in turn, receptors for specific phages, they would be forced to acquire CRISPR-Cas.}, }
@article {pmid36960537, year = {2023}, author = {Jeon, W and Jung, SY and Lee, CY and Kim, WT and Kim, H and Jang, KW and Lim, H and Lee, M and Jeong, DH and Kim, SD and Kim, IA and Choi, SH and Son, TG and Kim, KS}, title = {Evaluation of Radiation Sensitivity Differences in Mouse Liver Tumor Organoids Using CRISPR/Cas9-Mediated Gene Mutation.}, journal = {Technology in cancer research &amp; treatment}, volume = {22}, number = {}, pages = {15330338231165125}, doi = {10.1177/15330338231165125}, pmid = {36960537}, issn = {1533-0338}, mesh = {Mice ; Animals ; *CRISPR-Cas Systems/genetics ; Mice, Inbred C57BL ; *Liver Neoplasms/genetics/radiotherapy/metabolism ; Mutation ; Organoids/metabolism/pathology ; Adenosine Triphosphate ; }, abstract = {BACKGROUND: To assess the radiosensitivity of liver tumors harboring different genetic mutations, mouse liver tumors were generated in vivo through the hydrodynamic injection of clustered regularly interspaced short palindromic repeat/caspase 9 (CRISPR/Cas9) constructs encoding single-guide RNAs (sgRNAs) targeting Tp53, Pten, Nf1, Nf2, Tsc2, Cdkn2a, or Rb1.<br><br>METHODS: The plasmid vectors were delivered to the liver of adult C57BL/6 mice via hydrodynamic tail vein injection. The vectors were injected into 10 mice in each group. Organoids were generated from mouse liver tumors. The radiation response of the organoids was assessed using an ATP cell viability assay.<br><br>RESULTS: The mean survival period of mice injected with vectors targeting Nf2 (4.8 months) was lower than that of other mice. Hematoxylin and eosin staining, immunohistochemical (IHC) staining, and target sequencing analyses revealed that mouse liver tumors harbored the expected mutations. Tumor organoids were established from mouse liver tumors. Histological evaluation revealed marked morphological similarities between the mouse liver tumors and the generated tumor organoids. Moreover, IHC staining indicated that the parental tumor protein expression pattern was maintained in the organoids. The results of the ATP cell viability assay revealed that the tumor organoids with mutated Nf2 were more resistant to high-dose radiation than those with other gene mutations.<br><br>CONCLUSIONS: This study developed a radiation response assessment system for mouse tumors with mutant target genes using CRISPR/Cas9 and organoids. The Tp53 and Pten double mutation in combination with the Nf2 mutation increased the radiation resistance of tumors. The system used in this study can aid in elucidating the mechanism underlying differential intrinsic radiation sensitivity of individual tumors.}, }
@article {pmid36958270, year = {2023}, author = {Botelho, J and Tüffers, L and Fuss, J and Buchholz, F and Utpatel, C and Klockgether, J and Niemann, S and Tümmler, B and Schulenburg, H}, title = {Phylogroup-specific variation shapes the clustering of antimicrobial resistance genes and defence systems across regions of genome plasticity in Pseudomonas aeruginosa.}, journal = {EBioMedicine}, volume = {90}, number = {}, pages = {104532}, doi = {10.1016/j.ebiom.2023.104532}, pmid = {36958270}, issn = {2352-3964}, abstract = {BACKGROUND: Pseudomonas aeruginosa is an opportunistic pathogen consisting of three phylogroups (hereafter named A, B, and C). Here, we assessed phylogroup-specific evolutionary dynamics across available and also new P. aeruginosa genomes.<br><br>METHODS: In this genomic analysis, we first generated new genome assemblies for 18 strains of the major P.&#xa0;aeruginosa clone type (mPact) panel, comprising a phylogenetically diverse collection of clinical and environmental isolates for this species. Thereafter, we combined these new genomes with 1991 publicly available P.&#xa0;aeruginosa genomes for a phylogenomic and comparative analysis. We specifically explored to what extent antimicrobial resistance (AMR) genes, defence systems, and virulence genes vary in their distribution across regions of genome plasticity (RGPs) and "masked" (RGP-free) genomes, and to what extent this variation differs among the phylogroups.<br><br>FINDINGS: We found that members of phylogroup B possess larger genomes, contribute a comparatively larger number of pangenome families, and show lower abundance of CRISPR-Cas systems. Furthermore, AMR and defence systems are pervasive in RGPs and integrative and conjugative/mobilizable elements (ICEs/IMEs) from phylogroups A and B, and the abundance of these cargo genes is often significantly correlated. Moreover, inter- and intra-phylogroup interactions occur at the accessory genome level, suggesting frequent recombination events. Finally, we provide here the mPact panel of diverse P.&#xa0;aeruginosa strains that may serve as a valuable reference for functional analyses.<br><br>INTERPRETATION: Altogether, our results highlight distinct pangenome characteristics of the P.&#xa0;aeruginosa phylogroups, which are possibly influenced by variation in the abundance of CRISPR-Cas systems and are shaped by the differential distribution of other defence systems and AMR genes.<br><br>FUNDING: German Science Foundation, Max-Planck Society, Leibniz ScienceCampus Evolutionary Medicine of the Lung, BMBF program Medical Infection Genomics, Kiel Life Science Postdoc Award.}, }
@article {pmid36952190, year = {2023}, author = {Chornyi, S and Koster, J and Waterham, HR}, title = {Applying CRISPR-Cas9 Genome Editing to Study Genes Involved in Peroxisome Biogenesis or Peroxisomal Functions.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2643}, number = {}, pages = {233-245}, pmid = {36952190}, issn = {1940-6029}, mesh = {Animals ; Humans ; *Gene Editing ; *CRISPR-Cas Systems/genetics ; Peroxisomes/genetics ; Genome ; Base Sequence ; Mammals/genetics ; }, abstract = {The development and application of the CRISPR-Cas9 technology for genome editing of mammalian cells have opened up a wealth of possibilities for genetically modifying and manipulating human cells, and use in functional studies or therapeutic approaches.Here we describe the approach that we have been using successfully to generate multiple human cell lines with targeted (partial) gene deletions, i.e., knockout cells, or human cells with modified genomic nucleotide sequences, i.e., knock-in cells, in genes encoding known or putative proteins involved in peroxisome biogenesis or peroxisomal functions.}, }
@article {pmid36914213, year = {2023}, author = {Zhao, J and Zuo, S and Huang, L and Lian, J and Xu, Z}, title = {CRISPR-Cas12a-based genome editing and transcriptional repression for biotin synthesis in Pseudomonas mutabilis.}, journal = {Journal of applied microbiology}, volume = {134}, number = {3}, pages = {}, doi = {10.1093/jambio/lxad049}, pmid = {36914213}, issn = {1365-2672}, mesh = {*Gene Editing ; *CRISPR-Cas Systems ; Biotin/genetics/metabolism ; Plasmids ; }, abstract = {AIMS: To establish a dual-function clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12a system combined genome editing and transcriptional repression for multiplex metabolic engineering of Pseudomonas mutabilis.<br><br>MATERIALS AND RESULTS: This CRISPR-Cas12a system consisted of two plasmids that enabled single gene deletion, replacement, and inactivation with efficiency >90% for most targets within 5&#x2009;days. With the guidance of truncated crRNA containing 16&#x2009;bp spacer sequences, a catalytically active Cas12a could be employed to repress the expression of the reporter gene eGFP up to 66.6%. When bdhA deletion and eGFP repression were tested simultaneously by transforming a single crRNA plasmid and Cas12a plasmid, the knockout efficiency reached 77.8% and the expression of eGFP was decreased by >50%. Finally, the dual-functional system was demonstrated to increase the production of biotin by 3.84-fold, with yigM deletion and birA repression achieved simultaneously.<br><br>CONCLUSIONS: This CRISPR-Cas12a system is an efficient genome editing and regulation tool to facilitate the construction of P. mutabilis cell factories.}, }
@article {pmid36893808, year = {2023}, author = {Peri, KVR and Faria-Oliveira, F and Larsson, A and Plovie, A and Papon, N and Geijer, C}, title = {Split-marker-mediated genome editing improves homologous recombination frequency in the CTG clade yeast Candida intermedia.}, journal = {FEMS yeast research}, volume = {23}, number = {}, pages = {}, pmid = {36893808}, issn = {1567-1364}, mesh = {*Gene Editing ; *Saccharomycetales/genetics ; Homologous Recombination ; Candida albicans/genetics ; CRISPR-Cas Systems ; }, abstract = {Genome-editing toolboxes are essential for the exploration and exploitation of nonconventional yeast species as cell factories, as they facilitate both genome studies and metabolic engineering. The nonconventional yeast Candida intermedia is a biotechnologically interesting species due to its capacity to convert a wide range of carbon sources, including xylose and lactose found in forestry and dairy industry waste and side-streams, into added-value products. However, possibilities of genetic manipulation have so far been limited due to lack of molecular tools for this species. We describe here the development of a genome editing method for C. intermedia, based on electroporation and gene deletion cassettes containing the Candida albicans NAT1 dominant selection marker flanked by 1000 base pair sequences homologous to the target loci. Linear deletion cassettes targeting the ADE2 gene originally resulted in <1% targeting efficiencies, suggesting that C. intermedia mainly uses nonhomologous end joining for integration of foreign DNA fragments. By developing a split-marker based deletion technique for C. intermedia, we successfully improved the homologous recombination rates, achieving targeting efficiencies up to 70%. For marker-less deletions, we also employed the split-marker cassette in combination with a recombinase system, which enabled the construction of double deletion mutants via marker recycling. Overall, the split-marker technique proved to be a quick and reliable method for generating gene deletions in C. intermedia, which opens the possibility to uncover and enhance its cell factory potential.}, }
@article {pmid36623883, year = {2023}, author = {Yang, W and Zhao, H and Dou, Y and Wang, P and Chang, Q and Qiao, X and Wang, X and Xu, C and Zhang, Z and Zhang, L}, title = {CYP3A4 and CYP3A5 Expression is Regulated by CYP3A4*1G in CRISPR/Cas9-Edited HepG2 Cells.}, journal = {Drug metabolism and disposition: the biological fate of chemicals}, volume = {51}, number = {4}, pages = {492-498}, doi = {10.1124/dmd.122.001111}, pmid = {36623883}, issn = {1521-009X}, mesh = {Humans ; *Cytochrome P-450 CYP3A/genetics/metabolism ; Hep G2 Cells ; *CRISPR-Cas Systems/genetics ; Rifampin/pharmacology ; RNA, Messenger/genetics ; Genotype ; }, abstract = {Functional CYP3A4*1G (G>A, rs2242480) in cytochrome P450 3A4 (CYP3A4) regulates the drug-metabolizing enzyme CYP3A4 expression. The objective of this study was to investigate whether CYP3A4*1G regulates both basal and rifampicin (RIF)-induced expression and enzyme activity of CYP3A4 and CYP3A5 in gene-edited human HepG2 cells. CYP3A4*1G GG and AA genotype HepG2 cells were established using the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) single nucleotide polymorphism technology and homology-directed repair in the CYP3A4*1G GA HepG2 cell line. In CYP3A4*1G GG, GA, and AA HepG2 cells, CYP3A4*1G regulated expression of CYP3A4 and CYP3A5 mRNA and protein in an allele-dependent manner. Of note, significantly decreased expression level of CYP3A4 and CYP3A5 was observed in CYP3A4*1G AA HepG2 cells. Moreover, the results after RIF treatment showed that CYP3A4*1G decreased the induction level of CYP3A4 and CYP3A5 mRNA expression in CYP3A4*1G AA HepG2 cells. At the same time, CYP3A4*1G decreased CYP3A4 enzyme activity and tacrolimus metabolism, especially in CYP3A4*1G GA HepG2 cells. In summary, we successfully constructed CYP3A4*1G GG and AA homozygous HepG2 cell models and found that CYP3A4*1G regulates both basal and RIF-induced expression and enzyme activity of CYP3A4 and CYP3A5 in CRISPR/Cas9 CYP3A4*1G HepG2 cells. SIGNIFICANCE STATEMENT: Cytochrome P450 (CYP) 3A4*1G regulates both basal and rifampicin (RIF)-induced expression and enzyme activity of CYP3A4 and CYP3A5. This study successfully established CYP3A4*1G (G>A, rs2242480), GG, and AA HepG2 cell models using CRISPR/Cas9, thus providing a powerful tool for studying the mechanism by which CYP3A4*1G regulates the basal and RIF-induced expression of CYP3A4 and CYP3A5.}, }
@article {pmid36597709, year = {2023}, author = {Zhang, R and Zhang, S and Li, J and Gao, J and Song, G and Li, W and Geng, S and Liu, C and Lin, Y and Li, Y and Li, G}, title = {CRISPR/Cas9-targeted mutagenesis of TaDCL4, TaDCL5 and TaRDR6 induces male sterility in common wheat.}, journal = {Plant biotechnology journal}, volume = {21}, number = {4}, pages = {839-853}, pmid = {36597709}, issn = {1467-7652}, mesh = {Male ; Humans ; *Triticum/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Plant Breeding ; Plant Proteins/genetics/metabolism ; RNA, Small Interfering/genetics ; Mutagenesis/genetics ; Plants/genetics ; *Infertility, Male/genetics ; RNA, Plant/genetics ; Gene Expression Regulation, Plant ; }, abstract = {Phased, small interfering RNAs (phasiRNAs) are important for plant anther development, especially for male sterility. PhasiRNA biogenesis is dependent on genes like RNA polymerase 6 (RDR6), DICER-LIKE 4 (DCL4), or DCL5 to produce 21- or 24 nucleotide (nt) double-strand small RNAs. Here, we generated mutants of DCL4, DCL5 and RDR6 using CRISPR/Cas9 system and studied their effects on plant reproductive development and phasiRNA production in wheat. We found that RDR6 mutation caused sever consequence throughout plant development starting from seed germination and the dcl4 mutants grew weaker with thorough male sterility, while dcl5 plants developed normally but exhibited male sterility. Correspondingly, DCL4 and DCL5, respectively, specified 21- and 24-nt phasiRNA biogenesis, while RDR6 contributed to both. Also, the three key genes evolved differently in wheat, with TaDCL5-A/B becoming non-functioning and TaRDR6-A being lost after polyploidization. Furthermore, we found that PHAS genes (phasiRNA precursors) identified via phasiRNAs diverged rapidly among sub-genomes of polyploid wheat. Despite no similarity being found among phasiRNAs of grasses, their targets were enriched for similar biological functions. In light of the important roles of phasiRNA pathways in gametophyte development, genetic dissection of the function of key genes may help generate male sterile lines suitable for hybrid wheat breeding.}, }
@article {pmid36587283, year = {2023}, author = {Cheng, J and Hill, C and Han, Y and He, T and Ye, X and Shabala, S and Guo, G and Zhou, M and Wang, K and Li, C}, title = {New semi-dwarfing alleles with increased coleoptile length by gene editing of gibberellin 3-oxidase 1 using CRISPR-Cas9 in barley (Hordeum vulgare L.).}, journal = {Plant biotechnology journal}, volume = {21}, number = {4}, pages = {806-818}, pmid = {36587283}, issn = {1467-7652}, mesh = {*Gibberellins ; Cotyledon ; *Hordeum/genetics ; CRISPR-Cas Systems/genetics ; Oxidoreductases/genetics ; Alleles ; Gene Editing ; Seedlings/genetics ; }, abstract = {The green revolution was based on genetic modification of the gibberellin (GA) hormone system with "dwarfing" gene mutations that reduces GA signals, conferring shorter stature, thus enabling plant adaptation to modern farming conditions. Strong GA-related mutants with shorter stature often have reduced coleoptile length, discounting yield gain due to their unsatisfactory seedling emergence under drought conditions. Here we present gibberellin (GA) 3-oxidase1 (GA3ox1) as an alternative semi-dwarfing gene in barley that combines an optimal reduction in plant height without restricting coleoptile and seedling growth. Using large-scale field trials with an extensive collection of barley accessions, we showed that a natural GA3ox1 haplotype moderately reduced plant height by 5-10 cm. We used CRISPR/Cas9 technology, generated several novel GA3ox1 mutants and validated the function of GA3ox1. We showed that altered GA3ox1 activities changed the level of active GA isoforms and consequently increased coleoptile length by an average of 8.2 mm, which could provide essential adaptation to maintain yield under climate change. We revealed that CRISPR/Cas9-induced GA3ox1 mutations increased seed dormancy to an ideal level that could benefit the malting industry. We conclude that selecting HvGA3ox1 alleles offers a new opportunity for developing barley varieties with optimal stature, longer coleoptile and additional agronomic traits.}, }
@article {pmid36529912, year = {2023}, author = {Luo, W and Suzuki, R and Imai, R}, title = {Precise in planta genome editing via homology-directed repair in wheat.}, journal = {Plant biotechnology journal}, volume = {21}, number = {4}, pages = {668-670}, pmid = {36529912}, issn = {1467-7652}, mesh = {*Gene Editing ; *Triticum/genetics ; CRISPR-Cas Systems/genetics ; Recombinational DNA Repair ; }, }
@article {pmid36529879, year = {2023}, author = {Zhu, Y and Lin, Y and Fan, Y and Wang, Y and Li, P and Xiong, J and He, Y and Cheng, S and Ye, X and Wang, F and Goodrich, J and Zhu, JK and Wang, K and Zhang, CJ}, title = {CRISPR/Cas9-mediated restoration of Tamyb10 to create pre-harvest sprouting-resistant red wheat.}, journal = {Plant biotechnology journal}, volume = {21}, number = {4}, pages = {665-667}, pmid = {36529879}, issn = {1467-7652}, mesh = {*Triticum/genetics ; CRISPR-Cas Systems/genetics ; Genes, Plant/genetics ; Seeds ; *Biological Phenomena ; }, }
@article {pmid36125667, year = {2023}, author = {Zhang, Z and Wu, X and Yang, J and Liu, X and Liu, R and Song, Y}, title = {Highly efficient base editing in rabbit by using near-PAMless engineered CRISPR/Cas9 variants.}, journal = {Science China. Life sciences}, volume = {66}, number = {3}, pages = {635-638}, pmid = {36125667}, issn = {1869-1889}, mesh = {Animals ; Rabbits ; *CRISPR-Cas Systems/genetics ; *Gene Editing ; CRISPR-Associated Protein 9/genetics ; RNA, Guide, Kinetoplastida ; }, }
@article {pmid36952053, year = {2023}, author = {Huo, G and Shepherd, J and Pan, X}, title = {Craspase: A novel CRISPR/Cas dual gene editor.}, journal = {Functional &amp; integrative genomics}, volume = {23}, number = {2}, pages = {98}, pmid = {36952053}, issn = {1438-7948}, }
@article {pmid36950734, year = {2023}, author = {Daròs, JA and Pasin, F and Merwaiss, F}, title = {CRISPR-Cas-based plant genome engineering goes viral.}, journal = {Molecular plant}, volume = {}, number = {}, pages = {}, doi = {10.1016/j.molp.2023.03.010}, pmid = {36950734}, issn = {1752-9867}, }
@article {pmid36950281, year = {2023}, author = {Didara, Z and Reithofer, F and Zöttl, K and Jürets, A and Kiss, I and Witte, A and Klein, R}, title = {Inhibition of adenovirus replication by CRISPR-Cas9-mediated targeting of the viral E1A gene.}, journal = {Molecular therapy. Nucleic acids}, volume = {32}, number = {}, pages = {48-60}, pmid = {36950281}, issn = {2162-2531}, abstract = {DNA-targeting CRISPR-Cas systems are able to cleave dsDNA in mammalian cells. Accordingly, they have been employed to target the genomes of dsDNA viruses, mostly when present in cells in a non-replicative state with low copy numbers. However, the sheer amount of viral DNA produced within a very short time by certain lytically replicating viruses potentially brings the capacities of CRISPR-Cas systems to their limits. The accessibility of viral DNA replication sites, short time of accessibility of the DNA before encapsidation, or its complexation with shielding proteins are further potential hurdles. Adenoviruses are fast-replicating dsDNA viruses for which no approved antiviral therapy currently exists. We evaluated the potency of CRISPR-Cas9 in inhibiting the replication of human adenovirus 5 in vitro by targeting its master regulator E1A with a set of guide RNAs and observed a decrease in infectious virus particles by up to three orders of magnitude. Target DNA cleavage also negatively impacted the amount of viral DNA accumulated during the infection cycle. This outcome was mainly caused by specific deletions, inversions, and duplications occurring between target sites, which abolished most E1A functions in most cases. Additionally, we compared two strategies for multiplex gRNA expression and obtained comparable results.}, }
@article {pmid36949234, year = {2023}, author = {Ravikiran, KT and Thribhuvan, R and Sheoran, S and Kumar, S and Kushwaha, AK and Vineeth, TV and Saini, M}, title = {Tailoring crops with superior product quality through genome editing: an update.}, journal = {Planta}, volume = {257}, number = {5}, pages = {86}, pmid = {36949234}, issn = {1432-2048}, mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Plants, Genetically Modified/genetics ; Genome, Plant/genetics ; Plant Breeding/methods ; Crops, Agricultural/genetics ; }, abstract = {In this review, using genome editing, the quality trait alterations in important crops have been discussed, along with the challenges encountered to maintain the crop products' quality. The delivery of economic produce with superior quality is as important as high yield since it dictates consumer's acceptance and end use. Improving product quality of various agricultural and horticultural crops is one of the important targets of plant breeders across the globe. Significant achievements have been made in various crops using conventional plant breeding approaches, albeit, at a slower rate. To keep pace with ever-changing consumer tastes and preferences and industry demands, such efforts must be supplemented with biotechnological tools. Fortunately, many of the quality attributes are resultant of well-understood biochemical pathways with characterized genes encoding enzymes at each step. Targeted mutagenesis and transgene transfer have been instrumental in bringing out desired qualitative changes in crops but have suffered from various pitfalls. Genome editing, a technique for methodical and site-specific modification of genes, has revolutionized trait manipulation. With the evolution of versatile and cost effective CRISPR/Cas9 system, genome editing has gained significant traction and is being applied in several crops. The availability of whole genome sequences with the advent of next generation sequencing (NGS) technologies further enhanced the precision of these techniques. CRISPR/Cas9 system has also been utilized for desirable modifications in quality attributes of various crops such as rice, wheat, maize, barley, potato, tomato, etc. The present review summarizes salient findings and achievements of application of genome editing for improving product quality in various crops coupled with pointers for future research endeavors.}, }
@article {pmid36947920, year = {2023}, author = {Zhan, X and Zhou, J and Jiang, Y and An, P and Luo, B and Lan, F and Ying, B and Wu, Y}, title = {DNA tetrahedron-based CRISPR bioassay for treble-self-amplified and multiplex HPV-DNA detection with elemental tagging.}, journal = {Biosensors &amp; bioelectronics}, volume = {229}, number = {}, pages = {115229}, doi = {10.1016/j.bios.2023.115229}, pmid = {36947920}, issn = {1873-4235}, abstract = {Sensitive quantification of multiple analytes of interest is of great significance for clinical diagnosis. CRISPR Cas platforms offer a strategy for improving the specificity, sensitivity, and speed of nucleic acid-based diagnostics, while their multiplex analysis capability is still limited and challenging. Herein, we develop a novel DNA Tetrahedron (DTN)-supported biosensor based on the spatially separated CRISPR Cas self-amplification strategy and multiple-metal-nanoparticle tagging coupled with inductively coupled plasma mass spectrometry (ICP-MS) detection to improve the sensitivity and feasibility of the platform for multiplex detection of HPV-DNA (HPV-16, HPV-18 and HPV-52). Given target DNA induces robust trans-cleavage activity of the Cas12a/crRNA duplex, and the surrounding corresponding single-stranded DNA (ssDNA) linker are cleaved into short fragments that are unable to bond metal-nanoparticle probes ([197]Au, [107]Ag, [195]Pt) onto DTN modified magnetic beads probe (MBs-DTN), resulting in obvious ICP-MS signal change. Of note, compared with ssDNA functionalized MBs, a higher Signal-to-Noise Ratio was obtained by using MBs-DTN in our system, further amplifying the signal by regulating probes on the surface of MBs. As expected, the HPV-DNA could be detected with detection limits as low as 218 fM and be multiplexed assayed at one test with high accuracy and specificity by this proposed strategy. Furthermore, we demonstrated that the HPV-DNA in cervical swab samples could be detected, showing high consistency with DNA sequencing results. We believe that this work provides a promising option in designing CRISPR based multiplex detection system for high sensitivity, good specificity, and clinical molecular diagnostics.}, }
@article {pmid36914062, year = {2023}, author = {Huang, H and Zhang, W and Zhang, J and Zhao, A and Jiang, H}, title = {Epigenome editing based on CRISPR/dCas9[p300] facilitates transdifferentiation of human fibroblasts into Leydig-like cells.}, journal = {Experimental cell research}, volume = {425}, number = {2}, pages = {113551}, doi = {10.1016/j.yexcr.2023.113551}, pmid = {36914062}, issn = {1090-2422}, mesh = {Humans ; Male ; *Cell Transdifferentiation/genetics ; *Epigenome ; CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Cholesterol Side-Chain Cleavage Enzyme ; Testosterone ; Fibroblasts ; }, abstract = {Recently, Leydig cell (LCs) transplantation has a promising potential to treat male hypogonadism. However, the scarcity of seed cells is the actual barrier impeding the application of LCs transplantation. Utilizing the cutting-edge CRISPR/dCas9[VP64] technology, human foreskin fibroblasts (HFFs) were transdifferentiated into Leydig-like cells（iLCs） in previous study, but the efficiency of transdifferentiation is not very satisfactory. Therefore, this study was conducted to further optimize the CRISPR/dCas9 system for obtaining sufficient iLCs. First, the stable CYP11A1-Promoter-GFP-HFFs cell line was established by infecting HFFs with CYP11A1-Promoter-GFP lentiviral vectors, and then co-infected with dCas9[p300] and the combination of sgRNAs targeted to NR5A1, GATA4 and DMRT1. Next, this study adopted quantitative reverse transcription polymerase chain reaction (qRT-PCR), Western blot, and immunofluorescence to determine the efficiency of transdifferentiation, the generation of testosterone, the expression levels of steroidogenic biomarkers. Moreover, we utilized chromatin immuno-precipitation (ChIP) followed by quantitative polymerase chain reaction (ChIP-qPCR) to measure the levels of acetylation of targeted H3K27. The results revealed that advanced dCas9[p300] facilitated generation of iLCs. Moreover, the dCas9[p300]-mediated iLCs significantly expressed the steroidogenic biomarkers and produced more testosterone with or without LH treatment than the dCas9[VP64]-mediated. Additionally, preferred enrichment in H3K27ac at the promoters was detected only with dCas9[p300] treatment. The data provided here imply that the improved version of dCas9 can aid in the harvesting of iLCs, and will provide sufficient seed cells for cell transplantation treatment of androgen deficiency in the future.}, }
@article {pmid36945831, year = {2023}, author = {Liang, F and Dong, Z and Ye, J and Hu, W and Bhandari, RK and Mai, K and Wang, X}, title = {In vivo DNA methylation editing in zebrafish.}, journal = {Epigenetics}, volume = {18}, number = {1}, pages = {2192326}, doi = {10.1080/15592294.2023.2192326}, pmid = {36945831}, issn = {1559-2308}, mesh = {Animals ; *DNA Methylation ; *CRISPR-Cas Systems ; Gene Editing/methods ; Zebrafish/genetics ; Epigenome ; }, abstract = {The CRISPR/dCas9-based epigenome editing technique has driven much attention. Fused with a catalytic domain from Dnmt or Tet protein, the CRISPR/dCas9-DnmtCD or -TetCD systems possess the targeted DNA methylation editing ability and have established a series of in vitro and in vivo disease models. However, no publication has been reported on zebrafish (Danio rerio), an important animal model in biomedicine. The present study demonstrated that CRISPR/dCas9-Dnmt7 and -Tet2 catalytic domain fusions could site-specifically edit genomic DNA methylation in vivo in zebrafish and may serve as an efficient toolkit for DNA methylation editing in the zebrafish model.}, }
@article {pmid36944919, year = {2023}, author = {Chen, D and Liang, Y and Wang, H and Wang, H and Su, F and Zhang, P and Wang, S and Liu, W and Li, Z}, title = {CRISPR-Cas-Driven Single Micromotor (Cas-DSM) Enables Direct Detection of Nucleic Acid Biomarkers at the Single-Molecule Level.}, journal = {Analytical chemistry}, volume = {}, number = {}, pages = {}, doi = {10.1021/acs.analchem.2c05767}, pmid = {36944919}, issn = {1520-6882}, abstract = {The target-dependent endonuclease activity (also known as the trans-cleavage activity) of CRISPR-Cas systems has stimulated great interest in the development of nascent sensing strategies for nucleic acid diagnostics. Despite many attempts, the majority of the sensitive CRISPR-Cas diagnostics strategies mainly rely on nucleic acid preamplification, which generally needs complex probes/primers designs, multiple experimental steps, and a longer testing time, as well as introducing the risk of false-positive results. In this work, we propose the CRISPR-Cas-Driven Single Micromotor (Cas-DSM), which can directly detect the nucleic acid targets at a single-molecule level with high specificity. We have demonstrated that the Cas-DSM is a reliable and practical method for the quantitative detection of DNA/RNA in various complex clinical samples as well as in individual cells without any preamplification processes. Due to the excellent features of the CRISPR/Cas system, including constant temperature, simple design, high specificity, and flexible programmability, the Cas-DSM could serve as a simple and universal platform for nucleic acid detection. More importantly, this work will provide a breakthrough for the development of next-generation amplification-free CRISPR/Cas sensing toolboxes.}, }
@article {pmid36940764, year = {2023}, author = {Hussain, MS and Anand, V and Kumar, M}, title = {Functional PAM sequence for DNA interference by CRISPR-Cas I-B system of Leptospira interrogans and the role of LinCas11b encoded within lincas8b.}, journal = {International journal of biological macromolecules}, volume = {}, number = {}, pages = {124086}, doi = {10.1016/j.ijbiomac.2023.124086}, pmid = {36940764}, issn = {1879-0003}, abstract = {Pathogenic species of Leptospira are recalcitrant for genetic manipulation using conventional tools, and therefore there is a need to explore techniques of higher efficiency. Application of endogenous CRISPR-Cas tool is emerging and efficient; nevertheless, it is limited by a poor understanding of interference machinery in the bacterial genome and its associated protospacer adjacent motif (PAM). In this study, interference machinery of CRISPR-Cas subtype I-B (Lin_I-B) from L. interrogans was experimentally validated in E. coli using the various identified PAM (TGA, ATG, ATA). The overexpression of the Lin_I-B interference machinery in E. coli demonstrated that LinCas5, LinCas6, LinCas7, and LinCas8b can self-assemble on cognate CRISPR RNA to form an interference complex (LinCascade). Moreover, a robust interference of target plasmids containing a protospacer with a PAM suggested a functional LinCascade. We also recognized a small open reading frame within lincas8b that independently co-translates LinCas11b. A mutant variant of LinCascade[-Cas11b] that lacks LinCas11b co-expression erred to mount target plasmid interference. At the same time, LinCas11b complementation in LinCascade[-Cas11b] rescued target plasmid interference. Thus, the present study establishes Leptospira subtype I-B interference machinery to be functional and, soon, may pave the way for scientists to harness it as a programmable endogenous genetic manipulation tool.}, }
@article {pmid36940173, year = {2023}, author = {Suliman Maashi, M}, title = {CRISPR/Cas-based Aptasensor as an Innovative Sensing Approaches for Food Safety Analysis: Recent Progresses and New Horizons.}, journal = {Critical reviews in analytical chemistry}, volume = {}, number = {}, pages = {1-19}, doi = {10.1080/10408347.2023.2188955}, pmid = {36940173}, issn = {1547-6510}, abstract = {Food safety is one of the greatest public problems occurring around the world. Chemical, physical, and microbiological hazards could lead to food safety problems, which might occur at all stages of the supply chain. To tackle food safety problems and protect consumer health, specific, accurate, and rapid diagnosis techniques meeting various requirements are the imperative measures to ensure food safety. CRISPR-Cas system, a novel emerging technology, is effectively repurposed in (bio)sensing and has shown a tremendous capability to develop on-site and portable diagnostic methods with high specificity and sensitivity. Among numerous existing CRISPR/Cas systems, CRISPR/Cas13a and CRISPR/Cas12a are extensively employed in the design of biosensors, owing to their ability to cleave both non-target and target sequences. However, the specificity limitation in CRISPR/Cas has hindered its progress. Nowadays, nucleic acid aptamers recognized for their specificity and high-affinity characteristics for their analytes are incorporated into CRISPR/Cas systems. With the benefits of reproducibility, high durability, portability, facile operation, and cost-effectiveness, CRISPR/Cas-based aptasensing approaches are an ideal choice for fabricating highly specific point-of-need analytical tools with enhanced response signals. In the current study, we explore some of the most recent progress in the CRISPR/Cas-mediated aptasensors for detecting food risk factors including veterinary drugs, pesticide residues, pathogens, mycotoxins, heavy metals, illegal additives, food additives, and other contaminants. The nanomaterial engineering support with CRISPR/Cas aptasensors is also signified to achieve a hopeful perspective to provide new straightforward test kits toward trace amounts of different contaminants encountered in food samples.}, }
@article {pmid36939849, year = {2023}, author = {Kedlaya, MN and Puzhankara, L and Prasad, R and Raj, A}, title = {Periodontal Disease Pathogens, Pathogenesis, and Therapeutics: The CRISPR-Cas Effect.}, journal = {The CRISPR journal}, volume = {}, number = {}, pages = {}, doi = {10.1089/crispr.2022.0094}, pmid = {36939849}, issn = {2573-1602}, abstract = {Periodontal disease (PD) is an immune-inflammatory disease affecting the supporting structures of the teeth, which results in progressive destruction of the hard and soft tissues surrounding teeth, ultimately resulting in tooth loss. The primary etiological factor for this disease is the presence of pathogenic microorganisms. Pathogenic bacteria face antagonistic conditions and foreign DNA components during the infection stage and depend on defense mechanisms such as clustered regularly interspaced short palindromic repeats (CRISPR)-Cas to counter them. Virulence genes regulated by the CRISPR-Cas system are often expressed by bacteria as part of the stress response to the presence of stress conditions and foreign elements. There is ever-growing evidence regarding the role of CRISPR-Cas in virulence of periodontal pathogens. The same CRISPR-Cas system may also be targeted to reduce bacterial virulence and it may also be utilized to develop diagnostic and therapeutic strategies for prevention and control of PD progression. This review article describes the CRISPR-Cas systems in the periodontal dysbiotic microbial communities, their role in the virulence of periodontal pathogens, and their potential role in understanding the pathogenesis of periodontitis and treatment of PD.}, }
@article {pmid36914863, year = {2023}, author = {Ledford, H}, title = {Why CRISPR babies are still too risky - embryo studies highlight challenges.}, journal = {Nature}, volume = {615}, number = {7953}, pages = {568-569}, pmid = {36914863}, issn = {1476-4687}, mesh = {*Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *CRISPR-Cas Systems/genetics ; *Gene Editing/ethics ; Humans ; Infant, Newborn ; Risk Assessment ; *Embryo, Mammalian/metabolism ; Germ-Line Mutation ; }, }
@article {pmid36893726, year = {2023}, author = {Liu, Y and Ma, L and Liu, W and Xie, L and Wu, Q and Wang, Y and Zhou, Y and Zhang, Y and Jiao, B and He, Y}, title = {RPA-CRISPR/Cas12a Combined with Rolling Circle Amplification-Enriched DNAzyme: A Homogeneous Photothermal Sensing Strategy for Plant Pathogens.}, journal = {Journal of agricultural and food chemistry}, volume = {71}, number = {11}, pages = {4736-4744}, doi = {10.1021/acs.jafc.2c07965}, pmid = {36893726}, issn = {1520-5118}, mesh = {Humans ; *DNA, Catalytic ; Recombinases ; CRISPR-Cas Systems ; DNA ; DNA Primers ; Nucleic Acid Amplification Techniques/methods ; }, abstract = {Alternaria is an endemic fungus associated with brown spot disease, which is one of the most serious citrus diseases. In addition, the mycotoxins metabolized by Alternaria threaten human health seriously. Herein, a novel homogeneous and portable qualitative photothermal method based on recombinase polymerase amplification (RPA), CRISPR/Cas12a, and rolling circle amplification (RCA) for the detection of Alternaria is described. Using RCA primers as substrates for CRISPR/Cas12a trans-cleavage, the two systems, RPA-CRISPR/Cas12a and RCA-enriched G-quadruplex/hemin DNAzyme, are intelligently combined. Target DNA at fg/μL levels can be detected with high specificity. Additionally, the practicability of the proposed method is demonstrated by analyzing cultured Alternaria from different fruit and vegetable samples, as well as citrus fruit samples collected in the field. Furthermore, the implementation of this method does not require any sophisticated equipment and complicated washing steps. Therefore, it has great potential to screen Alternaria in poor laboratories.}, }
@article {pmid36774242, year = {2023}, author = {Papadopoulos, C and Albà, MM}, title = {Newly evolved genes in the human lineage are functional.}, journal = {Trends in genetics : TIG}, volume = {39}, number = {4}, pages = {235-236}, doi = {10.1016/j.tig.2023.02.001}, pmid = {36774242}, issn = {0168-9525}, mesh = {Animals ; Humans ; *Evolution, Molecular ; *Hominidae ; Genome ; Genomics ; CRISPR-Cas Systems ; }, abstract = {Genes restricted to a given species or lineage are mysterious. Many emerged de novo from ancestral noncoding genomic regions rather than from pre-existing genes. A new study by Vakirlis and colleagues shows that, in humans, many of these are associated with phenotypic effects, accelerating our understanding of their functional importance.}, }
@article {pmid36772799, year = {2023}, author = {Bachtarzi, H}, title = {Genome Editing: Moving Toward a New Era of Innovation, Development, and Approval.}, journal = {Human gene therapy}, volume = {34}, number = {5-6}, pages = {171-176}, doi = {10.1089/hum.2022.230}, pmid = {36772799}, issn = {1557-7422}, mesh = {*Gene Editing ; *Genetic Therapy ; Endonucleases/genetics ; Transcription Activator-Like Effector Nucleases/genetics ; Zinc Finger Nucleases/genetics ; CRISPR-Cas Systems ; }, abstract = {Therapeutic genome editing is currently reshaping and transforming the development of advanced therapies as more ex vivo and in vivo gene editing-based technologies are used to treat a broad range of debilitating and complex disorders. With first-generation gene editing modalities (notably those based on ZFNs, TALENs and CRISPR/Cas9), comes a new second-generation of gene editing-based therapeutics including base editing, prime editing and other nuclease-free genome editing modalities. Such ground-breaking innovative products warrant careful considerations from a product development and regulatory perspective, that take into account not only the common development considerations that apply to standard gene and cell therapy products, but also other specific considerations linked with the technology being used. This article sheds light into specific considerations for developing safe and effective in vivo and ex vivo genome editing medicines that will continue to push barriers even further for the cell and gene therapy field.}, }
@article {pmid36694939, year = {2023}, author = {Nishimura, A and Tanahashi, R and Oi, T and Kan, K and Takagi, H}, title = {Plasmid-free CRISPR/Cas9 genome editing in Saccharomyces cerevisiae.}, journal = {Bioscience, biotechnology, and biochemistry}, volume = {87}, number = {4}, pages = {458-462}, doi = {10.1093/bbb/zbad008}, pmid = {36694939}, issn = {1347-6947}, mesh = {*Gene Editing ; *Saccharomyces cerevisiae/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Plasmids/genetics ; Transfection ; }, abstract = {The current CRISPR/Cas9 systems in the yeast Saccharomyces cerevisiae cannot be considered a non-genetic modification technology because it requires the introduction of Cas9 and sgRNA into yeast cells using plasmid expression systems. Our present study showed that the yeast genome can be edited without plasmid expression systems by using a commercially available protein transfection reagent and chemically modified sgRNAs.}, }
@article {pmid36937767, year = {2023}, author = {Hu, M and Chen, S and Ni, Y and Wei, W and Mao, W and Ge, M and Qian, X}, title = {CRISPR/Cas9-mediated genome editing in vancomycin-producing strain Amycolatopsis keratiniphila.}, journal = {Frontiers in bioengineering and biotechnology}, volume = {11}, number = {}, pages = {1141176}, pmid = {36937767}, issn = {2296-4185}, abstract = {Amycolatopsis is an important source of diverse valuable bioactive natural products. The CRISPR/Cas-mediated gene editing tool has been established in some Amycolatopsis species and has accomplished the deletion of single gene or two genes. The goal of this study was to develop a high-efficient CRISPR/Cas9-mediated genome editing system in vancomycin-producing strain A. keratiniphila HCCB10007 and enhance the production of vancomycin by deleting the large fragments of ECO-0501 BGC. By adopting the promoters of gapdhp and ermE*p which drove the expressions of scocas9 and sgRNA, respectively, the all-in-one editing plasmid by homology-directed repair (HDR) precisely deleted the single gene gtfD and inserted the gene eGFP with the efficiency of 100%. Furthermore, The CRISPR/Cas9-mediated editing system successfully deleted the large fragments of cds13-17 (7.7 kb), cds23 (12.7 kb) and cds22-23 (21.2 kb) in ECO-0501 biosynthetic gene cluster (BGC) with high efficiencies of 81%-97% by selecting the sgRNAs with a suitable PAM sequence. Finally, a larger fragment of cds4-27 (87.5 kb) in ECO-0501 BGC was deleted by a dual-sgRNA strategy. The deletion of the ECO-0501 BGCs revealed a noticeable improvement of vancomycin production, and the mutants, which were deleted the ECO-0501 BGCs of cds13-17, cds22-23 and cds4-27, all achieved a 30%-40% increase in vancomycin yield. Therefore, the successful construction of the CRISPR/Cas9-mediated genome editing system and its application in large fragment deletion in A. keratiniphila HCCB10007 might provide a powerful tool for other Amycolatopsis species.}, }
@article {pmid36937752, year = {2023}, author = {Tang, Y and Wang, F and Wang, Y and Wang, Y and Liu, Y and Chen, Z and Li, W and Yang, S and Ma, L}, title = {In vitro characterization of a pAgo nuclease TtdAgo from Thermococcus thioreducens and evaluation of its effect in vivo.}, journal = {Frontiers in bioengineering and biotechnology}, volume = {11}, number = {}, pages = {1142637}, pmid = {36937752}, issn = {2296-4185}, abstract = {In spite of the development of genome-editing tools using CRISPR-Cas systems, highly efficient and effective genome-editing tools are still needed that use novel programmable nucleases such as Argonaute (Ago) proteins to accelerate the construction of microbial cell factories. In this study, a prokaryotic Ago (pAgo) from a hyperthermophilic archaeon Thermococcus thioreducens (TtdAgo) was characterized in vitro. Our results showed that TtdAgo has a typical DNA-guided DNA endonuclease activity, and the efficiency and accuracy of cleavage are modulated by temperature, divalent ions, and the phosphorylation and length of gDNAs and their complementarity to the DNA targets. TtdAgo can utilize 5'-phosphorylated (5'-P) or 5'- hydroxylated (5'-OH) DNA guides to cleave single-stranded DNA (ssDNA) at temperatures ranging from 30°C to 95°C in the presence of Mn[2+] or Mg[2+] and displayed no obvious preference for the 5'-end-nucleotide of the guide. In addition, single-nucleotide mismatches had little effects on cleavage efficiency, except for mismatches at position 4 or 8 that dramatically reduced target cleavage. Moreover, TtdAgo performed programmable cleavage of double-stranded DNA at 75°C. We further introduced TtdAgo into an industrial ethanologenic bacterium Zymomonas mobilis to evaluate its effect in vivo. Our preliminary results indicated that TtdAgo showed cell toxicity toward Z. mobilis, resulting in a reduced growth rate and final biomass. In conclusion, we characterized TtdAgo in vitro and investigated its effect on Z. mobilis in this study, which lays a foundation to develop Ago-based genome-editing tools for recalcitrant industrial microorganisms in the future.}, }
@article {pmid36935157, year = {2023}, author = {Deng, L and He, X and Liu, K and Li, Y and Xia, H and Qian, H and Lu, X and Mao, X and Xiang, Y}, title = {One-pot RPA-Cas12a assay for instant and visual detection of Burkholderia pseudomallei.}, journal = {Analytica chimica acta}, volume = {1252}, number = {}, pages = {341059}, doi = {10.1016/j.aca.2023.341059}, pmid = {36935157}, issn = {1873-4324}, mesh = {Humans ; *Burkholderia pseudomallei/genetics ; *Melioidosis/diagnosis/genetics/microbiology ; CRISPR-Cas Systems ; }, abstract = {Burkholderia pseudomallei is the causative agent of melioidosis, a potentially life-threatening infectious disease, and poses public health risks in endemic areas. Due to the high mortality, intrinsic antibiotic resistance, and atypical manifestations, establishing a rapid, accurate, and sensitive identification of B. pseudomallei enables earlier diagnosis, proper treatments, and better outcomes of melioidosis. Herein, we present a One-Pot CRISPR-integrated assay for Instant and Visual Detection (termed OPC-IVD) of B. pseudomallei. The integration of recombinase polymerase amplification and CRISPR-Cas12a recognition-activated trans-cleavage, achieved a true all-in-one single-tube reaction system, initiating the amplification and cleavage simultaneously, which realized a facile sample-to-answer assay. This approach could be performed with simplified DNA extraction and completed around 30 min by holding the reaction tube in the hand. The detection limit of our OPC-IVD was determined to be 2.19 copy/uL of plasmid DNA, 12.5 CFU/mL of B. pseudomallei, and 61.5 CFU/mL of bacteria in spiked blood samples, respectively. Furthermore, the introduction of internal amplification control effectively reduced the occurrence of false negatives, which was incorporated in the reaction system, and amplified simultaneously with the target and read by naked eyes. The assay exhibited 100% accuracy when evaluated in clinical isolates and samples. The streamlined workflow of our OPC-IVD of B. pseudomallei enables a field-deployable, instrument-free, and ultra-fast approach that can be utilized by non-expert personnel in the field of molecular diagnosis of melioidosis especially in under-resourced setting.}, }
@article {pmid36934130, year = {2023}, author = {Schary, Y and Rotem, I and Caller, T and Lewis, N and Shaihov-Teper, O and Brzezinski, RY and Lendengolts, D and Raanani, E and Sternik, L and Naftali-Shani, N and Leor, J}, title = {CRISPR-Cas9 editing of TLR4 to improve the outcome of cardiac cell therapy.}, journal = {Scientific reports}, volume = {13}, number = {1}, pages = {4481}, pmid = {36934130}, issn = {2045-2322}, mesh = {Humans ; Mice ; Animals ; *Toll-Like Receptor 4/genetics ; Cicatrix/pathology ; CRISPR-Cas Systems/genetics ; Cells, Cultured ; *Myocardial Infarction/genetics/therapy/pathology ; Pericardium/pathology ; Cell- and Tissue-Based Therapy ; Inflammation/pathology ; }, abstract = {Inflammation and fibrosis limit the reparative properties of human mesenchymal stromal cells (hMSCs). We hypothesized that disrupting the toll-like receptor 4 (TLR4) gene would switch hMSCs toward a reparative phenotype and improve the outcome of cell therapy for infarct repair. We developed and optimized an improved electroporation protocol for CRISPR-Cas9 gene editing. This protocol achieved a 68% success rate when applied to isolated hMSCs from the heart and epicardial fat of patients with ischemic heart disease. While cell editing lowered TLR4 expression in hMSCs, it did not affect classical markers of hMSCs, proliferation, and migration rate. Protein mass spectrometry analysis revealed that edited cells secreted fewer proteins involved in inflammation. Analysis of biological processes revealed that TLR4 editing reduced processes linked to inflammation and extracellular organization. Furthermore, edited cells expressed less NF-ƙB and secreted lower amounts of extracellular vesicles and pro-inflammatory and pro-fibrotic cytokines than unedited hMSCs. Cell therapy with both edited and unedited hMSCs improved survival, left ventricular remodeling, and cardiac function after myocardial infarction (MI) in mice. Postmortem histologic analysis revealed clusters of edited cells that survived in the scar tissue 28 days after MI. Morphometric analysis showed that implantation of edited cells increased the area of myocardial islands in the scar tissue, reduced the occurrence of transmural scar, increased scar thickness, and decreased expansion index. We show, for the first time, that CRISPR-Cas9-based disruption of the TLR4-gene reduces pro-inflammatory polarization of hMSCs and improves infarct healing and remodeling in mice. Our results provide a new approach to improving the outcomes of cell therapy for cardiovascular diseases.}, }
@article {pmid36932065, year = {2023}, author = {Karlikow, M and Amalfitano, E and Yang, X and Doucet, J and Chapman, A and Mousavi, PS and Homme, P and Sutyrina, P and Chan, W and Lemak, S and Yakunin, AF and Dolezal, AG and Kelley, S and Foster, LJ and Harpur, BA and Pardee, K}, title = {CRISPR-induced DNA reorganization for multiplexed nucleic acid detection.}, journal = {Nature communications}, volume = {14}, number = {1}, pages = {1505}, pmid = {36932065}, issn = {2041-1723}, mesh = {Animals ; DNA/genetics ; *Nucleic Acids ; Agriculture ; Head ; RNA/genetics ; CRISPR-Cas Systems/genetics ; Nucleic Acid Amplification Techniques ; *Biosensing Techniques ; }, abstract = {Nucleic acid sensing powered by the sequence recognition of CRIPSR technologies has enabled major advancement toward rapid, accurate and deployable diagnostics. While exciting, there are still many challenges facing their practical implementation, such as the widespread need for a PAM sequence in the targeted nucleic acid, labile RNA inputs, and limited multiplexing. Here we report FACT (Functionalized Amplification CRISPR Tracing), a CRISPR-based nucleic acid barcoding technology compatible with Cas12a and Cas13a, enabling diagnostic outputs based on cis- and trans-cleavage from any sequence. Furthermore, we link the activation of CRISPR-Cas12a to the expression of proteins through a Reprogrammable PAIRing system (RePAIR). We then combine FACT and RePAIR to create FACTOR (FACT on RePAIR), a CRISPR-based diagnostic, that we use to detect infectious disease in an agricultural use case: honey bee viral infection. With high specificity and accuracy, we demonstrate the potential of FACTOR to be applied to the sensing of any nucleic acid of interest.}, }
@article {pmid36930418, year = {2023}, author = {Thakur, N and Nigam, M and Awasthi, G and Shukla, A and Shah, AA and Negi, N and Khan, SA and Casini, R and Elansary, HO}, title = {Synergistic soil-less medium for enhanced yield of crops: a step towards incorporating genomic tools for attaining net zero hunger.}, journal = {Functional &amp; integrative genomics}, volume = {23}, number = {2}, pages = {86}, pmid = {36930418}, issn = {1438-7948}, mesh = {Humans ; *CRISPR-Cas Systems ; *Soil ; Proteomics ; Hunger ; Phylogeny ; Genome, Plant ; Plant Breeding/methods ; Crops, Agricultural/genetics ; Genomics ; }, abstract = {Globally, industrial farming endangers crucial ecological mechanisms upon which food production relies, while 815 million people are undernourished and a significant number are malnourished. Zero Hunger aims to concurrently solve global ecological sustainability and food security concerns. Recent breakthroughs in molecular tools and approaches have allowed scientists to detect and comprehend the nature and structure of agro-biodiversity at the molecular and genetic levels, providing us an advantage over traditional methods of crop breeding. These bioinformatics techniques let us optimize our target plants for our soil-less medium and vice versa. Most of the soil-borne and seed-borne diseases are the outcomes of non-treated seed and growth media, which are important factors in low productivity. The farmers do not consider these issues, thereby facing problems growing healthy crops and suffering economic losses. This study is going to help the farmers increase their eco-friendly, chemical residue-free, quality yield of crops and their economic returns. The present invention discloses a synergistic soil-less medium that consists of only four ingredients mixed in optimal ratios by weight: vermicompost (70-80%), vermiculite (10-15%), coco peat (10-15%), and Rhizobium (0-1%). The medium exhibits better physical and chemical characteristics than existing conventional media. The vermiculite to coco peat ratio is reduced, while the vermicompost ratio is increased, with the goals of lowering toxicity, increasing plant and water holding capacity, avoiding drying of the media, and conserving water. The medium provides balanced nutrition and proper ventilation for seed germination and the growth of seedlings. Rhizobium is also used to treat the plastic bags and seeds. The results clearly show that the current synergistic soil-less environment is best for complete plant growth. Securing genetic advantages via sexual recombination, induced random mutations, and transgenic techniques have been essential for the development of improved agricultural varieties. The recent availability of targeted genome-editing technology provides a new path for integrating beneficial genetic modifications into the most significant agricultural species on the planet. Clustered regularly interspaced short palindromic repeats and associated protein 9 (CRISPR/Cas9) has evolved into a potent genome-editing tool for imparting genetic modifications to crop species. In addition, the integration of analytical methods like population genomics, phylogenomics, and metagenomics addresses conservation problems, while whole genome sequencing has opened up a new dimension for explaining the genome architecture and its interactions with other species. The in silico genomic and proteomic investigation was also conducted to forecast future investigations for the growth of French beans on a synergistic soil-less medium with the purpose of studying how a blend of vermicompost, vermiculite, cocopeat, and Rhizobium secrete metal ions, and other chemical compounds into the soil-less medium and affect the development of our target plant as well as several other plants. This interaction was studied using functional and conserved region analysis, phylogenetic analysis, and docking tools.}, }
@article {pmid36930175, year = {2023}, author = {Du, SW and Palczewski, K}, title = {Eye on genome editing.}, journal = {The Journal of experimental medicine}, volume = {220}, number = {5}, pages = {}, doi = {10.1084/jem.20230146}, pmid = {36930175}, issn = {1540-9538}, mesh = {Mice ; Animals ; *Gene Editing/methods ; *Retinitis Pigmentosa/genetics/therapy ; Mutation/genetics ; Disease Models, Animal ; CRISPR-Cas Systems/genetics ; }, abstract = {CRISPR/Cas9 genome editing techniques have the potential to treat previously untreatable inherited genetic disorders of vision by correcting mutations that cause these afflictions. Using a prime editor, Qin et al. (2023. J. Exp. Med.https://doi.org/10.1084/jem.20220776) restored visual functions in a mouse model (rd10) of retinitis pigmentosa.}, }
@article {pmid36876906, year = {2023}, author = {Huang, M and Coral, D and Ardalani, H and Spegel, P and Saadat, A and Claussnitzer, M and Mulder, H and Franks, PW and Kalamajski, S}, title = {Identification of a weight loss-associated causal eQTL in MTIF3 and the effects of MTIF3 deficiency on human adipocyte function.}, journal = {eLife}, volume = {12}, number = {}, pages = {}, pmid = {36876906}, issn = {2050-084X}, support = {ERC-2015-CoG -681742 NASCENT/ERC_/European Research Council/International ; }, mesh = {Humans ; *Obesity/genetics/metabolism ; Causality ; Cell Line ; *Adipocytes/metabolism ; Weight Loss ; CRISPR-Cas Systems ; }, abstract = {Genetic variation at the MTIF3 (Mitochondrial Translational Initiation Factor 3) locus has been robustly associated with obesity in humans, but the functional basis behind this association is not known. Here, we applied luciferase reporter assay to map potential functional variants in the haplotype block tagged by rs1885988 and used CRISPR-Cas9 to edit the potential functional variants to confirm the regulatory effects on MTIF3 expression. We further conducted functional studies on MTIF3-deficient differentiated human white adipocyte cell line (hWAs-iCas9), generated through inducible expression of CRISPR-Cas9 combined with delivery of synthetic MTIF3-targeting guide RNA. We demonstrate that rs67785913-centered DNA fragment (in LD with rs1885988, r[2] > 0.8) enhances transcription in a luciferase reporter assay, and CRISPR-Cas9-edited rs67785913 CTCT cells show significantly higher MTIF3 expression than rs67785913 CT cells. Perturbed MTIF3 expression led to reduced mitochondrial respiration and endogenous fatty acid oxidation, as well as altered expression of mitochondrial DNA-encoded genes and proteins, and disturbed mitochondrial OXPHOS complex assembly. Furthermore, after glucose restriction, the MTIF3 knockout cells retained more triglycerides than control cells. This study demonstrates an adipocyte function-specific role of MTIF3, which originates in the maintenance of mitochondrial function, providing potential explanations for why MTIF3 genetic variation at rs67785913 is associated with body corpulence and response to weight loss interventions.}, }
@article {pmid36825467, year = {2023}, author = {Chen, Z and Zheng, S and Fu, C}, title = {Shotgun knockdown of RNA by CRISPR-Cas13d in fission yeast.}, journal = {Journal of cell science}, volume = {136}, number = {6}, pages = {}, doi = {10.1242/jcs.260769}, pmid = {36825467}, issn = {1477-9137}, mesh = {RNA/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Schizosaccharomyces/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Promoter Regions, Genetic/genetics ; Protein Serine-Threonine Kinases/metabolism ; *Schizosaccharomyces pombe Proteins/genetics/metabolism ; }, abstract = {The CRISPR-Cas13d system has a single small effector protein that targets RNA and does not require the presence of a protospacer flanking site in the targeted transcript. These features make CRISPR-Cas13d an attractive system for RNA manipulation. Here, we report the successful implementation of the CRISPR-Cas13d system in fission yeast for RNA knockdown. A high effectiveness of the CRISPR-Cas13d system was ensured by using an array of CRISPR RNAs (crRNAs) that are flanked by two self-cleaving ribozymes and are expressed from an RNA polymerase II promoter. Given the repressible nature of the promoter, RNA knockdown by the CRISPR-Cas13d system is reversible. Moreover, using the CRISPR-Cas13d system, we identified an effective crRNA array targeting the transcript of gfp and the effectiveness was demonstrated by successful knockdown of the transcripts of noc4-gfp, bub1-gfp and ade6-gfp. In principle, the effective GFP crRNA array allows knockdown of any transcript carrying the GFP sequences. This new CRISPR-Cas13d-based toolkit is expected to have a wide range of applications in many aspects of biology, including dissection of gene function and visualization of RNA.}, }
@article {pmid36651153, year = {2023}, author = {Dubois, M}, title = {Phishing alert! A Cas9-based method reveals the identity of promoter-bound transcription factors.}, journal = {Plant physiology}, volume = {191}, number = {3}, pages = {1462-1464}, pmid = {36651153}, issn = {1532-2548}, mesh = {*CRISPR-Cas Systems ; }, }
@article {pmid36305686, year = {2023}, author = {Wang, Z and He, Z and Liu, Z and Qu, M and Gao, C and Wang, C and Wang, Y}, title = {A reverse chromatin immunoprecipitation technique based on the CRISPR-dCas9 system.}, journal = {Plant physiology}, volume = {191}, number = {3}, pages = {1505-1519}, pmid = {36305686}, issn = {1532-2548}, mesh = {Reproducibility of Results ; *DNA ; *Plants ; Chromatin Immunoprecipitation/methods ; RNA ; CRISPR-Cas Systems/genetics ; }, abstract = {DNA-protein interaction is one of the most crucial interactions in biological processes. However, the technologies available to study DNA-protein interactions are all based on DNA hybridization; however, DNA hybridization is not highly specific and is relatively low in efficiency. RNA-guided DNA recognition is highly specific and efficient. To overcome the limitations of technologies based on DNA hybridization, we built a DNA-binding protein capture technology based on the clustered regularly interspaced palindromic repeats (CRISPR)-dead Cas9 (dCas9) system and transient genetic transformation, termed reverse chromatin immunoprecipitation based on CRISPR-dCas9 system (R-ChIP-dCas9). In this system, dCas9 was fused with Strep-Tag II to form a fusion protein for StrepTactin affinity purification. Transient transformation was performed for the expression of dCas9 and guide RNA (gRNA) to form the dCas9-gRNA complex in birch (Betula platyphylla) plants, which binds to the target genomic DNA region. The dCas9-gRNA-DNA complex was crosslinked, then the chromatin was sonicated into fragments, and purified using StrepTactin beads. The proteins binding to the target genomic DNA region were identified using mass spectrometry. Using this method, we determined the upstream regulators of a NAM, ATAF, and CUC (NAC) transcription factor (TF), BpNAC090, and 32 TFs potentially regulating BpNAC090 were identified. The reliability of R-ChIP-dCas9 was further confirmed by chromatin immunoprecipitation, electrophoretic mobility shift assays, and yeast one-hybrid. This technology can be adapted to various plant species and does not depend on the availability of a stable transformation system; therefore, it has wide application in identifying proteins bound to genomic DNA.}, }
@article {pmid35388882, year = {2023}, author = {Musunuru, K}, title = {CRISPR and cardiovascular diseases.}, journal = {Cardiovascular research}, volume = {119}, number = {1}, pages = {79-93}, doi = {10.1093/cvr/cvac048}, pmid = {35388882}, issn = {1755-3245}, mesh = {Animals ; Humans ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Cardiovascular Diseases/diagnosis/genetics/therapy ; Genetic Therapy ; Gene Editing ; *Muscular Dystrophy, Duchenne/genetics ; CRISPR-Cas Systems ; }, abstract = {CRISPR technologies have progressed by leaps and bounds over the past decade, not only having a transformative effect on biomedical research but also yielding new therapies that are poised to enter the clinic. In this review, I give an overview of (i) the various CRISPR DNA-editing technologies, including standard nuclease gene editing, base editing, prime editing, and epigenome editing, (ii) their impact on cardiovascular basic science research, including animal models, human pluripotent stem cell models, and functional screens, and (iii) emerging therapeutic applications for patients with cardiovascular diseases, focusing on the examples of hypercholesterolaemia, transthyretin amyloidosis, and Duchenne muscular dystrophy.}, }
@article {pmid36937283, year = {2023}, author = {Gu, B and Zhuo, C and Xu, X and El Bissati, K}, title = {Editorial: Molecular diagnostics for infectious diseases: Novel approaches, clinical applications and future challenges.}, journal = {Frontiers in microbiology}, volume = {14}, number = {}, pages = {1153827}, pmid = {36937283}, issn = {1664-302X}, }
@article {pmid36937152, year = {2023}, author = {Xiao, H and Hu, J and Huang, C and Feng, W and Liu, Y and Kumblathan, T and Tao, J and Xu, J and Le, XC and Zhang, H}, title = {CRISPR techniques and potential for the detection and discrimination of SARS-CoV-2 variants of concern.}, journal = {Trends in analytical chemistry : TRAC}, volume = {161}, number = {}, pages = {117000}, pmid = {36937152}, issn = {0165-9936}, abstract = {The continuing evolution of the SARS-CoV-2 virus has led to the emergence of many variants, including variants of concern (VOCs). CRISPR-Cas systems have been used to develop techniques for the detection of variants. These techniques have focused on the detection of variant-specific mutations in the spike protein gene of SARS-CoV-2. These sequences mostly carry single-nucleotide mutations and are difficult to differentiate using a single CRISPR-based assay. Here we discuss the specificity of the Cas9, Cas12, and Cas13 systems, important considerations of mutation sites, design of guide RNA, and recent progress in CRISPR-based assays for SARS-CoV-2 variants. Strategies for discriminating single-nucleotide mutations include optimizing the position of mismatches, modifying nucleotides in the guide RNA, and using two guide RNAs to recognize the specific mutation sequence and a conservative sequence. Further research is needed to confront challenges in the detection and differentiation of variants and sublineages of SARS-CoV-2 in clinical diagnostic and point-of-care applications.}, }
@article {pmid36934807, year = {2023}, author = {Marino, ND}, title = {Phage against the machine: discovery and mechanism of type V anti-CRISPRs.}, journal = {Journal of molecular biology}, volume = {}, number = {}, pages = {168054}, doi = {10.1016/j.jmb.2023.168054}, pmid = {36934807}, issn = {1089-8638}, abstract = {The discovery of diverse bacterial CRISPR-Cas systems has reignited interest in understanding bacterial defense pathways while yielding exciting new tools for genome editing. CRISPR-Cas systems are widely distributed in prokaryotes, found in 40% of bacteria and 90% of archaea, where they function as adaptive immune systems against bacterial viruses (phage) and other mobile genetic elements. In turn, phage have evolved inhibitors, called anti-CRISPR proteins, to prevent targeting. Type V CRISPR-Cas12 systems have emerged as a particularly exciting arena in this co-evolutionary arms race. Type V anti-CRISPRs have highly diverse and novel mechanisms of action, some of which appear to be unusually potent or widespread. In this review, we discuss the discovery and mechanism of these anti-CRISPRs as well as future areas for exploration.}, }
@article {pmid36933595, year = {2023}, author = {Bhatia, S and Pooja,  and Yadav, SK}, title = {CRISPR-Cas for genome editing: Classification, mechanism, designing and applications.}, journal = {International journal of biological macromolecules}, volume = {}, number = {}, pages = {124054}, doi = {10.1016/j.ijbiomac.2023.124054}, pmid = {36933595}, issn = {1879-0003}, abstract = {Clustered regularly interspersed short pallindromic repeats (CRISPR) and CRISPR associated proteins (Cas) system (CRISPR-Cas) came into light as prokaryotic defence mechanism for adaptive immune response. CRISPR-Cas works by integrating short sequences of the target genome (spacers) into the CRISPR locus. The locus containing spacers interspersed repeats is further expressed into small guide CRISPR RNA (crRNA) which is then deployed by the Cas proteins to evade the target genome. Based on the Cas proteins CRISPR-Cas is classified according to polythetic system of classification. The characteristic of the CRISPR-Cas9 system to target DNA sequences using programmable RNAs has opened new arenas due to which today CRISPR-Cas has evolved as cutting end technique in the field of genome editing. Here, we discuss about the evolution of CRISPR, its classification and various Cas systems including the designing and molecular mechanism of CRISPR-Cas. Applications of CRISPR-Cas as a genome editing tools are also highlighted in the areas such as agriculture, and anticancer therapy. Briefly discuss the role of CRISPR and its Cas systems in the diagnosis of COVID-19 and its possible preventive measures. The challenges in existing CRISP-Cas technologies and their potential solutions are also discussed briefly.}, }
@article {pmid36929199, year = {2023}, author = {Rostain, W and Grebert, T and Vyhovskyi, D and Pizarro, PT and Tshinsele-Van Bellingen, G and Cui, L and Bikard, D}, title = {Cas9 off-target binding to the promoter of bacterial genes leads to silencing and toxicity.}, journal = {Nucleic acids research}, volume = {}, number = {}, pages = {}, doi = {10.1093/nar/gkad170}, pmid = {36929199}, issn = {1362-4962}, support = {677823/ERC_/European Research Council/International ; 101044479/ERC_/European Research Council/International ; }, abstract = {Genetic tools derived from the Cas9 RNA-guided nuclease are providing essential capabilities to study and engineer bacteria. While the importance of off-target effects was noted early in Cas9's application to mammalian cells, off-target cleavage by Cas9 in bacterial genomes is easily avoided due to their smaller size. Despite this, several studies have reported experimental setups in which Cas9 expression was toxic, even when using the catalytic dead variant of Cas9 (dCas9). Specifically, dCas9 was shown to be toxic when in complex with guide RNAs sharing specific PAM (protospacer adjacent motif)-proximal sequence motifs. Here, we demonstrate that this toxicity is caused by off-target binding of Cas9 to the promoter of essential genes, with silencing of off-target genes occurring with as little as 4 nt of identity in the PAM-proximal sequence. Screens performed in various strains of Escherichia coli and other enterobacteria show that the nature of toxic guide RNAs changes together with the evolution of sequences at off-target positions. These results highlight the potential for Cas9 to bind to hundreds of off-target positions in bacterial genomes, leading to undesired effects. This phenomenon must be considered in the design and interpretation of CRISPR-Cas experiments in bacteria.}, }
@article {pmid36927663, year = {2023}, author = {Wu, ZS and Gao, Y and Du, YT and Dang, S and He, KM}, title = {The protocol of tagging endogenous proteins with fluorescent tags using CRISPR-Cas9 genome editing.}, journal = {Yi chuan = Hereditas}, volume = {45}, number = {2}, pages = {165-175}, doi = {10.16288/j.yczz.22-395}, pmid = {36927663}, issn = {0253-9772}, mesh = {Animals ; *Gene Editing/methods ; *CRISPR-Cas Systems ; CRISPR-Associated Protein 9/genetics ; Recombinational DNA Repair ; DNA ; Mammals/genetics ; }, abstract = {The currently widely used CRISPR-Cas9 genome editing technology enables the editing of target genes (knock-out or knock-in) with high accuracy and efficiency. Guided by the small guide RNA, the Cas9 nuclease induces a DNA double-strand break at the targeted genomic locus. The DNA double-strand break can be repaired by the homology-directed repair pathway in the presence of a repair template. With the repair template containing the coding sequence of a fluorescent tag, the targeted gene can be inserted with the sequence of a fluorescent tag at the designed position. The genome editing mediated labeling of endogenous proteins with fluorescent tags avoids the potential artifacts caused by gene overexpression and substantially improves the reproductivity of imaging experiments. This protocol focuses on creating mammalian cell lines with endogenous proteins tagged with fluorescent proteins or self-labeling protein tags using CRISPR-Cas9 genome editing.}, }
@article {pmid36927640, year = {2023}, author = {Liu, MZ and Wang, LR and Li, YM and Ma, XY and Han, HH and Li, DL}, title = {Generation of genetically modified rat models via the CRISPR/Cas9 technology.}, journal = {Yi chuan = Hereditas}, volume = {45}, number = {1}, pages = {78-87}, doi = {10.16288/j.yczz.22-354}, pmid = {36927640}, issn = {0253-9772}, mesh = {Rats ; Animals ; *CRISPR-Cas Systems ; *Gene Editing/methods ; DNA Breaks, Double-Stranded ; Recombinational DNA Repair ; DNA End-Joining Repair/genetics ; Mammals/genetics ; }, abstract = {The RNA-guided CRISPR/Cas9 genomic editing system consists of a single guide RNA (sgRNA) and a Cas9 nuclease. The two components form a complex in cells and target the genomic loci complementary to the sgRNA. The Cas9 nuclease cleaves the target site creating a double stranded DNA break (DSB). In mammalian cells, DSBs are often repaired via error prone non-homologous end joining (NHEJ) or via homology directed repair (HDR) with the presence of donor DNA templates. Micro-injection of the CRISPR/Cas9 system into the rat embryos enables generation of genetically modified rat models. Here, we describe a detailed protocol for creating gene knockout or knockin rat models via the CRISPR/Cas9 technology.}, }
@article {pmid36927528, year = {2023}, author = {D'Antonio, L and Fieni, C and Ciummo, SL and Vespa, S and Lotti, L and Sorrentino, C and Di Carlo, E}, title = {Inactivation of interleukin-30 in colon cancer stem cells via CRISPR/Cas9 genome editing inhibits their oncogenicity and improves host survival.}, journal = {Journal for immunotherapy of cancer}, volume = {11}, number = {3}, pages = {}, doi = {10.1136/jitc-2022-006056}, pmid = {36927528}, issn = {2051-1426}, mesh = {Mice ; Animals ; *Colonic Neoplasms/pathology ; Cell Line, Tumor ; Gene Editing ; CRISPR-Cas Systems/genetics ; Cell Proliferation ; Neoplastic Stem Cells/pathology ; *Colorectal Neoplasms/pathology ; Cell Transformation, Neoplastic/pathology ; Carcinogenesis/genetics ; Interleukins/genetics ; }, abstract = {BACKGROUND: Progression of colorectal cancer (CRC), a leading cause of cancer-related death worldwide, is driven by colorectal cancer stem cells (CR-CSCs), which are regulated by endogenous and microenvironmental signals. Interleukin (IL)-30 has proven to be crucial for CSC viability and tumor progression. Whether it is involved in CRC tumorigenesis and impacts clinical behavior is unknown.<br><br>METHODS: IL30 production and functions, in stem and non-stem CRC cells, were determined by western blot, immunoelectron microscopy, flow cytometry, cell viability and sphere formation assays. CRISPR/Cas9-mediated deletion of the IL30 gene, RNA-Seq and implantation of IL30 gene transfected or deleted CR-CSCs in NSG mice allowed to investigate IL30's role in CRC oncogenesis. Bioinformatics and immunopathology of CRC samples highlighted the clinical implications.<br><br>RESULTS: We demonstrated that both CR-CSCs and CRC cells express membrane-anchored IL30 that regulates their self-renewal, via WNT5A and RAB33A, and/or proliferation and migration, primarily by upregulating CXCR4 via STAT3, which are suppressed by IL30 gene deletion, along with WNT and RAS pathways. Deletion of IL30 gene downregulates the expression of proteases, such as MMP2 and MMP13, chemokine receptors, mostly CCR7, CCR3 and CXCR4, and growth and inflammatory mediators, including ANGPT2, CXCL10, EPO, IGF1 and EGF. These factors contribute to IL30-driven CR-CSC and CRC cell expansion, which is abrogated by their selective blockade. IL30 gene deleted CR-CSCs displayed reduced tumorigenicity and gave rise to slow-growing and low metastatic tumors in 80% of mice, which survived much longer than controls. Bioinformatics and CIBERSORTx of the 'Colorectal Adenocarcinoma TCGA Nature 2012' collection, and morphometric assessment of IL30 expression in clinical CRC samples revealed that the lack of IL30 in CRC and infiltrating leucocytes correlates with prolonged overall survival.<br><br>CONCLUSIONS: IL30 is a new CRC driver, since its inactivation, which disables oncogenic pathways and multiple autocrine loops, inhibits CR-CSC tumorigenicity and metastatic ability. The development of CRISPR/Cas9-mediated targeting of IL30 could improve the current therapeutic landscape of CRC.}, }
@article {pmid36927151, year = {2023}, author = {Wang, R and Zhang, R and Khodaverdian, A and Yosef, N}, title = {Theoretical guarantees for phylogeny inference from single-cell lineage tracing.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {120}, number = {12}, pages = {e2203352120}, doi = {10.1073/pnas.2203352120}, pmid = {36927151}, issn = {1091-6490}, mesh = {*CRISPR-Cas Systems/genetics ; Phylogeny ; Cell Lineage/genetics ; *Gene Editing ; CRISPR-Associated Protein 9/genetics ; }, abstract = {Lineage-tracing technologies based on Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated protein 9 (CRISPR-Cas9) genome editing have emerged as a powerful tool for investigating development in single-cell contexts, but exact reconstruction of the underlying clonal relationships in experiment is complicated by features of the data. These complications are functions of the experimental parameters in these systems, such as the Cas9 cutting rate, the diversity of indel outcomes, and the rate of missing data. In this paper, we develop two theoretically grounded algorithms for the reconstruction of the underlying single-cell phylogenetic tree as well as asymptotic bounds for the number of recording sites necessary for exact recapitulation of the ground truth phylogeny at high probability. In doing so, we explore the relationship between the problem difficulty and the experimental parameters, with implications for experimental design. Lastly, we provide simulations showing the empirical performance of these algorithms and showing that the trends in the asymptotic bounds hold empirically. Overall, this work provides a theoretical analysis of phylogenetic reconstruction in single-cell CRISPR-Cas9 lineage-tracing technologies.}, }
@article {pmid36925702, year = {2023}, author = {Meng, X and Wu, TG and Lou, QY and Niu, KY and Jiang, L and Xiao, QZ and Xu, T and Zhang, L}, title = {Optimization of CRISPR-Cas system for clinical cancer therapy.}, journal = {Bioengineering &amp; translational medicine}, volume = {8}, number = {2}, pages = {e10474}, pmid = {36925702}, issn = {2380-6761}, abstract = {Cancer is a genetic disease caused by alterations in genome and epigenome and is one of the leading causes for death worldwide. The exploration of disease development and therapeutic strategies at the genetic level have become the key to the treatment of cancer and other genetic diseases. The functional analysis of genes and mutations has been slow and laborious. Therefore, there is an urgent need for alternative approaches to improve the current status of cancer research. Gene editing technologies provide technical support for efficient gene disruption and modification in vivo and in vitro, in particular the use of clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems. Currently, the applications of CRISPR-Cas systems in cancer rely on different Cas effector proteins and the design of guide RNAs. Furthermore, effective vector delivery must be met for the CRISPR-Cas systems to enter human clinical trials. In this review article, we describe the mechanism of the CRISPR-Cas systems and highlight the applications of class II Cas effector proteins. We also propose a synthetic biology approach to modify the CRISPR-Cas systems, and summarize various delivery approaches facilitating the clinical application of the CRISPR-Cas systems. By modifying the CRISPR-Cas system and optimizing its in vivo delivery, promising and effective treatments for cancers using the CRISPR-Cas system are emerging.}, }
@article {pmid36925687, year = {2023}, author = {Khambhati, K and Bhattacharjee, G and Gohil, N and Dhanoa, GK and Sagona, AP and Mani, I and Bui, NL and Chu, DT and Karapurkar, JK and Jang, SH and Chung, HY and Maurya, R and Alzahrani, KJ and Ramakrishna, S and Singh, V}, title = {Phage engineering and phage-assisted CRISPR-Cas delivery to combat multidrug-resistant pathogens.}, journal = {Bioengineering &amp; translational medicine}, volume = {8}, number = {2}, pages = {e10381}, pmid = {36925687}, issn = {2380-6761}, abstract = {Antibiotic resistance ranks among the top threats to humanity. Due to the frequent use of antibiotics, society is facing a high prevalence of multidrug resistant pathogens, which have managed to evolve mechanisms that help them evade the last line of therapeutics. An alternative to antibiotics could involve the use of bacteriophages (phages), which are the natural predators of bacterial cells. In earlier times, phages were implemented as therapeutic agents for a century but were mainly replaced with antibiotics, and considering the menace of antimicrobial resistance, it might again become of interest due to the increasing threat of antibiotic resistance among pathogens. The current understanding of phage biology and clustered regularly interspaced short palindromic repeats (CRISPR) assisted phage genome engineering techniques have facilitated to generate phage variants with unique therapeutic values. In this review, we briefly explain strategies to engineer bacteriophages. Next, we highlight the literature supporting CRISPR-Cas9-assisted phage engineering for effective and more specific targeting of bacterial pathogens. Lastly, we discuss techniques that either help to increase the fitness, specificity, or lytic ability of bacteriophages to control an infection.}, }
@article {pmid36925313, year = {2023}, author = {Chen, H and Li, B and Shi, S and Zhou, T and Wang, X and Wang, Z and Zhou, X and Wang, M and Shi, W and Ren, L}, title = {Au-Fe3O4 nanozyme coupled with CRISPR-Cas12a for sensitive and visual antibiotic resistance diagnosing.}, journal = {Analytica chimica acta}, volume = {1251}, number = {}, pages = {341014}, doi = {10.1016/j.aca.2023.341014}, pmid = {36925313}, issn = {1873-4324}, mesh = {*Angiotensin Receptor Antagonists ; *Angiotensin-Converting Enzyme Inhibitors ; CRISPR-Cas Systems ; Ampicillin ; Drug Resistance, Microbial/genetics ; }, abstract = {The accumulation and spread of antibiotic resistance bacteria (ARB) in the environment may accelerate the formation of superbugs and seriously threaten the health of all living beings. The timeliness and accurate diagnosing of antibiotic resistance is essential to controlling the propagation of superbugs in the environment and formulating effective public health management programs. Herein, we developed a speedy, sensitive, accurate, and user-friendly colorimetric assay for antibiotic resistance, via a synergistic combination of the peroxidase-like property of the Au-Fe3O4 nanozyme and the specific gene identification capability of the CRISPR-Cas12a. Once the CRISPR-Cas12a system recognizes a target resistance gene, it activates its trans-cleavage activity and subsequently releases the Au-Fe3O4 nanozymes, which oxidizes the 3,3,5,5-tetramethylbenzidine (TMB) with color change from transparent to blue. The diagnosing signals could be captured and analyzed by a smartphone. This method detected kanamycin-resistance genes, ampicillin-resistance genes, and chloramphenicol-resistance genes by simple operation steps with high sensitivity (<0.1 CFU μL[-1]) and speediness (<1 h). This approach may prove easy for the accurate and sensitive diagnosis of the ARGs or ARB in the field, thus surveilling and controlling the microbial water quality flexibly and efficiently.}, }
@article {pmid36925288, year = {2023}, author = {Niu, C and Liu, J and Xing, X and Zhang, C}, title = {CRISPR-Cas12a-assisted elimination of the non-specific signal from non-specific amplification in the Exponential Amplification Reaction.}, journal = {Analytica chimica acta}, volume = {1251}, number = {}, pages = {340998}, doi = {10.1016/j.aca.2023.340998}, pmid = {36925288}, issn = {1873-4324}, mesh = {*CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods ; }, abstract = {Non-specific amplification is a major problem in nucleic acid amplification resulting in false-positive results, especially for exponential amplification reactions (EXPAR). Although efforts were made to suppress the influence of non-specific amplification, such as chemical blocking of the template's 3'-ends and sequence-independent weakening of template-template interactions, it is still a common problem in many conventional EXPAR reactions. In this study, we propose a novel strategy to eliminate the non-specific signal from non-specific amplification by integrating the CRISPR-Cas12a system into two-templates EXPAR. An EXPAR-Cas12a strategy named EXPCas was developed, where the Cas12a system acted as a filter to filter out non-specific amplificons in EXPAR, suppressing and eliminating the influence of non-specific amplification. As a result, the signal-to-background ratio was improved from 1.3 to 15.4 using this method. With microRNA-21 (miRNA-21) as a target, the detection can be finished in 40 min with a LOD of 103 fM and no non-specific amplification was observed.}, }
@article {pmid36923939, year = {2023}, author = {Yin, Z and Su, R and Ge, L and Wang, X and Yang, J and Huang, G and Li, C and Liu, Y and Zhang, K and Deng, L and Fei, J}, title = {Single-cell resolution reveals RalA GTPase expanding hematopoietic stem cells and facilitating of BCR-ABL1-driven leukemogenesis in a CRISPR/Cas9 gene editing mouse model.}, journal = {International journal of biological sciences}, volume = {19}, number = {4}, pages = {1211-1227}, pmid = {36923939}, issn = {1449-2288}, mesh = {Humans ; Mice ; Animals ; *CRISPR-Cas Systems ; GTP Phosphohydrolases/metabolism ; Gene Editing ; Hematopoietic Stem Cells/metabolism/pathology ; *Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics/drug therapy/pathology ; Carcinogenesis/genetics ; Neoplastic Stem Cells/metabolism ; ral GTP-Binding Proteins/genetics/metabolism ; }, abstract = {BCR-ABL oncogene-mediated Philadelphia chromosome-positive (Ph+) chronic myeloid leukemia (CML) is suggested to originate from leukemic stem cells (LSCs); however, factors regulating self-renewal of LSC and normal hematopoietic stem cells (HSCs) are largely unclear. Here, we show that RalA, a small GTPase in the Ras downstream signaling pathway, has a critical effect on regulating the self-renewal of LSCs and HSCs. A RalA knock-in mouse model (RalA[Rosa26-Tg/+]) was initially constructed on the basis of the Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 (CRISPR/Cas9) assay to analyze normal hematopoietic differentiation frequency using single-cell resolution and flow cytometry. RalA overexpression promoted cell cycle progression and increased the frequency of granulocyte-monocyte progenitors (GMPs), HSCs and multipotent progenitors (MPPs). The uniform manifold approximation and projection (UMAP) plot revealed heterogeneities in HSCs and progenitor cells (HSPCs) and identified the subclusters of HSCs and GMPs with a distinct molecular signature. RalA also promoted BCR-ABL-induced leukemogenesis and self-renewal of primary LSCs and shortened the survival of leukemic mice. RalA knockdown prolonged survival and promoted sensitivity to imatinib in a patient-derived tumor xenograft model. Immunoprecipitation plus single-cell RNA sequencing of the GMP population confirmed that RalA induced this effect by interacting with RAC1. RAC1 inhibition by azathioprine effectively reduced the self-renewal, colony formation ability of LSCs and prolonged the survival in BCR-ABL1-driven RalA overexpression CML mice. Collectively, RalA was detected to be a vital factor that regulates the abilities of HSCs and LSCs, thus facilitating BCR-ABL-triggered leukemia in mice. RalA inhibition serves as the therapeutic approach to eradicate LSCs in CML.}, }
@article {pmid36922689, year = {2023}, author = {Fudge, JB}, title = {Cardiac defect corrected in vivo with CRISPR.}, journal = {Nature biotechnology}, volume = {41}, number = {3}, pages = {323}, doi = {10.1038/s41587-023-01721-y}, pmid = {36922689}, issn = {1546-1696}, mesh = {*Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *CRISPR-Cas Systems/genetics ; Gene Editing ; }, }
@article {pmid36842376, year = {2023}, author = {Dyke, E and Bijnagte-Schoenmaker, C and Wu, KM and Oudakker, A and Roepman, R and Nadif Kasri, N}, title = {Generation of induced pluripotent stem cell line carrying frameshift variants in NPHP1 (UCSFi001-A-68) using CRISPR/Cas9.}, journal = {Stem cell research}, volume = {68}, number = {}, pages = {103053}, doi = {10.1016/j.scr.2023.103053}, pmid = {36842376}, issn = {1876-7753}, mesh = {*Induced Pluripotent Stem Cells/metabolism ; CRISPR-Cas Systems/genetics ; Frameshift Mutation ; Adaptor Proteins, Signal Transducing/genetics/metabolism ; }, abstract = {NPHP1 (Nephrocystin 1) is a protein that localizes to the transition zone of the cilium, a small organelle that projects from the plasma membrane of most cells and allows for integration and coordination of signalling pathways during development and homeostasis. Loss of NPHP1 function due to biallelic NPHP1 gene mutations can lead to the development of ciliopathies - a heterogeneous spectra of disorders characterized by ciliary dysfunction. Here we report the generation of an NPHP1-null hiPSC line (UCSFi001-A-68) via CRISPR/Cas9-mediated non-homologous end joining in the UCSFi001-A background, for study of the role that this protein plays in different tissues.}, }
@article {pmid36825354, year = {2023}, author = {Zhu, Y and Champer, J}, title = {Simulations Reveal High Efficiency and Confinement of a Population Suppression CRISPR Toxin-Antidote Gene Drive.}, journal = {ACS synthetic biology}, volume = {12}, number = {3}, pages = {809-819}, doi = {10.1021/acssynbio.2c00611}, pmid = {36825354}, issn = {2161-5063}, mesh = {Humans ; *Antidotes ; *Gene Drive Technology ; CRISPR-Cas Systems/genetics ; }, abstract = {Though engineered gene drives hold great promise for spreading through and suppressing populations of disease vectors or invasive species, complications such as resistance alleles and spatial population structure can prevent their success. Additionally, most forms of suppression drives, such as homing drives or driving Y chromosomes, will generally spread uncontrollably between populations with even small levels of migration. The previously proposed CRISPR-based toxin-antidote system called toxin-antidote dominant embryo (TADE) suppression drive could potentially address the issues of confinement and resistance. However, it is a relatively weak form of drive compared to homing drives, which might make it particularly vulnerable to spatial population structure. In this study, we investigate TADE suppression drive using individual-based simulations in a continuous spatial landscape. We find that the drive is actually more confined than in simple models without space, even in its most efficient form with low cleavage rate in embryos from maternally deposited Cas9. Furthermore, the drive performed well in continuous space scenarios if the initial release requirements were met, suppressing the population in a timely manner without being severely affected by chasing, a phenomenon in which wild-type individuals avoid the drive by recolonizing empty areas. At higher embryo cut rates, the drive loses its ability to spread, but a single, widespread release can often still induce rapid population collapse. Thus, if TADE suppression gene drives can be successfully constructed, they may play an important role in control of disease vectors and invasive species when stringent confinement to target populations is desired.}, }
@article {pmid36807980, year = {2023}, author = {Du, L and Zhang, D and Luo, Z and Lin, Z}, title = {Molecular basis of stepwise cyclic tetra-adenylate cleavage by the type III CRISPR ring nuclease Crn1/Sso2081.}, journal = {Nucleic acids research}, volume = {51}, number = {5}, pages = {2485-2495}, pmid = {36807980}, issn = {1362-4962}, mesh = {*Clustered Regularly Interspaced Short Palindromic Repeats ; Second Messenger Systems ; Signal Transduction ; Endonucleases/metabolism ; Ions/metabolism ; CRISPR-Cas Systems ; *CRISPR-Associated Proteins/metabolism ; }, abstract = {The cyclic oligoadenylates (cOAs) act as second messengers of the type III CRISPR immunity system through activating the auxiliary nucleases for indiscriminate RNA degradation. The cOA-degrading nucleases (ring nucleases) provide an 'off-switch' regulation of the signaling, thereby preventing cell dormancy or cell death. Here, we describe the crystal structures of the founding member of CRISPR-associated ring nuclease 1 (Crn1) Sso2081 from Saccharolobus solfataricus, alone, bound to phosphate ions or cA4 in both pre-cleavage and cleavage intermediate states. These structures together with biochemical characterizations establish the molecular basis of cA4 recognition and catalysis by Sso2081. The conformational changes in the C-terminal helical insert upon the binding of phosphate ions or cA4 reveal a gate-locking mechanism for ligand binding. The critical residues and motifs identified in this study provide a new insight to distinguish between cOA-degrading and -nondegrading CARF domain-containing proteins.}, }
@article {pmid36805322, year = {2023}, author = {Ham, O and Kim, S and Lee, Y and Lee, MO}, title = {Generation of telomeric repeat binding factor 1 (TRF1)-knockout human embryonic stem cell lines, KRIBBe010-A-95, KRIBBe010-A-96, and KRIBBe010-A-97, using CRISPR/Cas9 technology.}, journal = {Stem cell research}, volume = {68}, number = {}, pages = {103045}, doi = {10.1016/j.scr.2023.103045}, pmid = {36805322}, issn = {1876-7753}, mesh = {Humans ; *Telomere/genetics/metabolism ; CRISPR-Cas Systems/genetics ; *Human Embryonic Stem Cells/metabolism ; Telomeric Repeat Binding Protein 1/genetics/metabolism ; Cell Line ; }, abstract = {Telomeric repeat binding factor 1 (TRF1) plays an essential role in maintaining telomere length. Here, we established TRF1-knockout human pluripotent stem cells (hPSCs; hTRF1-KO) using the CRISPR/Cas9 technology. The hTRF1-KO cell lines expressed pluripotency markers and demonstrated a normal karyotype (46, XX) and DNA profile. In addition, hTRF1-KOcells spontaneously differentiated into all three germ layers in vitro. Thus, these cell lines could be useful models in various research fields.}, }
@article {pmid36802585, year = {2023}, author = {Huan, YW and Torraca, V and Brown, R and Fa-Arun, J and Miles, SL and Oyarzún, DA and Mostowy, S and Wang, B}, title = {P1 Bacteriophage-Enabled Delivery of CRISPR-Cas9 Antimicrobial Activity Against Shigella flexneri.}, journal = {ACS synthetic biology}, volume = {12}, number = {3}, pages = {709-721}, doi = {10.1021/acssynbio.2c00465}, pmid = {36802585}, issn = {2161-5063}, support = {206444/Z/17/Z/WT_/Wellcome Trust/United Kingdom ; }, mesh = {*CRISPR-Cas Systems/genetics ; *Anti-Infective Agents ; Gene Editing ; Bacteriophage P1/genetics ; Zebrafish/genetics ; Shigella flexneri/genetics ; Animals ; }, abstract = {The discovery of clustered, regularly interspaced, short palindromic repeats (CRISPR) and the Cas9 RNA-guided nuclease provides unprecedented opportunities to selectively kill specific populations or species of bacteria. However, the use of CRISPR-Cas9 to clear bacterial infections in vivo is hampered by the inefficient delivery of cas9 genetic constructs into bacterial cells. Here, we use a broad-host-range P1-derived phagemid to deliver the CRISPR-Cas9 chromosomal-targeting system into Escherichia coli and the dysentery-causing Shigella flexneri to achieve DNA sequence-specific killing of targeted bacterial cells. We show that genetic modification of the helper P1 phage DNA packaging site (pac) significantly enhances the purity of packaged phagemid and improves the Cas9-mediated killing of S. flexneri cells. We further demonstrate that P1 phage particles can deliver chromosomal-targeting cas9 phagemids into S. flexneri in vivo using a zebrafish larvae infection model, where they significantly reduce the bacterial load and promote host survival. Our study highlights the potential of combining P1 bacteriophage-based delivery with the CRISPR chromosomal-targeting system to achieve DNA sequence-specific cell lethality and efficient clearance of bacterial infection.}, }
@article {pmid36801567, year = {2023}, author = {Stępniewski, J and Jeż, M and Dulak, J}, title = {Generation of miR-15a/16-1 cluster-deficient human induced pluripotent stem cell line (DMBi001-A-2) using CRISPR/Cas9 gene editing.}, journal = {Stem cell research}, volume = {68}, number = {}, pages = {103046}, doi = {10.1016/j.scr.2023.103046}, pmid = {36801567}, issn = {1876-7753}, mesh = {Humans ; Animals ; Mice ; Gene Editing ; *Induced Pluripotent Stem Cells/metabolism ; CRISPR-Cas Systems/genetics ; *MicroRNAs/genetics/metabolism ; Cardiomegaly ; }, abstract = {miR-15a/16-1 cluster, composed of MIR15A and MIR16-1 genes located in close proximity on chromosome 13 was described to regulate post-natal cell cycle withdrawal of cardiomyocytes in mice. In humans, on the other hand, the level of miR-15a-5p and miR-16-p was negatively associated with the severity of cardiac hypertrophy. Therefore, to better understand the role of these microRNAs in human cardiomyocytes in regard to their proliferative potential and hypertrophic growth, we generated hiPSC line with complete deletion of miR-15a/16-1 cluster using CRISPR/Cas9 gene editing. Obtained cells demonstrate expression of pluripotency markers, differentiation capacity into all three germ layers and normal karyotype.}, }
@article {pmid36787424, year = {2023}, author = {Jeong, SH and Kim, HJ and Lee, SJ}, title = {New Target Gene Screening Using Shortened and Random sgRNA Libraries in Microbial CRISPR Interference.}, journal = {ACS synthetic biology}, volume = {12}, number = {3}, pages = {800-808}, doi = {10.1021/acssynbio.2c00595}, pmid = {36787424}, issn = {2161-5063}, mesh = {*CRISPR-Cas Systems/genetics ; *Escherichia coli/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Xylose ; Nucleotides ; }, abstract = {CRISPR interference (CRISPRi) screening has been used for identification of target genes related to specific phenotypes using single-molecular guide RNA (sgRNA) libraries. In CRISPRi screening, the sizes of random sgRNA libraries contained with the original target recognition sequences are large (∼10[12]). Here, we demonstrated that the length of the target recognition sequence (TRS) can be shortened in sgRNAs from the original 20 nucleotides (N20) to 9 nucleotides (N9) that is still sufficient for dCas9 to repress target genes in the xylose operon of Escherichia coli, regardless of binding to a promoter or open reading frame region. Based on the results, we constructed random sgRNA plasmid libraries with 5'-shortened TRS lengths, and identified xylose metabolic target genes by Sanger sequencing of sgRNA plasmids purified from Xyl[-] phenotypic cells. Next, the random sgRNA libraries were harnessed to screen for target genes to enhance violacein pigment production in synthetic E. coli cells. Seventeen target genes were selected by analyzing the redundancy of the TRS in sgRNA plasmids in dark purple colonies. Among them, seven genes (tyrR, pykF, cra, ptsG, pykA, sdaA, and tnaA) have been known to increase the intracellular l-tryptophan pool, the precursor of a violacein. Seventeen cells with a single deletion of each target gene exhibited a significant increase in violacein production. These results indicate that using shortened random TRS libraries for CRISPRi can be simple and cost-effective for phenotype-based target gene screening.}, }
@article {pmid36669880, year = {2023}, author = {Nguyen, TTT and Tanaka, Y and Sanada, M and Hosaka, M and Tamai, M and Kagami, K and Komatsu, C and Somazu, S and Harama, D and Kasai, S and Watanabe, A and Akahane, K and Goi, K and Inukai, T}, title = {CRISPR/Cas9-Mediated Induction of Relapse-Specific NT5C2 and PRPS1 Mutations Confers Thiopurine Resistance as a Relapsed Lymphoid Leukemia Model.}, journal = {Molecular pharmacology}, volume = {103}, number = {4}, pages = {199-210}, doi = {10.1124/molpharm.122.000546}, pmid = {36669880}, issn = {1521-0111}, mesh = {Humans ; *Mercaptopurine/pharmacology ; CRISPR-Cas Systems/genetics ; Mutation ; *Precursor Cell Lymphoblastic Leukemia-Lymphoma/drug therapy/genetics ; Recurrence ; 5'-Nucleotidase/genetics/metabolism/therapeutic use ; Ribose-Phosphate Pyrophosphokinase/genetics/metabolism ; }, abstract = {6-Mercaptopurine (6-MP) is a key component in maintenance therapy for childhood acute lymphoblastic leukemia (ALL). Recent next-generation sequencing analysis of childhood ALL clarified the emergence of the relapse-specific mutations of the NT5C2 and PRPS1 genes, which are involved in thiopurine metabolism. In this scenario, minor clones of leukemia cells could acquire the 6-MP-resistant phenotype as a result of the NT5C2 or PRPS1 mutation during chemotherapy (including 6-MP treatment) and confer disease relapse after selective expansion. Thus, to establish new therapeutic modalities overcoming 6-MP resistance in relapsed ALL, human leukemia models with NT5C2 and PRPS1 mutations in the intrinsic genes are urgently required. Here, mimicking the initiation process of the above clinical course, we sought to induce two relapse-specific hotspot mutations (R39Q mutation of the NT5C2 gene and S103N mutation of the PRPS1 gene) into a human lymphoid leukemia cell line by homologous recombination (HR) using the CRISPR/Cas9 system. After 6-MP selection of the cells transfected with Cas9 combined with single-guide RNA and donor DNA templates specific for either of those two mutations, we obtained the sublines with the intended NT5C2-R39Q and PRPS1-S103N mutation as a result of HR. Moreover, diverse in-frame small insertion/deletions were also confirmed in the 6-MP-resistant sublines at the target sites of the NT5C2 and PRPS1 genes as a result of nonhomologous end joining. These sublines are useful for molecular pharmacological evaluation of the NT5C2 and PRPS1 gene mutations in the 6-MP sensitivity and development of therapy overcoming the thiopurine resistance of leukemia cells. SIGNIFICANCE STATEMENT: Mimicking the initiation process of relapse-specific mutations of the NT5C2 and PRPS1 genes in childhood acute lymphoblastic leukemia treated with 6-mercaptopurine (6-MP), this study sought to introduce NT5C2-R39Q and PRPS1-S103N mutations into a human lymphoid leukemia cell line by homologous recombination using the CRISPR/Cas9 system. In the resultant 6-MP-resistant sublines, the intended mutations and diverse in-frame small insertions/deletions were confirmed, indicating that the obtained sublines are useful for molecular pharmacological evaluation of the NT5C2 and PRPS1 gene mutations.}, }
@article {pmid36620901, year = {2023}, author = {Lackner, M and Helmbrecht, N and Pääbo, S and Riesenberg, S}, title = {Detection of unintended on-target effects in CRISPR genome editing by DNA donors carrying diagnostic substitutions.}, journal = {Nucleic acids research}, volume = {51}, number = {5}, pages = {e26}, pmid = {36620901}, issn = {1362-4962}, mesh = {Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; DNA/genetics ; Nucleotides ; }, abstract = {CRISPR nucleases can introduce double-stranded DNA breaks in genomes at positions specified by guide RNAs. When repaired by the cell, this may result in the introduction of insertions and deletions or nucleotide substitutions provided by exogenous DNA donors. However, cellular repair can also result in unintended on-target effects, primarily larger deletions and loss of heterozygosity due to gene conversion. Here we present a strategy that allows easy and reliable detection of unintended on-target effects as well as the generation of control cells that carry wild-type alleles but have demonstratively undergone genome editing at the target site. Our 'sequence-ascertained favorable editing' (SAFE) donor approach relies on the use of DNA donor mixtures containing the desired nucleotide substitutions or the wild-type alleles together with combinations of additional 'diagnostic' substitutions unlikely to have any effects. Sequencing of the target sites then results in that two different sequences are seen when both chromosomes are edited with 'SAFE' donors containing different sets of substitutions, while a single sequence indicates unintended effects such as deletions or gene conversion. We analyzed more than 850 human embryonic stem cell clones edited with 'SAFE' donors and detect all copy number changes and almost all clones with gene conversion.}, }
@article {pmid36259148, year = {2023}, author = {Zulhussnain, M and Zahoor, MK and Ranian, K and Ahmad, A and Jabeen, F}, title = {CRISPR Cas9 mediated knockout of sex determination pathway genes in Aedes aegypti.}, journal = {Bulletin of entomological research}, volume = {113}, number = {2}, pages = {243-252}, doi = {10.1017/S0007485322000505}, pmid = {36259148}, issn = {1475-2670}, mesh = {Male ; Female ; Animals ; *Aedes/genetics ; CRISPR-Cas Systems ; Mosquito Vectors/genetics ; Protein Isoforms/genetics ; *Dengue ; }, abstract = {The vector role of Aedes aegypti for viral diseases including dengue and dengue hemorrhagic fever makes it imperative for its proper control. Despite various adopted control strategies, genetic control measures have been recently focused against this vector. CRISPR Cas9 system is a recent and most efficient gene editing tool to target the sex determination pathway genes in Ae. aegypti. In the present study, CRISPR Cas9 system was used to knockout Ae. aegypti doublesex (Aaedsx) and Ae. aegypti sexlethal (AaeSxl) genes in Ae. aegypti embryos. The injection mixes with Cas9 protein (333 ng ul[-1]) and gRNAs (each at 100 ng ul[-1]) were injected into eggs. Injected eggs were allowed to hatch at 26 ± 1°C, 60 ± 10% RH. The survival and mortality rate was recorded in knockout Aaedsx and AaeSxl. The results revealed that knockout produced low survival and high mortality. A significant percentage of eggs (38.33%) did not hatch as compared to control groups (P value 0.00). Highest larval mortality (11.66%) was found in the knockout of Aaedsx female isoform, whereas, the emergence of only male adults also showed that the knockout of Aaedsx (female isoform) does not produce male lethality. The survival (3.33%) of knockout for AaeSxl eggs to the normal adults suggested further study to investigate AaeSxl as an efficient upstream of Aaedsx to target for sex transformation in Ae. aegypti mosquitoes.}, }
@article {pmid36229610, year = {2023}, author = {Lee, S and Lee, H and Baek, G and Kim, JS}, title = {Precision mitochondrial DNA editing with high-fidelity DddA-derived base editors.}, journal = {Nature biotechnology}, volume = {41}, number = {3}, pages = {378-386}, pmid = {36229610}, issn = {1546-1696}, mesh = {Humans ; *DNA, Mitochondrial/genetics ; *Gene Editing/methods ; Mitochondria/metabolism ; Mutation ; Cytosine/metabolism ; CRISPR-Cas Systems ; }, abstract = {Bacterial toxin DddA-derived cytosine base editors (DdCBEs)-composed of split DddAtox (a cytosine deaminase specific to double-stranded DNA), custom-designed TALE (transcription activator-like effector) DNA-binding proteins, and a uracil glycosylase inhibitor-enable mitochondrial DNA (mtDNA) editing in human cells, which may pave the way for therapeutic correction of pathogenic mtDNA mutations in patients. The utility of DdCBEs has been limited by off-target activity, which is probably caused by spontaneous assembly of the split DddAtox deaminase enzyme, independent of DNA-binding interactions. We engineered high-fidelity DddA-derived cytosine base editors (HiFi-DdCBEs) with minimal off-target activity by substituting alanine for amino acid residues at the interface between the split DddAtox halves. The resulting domains cannot form a functional deaminase without binding of their linked TALE proteins at adjacent sites on DNA. Whole mitochondrial genome sequencing shows that, unlike conventional DdCBEs, which induce hundreds of unwanted off-target C-to-T conversions in human mtDNA, HiFi-DdCBEs are highly efficient and precise, avoiding collateral off-target mutations, and as such, they will probably be desirable for therapeutic applications.}, }
@article {pmid36203014, year = {2023}, author = {Christie, KA and Guo, JA and Silverstein, RA and Doll, RM and Mabuchi, M and Stutzman, HE and Lin, J and Ma, L and Walton, RT and Pinello, L and Robb, GB and Kleinstiver, BP}, title = {Precise DNA cleavage using CRISPR-SpRYgests.}, journal = {Nature biotechnology}, volume = {41}, number = {3}, pages = {409-416}, pmid = {36203014}, issn = {1546-1696}, support = {R35 HG010717/HG/NHGRI NIH HHS/United States ; P01 HL142494/HL/NHLBI NIH HHS/United States ; }, mesh = {*CRISPR-Cas Systems/genetics ; *DNA Cleavage ; RNA, Guide, Kinetoplastida/genetics ; DNA/genetics ; Gene Editing/methods ; }, abstract = {Methods for in vitro DNA cleavage and molecular cloning remain unable to precisely cleave DNA directly adjacent to bases of interest. Restriction enzymes (REs) must bind specific motifs, whereas wild-type CRISPR-Cas9 or CRISPR-Cas12 nucleases require protospacer adjacent motifs (PAMs). Here we explore the utility of our previously reported near-PAMless SpCas9 variant, named SpRY, to serve as a universal DNA cleavage tool for various cloning applications. By performing SpRY DNA digests (SpRYgests) using more than 130 guide RNAs (gRNAs) sampling a wide diversity of PAMs, we discovered that SpRY is PAMless in vitro and can cleave DNA at practically any sequence, including sites refractory to cleavage with wild-type SpCas9. We illustrate the versatility and effectiveness of SpRYgests to improve the precision of several cloning workflows, including those not possible with REs or canonical CRISPR nucleases. We also optimize a rapid and simple one-pot gRNA synthesis protocol to streamline SpRYgest implementation. Together, SpRYgests can improve various DNA engineering applications that benefit from precise DNA breaks.}, }
@article {pmid36163548, year = {2023}, author = {Grünewald, J and Miller, BR and Szalay, RN and Cabeceiras, PK and Woodilla, CJ and Holtz, EJB and Petri, K and Joung, JK}, title = {Engineered CRISPR prime editors with compact, untethered reverse transcriptases.}, journal = {Nature biotechnology}, volume = {41}, number = {3}, pages = {337-343}, pmid = {36163548}, issn = {1546-1696}, support = {R35 GM118158/GM/NIGMS NIH HHS/United States ; RM1 HG009490/HG/NHGRI NIH HHS/United States ; }, mesh = {Animals ; Mice ; Humans ; *RNA-Directed DNA Polymerase/genetics ; *Moloney murine leukemia virus/genetics ; Gene Editing ; CRISPR-Cas Systems/genetics ; }, abstract = {The CRISPR prime editor PE2 consists of a Streptococcus pyogenes Cas9 nickase (nSpCas9) fused at its C-terminus to a Moloney murine leukemia virus reverse transcriptase (MMLV-RT). Here we show that separated nSpCas9 and MMLV-RT proteins function as efficiently as intact PE2 in human cells. We use this Split-PE system to rapidly identify and engineer more compact prime editor architectures that also broaden the types of RTs used for prime editing.}, }
@article {pmid36925473, year = {2023}, author = {Zakrzewska, M and Burmistrz, M}, title = {Mechanisms regulating the CRISPR-Cas systems.}, journal = {Frontiers in microbiology}, volume = {14}, number = {}, pages = {1060337}, pmid = {36925473}, issn = {1664-302X}, abstract = {The CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats- CRISPR associated proteins) is a prokaryotic system that enables sequence specific recognition and cleavage of nucleic acids. This is possible due to cooperation between CRISPR array which contains short fragments of DNA called spacers that are complimentary to the targeted nucleic acid and Cas proteins, which take part in processes of: acquisition of new spacers, processing them into their functional form as well as recognition and cleavage of targeted nucleic acids. The primary role of CRISPR-Cas systems is to provide their host with an adaptive and hereditary immunity against exogenous nucleic acids. This system is present in many variants in both Bacteria and Archea. Due to its modular structure, and programmability CRISPR-Cas system become attractive tool for modern molecular biology. Since their discovery and implementation, the CRISPR-Cas systems revolutionized areas of gene editing and regulation of gene expression. Although our knowledge on how CRISPR-Cas systems work has increased rapidly in recent years, there is still little information on how these systems are controlled and how they interact with other cellular mechanisms. Such regulation can be the result of both auto-regulatory mechanisms as well as exogenous proteins of phage origin. Better understanding of these interaction networks would be beneficial for optimization of current and development of new CRISPR-Cas-based tools. In this review we summarize current knowledge on the various molecular mechanisms that affect activity of CRISPR-Cas systems.}, }
@article {pmid36925471, year = {2023}, author = {Cerbino, GN and Traglia, GM and Ayala Nuñez, T and Parmeciano Di Noto, G and Ramírez, MS and Centrón, D and Iriarte, A and Quiroga, C}, title = {Comparative genome analysis of the genus Shewanella unravels the association of key genetic traits with known and potential pathogenic lineages.}, journal = {Frontiers in microbiology}, volume = {14}, number = {}, pages = {1124225}, pmid = {36925471}, issn = {1664-302X}, abstract = {Shewanella spp. are Gram-negative rods widely disseminated in aquatic niches that can also be found in human-associated environments. In recent years, reports of infections caused by these bacteria have increased significantly. Mobilome and resistome analysis of a few species showed that they are versatile; however, comprehensive comparative studies in the genus are lacking. Here, we analyzed the genetic traits of 144 genomes from Shewanella spp. isolates focusing on the mobilome, resistome, and virulome to establish their evolutionary relationship and detect unique features based on their genome content and habitat. Shewanella spp. showed a great diversity of mobile genetic elements (MGEs), most of them associated with monophyletic lineages of clinical isolates. Furthermore, 79/144 genomes encoded at least one antimicrobial resistant gene with their highest occurrence in clinical-related lineages. CRISPR-Cas systems, which confer immunity against MGEs, were found in 41 genomes being I-E and I-F the more frequent ones. Virulome analysis showed that all Shewanella spp. encoded different virulence genes (motility, quorum sensing, biofilm, adherence, etc.) that may confer adaptive advantages for survival against hosts. Our data revealed that key accessory genes are frequently found in two major clinical-related groups, which encompass the opportunistic pathogens Shewanella algae and Shewanella xiamenensis together with several other species. This work highlights the evolutionary nature of Shewanella spp. genomes, capable of acquiring different key genetic traits that contribute to their adaptation to different niches and facilitate the emergence of more resistant and virulent isolates that impact directly on human and animal health.}, }
@article {pmid36905119, year = {2023}, author = {Pedrazzoli, E and Bianchi, A and Umbach, A and Amistadi, S and Brusson, M and Frati, G and Ciciani, M and Badowska, KA and Arosio, D and Miccio, A and Cereseto, A and Casini, A}, title = {An optimized SpCas9 high-fidelity variant for direct protein delivery.}, journal = {Molecular therapy : the journal of the American Society of Gene Therapy}, volume = {}, number = {}, pages = {}, doi = {10.1016/j.ymthe.2023.03.007}, pmid = {36905119}, issn = {1525-0024}, abstract = {Electroporation of the Cas9 ribonucleoprotein (RNP) complex offers the advantage of preventing off-target cleavages and potential immune responses produced by long-term expression of the nuclease. Nevertheless, the majority of engineered high-fidelity Streptococcus pyogenes Cas9 (SpCas9) variants are less active than the wild-type enzyme and are not compatible with RNP delivery. Building on our previous studies on evoCas9, we developed a high-fidelity SpCas9 variant suitable for RNP delivery. The editing efficacy and precision of the recombinant high-fidelity Cas9 (rCas9HF), characterized by the K526D substitution, was compared with the R691A mutant (HiFi Cas9), which is currently the only available high-fidelity Cas9 that can be used as an RNP. The comparative analysis was extended to gene substitution experiments where the two high fidelities were used in combination with a DNA donor template, generating different ratios of non-homologous end joining (NHEJ) versus homology-directed repair (HDR) for precise editing. The analyses revealed a heterogeneous efficacy and precision indicating different targeting capabilities between the two variants throughout the genome. The development of rCas9HF, characterized by an editing profile diverse from the currently used HiFi Cas9 in RNP electroporation, increases the genome editing solutions for the highest precision and efficient applications.}, }
@article {pmid36925314, year = {2023}, author = {Wang, X and Mu, X and Li, J and Liu, G and Zhao, S and Tian, J}, title = {A novel nanoparticle surface-constrained CRISPR-Cas12a 3D DNA walker-like nanomachines for sensitive and stable miRNAs detection.}, journal = {Analytica chimica acta}, volume = {1251}, number = {}, pages = {340950}, doi = {10.1016/j.aca.2023.340950}, pmid = {36925314}, issn = {1873-4324}, abstract = {The CRISPR-Cas system has broad prospects as a new type of nucleic acid signal amplification technology based on the trans-cleavage activity of Cas12a to single-stranded DNA, but the trans-cleavage reaction efficiency is relatively low in solution. In order to overcome this negative factor, a new 3D DNA nanomachine whose CRISPR-Cas12a is limited to the surface of nanoparticles is used for sensitive and stable detection of miRNA. By loading Cas12a activator onto spherical nucleic acid (SNA), the CRISPR-Cas12a activator system on the surface of Au nanoparticles (AuNPs) acts as a walker to carry out continuous recognition-walking-cutting reaction on the surface of AuNPs, which enhances the trans-cleavage activity of Cas12a to SNAs. Benefiting from the confinement effect of spherical nucleic acids surface, a 3D DNA nanomachine has been developed for the detection of miRNA-21, which has achieved high sensitivity and accuracy, and the detection limit is able to reach 8.0 pM. This new 3D DNA walker-like nanomachine provided another insight for future bioanalysis and early clinical diagnoses of disease and liquid biopsy.}, }
@article {pmid36922985, year = {2023}, author = {Mikkelsen, NS and Hernandez, SS and Jensen, TI and Schneller, JL and Bak, RO}, title = {Enrichment of transgene integrations by transient CRISPR activation of a silent reporter gene.}, journal = {Molecular therapy. Methods &amp; clinical development}, volume = {29}, number = {}, pages = {1-16}, pmid = {36922985}, issn = {2329-0501}, abstract = {CRISPR-Cas-mediated site-specific integration of transgenes by homology-directed repair (HDR) is challenging, especially in primary cells, where inferior editing efficiency may impede the development of gene- and cellular therapies. Various strategies for enrichment of cells with transgene integrations have been developed, but most strategies either generate unwanted genomic scars or rely on permanent integration and expression of a reporter gene used for selection. However, stable expression of a reporter gene may perturb cell homeostasis and function. Here we develop a broadly applicable and versatile enrichment strategy by harnessing the capability of CRISPR activation (CRISPRa) to transiently induce expression of a therapeutically relevant reporter gene used for immunomagnetic enrichment. This strategy is readily adaptable to primary human T cells and CD34+ hematopoietic stem and progenitor cells (HSPCs), where enrichment of 1.8- to 3.3-fold and 3.2- to 3.6-fold was achieved, respectively. Furthermore, chimeric antigen receptor (CAR) T cells were enriched 2.5-fold and demonstrated improved cytotoxicity over non-enriched CAR T cells. Analysis of HDR integrations showed a proportion of cells harboring deletions of the transgene cassette arising either from impartial HDR or truncated adeno-associated virus (AAV) vector genomes. Nonetheless, this novel enrichment strategy expands the possibility to enrich for transgene integrations in research settings and in gene and cellular therapies.}, }
@article {pmid36917574, year = {2023}, author = {Jianwei, L and Jobichen, C and Machida, S and Meng, S and Read, RJ and Hongying, C and Jian, S and Yuan, YA and Sivaraman, J}, title = {Structures of apo Cas12a and its complex with crRNA and DNA reveal the dynamics of ternary complex formation and target DNA cleavage.}, journal = {PLoS biology}, volume = {21}, number = {3}, pages = {e3002023}, pmid = {36917574}, issn = {1545-7885}, mesh = {*CRISPR-Cas Systems ; *RNA, Guide, CRISPR-Cas Systems ; DNA Cleavage ; RNA/chemistry ; DNA/chemistry ; Bacterial Proteins/metabolism ; }, abstract = {Cas12a is a programmable nuclease for adaptive immunity against invading nucleic acids in CRISPR-Cas systems. Here, we report the crystal structures of apo Cas12a from Lachnospiraceae bacterium MA2020 (Lb2) and the Lb2Cas12a+crRNA complex, as well as the cryo-EM structure and functional studies of the Lb2Cas12a+crRNA+DNA complex. We demonstrate that apo Lb2Cas12a assumes a unique, elongated conformation, whereas the Lb2Cas12a+crRNA binary complex exhibits a compact conformation that subsequently rearranges to a semi-open conformation in the Lb2Cas12a+crRNA+DNA ternary complex. Notably, in solution, apo Lb2Cas12a is dynamic and can exist in both elongated and compact forms. Residues from Met493 to Leu523 of the WED domain undergo major conformational changes to facilitate the required structural rearrangements. The REC lobe of Lb2Cas12a rotates 103° concomitant with rearrangement of the hinge region close to the WED and RuvC II domains to position the RNA-DNA duplex near the catalytic site. Our findings provide insight into crRNA recognition and the mechanism of target DNA cleavage.}, }
@article {pmid36917274, year = {2023}, author = {Spasskaya, DS and Davletshin, AI and Bachurin, SS and Tutyaeva, VV and Garbuz, DG and Karpov, DS}, title = {Improving the on-target activity of high-fidelity Cas9 editors by combining rational design and random mutagenesis.}, journal = {Applied microbiology and biotechnology}, volume = {}, number = {}, pages = {}, pmid = {36917274}, issn = {1432-0614}, abstract = {Genomic and post-genomic editors based on CRISPR/Cas systems are widely used in basic research and applied sciences, including human gene therapy. Most genome editing tools are based on the CRISPR/Cas9 type IIA system from Streptococcus pyogenes. Unfortunately, a number of drawbacks have hindered its application in therapeutic approaches, the most serious of which is the relatively high level of off-targets. To overcome this obstacle, various high-fidelity Cas9 variants have been created. However, they show reduced on-target activity compared to wild-type Cas9 possibly due to increased sensitivity to eukaryotic chromatin. Here, we combined a rational approach with random mutagenesis to create a set of new Cas9 variants showing high specificity and increased activity in Saccharomyces cerevisiae yeast. Moreover, a novel mutation in the PAM (protospacer adjacent motif)-interacting Cas9 domain was found, which increases the on-target activity of high-fidelity Cas9 variants while retaining their high specificity. The obtained data suggest that this mutation acts by weakening the eukaryotic chromatin barrier for Cas9 and rearranging the RuvC active center. Improved Cas9 variants should further advance genome and post-genome editing technologies. KEY POINTS: • D147Y and P411T mutations increase the activity of high-fidelity Cas9 variants. • The new L1206P mutation further increases the activity of high-fidelity Cas9 variants. • The L1206P mutation weakens the chromatin barrier for Cas9 editors.}, }
@article {pmid36914349, year = {2023}, author = {Wang, D and Fletcher, GC and Gagic, D and On, SLW and Palmer, JS and Flint, SH}, title = {Comparative genome identification of accessory genes associated with strong biofilm formation in Vibrio parahaemolyticus.}, journal = {Food research international (Ottawa, Ont.)}, volume = {166}, number = {}, pages = {112605}, doi = {10.1016/j.foodres.2023.112605}, pmid = {36914349}, issn = {1873-7145}, mesh = {*Vibrio parahaemolyticus/genetics ; Biofilms ; Genomics ; Operon ; Cellulose ; }, abstract = {Vibrio parahaemolyticus biofilms on the seafood processing plant surfaces are a potential source of seafood contamination and subsequent food poisoning. Strains differ in their ability to form biofilm, but little is known about the genetic characteristics responsible for biofilm development. In this study, pangenome and comparative genome analysis of V. parahaemolyticus strains reveals genetic attributes and gene repertoire that contribute to robust biofilm formation. The study identified 136 accessory genes that were exclusively present in strong biofilm forming strains and these were functionally assigned to the Gene Ontology (GO) pathways of cellulose biosynthesis, rhamnose metabolic and catabolic processes, UDP-glucose processes and O antigen biosynthesis (p < 0.05). Strategies of CRISPR-Cas defence and MSHA pilus-led attachment were implicated via Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation. Higher levels of horizontal gene transfer (HGT) were inferred to confer more putatively novel properties on biofilm-forming V. parahaemolyticus. Furthermore, cellulose biosynthesis, a neglected potential virulence factor, was identified as being acquired from within the order Vibrionales. The cellulose synthase operons in V. parahaemolyticus were examined for their prevalence (22/138, 15.94 %) and were found to consist of the genes bcsG, bcsE, bcsQ, bcsA, bcsB, bcsZ, bcsC. This study provides insights into robust biofilm formation of V. parahaemolyticus at the genomic level and facilitates: identification of key attributes for robust biofilm formation, elucidation of biofilm formation mechanisms and development of potential targets for novel control strategies of persistent V. parahaemolyticus.}, }
@article {pmid36913066, year = {2023}, author = {Adeyinka, OS and Tabassum, B and Koloko, BL and Ogungbe, IV}, title = {Enhancing the quality of staple food crops through CRISPR/Cas-mediated site-directed mutagenesis.}, journal = {Planta}, volume = {257}, number = {4}, pages = {78}, pmid = {36913066}, issn = {1432-2048}, mesh = {*CRISPR-Cas Systems/genetics ; *Plant Breeding ; Gene Editing ; Crops, Agricultural/genetics ; Genome, Plant/genetics ; Mutagenesis, Site-Directed ; Plants, Genetically Modified/genetics ; }, abstract = {The enhancement of CRISPR-Cas gene editing with robust nuclease activity promotes genetic modification of desirable agronomic traits, such as resistance to pathogens, drought tolerance, nutritional value, and yield-related traits in crops. The genetic diversity of food crops has reduced tremendously over the past twelve millennia due to plant domestication. This reduction presents significant challenges for the future especially considering the risks posed by global climate change to food production. While crops with improved phenotypes have been generated through crossbreeding, mutation breeding, and transgenic breeding over the years, improving phenotypic traits through precise genetic diversification has been challenging. The challenges are broadly associated with the randomness of genetic recombination and conventional mutagenesis. This review highlights how emerging gene-editing technologies reduce the burden and time necessary for developing desired traits in plants. Our focus is to provide readers with an overview of the advances in CRISPR-Cas-based genome editing for crop improvement. The use of CRISPR-Cas systems in generating genetic diversity to enhance the quality and nutritional value of staple food crops is discussed. We also outlined recent applications of CRISPR-Cas in developing pest-resistant crops and removing unwanted traits, such as allergenicity from crops. Genome editing tools continue to evolve and present unprecedented opportunities to enhance crop germplasm via precise mutations at the desired loci of the plant genome.}, }
@article {pmid36801336, year = {2023}, author = {Chen, J and Shi, G and Yan, C}, title = {Portable biosensor for on-site detection of kanamycin in water samples based on CRISPR-Cas12a and an off-the-shelf glucometer.}, journal = {The Science of the total environment}, volume = {872}, number = {}, pages = {162279}, doi = {10.1016/j.scitotenv.2023.162279}, pmid = {36801336}, issn = {1879-1026}, mesh = {*Kanamycin ; CRISPR-Cas Systems ; Reproducibility of Results ; beta-Fructofuranosidase ; Anti-Bacterial Agents ; Water ; *Biosensing Techniques ; }, abstract = {On-site and cost-effective monitoring of antibiotic residue in water samples using a ubiquitous device that is readily available to the general public is a big challenge. Herein, we developed a portable biosensor for kanamycin (KAN) detection based on a glucometer and CRISPR-Cas12a. The aptamer-KAN interactions liberate the trigger C strand, which can initiate the hairpin assembly to produce numerous double-stranded DNA. After recognition by CRISPR-Cas12a, Cas12a can cleave the magnetic bead and invertase-modified single-stranded DNA. After magnetic separation, the invertase can convert sucrose into glucose, which can be quantified by a glucometer. The linear range of the glucometer biosensor is from 1 pM to 100 nM and the detection limit is 1 pM. The biosensor also exhibited high selectivity and the nontarget antibiotics had no significant interference with KAN detection. The sensing system is robust and can work in complex samples with excellent accuracy and reliability. The recovery values were in the range of 89-107.2 % for water samples and 86-106.5 % for milk samples. The relative standard deviation (RSD) was below 5 %. With the advantages of simple operation, low cost, and easy accessibility to the public, this portable pocket-sized sensor can realize the on-site detection of antibiotic residue in resource-limited settings.}, }
@article {pmid36518081, year = {2023}, author = {Abhinandan, K and Hickerson, NMN and Lan, X and Samuel, MA}, title = {Disabling of ARC1 through CRISPR-Cas9 leads to a complete breakdown of self-incompatibility responses in Brassica napus.}, journal = {Plant communications}, volume = {4}, number = {2}, pages = {100504}, doi = {10.1016/j.xplc.2022.100504}, pmid = {36518081}, issn = {2590-3462}, mesh = {*Brassica napus/genetics/metabolism ; CRISPR-Cas Systems/genetics ; *Arabidopsis/metabolism ; Plant Proteins/genetics/metabolism ; }, }
@article {pmid36857724, year = {2023}, author = {Ma, R and Jiang, J and Ya, Y and Lin, Y and Zhou, Y and Wu, Y and Tan, X and Huang, K and Du, F and Xu, J}, title = {A carbon dot-based nanoscale covalent organic framework as a new emitter combined with a CRISPR/Cas12a-mediated electrochemiluminescence biosensor for ultrasensitive detection of bisphenol A.}, journal = {The Analyst}, volume = {148}, number = {6}, pages = {1362-1370}, doi = {10.1039/d3an00024a}, pmid = {36857724}, issn = {1364-5528}, mesh = {Carbon ; *Metal-Organic Frameworks ; CRISPR-Cas Systems ; Luminescent Measurements ; *Biosensing Techniques ; Electrochemical Techniques ; Limit of Detection ; }, abstract = {Exploring new highly efficient electrochemiluminescence (ECL) luminophores is a necessary condition for developing ultrasensitive ECL biosensors. Therefore, a luminescent carbon dot-based covalent organic framework (CD-COF) was prepared using aldehyde-based carbon dots (CDs) and 1,3,5-tris (4-aminophenyl) benzene (TPB). Because the CD-COF made the regular arrangement of CDs conducive to improving the ECL response, CD-COF had a higher ECL intensity and efficiency than CDs. What's more, the ECL intensity of the CD-COF/S2O8[2-]/Bu4N[+] system was about 2.98, 7.50, and 28.08 times higher than those of the CD-COF/S2O8[2-], CDs/S2O8[2-] and S2O8[2-] systems, respectively. Considering the remarkable ECL performance, the CD-COF/S2O8[2-]/Bu4N[+] system was employed combined with the CRISPR/Cas12a trans-cutting strategy to construct an "off-on" ECL biosensor for BPA detection. The proposed ECL biosensor exhibited excellent performance with a wide linear range from 1.0 × 10[-14] mol L[-1] to 1.0 × 10[-5] mol L[-1] with a low detection limit of 2.21 fM (S/N = 3) under the optimized conditions. The biosensor demonstrated that CD-COF can be used as an efficient ECL emitter, thus expanding the application field of COFs. In addition, the good stability and specificity of the biosensor enabled the rapid detection of BPA, which will provide valuable insights into promising ultrasensitive ECL biosensors.}, }
@article {pmid36848598, year = {2023}, author = {Puppulin, L and Ishikawa, J and Sumino, A and Marchesi, A and Flechsig, H and Umeda, K and Kodera, N and Nishimasu, H and Shibata, M}, title = {Dynamics of Target DNA Binding and Cleavage by Staphylococcus aureus Cas9 as Revealed by High-Speed Atomic Force Microscopy.}, journal = {ACS nano}, volume = {17}, number = {5}, pages = {4629-4641}, doi = {10.1021/acsnano.2c10709}, pmid = {36848598}, issn = {1936-086X}, mesh = {*CRISPR-Cas Systems ; *Staphylococcus aureus/metabolism ; Microscopy, Atomic Force ; Gene Editing/methods ; DNA/chemistry ; }, abstract = {Programmable DNA binding and cleavage by CRISPR-Cas9 has revolutionized the life sciences. However, the off-target cleavage observed in DNA sequences with some homology to the target still represents a major limitation for a more widespread use of Cas9 in biology and medicine. For this reason, complete understanding of the dynamics of DNA binding, interrogation and cleavage by Cas9 is crucial to improve the efficiency of genome editing. Here, we use high-speed atomic force microscopy (HS-AFM) to investigate Staphylococcus aureus Cas9 (SaCas9) and its dynamics of DNA binding and cleavage. Upon binding to single-guide RNA (sgRNA), SaCas9 forms a close bilobed structure that transiently and flexibly adopts also an open configuration. The SaCas9-mediated DNA cleavage is characterized by release of cleaved DNA and immediate dissociation, confirming that SaCas9 operates as a multiple turnover endonuclease. According to present knowledge, the process of searching for target DNA is mainly governed by three-dimensional diffusion. Independent HS-AFM experiments show a potential long-range attractive interaction between SaCas9-sgRNA and its target DNA. The interaction precedes the formation of the stable ternary complex and is observed exclusively in the vicinity of the protospacer-adjacent motif (PAM), up to distances of several nanometers. The direct visualization of the process by sequential topographic images suggests that SaCas9-sgRNA binds to the target sequence first, while the following binding of the PAM is accompanied by local DNA bending and formation of the stable complex. Collectively, our HS-AFM data reveal a potential and unexpected behavior of SaCas9 during the search for DNA targets.}, }
@article {pmid36641494, year = {2023}, author = {Senthilnathan, R and Ilangovan, I and Kunale, M and Easwaran, N and Ramamoorthy, S and Veeramuthu, A and Kodiveri Muthukaliannan, G}, title = {An update on CRISPR-Cas12 as a versatile tool in genome editing.}, journal = {Molecular biology reports}, volume = {50}, number = {3}, pages = {2865-2881}, pmid = {36641494}, issn = {1573-4978}, mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Genetic Engineering ; Mutagenesis ; Mutation ; }, abstract = {Gene editing techniques, which help in modification of any DNA sequence at ease, have revolutionized the world of Genetic engineering. Although there are other gene-editing techniques, CRISPR has emerged as the chief and most preferred tool due to its simplicity and capacity to execute effective gene editing in a wide range of organisms. Although Cas9 has widely been employed for genetic modification over the years, Cas12 systems have lately emerged as a viable option. This review primarily focuses on assessing Cas12-mediated mutagenesis and elucidating the editing efficacy of both Cpf1 (Cas12a) and C2c1 (Cas12b) systems in microbes, plants, and other species. Also, we reviewed several genetic alterations that have been performed with these Cas12 systems to improve editing efficiency. Furthermore, the experimental benefits and applications of Cas12 systems are highlighted in this study.}, }
@article {pmid36550260, year = {2023}, author = {Han, JY and Seo, J and Choi, Y and Im, W and Ban, JJ and Sung, JJ}, title = {CRISPR-Cas9 mediated genome editing of Huntington's disease neurospheres.}, journal = {Molecular biology reports}, volume = {50}, number = {3}, pages = {2127-2136}, pmid = {36550260}, issn = {1573-4978}, mesh = {Mice ; Animals ; *Brain-Derived Neurotrophic Factor/genetics/metabolism ; *Huntington Disease/metabolism ; Mice, Transgenic ; Gene Editing ; CRISPR-Cas Systems/genetics ; Disease Models, Animal ; }, abstract = {BACKGROUND: Huntington's disease (HD) is a fatal genetic disease caused by polyglutamine aggregation encoded by an expanded CAG repeat in the huntingtin gene (HTT). In this study, we cultured neurospheres derived from R6/2 mice, a representative animal model of HD, as an in vitro model. GuideRNAs were designed to induce large deletion or frameshift indel mutation of CAG expansion. These gRNAs and Cas9 were delivered to the R6/2 neurospheres and disease-related phenotypes were observed.<br><br>METHODS AND RESULTS: Deletion or indel mutation of the CAG repeat was confirmed by PCR, T7E1 assay and sequencing of the edited neurospheres. Edited neurospheres showed decreased polyglutamine aggregation compared with control HD neurospheres. In the edited neurosphere, we confirmed the upregulation of peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1&#x3b1;) and brain-derived neurotrophic factor (BDNF), whose reduced expressions are closely involved in the disease progression. In addition, flow cytometry result showed an increase in cell viability with an overall decrease in necrotic and apoptotic populations among edited R6/2 neurospheres. Additional siRNA experiments confirmed that the increased viability was decreased through inhibition of PGC-1&#x3b1; or BDNF.<br><br>CONCLUSION: Our study confirmed that CAG repeat of R6/2 mouse-derived neurospheres can be edited through CRISPR-Cas9. Editing of CAG repeat sequence decreases polyglutamine aggregation and cellular apoptosis of HD neurospheres, which may be related to the increased expressions of PGC-1&#x3b1; and BDNF.&#xa0;Our data provide the evidence that CRISPR-Cas9 mediated genome editing has therapeutic potential on HD neuronal cells.}, }
@article {pmid36428245, year = {2023}, author = {Zhang, Y and Blahut-Beatty, L and Zheng, S and Clough, SJ and Simmonds, DH}, title = {The Role of a Soybean 14-3-3 Gene (Glyma05g29080) on White Mold Resistance and Nodulation Investigations Using CRISPR-Cas9 Editing and RNA Silencing.}, journal = {Molecular plant-microbe interactions : MPMI}, volume = {36}, number = {3}, pages = {159-164}, doi = {10.1094/MPMI-07-22-0157-R}, pmid = {36428245}, issn = {0894-0282}, mesh = {*CRISPR-Cas Systems/genetics ; RNA Interference ; *Soybeans/genetics ; }, abstract = {The role of a soybean 14-3-3 gene (Glyma05g29080) in defense against white mold and in nodulation was investigated by loss-of-gene-function with CRISPR-Cas9 editing and silencing of RNA interference (RNAi). Particle bombardment was used to introduce the CRISPR expression cassette to target the soybean 14-3-3 gene and an RNAi construct to silence gene transcription. Transmission of the edited 14-3-3 gene and the RNAi construct was confirmed in their respective progeny. The recovered transgenic plants and their progeny were significantly more susceptible to Sclerotinia sclerotiorum infection and showed a significant reduction in nodulation, thus confirming the role of the 14-3-3 gene (Glyma05g29080) in both nodulation and defense.}, }
@article {pmid36912935, year = {2023}, author = {Timberlake, AT}, title = {Molecular scalpels: the future of pediatric craniofacial surgery?.}, journal = {Plastic and reconstructive surgery}, volume = {}, number = {}, pages = {}, doi = {10.1097/PRS.0000000000010402}, pmid = {36912935}, issn = {1529-4242}, abstract = {CRISPR-Cas genome editing tools are among the most substantial advances in the life sciences in modern history. Single dose gene therapies to correct pathogenic mutations have moved quickly from bench to bedside, with several therapeutics designed by CRISPR pioneers entering various stages of clinical investigation. Applications of these genetic technologies are poised to reshape the practice of both medicine and surgery. Many of the most morbid conditions treated by craniofacial surgeons are syndromic craniosynostoses caused by mutations in fibroblast growth factor receptor (FGFR) genes, including Apert, Pfeiffer, Crouzon, and Muenke syndromes. The fact that pathogenic mutations in these genes are recurrent in the majority of affected families presents a unique opportunity to develop "off the shelf" gene editing therapies to correct these mutations in affected children. The therapeutic potential of these interventions could reshape pediatric craniofacial surgery, potentially first eliminating the need for midface advancement procedures in affected children.}, }
@article {pmid36912817, year = {2023}, author = {Wiegand, T and Wilkinson, R and Santiago-Frangos, A and Lynes, M and Hatzenpichler, R and Wiedenheft, B}, title = {Functional and Phylogenetic Diversity of Cas10 Proteins.}, journal = {The CRISPR journal}, volume = {}, number = {}, pages = {}, doi = {10.1089/crispr.2022.0085}, pmid = {36912817}, issn = {2573-1602}, abstract = {Cas10 proteins are large subunits of type III CRISPR RNA (crRNA)-guided surveillance complexes, many of which have nuclease and cyclase activities. Here, we use computational and phylogenetic methods to identify and analyze 2014 Cas10 sequences from genomic and metagenomic databases. Cas10 proteins cluster into five distinct clades that mirror previously established CRISPR-Cas subtypes. Most Cas10 proteins (85.0%) have conserved polymerase active-site motifs, while HD-nuclease domains are less well conserved (36.0%). We identify Cas10 variants that are split over multiple genes or genetically fused to nucleases activated by cyclic nucleotides (i.e., NucC) or components of toxin-antitoxin systems (i.e., AbiEii). To clarify the functional diversification of Cas10 proteins, we cloned, expressed, and purified five representatives from three phylogenetically distinct clades. None of the Cas10s are functional cyclases in isolation, and activity assays performed with polymerase domain active site mutants indicate that previously reported Cas10 DNA-polymerase activity may be a result of contamination. Collectively, this work helps clarify the phylogenetic and functional diversity of Cas10 proteins in type III CRISPR systems.}, }
@article {pmid36910714, year = {2023}, author = {Jang, G and Shin, HR and Do, HS and Kweon, J and Hwang, S and Kim, S and Heo, SH and Kim, Y and Lee, BH}, title = {Therapeutic gene correction for Lesch-Nyhan syndrome using CRISPR-mediated base and prime editing.}, journal = {Molecular therapy. Nucleic acids}, volume = {31}, number = {}, pages = {586-595}, pmid = {36910714}, issn = {2162-2531}, abstract = {Lesch-Nyhan syndrome (LNS) is inherited as an X-linked recessive genetic disorder caused by mutations in hypoxanthine-guanine phosphoribosyl transferase 1 (HPRT1). Patients with LNS show various clinical phenotypes, including hyperuricemia, gout, devastating behavioral abnormality, intellectual disability, and self-harm. Although uric acid overproduction can be modulated with the xanthine oxidase inhibitor allopurinol, there exists no treatment for behavioral and neurological manifestations of LNS. In the current study, CRISPR-mediated base editors (BEs) and prime editors (PEs) were utilized to generate LNS-associated disease models and correct the disease models for therapeutic approach. Cytosine BEs (CBEs) were used to induce c.430C>T and c.508C>T mutations in HAP1 cells, and then adenine BEs (ABEs) were used to correct these mutations without DNA cleavage. PEs induced a c.333_334ins(A) mutation, identified in a Korean patient with LNS, in HAP1 cells, which was corrected in turn by PEs. Furthermore, improved PEs corrected the same mutation in LNS patient-derived fibroblasts by up to 14% without any unwanted mutations. These results suggest that CRISPR-mediated BEs and PEs would be suggested as a potential therapeutic strategy of this extremely rare, devastating genetic disease.}, }
@article {pmid36905519, year = {2023}, author = {Freund, EC and Haag, SM and Haley, B and Murthy, A}, title = {Optimized Nonviral Gene Disruption in Primary Murine and Human Myeloid Cells.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2618}, number = {}, pages = {201-217}, pmid = {36905519}, issn = {1940-6029}, mesh = {Humans ; Mice ; Animals ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Electroporation ; Genetic Engineering ; Macrophages ; }, abstract = {Genetically engineered myeloid cells such as monocytes, macrophages, and dendritic cells have broad applications in basic and translational research. Their central roles in innate and adaptive immunity make them attractive as putative therapeutic cell products. However, efficient gene editing of primary myeloid cells presents unique challenges owing to their sensitivity to foreign nucleic acids and poor editing efficiencies using current methodologies (Hornung et al., Science 314:994-997, 2006; Coch et al., PLoS One 8:e71057, 2013; Bartok and Hartmann, Immunity 53:54-77, 2020; Hartmann, Adv Immunol 133:121-169, 2017; Bobadilla et al., Gene Ther 20:514-520, 2013; Schlee and Hartmann, Nat Rev Immunol 16:566-580, 2016; Leyva et al., BMC Biotechnol 11:13, 2011). This chapter describes nonviral CRISPR-mediated gene knockout in primary human and murine monocytes as well as monocyte-derived or bone marrow-derived macrophages and dendritic cells. Electroporation-mediated delivery of recombinant Cas9 complexed with synthetic guide RNAs can be applied for population-level disruption of single or multiple gene targets.}, }
@article {pmid36902207, year = {2023}, author = {Ueda, J and Yamazaki, T and Funakoshi, H}, title = {Toward the Development of Epigenome Editing-Based Therapeutics: Potentials and Challenges.}, journal = {International journal of molecular sciences}, volume = {24}, number = {5}, pages = {}, pmid = {36902207}, issn = {1422-0067}, mesh = {*Epigenome ; *Gene Editing ; Epigenesis, Genetic ; DNA Methylation ; Chromatin ; CRISPR-Cas Systems ; }, abstract = {The advancement in epigenetics research over the past several decades has led to the potential application of epigenome-editing technologies for the treatment of various diseases. In particular, epigenome editing is potentially useful in the treatment of genetic and other related diseases, including rare imprinted diseases, as it can regulate the expression of the epigenome of the target region, and thereby the causative gene, with minimal or no modification of the genomic DNA. Various efforts are underway to successfully apply epigenome editing in vivo, such as improving target specificity, enzymatic activity, and drug delivery for the development of reliable therapeutics. In this review, we introduce the latest findings, summarize the current limitations and future challenges in the practical application of epigenome editing for disease therapy, and introduce important factors to consider, such as chromatin plasticity, for a more effective epigenome editing-based therapy.}, }
@article {pmid36902202, year = {2023}, author = {Zhou, J and Li, Z and Li, Y and Zhao, Q and Luan, X and Wang, L and Liu, Y and Liu, H and Zhang, J and Yao, D}, title = {Effects of Different Gene Editing Modes of CRISPR/Cas9 on Soybean Fatty Acid Anabolic Metabolism Based on GmFAD2 Family.}, journal = {International journal of molecular sciences}, volume = {24}, number = {5}, pages = {}, pmid = {36902202}, issn = {1422-0067}, mesh = {*Gene Editing/methods ; *Soybeans/genetics ; CRISPR-Cas Systems ; Oleic Acid/metabolism ; Fatty Acid Desaturases/metabolism ; Plants, Genetically Modified/genetics ; Fatty Acids/metabolism ; }, abstract = {Δ[12]-fatty acid dehydrogenase (FAD2) is the essential enzyme responsible for catalyzing the formation of linoleic acid from oleic acid. CRISPR/Cas9 gene editing technology has been an essential tool for molecular breeding in soybeans. To evaluate the most suitable type of gene editing in soybean fatty acid synthesis metabolism, this study selected five crucial enzyme genes of the soybean FAD2 gene family-GmFAD2-1A, GmFAD2-1B, GmFAD2-2A, GmFAD2-2B, and GmFAD2-2C-and created a CRISPR/Cas9-mediated single gene editing vector system. The results of Sanger sequencing showed that 72 transformed plants positive for T1 generation were obtained using Agrobacterium-mediated transformation, of which 43 were correctly edited plants, with the highest editing efficiency of 88% for GmFAD2-2A. The phenotypic analysis revealed that the oleic acid content of the progeny of GmFAD2-1A gene-edited plants had a higher increase of 91.49% when compared to the control JN18, and the rest of the gene-edited plants in order were GmFAD2-2A, GmFAD2-1B, GmFAD2-2C, and GmFAD2-2B. The analysis of gene editing type has indicated that base deletions greater than 2bp were the predominant editing type in all editing events. This study provides ideas for the optimization of CRISPR/Cas9 gene editing technology and the development of new tools for precise base editing in the future.}, }
@article {pmid36901914, year = {2023}, author = {Ivanov, AV and Safenkova, IV and Biketov, SF and Zherdev, AV and Dzantiev, BB}, title = {Engineering of DNA Structures Attached to Magnetic Particles for Effective Trans- and Cis-Cleavage in Cas12-Based Biosensors.}, journal = {International journal of molecular sciences}, volume = {24}, number = {5}, pages = {}, pmid = {36901914}, issn = {1422-0067}, mesh = {*DNA/chemistry ; Endonucleases/metabolism ; Oligonucleotides ; *Biosensing Techniques ; Magnetic Phenomena ; CRISPR-Cas Systems ; }, abstract = {Sequence-specific endonuclease Cas12-based biosensors have rapidly evolved as a strong tool to detect nucleic acids. Magnetic particles (MPs) with attached DNA structures could be used as a universal platform to manipulate the DNA-cleavage activity of Cas12. Here, we propose nanostructures of trans- and cis-DNA targets immobilized on the MPs. The main advantage of the nanostructures is a rigid double-stranded DNA adaptor that distances the cleavage site from the MP surface to ensure maximum Cas12 activity. Adaptors with different lengths were compared by detecting the cleavage by fluorescence and gel electrophoresis of the released DNA fragments. The length-dependent effects for cleavage on the MPs' surface were found both for cis- and trans-targets. For trans-DNA targets with a cleavable 15-dT tail, the results showed that the optimal range of the adaptor length was 120-300 bp. For cis-targets, we varied the length and location of the adaptor (at the PAM or spacer ends) to estimate the effect of the MP's surface on the PAM-recognition process or R-loop formation. The sequential arrangement of an adaptor, PAM, and a spacer was preferred and required the minimum adaptor length of 3 bp. Thus, with cis-cleavage, the cleavage site can be located closer to the surface of the MPs than with trans-cleavage. The findings provide solutions for efficient Cas12-based biosensors using surface-attached DNA structures.}, }
@article {pmid36901844, year = {2023}, author = {Wang, W and Huang, P and Dai, W and Tang, H and Qiu, Y and Chang, Y and Han, Z and Li, X and Du, L and Ye, X and Zou, C and Wang, K}, title = {Application of Nicotinamide to Culture Medium Improves the Efficiency of Genome Editing in Hexaploid Wheat.}, journal = {International journal of molecular sciences}, volume = {24}, number = {5}, pages = {}, pmid = {36901844}, issn = {1422-0067}, mesh = {*Gene Editing/methods ; *Triticum/genetics ; CRISPR-Cas Systems ; Histones/genetics ; Mutation ; }, abstract = {Histone acetylation is the earliest and most well-characterized of post-translation modifications. It is mediated by histone acetyltransferases (HAT) and histone deacetylases (HDAC). Histone acetylation could change the chromatin structure and status and further regulate gene transcription. In this study, nicotinamide, a histone deacetylase inhibitor (HDACi), was used to enhance the efficiency of gene editing in wheat. Transgenic immature and mature wheat embryos harboring a non-mutated GUS gene, the Cas9 and a GUS-targeting sgRNA were treated with nicotinamide in two concentrations (2.5 and 5 mM) for 2, 7, and 14 days in comparison with a no-treatment control. The nicotinamide treatment resulted in GUS mutations in up to 36% of regenerated plants, whereas no mutants were obtained from the non-treated embryos. The highest efficiency was achieved when treated with 2.5 mM nicotinamide for 14 days. To further validate the impact of nicotinamide treatment on the effectiveness of genome editing, the endogenous TaWaxy gene, which is responsible for amylose synthesis, was tested. Utilizing the aforementioned nicotinamide concentration to treat embryos containing the molecular components for editing the TaWaxy gene, the editing efficiency could be increased to 30.3% and 13.3%, respectively, for immature and mature embryos in comparison to the 0% efficiency observed in the control group. In addition, nicotinamide treatment during transformation progress could also improve the efficiency of genome editing approximately threefold in a base editing experiment. Nicotinamide, as a novel approach, may be employed to improve the editing efficacy of low-efficiency genome editing tools such as base editing and prime editing (PE) systems in wheat.}, }
@article {pmid36898814, year = {2023}, author = {Dou, B and Zhang, Y and Gao, H and Zhang, S and Zheng, J and Lu, X and Liu, S and Zhou, H and Hun, X}, title = {CRISPR/Cas12a-based MUSCA-PEC strategy for HSV-1 assay.}, journal = {Analytica chimica acta}, volume = {1250}, number = {}, pages = {340955}, doi = {10.1016/j.aca.2023.340955}, pmid = {36898814}, issn = {1873-4324}, mesh = {Humans ; CRISPR-Cas Systems ; *Herpesvirus 1, Human ; Alkaline Phosphatase ; Biological Assay ; Coloring Agents ; *Biosensing Techniques ; }, abstract = {In the photoelectrochemical sensing, constant potential excitation to get the photoelectrochemical signal is the main excitation signal mode. Novel method for photoelectrochemical signal obtaining is needed. Inspired by this ideal, a photoelectrochemical strategy for Herpes simplex virus (HSV-1) detection with multiple potential step chronoamperometry (MUSCA) pattern was fabricated using CRISPR/Cas12a cleavage coupled with entropy-driven target recycling. In the presence of target, HSV-1, the Cas12a was activated by the H1-H2 complex obtained by entropy-driven, then digesting the circular fragment of csRNA to expose single-stranded crRNA2 and alkaline phosphatase (ALP). The inactive Cas12a was self-assembled with crRNA2 and activated again with the help of assistant dsDNA. After multiple rounds of CRISPR/Cas12a cleavage and magnetic separation, MUSCA, as a signal amplifier, collected the enhanced photocurrent responses generated by catalyzed p-Aminophenol (p-AP). Different from the reported signal enhancement strategies based on photoactive nanomaterials and sensing mechanisms, MUSCA technique endowed the strategy with unique advantages of direct, fast and ultrasensitive. A superior detection limit of 3 aM toward HSV-1 was achieved. This strategy was successfully applied for HSV-1 detection in Human serum samples. The combination of MUSCA technique and CRISPR/Cas12a assay brings broader potential prospect for the detection of nucleic acids.}, }
@article {pmid36858011, year = {2023}, author = {Li, M and Luo, N and Liao, X and Zou, L}, title = {Proximity hybridization-regulated CRISPR/Cas12a-based dual signal amplification strategy for sensitive detection of circulating tumor DNA.}, journal = {Talanta}, volume = {257}, number = {}, pages = {124395}, doi = {10.1016/j.talanta.2023.124395}, pmid = {36858011}, issn = {1873-3573}, mesh = {Humans ; *Circulating Tumor DNA/genetics ; CRISPR-Cas Systems ; Nucleic Acid Hybridization/methods ; DNA ; *Neoplasms ; *Biosensing Techniques/methods ; }, abstract = {Circulating tumor DNA (ctDNA) is regarded as an ideal candidate biomarker for the non-invasive diagnosis of cancer. However, the lack of convenient and reliable detection methods for ctDNA restricts its clinical application. Herein, we developed a dual signal amplification strategy for sensitive detection of ctDNA based on hybridization chain reaction (HCR) and proximity hybridization-regulated CRISPR/Cas12a. The ctDNA initiates HCR through the continuous hybridization of two hairpin probes (H1 and H2), yielding long nicked double-stranded DNA nanowires composed of numerous split segments, which are successively connected to activate the trans-cleavage activity of CRISPR/Cas12a. In this case, the doubly labeled single-stranded DNA reporter can be cleaved to produce a strong fluorescent signal. Owing to the dual amplification of HCR and CRISPR/Cas12a, this strategy exhibits high sensitivity toward ctDNA with a low detection limit of 5.43 fM. Moreover, the proposed method was successfully applied for ctDNA detection in serum samples with satisfactory results, which has great potential in the clinical diagnosis of cancer.}, }
@article {pmid36841059, year = {2023}, author = {Zhang, L and Bai, T and Wu, H}, title = {sgRNA-2wPSM: Identify sgRNAs on-target activity by combining two-window-based position specific mismatch and synthetic minority oversampling technique.}, journal = {Computers in biology and medicine}, volume = {155}, number = {}, pages = {106489}, doi = {10.1016/j.compbiomed.2022.106489}, pmid = {36841059}, issn = {1879-0534}, mesh = {*CRISPR-Cas Systems ; *Gene Editing ; Algorithms ; Base Sequence ; Support Vector Machine ; }, abstract = {sgRNAs on-target activity prediction is a critical step in the CRISPR-Cas9 system. Due to its importance to RNA function research and genome editing application, some computational methods were introduced, treating it as a binary classification task or a regression task. Among these methods, sgRNA-PSM is a state-of-the-art method. In this work, we improved this method by proposing a new feature extraction method called two-window-based PSM, which divides the DNA sequences into two non-overlapping segments so as to extract different patterns in the two different segments. The two-window-based PSM were fed into Support Vector Machines (SVMs), and a new method called sgRNA-2wPSM was proposed. Furthermore, a new oversampling method called SCORE-SVM-SMOTE was proposed to solve the imbalanced training set problem based on the SVM-SMOTE algorithm. Results on the benchmark datasets indicated that sgRNA-2wPSM is superior to other methods.}, }
@article {pmid36827793, year = {2023}, author = {Liu, D and Sun, XM and Zhu, L and Li, CY}, title = {Using time-shared scanning optical tweezers assisted two-photon fluorescence imaging to establish a versatile CRISPR/Cas12a-mediated biosensor.}, journal = {Biosensors &amp; bioelectronics}, volume = {227}, number = {}, pages = {115158}, doi = {10.1016/j.bios.2023.115158}, pmid = {36827793}, issn = {1873-4235}, mesh = {Optical Tweezers ; CRISPR-Cas Systems ; *Biosensing Techniques ; Optical Imaging ; *DNA, Catalytic ; }, abstract = {Based on the admirable precision to identify target nucleic acids and the particular trans-cleavage feature, CRISPR/Cas12a system is a useful means to further improve the sensing accuracy and the design flexibility of fluorescence biosensors. However, the current construction concepts still suffer from insufficient sensitivity, unsuitable for complicated real samples and limited detection species. In this work, much efforts are achieved to address these obstacles. At first, we adopt a microsphere sustained signal enrichment, under which a home-made time-shared scanning optical tweezers assisted fluorescence imaging is employed to guarantee a stable excitation and also realize multiflux measurement. Furthermore, by taking advantage of the low background merit of the near-infrared light excited two-photon fluorescence, a commendable anti-interference capability is endowed to operate in complex media. After utilizing a functional DNA (e.g. aptamer and DNAzyme) regulated mediation pathway to respond non-nucleic acid analytes (alpha fetal protein and Pb[2+]), the newly-established CRISPR/Cas12a-mediated fluorescence biosensor is found to display favorable assay performance. More importantly, our analytical methodology can act as a versatile and reliable toolbox in various applications such as disease diagnosis and environmental analysis, propelling the development of CRISPR system in biosensing field.}, }
@article {pmid36809733, year = {2023}, author = {Liu, W and Zhu, X and Li, L and Wang, S and Li, CZ and Liang, T}, title = {Reining in Cas13a activity with N-terminal removable tags expands Cas13a based molecular sensing and enables precise gene interference.}, journal = {Biosensors &amp; bioelectronics}, volume = {227}, number = {}, pages = {115138}, doi = {10.1016/j.bios.2023.115138}, pmid = {36809733}, issn = {1873-4235}, mesh = {*CRISPR-Cas Systems ; *Biosensing Techniques ; RNA/genetics ; Peptide Hydrolases/genetics ; }, abstract = {Activation of Cas13 is exclusively dependent on crRNA-target RNA hybridization according to the canonical mode of Cas13 action. Upon activation Cas13 can cleave both target RNA and any surrounding RNA. The latter has been well adopted by therapeutic gene interference and biosensor development. This work for the first time, rationale designs and validates a multi-component controlled activation system of Cas13 by N-terminus tagging. A composite SUMO tag comprised of His, Twinstrep, and Smt3 tags fully suppresses target dependent activation of Cas13a by interfering with crRNA docking. The suppression releases upon proteases mediated proteolytic cleavage. The modular composition of the composite tag can be altered to fulfill customized response to alternative proteases. The biosensor SUMO-Cas13a is able to resolve a broad concentration range of protease Ulp1 with a calculated LOD of 48.8pg/μL in aqueous buffer. Further, in accordance with this finding Cas13a was successfully programmed to exert target gene knock down preferentially in SUMO protease high cell types. In summary the discovered regulatory component not only fulfills Cas13a based protease detection for the first time, but also delivers a novel strategy for multi-component controlled activation of Cas13a toward temporal and spacial precision.}, }
@article {pmid36801553, year = {2023}, author = {Wang, X and Chen, Y and Ma, L and Han, Z and Liu, Y and Qiao, J}, title = {An amplification-free CRISPR/Cas12a-based fluorescence assay for ultrasensitive detection of nuclease activity.}, journal = {Talanta}, volume = {257}, number = {}, pages = {124329}, doi = {10.1016/j.talanta.2023.124329}, pmid = {36801553}, issn = {1873-3573}, mesh = {Humans ; *CRISPR-Cas Systems ; *Biological Assay ; DNA, Single-Stranded ; Deoxyribonuclease I ; Endonucleases ; RNA, Guide, CRISPR-Cas Systems ; Ribonuclease H ; }, abstract = {Nuclease, such as RNase H and DNase I, plays key roles in plenty of cellular processes and could be potential therapeutic target for drug development. It is necessary to establish rapid and simple-to-use methods to detect nuclease activity. Herein, we develop a Cas12a-based fluorescence assay without any nucleic acid amplification steps for ultrasensitive detection of RNase H or DNase I activity. By our design, the pre-assembled crRNA/ssDNA duplex triggered the cleavage of fluorescent probes in the presence of Cas12a enzymes. However, the crRNA/ssDNA duplex was selectively digested with the addition of RNase H or DNase I, which leaded to fluorescence intensity changes. Under optimized conditions, the method exhibited good analytical performance, achieving a limit of detection (LOD) as low as 0.0082 U/mL for RNase H and 0.13 U/mL for DNase I, respectively. The method was feasible for analysis of RNase H in human serum and cell lysates, as well as for screening of enzyme inhibitors. Moreover, it can be adopted to image RNase H activity in living cells. Together, this study provides a facile platform for nuclease detection and could be expanded for other biomedical research and clinical diagnostics.}, }
@article {pmid36371609, year = {2023}, author = {Zhao, J and Tan, Y and Jiang, Y and Zhu-Salzman, K and Xiao, L}, title = {CRISPR/Cas9-mediated methoprene-tolerant 1 knockout results in precocious metamorphosis of beet armyworm (Spodoptera exigua) only at the late larval stage.}, journal = {Insect molecular biology}, volume = {32}, number = {2}, pages = {132-142}, doi = {10.1111/imb.12819}, pmid = {36371609}, issn = {1365-2583}, mesh = {Animals ; *Methoprene ; Larva ; Spodoptera/genetics ; *Beta vulgaris/genetics/metabolism ; CRISPR-Cas Systems ; Metamorphosis, Biological ; Juvenile Hormones/metabolism ; Insecta/genetics ; Pupa ; Insect Proteins/metabolism ; Gene Expression Regulation, Developmental ; }, abstract = {Juvenile hormone (JH) controls almost every aspect of an insect, especially metamorphosis. Since RNA interference works on transcripts and is often insufficient in Lepidoptera, how JH affects larval development in these insects is not well studied. Using the CRISPR/Cas9 technique, we knocked out Spodoptera exigua methoprene-tolerant 1 (SeMet1) gene of beet armyworm by modifying two sites in the coding region. However, SeMet1 knockout did not affect egg hatch rate or larval development at L1-L3 stages. In contrast to the consistent five larval instars of the control group, L4 SeMet1 mutants began to show signs of precocious metamorphosis, that is, small patches of pupal cuticle. Most L4 and all L5 SeMet1 mutants died for failing to shed their mosaic cuticles. RNA-seq indicated that most genes encoding pupal cuticle proteins and chitinase genes were altered in SeMet1 mutant L4 larvae. SeKr-h1, a key transcription factor in JH action was significantly down-regulated in L3-L5 larvae, while SeBR-C, a pupal indicator was only upregulated in L4-L5 larvae. These results suggested that S. exigua larvae may initially develop independently of JH, and involve SeMet1 in transducing JH signalling, leading to controlled larval metamorphosis at the late larval stage. We believe our findings will enhance better understanding of JH regulation of larval development.}, }
@article {pmid36910196, year = {2023}, author = {Kogawa, M and Nishikawa, Y and Saeki, T and Yoda, T and Arikawa, K and Takeyama, H and Hosokawa, M}, title = {Revealing within-species diversity in uncultured human gut bacteria with single-cell long-read sequencing.}, journal = {Frontiers in microbiology}, volume = {14}, number = {}, pages = {1133917}, pmid = {36910196}, issn = {1664-302X}, abstract = {Obtaining complete and accurate bacterial genomes is vital for studying the characteristics of uncultured bacteria. Single-cell genomics is a promising approach for the culture-independent recovery of bacterial genomes from individual cells. However, single-amplified genomes (SAGs) often have fragmented and incomplete sequences due to chimeric and biased sequences introduced during the genome amplification process. To address this, we developed a single-cell amplified genome long-read assembly (scALA) workflow to construct complete circular SAGs (cSAGs) from long-read single-cell sequencing data of uncultured bacteria. We used the SAG-gel platform, which is both cost-effective and high-throughput, to obtain hundreds of short-read and long-read sequencing data for specific bacterial strains. The scALA workflow generated cSAGs by repeated in silico processing for sequence bias reduction and contig assembly. From 12 human fecal samples, including two cohabitant groups, scALA generated 16 cSAGs of three specifically targeted bacterial species: Anaerostipes hadrus, Agathobacter rectalis, and Ruminococcus gnavus. We discovered strain-specific structural variations shared among cohabiting hosts, while all cSAGs of the same species showed high homology in aligned genomic regions. A. hadrus cSAGs exhibited 10 kbp-long phage insertions, various saccharide metabolic capabilities, and different CRISPR-Cas systems in each strain. The sequence similarity of A. hadrus genomes did not necessarily correspond with orthologous functional genes, while host geographical regionality seemed to be highly related to gene possession. scALA allowed us to obtain closed circular genomes of specifically targeted bacteria from human microbiota samples, leading to an understanding of within-species diversities, including structural variations and linking mobile genetic elements, such as phages, to hosts. These analyses provide insight into microbial evolution, the adaptation of the community to environmental changes, and interactions with hosts. cSAGs constructed using this method can expand bacterial genome databases and our understanding of within-species diversities in uncultured bacteria.}, }
@article {pmid36909407, year = {2023}, author = {Majeed, Y and Zhu, X and Zhang, N and Ul-Ain, N and Raza, A and Haider, FU and Si, H}, title = {Harnessing the role of mitogen-activated protein kinases against abiotic stresses in plants.}, journal = {Frontiers in plant science}, volume = {14}, number = {}, pages = {932923}, pmid = {36909407}, issn = {1664-462X}, abstract = {Crop plants are vulnerable to various biotic and abiotic stresses, whereas plants tend to retain their physiological mechanisms by evolving cellular regulation. To mitigate the adverse effects of abiotic stresses, many defense mechanisms are induced in plants. One of these mechanisms is the mitogen-activated protein kinase (MAPK) cascade, a signaling pathway used in the transduction of extracellular stimuli into intercellular responses. This stress signaling pathway is activated by a series of responses involving MAPKKKs→MAPKKs→MAPKs, consisting of interacting proteins, and their functions depend on the collaboration and activation of one another by phosphorylation. These proteins are key regulators of MAPK in various crop plants under abiotic stress conditions and also related to hormonal responses. It is revealed that in response to stress signaling, MAPKs are characterized as multigenic families and elaborate the specific stimuli transformation as well as the antioxidant regulation system. This pathway is directed by the framework of proteins and stopping domains confer the related associates with unique structure and functions. Early studies of plant MAPKs focused on their functions in model plants. Based on the results of whole-genome sequencing, many MAPKs have been identified in plants, such as Arbodiposis, tomato, potato, alfalfa, poplar, rice, wheat, maize, and apple. In this review, we summarized the recent work on MAPK response to abiotic stress and the classification of MAPK cascade in crop plants. Moreover, we highlighted the modern research methodologies such as transcriptomics, proteomics, CRISPR/Cas technology, and epigenetic studies, which proposed, identified, and characterized the novel genes associated with MAPKs and their role in plants under abiotic stress conditions. In-silico-based identification of novel MAPK genes also facilitates future research on MAPK cascade identification and function in crop plants under various stress conditions.}, }
@article {pmid36909403, year = {2023}, author = {Tiwari, JK and Singh, AK and Behera, TK}, title = {CRISPR/Cas genome editing in tomato improvement: Advances and applications.}, journal = {Frontiers in plant science}, volume = {14}, number = {}, pages = {1121209}, pmid = {36909403}, issn = {1664-462X}, abstract = {The narrow genetic base of tomato poses serious challenges in breeding. Hence, with the advent of clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein9 (CRISPR/Cas9) genome editing, fast and efficient breeding has become possible in tomato breeding. Many traits have been edited and functionally characterized using CRISPR/Cas9 in tomato such as plant architecture and flower characters (e.g. leaf, stem, flower, male sterility, fruit, parthenocarpy), fruit ripening, quality and nutrition (e.g., lycopene, carotenoid, GABA, TSS, anthocyanin, shelf-life), disease resistance (e.g. TYLCV, powdery mildew, late blight), abiotic stress tolerance (e.g. heat, drought, salinity), C-N metabolism, and herbicide resistance. CRISPR/Cas9 has been proven in introgression of de novo domestication of elite traits from wild relatives to the cultivated tomato and vice versa. Innovations in CRISPR/Cas allow the use of online tools for single guide RNA design and multiplexing, cloning (e.g. Golden Gate cloning, GoldenBraid, and BioBrick technology), robust CRISPR/Cas constructs, efficient transformation protocols such as Agrobacterium, and DNA-free protoplast method for Cas9-gRNAs ribonucleoproteins (RNPs) complex, Cas9 variants like PAM-free Cas12a, and Cas9-NG/XNG-Cas9, homologous recombination (HR)-based gene knock-in (HKI) by geminivirus replicon, and base/prime editing (Target-AID technology). This mini-review highlights the current research advances in CRISPR/Cas for fast and efficient breeding of tomato.}, }
@article {pmid36907678, year = {2023}, author = {Duan, W and Urani, E and Mattson, MP}, title = {The potential of gene editing for Huntington's disease.}, journal = {Trends in neurosciences}, volume = {}, number = {}, pages = {}, doi = {10.1016/j.tins.2023.02.005}, pmid = {36907678}, issn = {1878-108X}, abstract = {Huntington's disease (HD) is a dominantly inherited neurodegenerative disorder caused by a trinucleotide repeat expansion in the huntingtin gene resulting in long stretches of polyglutamine repeats in the huntingtin protein. The disease involves progressive degeneration of neurons in the striatum and cerebral cortex resulting in loss of control of motor function, psychiatric problems, and cognitive deficits. There are as yet no treatments that can slow disease progression in HD. Recent advances in gene editing using clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) systems and demonstrations of their ability to correct gene mutations in animal models of a range of diseases suggest that gene editing may prove effective in preventing or ameliorating HD. Here we describe (i) potential CRISPR-Cas designs and cellular delivery methods for the correction of mutant genes that cause inherited diseases, and (ii) recent preclinical findings demonstrating the efficacy of such gene-editing approaches in animal models, with a focus on HD.}, }
@article {pmid36905332, year = {2023}, author = {Matsumoto, D and Kishi, K and Matsugi, E and Inoue, Y and Nigorikawa, K and Nomura, W}, title = {Cas9-Geminin and Cdt1-fused anti-CRISPR protein synergistically increase editing accuracy.}, journal = {FEBS letters}, volume = {}, number = {}, pages = {}, doi = {10.1002/1873-3468.14608}, pmid = {36905332}, issn = {1873-3468}, abstract = {Genome editing with CRISPR-Cas9, particularly for therapeutic purposes, should be accomplished via the homology-directed repair (HDR) pathway, which exhibits greater precision than other pathways. However, one of the issues to be solved is that genome editing efficiency with HDR is generally low. A Streptococcus pyogenes Cas9 (SpyCas9) fusion with human Geminin (Cas9-Gem) reportedly increases HDR efficiency slightly. In contrast, we found that regulation of SpyCas9 activity with an anti-CRISPR protein (AcrIIA4) fused to Chromatin licensing and DNA replication factor 1 (Cdt1) significantly increases HDR efficiency and reduces off-target effects. Here, another anti-CRISPR protein, AcrIIA5, was applied, and the combined use of Cas9-Gem and Anti-CRISPR+Cdt1 showed synergistic enhancement of HDR efficiency. The method may be applicable to various anti-CRISPR/CRISPR-Cas combinations.}, }
@article {pmid36903878, year = {2023}, author = {Kong, Q and Li, J and Wang, S and Feng, X and Shou, H}, title = {Combination of Hairy Root and Whole-Plant Transformation Protocols to Achieve Efficient CRISPR/Cas9 Genome Editing in Soybean.}, journal = {Plants (Basel, Switzerland)}, volume = {12}, number = {5}, pages = {}, pmid = {36903878}, issn = {2223-7747}, abstract = {The new gene-editing technology CRISPR/Cas system has been widely used for genome engineering in various organisms. Since the CRISPR/Cas gene-editing system has a certain possibility of low efficiency and the whole plant transformation of soybean is time-consuming and laborious, it is important to evaluate the editing efficiency of designed CRISPR constructs before the stable whole plant transformation process starts. Here, we provide a modified protocol for generating transgenic hairy soybean roots to assess the efficiency of guide RNA (gRNA) sequences of the CRISPR/Cas constructs within 14 days. The cost- and space-effective protocol was first tested in transgenic soybean harboring the GUS reporter gene for the efficiency of different gRNA sequences. Targeted DNA mutations were detected in 71.43-97.62% of the transgenic hairy roots analyzed as evident by GUS staining and DNA sequencing of the target region. Among the four designed gene-editing sites, the highest editing efficiency occurred at the 3' terminal of the GUS gene. In addition to the reporter gene, the protocol was tested for the gene-editing of 26 soybean genes. Among the gRNAs selected for stable transformation, the editing efficiency of hairy root transformation and stable transformation ranged from 5% to 88.8% and 2.7% to 80%, respectively. The editing efficiencies of stable transformation were positively correlated with those of hairy root transformation with a Pearson correlation coefficient (r) of 0.83. Our results demonstrated that soybean hairy root transformation could rapidly assess the efficiency of designed gRNA sequences on genome editing. This method can not only be directly applied to the functional study of root-specific genes, but more importantly, it can be applied to the pre-screening of gRNA in CRISPR/Cas gene editing.}, }
@article {pmid36902028, year = {2023}, author = {Liu, B and Song, W and Wang, L and Wu, Y and Xu, X and Niu, X and Huang, S and Liu, Y and Tang, W}, title = {dCas9-BE3 and dCas12a-BE3 Systems Mediated Base Editing in Kiwifruit Canker Causal Agent Pseudomonas syringae pv. actinidiae.}, journal = {International journal of molecular sciences}, volume = {24}, number = {5}, pages = {}, pmid = {36902028}, issn = {1422-0067}, abstract = {Pseudomonas syringae pv. actinidiae (Psa) causes bacterial canker of kiwifruit with heavy economic losses. However, little is known about the pathogenic genes of Psa. CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas-mediated genome editing technology has dramatically facilitated the characterization of gene function in various organisms. However, CRISPR genome editing could not be efficiently employed in Psa due to lacking homologous recombination repair. The base editor (BE) system, which depends on CRISPR/Cas, directly induces single nucleoside C to T without homology recombination repair. Here, we used dCas9-BE3 and dCas12a-BE3 systems to create substitutions of C to T and to convert CAG/CAA/CGA codons to stop codons (TAG/TAA/TGA) in Psa. The dCas9-BE3 system-induced single C-to-T conversion frequency of 3 to 10 base positions ranged from 0% to 100%, with a mean of 77%. The dCas12a-BE3 system-induced single C-to-T conversion frequency of 8 to 14 base positions in the spacer region ranged from 0% to 100%, with a mean of 76%. In addition, a relatively saturated Psa gene knockout system covering more than 95% of genes was developed based on dCas9-BE3 and dCas12a-BE3, which could knock out two or three genes at the same time in the Psa genome. We also found that hopF2 and hopAO2 were involved in the Psa virulence of kiwifruit. The HopF2 effector can potentially interact with proteins such as RIN, MKK5, and BAK1, while the HopAO2 effector can potentially interact with the EFR protein to reduce the host's immune response. In conclusion, for the first time, we established a PSA.AH.01 gene knockout library that may promote research on elucidating the gene function and pathogenesis of Psa.}, }
@article {pmid36901793, year = {2023}, author = {Wang, Y and Fan, H and Tong, Y}, title = {Unveil the Secret of the Bacteria and Phage Arms Race.}, journal = {International journal of molecular sciences}, volume = {24}, number = {5}, pages = {}, pmid = {36901793}, issn = {1422-0067}, abstract = {Bacteria have developed different mechanisms to defend against phages, such as preventing phages from being adsorbed on the surface of host bacteria; through the superinfection exclusion (Sie) block of phage's nucleic acid injection; by restricting modification (R-M) systems, CRISPR-Cas, aborting infection (Abi) and other defense systems to interfere with the replication of phage genes in the host; through the quorum sensing (QS) enhancement of phage's resistant effect. At the same time, phages have also evolved a variety of counter-defense strategies, such as degrading extracellular polymeric substances (EPS) that mask receptors or recognize new receptors, thereby regaining the ability to adsorb host cells; modifying its own genes to prevent the R-M systems from recognizing phage genes or evolving proteins that can inhibit the R-M complex; through the gene mutation itself, building nucleus-like compartments or evolving anti-CRISPR (Acr) proteins to resist CRISPR-Cas systems; and by producing antirepressors or blocking the combination of autoinducers (AIs) and its receptors to suppress the QS. The arms race between bacteria and phages is conducive to the coevolution between bacteria and phages. This review details bacterial anti-phage strategies and anti-defense strategies of phages and will provide basic theoretical support for phage therapy while deeply understanding the interaction mechanism between bacteria and phages.}, }
@article {pmid36894612, year = {2023}, author = {Pavlou, S and Foskolou, S and Patikas, N and Field, SF and Papachristou, EK and Santos, C and Edwards, AR and Kishore, K and Ansari, R and Rajan, SS and Fernandes, HJR and Metzakopian, E}, title = {CRISPR-Cas9 genetic screen leads to the discovery of L-Moses, a KAT2B inhibitor that attenuates Tunicamycin-mediated neuronal cell death.}, journal = {Scientific reports}, volume = {13}, number = {1}, pages = {3934}, pmid = {36894612}, issn = {2045-2322}, mesh = {Humans ; Tunicamycin/pharmacology ; *CRISPR-Cas Systems ; *Endoplasmic Reticulum/metabolism ; Cell Death ; Endoplasmic Reticulum Stress ; Dopaminergic Neurons/metabolism ; Apoptosis ; p300-CBP Transcription Factors/metabolism ; }, abstract = {Accumulation of aggregated and misfolded proteins, leading to endoplasmic reticulum stress and activation of the unfolded protein response, is a hallmark of several neurodegenerative disorders, including Alzheimer's and Parkinson's disease. Genetic screens are powerful tools that are proving invaluable in identifying novel modulators of disease associated processes. Here, we performed a loss-of-function genetic screen using a human druggable genome library, followed by an arrayed-screen validation, in human iPSC-derived cortical neurons. We identified and genetically validated 13 genes, whose knockout was neuroprotective against Tunicamycin, a glycoprotein synthesis inhibitor widely used to induce endoplasmic reticulum stress. We also demonstrated that pharmacological inhibition of KAT2B, a lysine acetyltransferase identified by our genetic screens, by L-Moses, attenuates Tunicamycin-mediated neuronal cell death and activation of CHOP, a key pro-apoptotic member of the unfolded protein response in both cortical and dopaminergic neurons. Follow-up transcriptional analysis suggested that L-Moses provided neuroprotection by partly reversing the transcriptional changes caused by Tunicamycin. Finally, L-Moses treatment attenuated total protein levels affected by Tunicamycin, without affecting their acetylation profile. In summary, using an unbiased approach, we identified KAT2B and its inhibitor, L-Moses, as potential therapeutic targets for neurodegenerative diseases.}, }
@article {pmid36762651, year = {2023}, author = {Komori, T and Hata, S and Mabuchi, A and Genova, M and Harada, T and Fukuyama, M and Chinen, T and Kitagawa, D}, title = {A CRISPR-del-based pipeline for complete gene knockout in human diploid cells.}, journal = {Journal of cell science}, volume = {136}, number = {6}, pages = {}, doi = {10.1242/jcs.260000}, pmid = {36762651}, issn = {1477-9137}, mesh = {Humans ; Gene Knockout Techniques ; *CRISPR-Cas Systems/genetics ; *Diploidy ; INDEL Mutation/genetics ; Cell Line ; Gene Editing/methods ; }, abstract = {The advance of CRISPR/Cas9 technology has enabled us easily to generate gene knockout cell lines by introducing insertion-deletion mutations (indels) at the target site via the error-prone non-homologous end joining repair system. Frameshift-promoting indels can disrupt gene functions by generation of a premature stop codon. However, there is growing evidence that targeted genes are not always knocked out by the indel-based gene disruption. Here, we established a pipeline of CRISPR-del, which induces a large chromosomal deletion by cutting two different target sites, to perform 'complete' gene knockout efficiently in human diploid cells. Quantitative analyses show that the frequency of gene deletion with this approach is much higher than that of conventional CRISPR-del methods. The lengths of the deleted genomic regions demonstrated in this study are longer than those of 95% of the human protein-coding genes. Furthermore, the pipeline enabled the generation of a model cell line having a bi-allelic cancer-associated chromosomal deletion. Overall, these data lead us to propose that the CRISPR-del pipeline is an efficient and practical approach for producing 'complete' gene knockout cell lines in human diploid cells.}, }
@article {pmid36649793, year = {2023}, author = {Dickson, A and Mullooly, N and Serrano, A and Escudero-Ibarz, L and Wiggins, C and Gianni, D}, title = {Highly scalable arrayed CRISPR mediated gene silencing in primary lung small airway epithelial cells.}, journal = {SLAS discovery : advancing life sciences R &amp; D}, volume = {28}, number = {2}, pages = {29-35}, doi = {10.1016/j.slasd.2023.01.003}, pmid = {36649793}, issn = {2472-5560}, mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing ; Gene Silencing ; Lung ; Epithelial Cells/metabolism ; }, abstract = {Small airway epithelial cells (SAECs) play a central role in the pathogenesis of lung diseases and are now becoming a crucial cellular model for target identification and validation in drug discovery. However, primary cell lines such as SAECs are often difficult to transfect using traditional lipofection methods; therefore, gene editing using CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 is often carried out through ribonucleoprotein (RNP) electroporation. Here we have established a robust, scalable, and automated arrayed CRISPR nuclease (CRISPRn) screening workflow for SAECs which can be combined with a myriad of disease-specific endpoint assays.}, }
@article {pmid36508706, year = {2023}, author = {Hardouin, G and Antoniou, P and Martinucci, P and Felix, T and Manceau, S and Joseph, L and Masson, C and Scaramuzza, S and Ferrari, G and Cavazzana, M and Miccio, A}, title = {Adenine base editor-mediated correction of the common and severe IVS1-110 (G&gt;A) β-thalassemia mutation.}, journal = {Blood}, volume = {141}, number = {10}, pages = {1169-1179}, doi = {10.1182/blood.2022016629}, pmid = {36508706}, issn = {1528-0020}, mesh = {Humans ; *beta-Thalassemia/genetics/therapy ; Gene Editing ; CRISPR-Cas Systems ; Mutation ; beta-Globins/genetics ; }, abstract = {β-Thalassemia (BT) is one of the most common genetic diseases worldwide and is caused by mutations affecting β-globin production. The only curative treatment is allogenic hematopoietic stem/progenitor cells (HSPCs) transplantation, an approach limited by compatible donor availability and immunological complications. Therefore, transplantation of autologous, genetically-modified HSPCs is an attractive therapeutic option. However, current gene therapy strategies based on the use of lentiviral vectors are not equally effective in all patients and CRISPR/Cas9 nuclease-based strategies raise safety concerns. Thus, base editing strategies aiming to correct the genetic defect in patients' HSPCs could provide safe and effective treatment. Here, we developed a strategy to correct one of the most prevalent BT mutations (IVS1-110 [G>A]) using the SpRY-ABE8e base editor. RNA delivery of the base editing system was safe and led to ∼80% of gene correction in the HSPCs of patients with BT without causing dangerous double-strand DNA breaks. In HSPC-derived erythroid populations, this strategy was able to restore β-globin production and correct inefficient erythropoiesis typically observed in BT both in vitro and in vivo. In conclusion, this proof-of-concept study paves the way for the development of a safe and effective autologous gene therapy approach for BT.}, }
@article {pmid36434096, year = {2023}, author = {Jin, S and Lin, Q and Gao, Q and Gao, C}, title = {Optimized prime editing in monocot plants using PlantPegDesigner and engineered plant prime editors (ePPEs).}, journal = {Nature protocols}, volume = {18}, number = {3}, pages = {831-853}, pmid = {36434096}, issn = {1750-2799}, mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; Genome, Plant ; Plants/genetics ; DNA ; RNA, Guide, Kinetoplastida ; }, abstract = {Prime editors (PEs), which can install desired base edits without donor DNA or double-strand breaks, have been used in plants and can, in principle, accelerate crop improvement and breeding. However, their editing efficiency in plants is generally low. Optimizing the prime editing guide RNA (pegRNA) by designing the sequence on the basis of melting temperature, using dual-pegRNAs and engineering PEs have all been shown to enhance PE efficiency. In addition, an automated pegRNA design platform, PlantPegDesigner, has been developed on the basis of rice prime editing experimental data. In this protocol, we present detailed protocols for designing and optimizing pegRNAs using PlantPegDesigner, constructing engineered plant PE vectors with enhanced editing efficiency for prime editing, evaluating prime editing efficiencies using a reporter system and comparing the effectiveness and byproducts of PEs by deep amplicon sequencing. Using this protocol, researchers can construct optimized pegRNAs for prime editing in 4-7 d and obtain prime-edited rice or wheat plants within 3 months.}, }
@article {pmid35915232, year = {2023}, author = {Hu, X and Zhang, B and Li, X and Li, M and Wang, Y and Dan, H and Zhou, J and Wei, Y and Ge, K and Li, P and Song, Z}, title = {The application and progression of CRISPR/Cas9 technology in ophthalmological diseases.}, journal = {Eye (London, England)}, volume = {37}, number = {4}, pages = {607-617}, pmid = {35915232}, issn = {1476-5454}, mesh = {Humans ; *CRISPR-Cas Systems ; Gene Editing ; Genetic Therapy ; *Retinitis Pigmentosa/genetics ; }, abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease (Cas) system is an adaptive immune defence system that has gradually evolved in bacteria and archaea to combat invading viruses and exogenous DNA. Advances in technology have enabled researchers to enhance their understanding of the immune process in vivo and its potential for use in genome editing. Thus far, applications of CRISPR/Cas9 genome editing technology in ophthalmology have included gene therapy for corneal dystrophy, glaucoma, congenital cataract, Leber's congenital amaurosis, retinitis pigmentosa, Usher syndrome, fundus neovascular disease, proliferative vitreoretinopathy, retinoblastoma and other eye diseases. Additionally, the combination of CRISPR/Cas9 genome editing technology with adeno-associated virus vector and inducible pluripotent stem cells provides further therapeutic avenues for the treatment of eye diseases. Nonetheless, many challenges remain in the development of clinically feasible retinal genome editing therapy. This review discusses the development, as well as mechanism of CRISPR/Cas9 and its applications and challenges in gene therapy for eye diseases.}, }
@article {pmid36897939, year = {2023}, author = {Elmore, JR and Dexter, GN and Baldino, H and Huenemann, JD and Francis, R and Peabody, GL and Martinez-Baird, J and Riley, LA and Simmons, T and Coleman-Derr, D and Guss, AM and Egbert, RG}, title = {High-throughput genetic engineering of nonmodel and undomesticated bacteria via iterative site-specific genome integration.}, journal = {Science advances}, volume = {9}, number = {10}, pages = {eade1285}, doi = {10.1126/sciadv.ade1285}, pmid = {36897939}, issn = {2375-2548}, abstract = {Efficient genome engineering is critical to understand and use microbial functions. Despite recent development of tools such as CRISPR-Cas gene editing, efficient integration of exogenous DNA with well-characterized functions remains limited to model bacteria. Here, we describe serine recombinase-assisted genome engineering, or SAGE, an easy-to-use, highly efficient, and extensible technology that enables selection marker-free, site-specific genome integration of up to 10 DNA constructs, often with efficiency on par with or superior to replicating plasmids. SAGE uses no replicating plasmids and thus lacks the host range limitations of other genome engineering technologies. We demonstrate the value of SAGE by characterizing genome integration efficiency in five bacteria that span multiple taxonomy groups and biotechnology applications and by identifying more than 95 heterologous promoters in each host with consistent transcription across environmental and genetic contexts. We anticipate that SAGE will rapidly expand the number of industrial and environmental bacteria compatible with high-throughput genetics and synthetic biology.}, }
@article {pmid36897609, year = {2023}, author = {Yang, K and Qian, J and Zhang, C and Wang, Z and Huang, Q and Shi, G and Zhang, Z and Yang, Y and Han, X}, title = {Biogenic materials for CRISPR delivery and therapeutics.}, journal = {Biomaterials science}, volume = {}, number = {}, pages = {}, doi = {10.1039/d2bm02169b}, pmid = {36897609}, issn = {2047-4849}, abstract = {CRISPR, as an emerging gene-editing technology, has been widely used in multidisciplinary fields, including genetic diseases and some cancers. However, it remains a challenge to efficiently deliver CRISPR for safe and efficient genome editing. Currently, biomimetic materials have become an attractive delivery strategy for CRISPR-mediated genome editing due to their low immunogenicity and application safety. The biomimetic materials delivery is involved in the improvement of cellular uptake of nanoparticle vectors, and the gene editing efficiency. In this review, we summarize the current delivery strategies of CRISPR/Cas systems based on biogenic materials such as viruses, bacteria, cells, bioactive substances, etc., focusing on the potential applications in disease research and therapy. Finally, the prospects and limitations of CRISPR-based systems in therapeutics are discussed.}, }
@article {pmid36896234, year = {2023}, author = {Petersen, B}, title = {Editorial: Genetic engineering in farm animals.}, journal = {Frontiers in genetics}, volume = {14}, number = {}, pages = {1155201}, pmid = {36896234}, issn = {1664-8021}, }
@article {pmid36896016, year = {2023}, author = {Hu, M and Bao, W and Peng, Q and Hu, W and Yang, X and Xiang, Y and Yan, X and Li, M and Xu, P and He, Q and Yang, S}, title = {Metabolic engineering of Zymomonas mobilis for co-production of D-lactic acid and ethanol using waste feedstocks of molasses and corncob residue hydrolysate.}, journal = {Frontiers in bioengineering and biotechnology}, volume = {11}, number = {}, pages = {1135484}, pmid = {36896016}, issn = {2296-4185}, abstract = {Lactate is the precursor for polylactide. In this study, a lactate producer of Z. mobilis was constructed by replacing ZMO0038 with LmldhA gene driven by a strong promoter PadhB, replacing ZMO1650 with native pdc gene driven by Ptet, and replacing native pdc with another copy of LmldhA driven by PadhB to divert carbon from ethanol to D-lactate. The resultant strain ZML-pdc-ldh produced 13.8 ± 0.2 g/L lactate and 16.9 ± 0.3 g/L ethanol using 48 g/L glucose. Lactate production of ZML-pdc-ldh was further investigated after fermentation optimization in pH-controlled fermenters. ZML-pdc-ldh produced 24.2 ± 0.6 g/L lactate and 12.9 ± 0.8 g/L ethanol as well as 36.2 ± 1.0 g/L lactate and 40.3 ± 0.3 g/L ethanol, resulting in total carbon conversion rate of 98.3% ± 2.5% and 96.2% ± 0.1% with final product productivity of 1.9 ± 0.0 g/L/h and 2.2 ± 0.0 g/L/h in RMG5 and RMG12, respectively. Moreover, ZML-pdc-ldh produced 32.9 ± 0.1 g/L D-lactate and 27.7 ± 0.2 g/L ethanol as well as 42.8 ± 0.0 g/L D-lactate and 53.1 ± 0.7 g/L ethanol with 97.1% ± 0.0% and 99.1% ± 0.8% carbon conversion rate using 20% molasses or corncob residue hydrolysate, respectively. Our study thus demonstrated that it is effective for lactate production by fermentation condition optimization and metabolic engineering to strengthen heterologous ldh expression while reducing the native ethanol production pathway. The capability of recombinant lactate-producer of Z. mobilis for efficient waste feedstock conversion makes it a promising biorefinery platform for carbon-neutral biochemical production.}, }
@article {pmid36895064, year = {2023}, author = {Li, ZH and Wang, J and Xu, JP and Wang, J and Yang, X}, title = {Recent advances in CRISPR-based genome editing technology and its applications in cardiovascular research.}, journal = {Military Medical Research}, volume = {10}, number = {1}, pages = {12}, pmid = {36895064}, issn = {2054-9369}, abstract = {The rapid development of genome editing technology has brought major breakthroughs in the fields of life science and medicine. In recent years, the clustered regularly interspaced short palindromic repeats (CRISPR)-based genome editing toolbox has been greatly expanded, not only with emerging CRISPR-associated protein (Cas) nucleases, but also novel applications through combination with diverse effectors. Recently, transposon-associated programmable RNA-guided genome editing systems have been uncovered, adding myriads of potential new tools to the genome editing toolbox. CRISPR-based genome editing technology has also revolutionized cardiovascular research. Here we first summarize the advances involving newly identified Cas orthologs, engineered variants and novel genome editing systems, and then discuss the applications of the CRISPR-Cas systems in precise genome editing, such as base editing and prime editing. We also highlight recent progress in cardiovascular research using CRISPR-based genome editing technologies, including the generation of genetically modified in vitro and animal models of cardiovascular diseases (CVD) as well as the applications in treating different types of CVD. Finally, the current limitations and future prospects of genome editing technologies are discussed.}, }
@article {pmid36893938, year = {2023}, author = {Hwang, S and Maxwell, KL}, title = {Diverse mechanisms of CRISPR-Cas9 inhibition by type II anti-CRISPR proteins.}, journal = {Journal of molecular biology}, volume = {}, number = {}, pages = {168041}, doi = {10.1016/j.jmb.2023.168041}, pmid = {36893938}, issn = {1089-8638}, abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas (CRISPR-associated proteins) systems provide bacteria and archaea with an adaptive immune response against invasion by mobile genetic elements like phages, plasmids, and transposons. These systems have been repurposed as very powerful biotechnological tools for gene editing applications in both bacterial and eukaryotic systems. The discovery of natural off-switches for CRISPR-Cas systems, known as anti-CRISPR proteins, provided a mechanism for controlling CRISPR-Cas activity and opened avenues for the development of more precise editing tools. In this review, we focus on the inhibitory mechanisms of anti-CRISPRs that are active against type II CRISPR-Cas systems and briefly discuss their biotechnological applications.}, }
@article {pmid36893718, year = {2023}, author = {Yang, H and Ledesma-Amaro, R and Gao, H and Ren, Y and Deng, R}, title = {CRISPR-based biosensors for pathogenic biosafety.}, journal = {Biosensors &amp; bioelectronics}, volume = {228}, number = {}, pages = {115189}, doi = {10.1016/j.bios.2023.115189}, pmid = {36893718}, issn = {1873-4235}, abstract = {Pathogenic biosafety is a worldwide concern. Tools for analyzing pathogenic biosafety, that are precise, rapid and field-deployable, are highly demanded. Recently developed biotechnological tools, especially those utilizing CRISPR/Cas systems which can couple with nanotechnologies, have enormous potential to achieve point-of-care (POC) testing for pathogen infection. In this review, we first introduce the working principle of class II CRISPR/Cas system for detecting nucleic acid and non-nucleic acid biomarkers, and highlight the molecular assays that leverage CRISPR technologies for POC detection. We summarize the application of CRISPR tools in detecting pathogens, including pathogenic bacteria, viruses, fungi and parasites and their variants, and highlight the profiling of pathogens' genotypes or phenotypes, such as the viability, and drug-resistance. In addition, we discuss the challenges and opportunities of CRISPR-based biosensors in pathogenic biosafety analysis.}, }
@article {pmid36881658, year = {2023}, author = {Yau, WW and Chen, GB and Zhou, J and Francisco, JC and Thimmukonda, NK and Li, S and Singh, BK and Yen, PM}, title = {Nicotinamide riboside rescues dysregulated glycolysis and fatty acid β-oxidation in a human hepatic cell model of Citrin deficiency.}, journal = {Human molecular genetics}, volume = {}, number = {}, pages = {}, doi = {10.1093/hmg/ddad018}, pmid = {36881658}, issn = {1460-2083}, abstract = {Citrin Deficiency (CD) is an inborn error of metabolism caused by loss-of-function of the mitochondrial aspartate/glutamate transporter, CITRIN, which is involved in both the urea cycle and malate aspartate shuttle. Patients with CD develop hepatosteatosis and hyperammonemia but there is no effective therapy for CD. Currently, there are no animal models that faithfully recapitulate the human CD phenotype. Accordingly, we generated a CITRIN knockout HepG2 cell line using CRISPR/Cas 9 genome editing technology to study metabolic and cell signaling defects in CD. CITRIN KO cells showed increased ammonia accumulation, higher cytosolic NADH/NAD+ ratio and reduced glycolysis. Surprisingly, these cells showed impaired fatty acid metabolism and mitochondrial activity. CITRIN KO cells also displayed increased cholesterol and bile acid metabolism resembling those observed in CD patients. Remarkably, normalizing cytosolic NADH:NAD+ ratio by nicotinamide riboside (NR) increased glycolysis and fatty acid oxidation but had no effect on the hyperammonemia suggesting the urea cycle defect was independent of the aspartate/malate shuttle defect of CD. The correction of glycolysis and fatty acid metabolism defects in CITRIN KO cells by reducing cytoplasmic NADH:NAD+ levels suggests this may be a novel strategy to treat some of the metabolic defects of CD and other mitochondrial diseases.}, }
@article {pmid36881621, year = {2023}, author = {Chauhan, VP and Sharp, PA and Langer, R}, title = {Altered DNA repair pathway engagement by engineered CRISPR-Cas9 nucleases.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {120}, number = {11}, pages = {e2300605120}, doi = {10.1073/pnas.2300605120}, pmid = {36881621}, issn = {1091-6490}, mesh = {*CRISPR-Cas Systems/genetics ; *INDEL Mutation ; Mutation ; Culture ; DNA End-Joining Repair/genetics ; Endonucleases/genetics ; }, abstract = {CRISPR-Cas9 introduces targeted DNA breaks that engage competing DNA repair pathways, producing a spectrum of imprecise insertion/deletion mutations (indels) and precise templated mutations (precise edits). The relative frequencies of these pathways are thought to primarily depend on genomic sequence and cell state contexts, limiting control over mutational outcomes. Here, we report that engineered Cas9 nucleases that create different DNA break structures engage competing repair pathways at dramatically altered frequencies. We accordingly designed a Cas9 variant (vCas9) that produces breaks which suppress otherwise dominant nonhomologous end-joining (NHEJ) repair. Instead, breaks created by vCas9 are predominantly repaired by pathways utilizing homologous sequences, specifically microhomology-mediated end-joining (MMEJ) and homology-directed repair (HDR). Consequently, vCas9 enables efficient precise editing through HDR or MMEJ while suppressing indels caused by NHEJ in dividing and nondividing cells. These findings establish a paradigm of targeted nucleases custom-designed for specific mutational applications.}, }
@article {pmid36881252, year = {2023}, author = {Zha, MJ and Cai, CE and He, PM}, title = {Outlook on the Security and Potential Improvements of CRISPR-Cas9.}, journal = {Molecular biotechnology}, volume = {}, number = {}, pages = {}, pmid = {36881252}, issn = {1559-0305}, abstract = {Gene editing technology is regarded as a good news to save patients with genetic diseases because of its significant function of specifically changing genetic information. From zinc-finger proteins to transcription activator-like effector protein nucleases gene editing tools are constantly updated. At the same time, scientists are constantly developing a variety of new gene editing therapy strategies, in order to promote gene editing therapy from various aspects and realize the maturity of the technology as soon as possible. In 2016, CRISPR-Cas9-mediated CAR-T therapy was the first to enter the clinical trial stage, indicating that the use of CRISPR-Cas system as the blade of the genetic lancet to save patients is officially on the schedule. The first challenge to achieve this exciting goal is to improve the security of the technology. This review will introduce the gene security issues faced by the CRISPR system as a clinical treatment tool, the current safer delivery methods and the newly developed CRISPR editing tools with higher precision. Many reviews summarize the means of improving the security of gene editing therapy and the comprehensive delivery method, while few articles focus on the threat of gene editing to the genomic security of the treatment target. Therefore, this review focuses on the risks brought by gene editing therapy to the patient genome, which provides a broader perspective for exploring and improving the security of gene editing therapy from two aspects of delivery system and CRISPR editing tools.}, }
@article {pmid36880733, year = {2023}, author = {Popescu, B and Shannon, K}, title = {Sidestepping SHP2 inhibition.}, journal = {The Journal of experimental medicine}, volume = {220}, number = {5}, pages = {}, doi = {10.1084/jem.20230082}, pmid = {36880733}, issn = {1540-9538}, mesh = {*CRISPR-Cas Systems/genetics ; *Mitogen-Activated Protein Kinases ; }, abstract = {Allosteric SHP2 inhibitors are a novel class of compounds that target hyperactive Ras/Mitogen Activated Protein Kinase (MAPK) signaling. In this issue of JEM, Wei et al. (2023. J. Exp. Med.https://doi.org/10.1084/jem.20221563) report a genome-wide CRISPR/Cas9 knockout screen that uncovered novel mechanisms of adaptive resistance to pharmacologic inhibition of SHP2.}, }
@article {pmid36880696, year = {2023}, author = {Hernandes-Lopes, J and Yassitepe, JECT and Koltun, A and Pauwels, L and Silva, VCHD and Dante, RA and Gerhardt, IR and Arruda, P}, title = {Genome editing in maize: Toward improving complex traits in a global crop.}, journal = {Genetics and molecular biology}, volume = {46}, number = {1 Suppl 1}, pages = {e20220217}, pmid = {36880696}, issn = {1415-4757}, abstract = {Recent advances in genome editing have enormously enhanced the effort to develop biotechnology crops for more sustainable food production. CRISPR/Cas, the most versatile genome-editing tool, has shown the potential to create genome modifications that range from gene knockout and gene expression pattern modulations to allele-specific changes in order to design superior genotypes harboring multiple improved agronomic traits. However, a frequent bottleneck is the delivery of CRISPR/Cas to crops that are less amenable to transformation and regeneration. Several technologies have recently been proposed to overcome transformation recalcitrance, including HI-Edit/IMGE and ectopic/transient expression of genes encoding morphogenic regulators. These technologies allow the eroding of the barriers that make crops inaccessible for genome editing. In this review, we discuss the advances in genome editing in crops with a particular focus on the use of technologies to improve complex traits such as water use efficiency, drought stress, and yield in maize.}, }
@article {pmid36880183, year = {2023}, author = {Punetha, M and Saini, S and Chaudhary, S and S Yadav, P and Whitworth, K and Green, J and Kumar, D and Kues, W}, title = {Induced Pluripotent Stem Cells in the Era of Precise Genome Editing.}, journal = {Current stem cell research &amp; therapy}, volume = {}, number = {}, pages = {}, doi = {10.2174/1574888X18666230307115326}, pmid = {36880183}, issn = {2212-3946}, abstract = {Genome editing has enhanced our ability to understand the role of genetics in a number of diseases by facilitating the development of more precise cellular and animal models to study pathophysiological processes. These advances have shown extraordinary promise in a multitude of areas, from basic research to applied bioengineering and biomedical research. Induced pluripotent stem cells (iPSCs) are known for their high replicative capacity and are excellent targets for genetic manipulation as they can be clonally expanded from a single cell without compromising their pluripotency. Clustered, regularly interspaced short palindromic repeats (CRISPR) and CRISPR/Cas RNA-guided nucleases have rapidly become the method of choice for gene editing due to their high specificity, simplicity, low cost, and versatility. Coupling the cellular versatility of iPSCs differentiation with CRISPR/Cas9-mediated genome editing technology can be an effective experimental technique for providing new insights into the therapeutic use of this technology. However, before using these techniques for gene therapy, their therapeutic safety and efficacy following models need to be assessed. In this review, we cover the remarkable progress that has been made in the use of genome editing tools in iPSCs, their applications in disease research and gene therapy as well as the hurdles that remain in the actual implementation of CRISPR/Cas systems.}, }
@article {pmid36878916, year = {2023}, author = {Park, J and Cui, G and Lee, H and Jeong, H and Kwak, JJ and Lee, J and Byeon, SH}, title = {CRISPR/Cas9 mediated specific ablation of vegfa in retinal pigment epithelium efficiently regresses choroidal neovascularization.}, journal = {Scientific reports}, volume = {13}, number = {1}, pages = {3715}, pmid = {36878916}, issn = {2045-2322}, mesh = {Humans ; Animals ; Mice ; Retinal Pigment Epithelium ; CRISPR-Cas Systems ; *Choroidal Neovascularization/genetics/therapy ; Retina ; *Craniocerebral Trauma ; Disease Models, Animal ; Vascular Endothelial Growth Factor A/genetics ; }, abstract = {The CRISPR/Cas9 system easily edits target genes in various organisms and is used to treat human diseases. In most therapeutic CRISPR studies, ubiquitously expressed promoters, such as CMV, CAG, and EF1α, are used; however, gene editing is sometimes necessary only in specific cell types relevant to the disease. Therefore, we aimed to develop a retinal pigment epithelium (RPE)-specific CRISPR/Cas9 system. We developed a CRISPR/Cas9 system that operates only in retinal pigment epithelium (RPE) by expressing Cas9 under the RPE-specific vitelliform macular dystrophy 2 promoter (pVMD2). This RPE-specific CRISPR/pVMD2-Cas9 system was tested in human retinal organoid and mouse model. We confirmed that this system works specifically in the RPE of human retinal organoids and mouse retina. In addition, the RPE-specific Vegfa ablation using the novel CRISPR-pVMD2-Cas9 system caused regression of choroidal neovascularization (CNV) without unwanted knock-out in the neural retina in laser-induced CNV mice, which is a widely used animal model of neovascular age-related macular degeneration. RPE-specific Vegfa knock-out (KO) and ubiquitous Vegfa KO were comparable in the efficient regression of CNV. The promoter substituted, cell type-specific CRISPR/Cas9 systems can be used in specific 'target cell' therapy, which edits genes while reducing unwanted off- 'target cell' effects.}, }
@article {pmid36812357, year = {2023}, author = {Jiang, Y and Zheng, C and Jin, M and Zhou, R and Wu, Q and Huang, F and Lou, Y and Zheng, L}, title = {An Ultrasensitive Colorimetric Foodborne Pathogenic Detection Method Using a CRISPR/Cas12a Mediated Strand Displacement/Hybridization Chain Reaction.}, journal = {Journal of agricultural and food chemistry}, volume = {71}, number = {9}, pages = {4193-4200}, doi = {10.1021/acs.jafc.2c08888}, pmid = {36812357}, issn = {1520-5118}, mesh = {*Colorimetry/methods ; Hydrogen Peroxide ; CRISPR-Cas Systems ; DNA ; *DNA, Catalytic/genetics ; }, abstract = {Accurate, rapid, and sensitive pathogenic detections play an important role in food safety. Herein, we developed a novel CRISPR/Cas12a mediated strand displacement/hybridization chain reaction (CSDHCR) nucleic acid assay for foodborne pathogenic colorimetric detection. A biotinylated DNA toehold is coupled on avidin magnetic beads and acts as an initiator strand to trigger the SDHCR. The SDHCR amplification allowed the formation of long hemin/G-quadruplex-based DNAzyme products to catalyze the TMB-H2O2 reaction. In the presence of the DNA targets, the trans-cleavage activity of CRISPR/Cas12a was activated to cleave the initiator DNA, resulting in the failure of SDHCR and no color change. Under optimal conditions, the CSDHCR has a satisfactory linear detection of DNA targets with a regression equation Y = 0.0531*X - 0.0091 (R[2] = 0.9903) in the range of 10 fM to 1 nM, and the limit of detection was determined as 4.54 fM. In addition, Vibrio vulnificus, one foodborne pathogen, was used to verify the practical application of the method, and it showed satisfactory specificity and sensitivity with a limit of detection at 1.0 × 10[0] CFU/mL coupling with recombinase polymerase amplification. Our proposed CSDHCR biosensor could be a promising alternative method for ultrasensitive and visual detection of nucleic acids and the practical application of foodborne pathogens.}, }
@article {pmid36811465, year = {2023}, author = {Xu, Y and Tian, N and Shi, H and Zhou, C and Wang, Y and Liang, FS}, title = {A Split CRISPR/Cas13b System for Conditional RNA Regulation and Editing.}, journal = {Journal of the American Chemical Society}, volume = {145}, number = {9}, pages = {5561-5569}, doi = {10.1021/jacs.3c01087}, pmid = {36811465}, issn = {1520-5126}, mesh = {*RNA/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; CRISPR-Cas Systems/genetics ; }, abstract = {The CRISPR/Cas13b system has been demonstrated as a robust tool for versatile RNA studies and relevant applications. New strategies enabling precise control of Cas13b/dCas13b activities and minimal interference with native RNA activities will further facilitate the understanding and regulation of RNA functions. Here, we engineered a split Cas13b system that can be conditionally activated and deactivated under the induction of abscisic acid (ABA), which achieved the downregulation of endogenous RNAs in dosage- and time-dependent manners. Furthermore, an ABA inducible split dCas13b system was generated to achieve temporally controlled deposition of m[6]A at specific sites on cellular RNAs through conditional assembly and disassembly of split dCas13b fusion proteins. We also showed that the activities of split Cas13b/dCas13b systems can be modulated by light via using a photoactivatable ABA derivative. Overall, these split Cas13b/dCas13b platforms expand the existing repertoire of the CRISPR and RNA regulation toolkit to achieve targeted manipulation of RNAs in native cellular environments with minimal functional disruption to these endogenous RNAs.}, }
@article {pmid36878037, year = {2023}, author = {Zhu, Y and Yin, S and Li, Z}, title = {Mechanism of inhibition of CRISPR-Cas9 by anti-CRISPR protein AcrIIC1.}, journal = {Biochemical and biophysical research communications}, volume = {654}, number = {}, pages = {34-39}, doi = {10.1016/j.bbrc.2023.02.065}, pmid = {36878037}, issn = {1090-2104}, abstract = {CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR associated) systems are bacterial and archaeal defense mechanisms against invading phages and viruses. To overcome these defenses, phages and other mobile genetic elements (MGEs) have evolved multiple anti-CRISPR proteins (Acrs) that can inhibit the function of CRISPR-Cas systems. The AcrIIC1 protein has been shown to be able to inhibit the activity of Neisseria meningitidis Cas9 (NmeCas9) in both bacteria and human cells. Here, we solve the structure of AcrIIC1 in complex with the HNH domain of NmeCas9 using X-ray crystallography. The structure shows that AcrIIC1 binds to the catalytic sites of the HNH domain, preventing it from accessing the DNA target. In addition, our biochemical data show that AcrIIC1 is a broad-spectrum inhibitor targeting Cas9 enzymes from different subtypes. Taken together, the structure and biochemical analysis reveal the molecular mechanism of AcrIIC1-mediated Cas9 inhibition and provide new insights into regulatory tools for Cas9-based applications.}, }
@article {pmid36802524, year = {2023}, author = {Shen, B and Li, L and Liu, C and Li, X and Li, X and Cheng, X and Wu, H and Yang, T and Cheng, W and Ding, S}, title = {Mesoporous Nanozyme-Enhanced DNA Tetrahedron Electrochemiluminescent Biosensor with Three-Dimensional Walking Nanomotor-Mediated CRISPR/Cas12a for Ultrasensitive Detection of Exosomal microRNA.}, journal = {Analytical chemistry}, volume = {95}, number = {9}, pages = {4486-4495}, doi = {10.1021/acs.analchem.2c05217}, pmid = {36802524}, issn = {1520-6882}, mesh = {Humans ; *MicroRNAs/analysis ; CRISPR-Cas Systems ; DNA/chemistry ; Photometry ; *Biosensing Techniques/methods ; }, abstract = {Exosomal microRNAs (exomiRNAs) have emerged as ideal biomarkers for early clinical diagnostics. The accurate detection of exomiRNAs plays a crucial role in facilitating clinical applications. Herein, an ultrasensitive electrochemiluminescent (ECL) biosensor was constructed using three-dimensional (3D) walking nanomotor-mediated CRISPR/Cas12a and tetrahedral DNA nanostructures (TDNs)-modified nanoemitters (TCPP-Fe@HMUiO@Au-ABEI) for exomiR-155 detection. Initially, the 3D walking nanomotor-mediated CRISPR/Cas12a strategy could effectively convert the target exomiR-155 into amplified biological signals for improving the sensitivity and specificity. Then, TCPP-Fe@HMUiO@Au nanozymes with excellent catalytic performance were used to amplify ECL signals because of the enhanced mass transfer and increased catalytic active sites, originating from its high surface areas (601.83 m[2]/g), average pore size (3.46 nm), and large pore volumes (0.52 cm[3]/g). Meanwhile, the TDNs as the scaffold to fabricate "bottom-up" anchor bioprobes could improve the trans-cleavage efficiency of Cas12a. Consequently, this biosensor achieved the limit of detection down to 273.20 aM ranging from 1.0 fM to 1.0 nM. Furthermore, the biosensor could discriminate breast cancer patients evidently by analyzing exomiR-155, and these results conformed to that of qRT-PCR. Thus, this work provides a promising tool for early clinical diagnostics.}, }
@article {pmid36877024, year = {2023}, author = {Bleriot, I and Blasco, L and Pacios, O and Fernández-García, L and López, M and Ortiz-Cartagena, C and Barrio-Pujante, A and Fernández-Cuenca, F and Pascual, &#xc1; and Martínez-Martínez, L and Oteo-Iglesias, J and Tomás, M}, title = {Proteomic Study of the Interactions between Phages and the Bacterial Host Klebsiella pneumoniae.}, journal = {Microbiology spectrum}, volume = {}, number = {}, pages = {e0397422}, doi = {10.1128/spectrum.03974-22}, pmid = {36877024}, issn = {2165-0497}, abstract = {Phages and bacteria have acquired resistance mechanisms for protection. In this context, the aims of the present study were to analyze the proteins isolated from 21 novel lytic phages of Klebsiella pneumoniae in search of defense mechanisms against bacteria and also to determine the infective capacity of the phages. A proteomic study was also conducted to investigate the defense mechanisms of two clinical isolates of K. pneumoniae infected by phages. For this purpose, the 21 lytic phages were sequenced and de novo assembled. The host range was determined in a collection of 47 clinical isolates of K. pneumoniae, revealing the variable infective capacity of the phages. Genome sequencing showed that all of the phages were lytic phages belonging to the order Caudovirales. Phage sequence analysis revealed that the proteins were organized in functional modules within the genome. Although most of the proteins have unknown functions, multiple proteins were associated with defense mechanisms against bacteria, including the restriction-modification system, the toxin-antitoxin system, evasion of DNA degradation, blocking of host restriction and modification, the orphan CRISPR-Cas system, and the anti-CRISPR system. Proteomic study of the phage-host interactions (i.e., between isolates K3574 and K3320, which have intact CRISPR-Cas systems, and phages vB_KpnS-VAC35 and vB_KpnM-VAC36, respectively) revealed the presence of several defense mechanisms against phage infection (prophage, defense/virulence/resistance, oxidative stress and plasmid proteins) in the bacteria, and of the Acr candidate (anti-CRISPR protein) in the phages. IMPORTANCE Researchers, including microbiologists and infectious disease specialists, require more knowledge about the interactions between phages and their bacterial hosts and about their defense mechanisms. In this study, we analyzed the molecular mechanisms of viral and bacterial defense in phages infecting clinical isolates of K. pneumoniae. Viral defense mechanisms included restriction-modification system evasion, the toxin-antitoxin (TA) system, DNA degradation evasion, blocking of host restriction and modification, and resistance to the abortive infection system, anti-CRISPR and CRISPR-Cas systems. Regarding bacterial defense mechanisms, proteomic analysis revealed expression of proteins involved in the prophage (FtsH protease modulator), plasmid (cupin phosphomannose isomerase protein), defense/virulence/resistance (porins, efflux pumps, lipopolysaccharide, pilus elements, quorum network proteins, TA systems, and methyltransferases), oxidative stress mechanisms, and Acr candidates (anti-CRISPR protein). The findings reveal some important molecular mechanisms involved in the phage-host bacterial interactions; however, further study in this field is required to improve the efficacy of phage therapy.}, }
@article {pmid36876071, year = {2023}, author = {Kaltenbrunner, A and Reimann, V and Hoffmann, UA and Aoyagi, T and Sakata, M and Nimura-Matsune, K and Watanabe, S and Steglich, C and Wilde, A and Hess, WR}, title = {Regulation of pSYSA defense plasmid copy number in Synechocystis through RNase E and a highly transcribed asRNA.}, journal = {Frontiers in microbiology}, volume = {14}, number = {}, pages = {1112307}, pmid = {36876071}, issn = {1664-302X}, abstract = {Synthetic biology approaches toward the development of cyanobacterial producer strains require the availability of appropriate sets of plasmid vectors. A factor for the industrial usefulness of such strains is their robustness against pathogens, such as bacteriophages infecting cyanobacteria. Therefore, it is of great interest to understand the native plasmid replication systems and the CRISPR-Cas based defense mechanisms already present in cyanobacteria. In the model cyanobacterium Synechocystis sp. PCC 6803, four large and three smaller plasmids exist. The ~100 kb plasmid pSYSA is specialized in defense functions by encoding all three CRISPR-Cas systems and several toxin-antitoxin systems. The expression of genes located on pSYSA depends on the plasmid copy number in the cell. The pSYSA copy number is positively correlated with the expression level of the endoribonuclease E. As molecular basis for this correlation we identified the RNase E-mediated cleavage within the pSYSA-encoded ssr7036 transcript. Together with a cis-encoded abundant antisense RNA (asRNA1), this mechanism resembles the control of ColE1-type plasmid replication by two overlapping RNAs, RNA I and II. In the ColE1 mechanism, two non-coding RNAs interact, supported by the small protein Rop, which is encoded separately. In contrast, in pSYSA the similar-sized protein Ssr7036 is encoded within one of the interacting RNAs and it is this mRNA that likely primes pSYSA replication. Essential for plasmid replication is furthermore the downstream encoded protein Slr7037 featuring primase and helicase domains. Deletion of slr7037 led to the integration of pSYSA into the chromosome or the other large plasmid pSYSX. Moreover, the presence of slr7037 was required for successful replication of a pSYSA-derived vector in another model cyanobacterium, Synechococcus elongatus PCC 7942. Therefore, we annotated the protein encoded by slr7037 as Cyanobacterial Rep protein A1 (CyRepA1). Our findings open new perspectives on the development of shuttle vectors for genetic engineering of cyanobacteria and of modulating the activity of the entire CRISPR-Cas apparatus in Synechocystis sp. PCC 6803.}, }
@article {pmid36874963, year = {2023}, author = {Elston, KM and Maeda, GP and Perreau, J and Barrick, JE}, title = {Addressing the challenges of symbiont-mediated RNAi in aphids.}, journal = {PeerJ}, volume = {11}, number = {}, pages = {e14961}, pmid = {36874963}, issn = {2167-8359}, mesh = {Animals ; Bees ; *Aphids ; RNA Interference ; Agriculture ; Biological Assay ; Endonucleases ; Escherichia coli ; RNA, Double-Stranded ; }, abstract = {Because aphids are global agricultural pests and models for bacterial endosymbiosis, there is a need for reliable methods to study and control their gene function. However, current methods available for aphid gene knockout and knockdown of gene expression are often unreliable and time consuming. Techniques like CRISPR-Cas genome editing can take several months to achieve a single gene knockout because they rely on aphids going through a cycle of sexual reproduction, and aphids often lack strong, consistent levels of knockdown when fed or injected with molecules that induce an RNA interference (RNAi) response. In the hopes of addressing these challenges, we attempted to adapt a new method called symbiont-mediated RNAi (smRNAi) for use in aphids. smRNAi involves engineering a bacterial symbiont of the insect to continuously supply double-stranded RNA (dsRNA) inside the insect body. This approach has been successful in thrips, kissing bugs, and honeybees. We engineered the laboratory Escherichia coli strain HT115 and the native aphid symbiont Serratia symbiotica CWBI-2.3[T] to produce dsRNA inside the gut of the pea aphid (Acyrthosiphon pisum) targeting salivary effector protein (C002) or ecdysone receptor genes. For C002 assays, we also tested co-knockdown with an aphid nuclease (Nuc1) to reduce RNA degradation. However, we found that smRNAi was not a reliable method for aphid gene knockdown under our conditions. We were unable to consistently achieve the expected phenotypic changes with either target. However, we did see indications that elements of the RNAi pathway were modestly upregulated, and expression of some targeted genes appeared to be somewhat reduced in some trials. We conclude with a discussion of the possible avenues through which smRNAi, and aphid RNAi in general, could be improved in the future.}, }
@article {pmid36873375, year = {2023}, author = {Ewaisha, R and Anderson, KS}, title = {Immunogenicity of CRISPR therapeutics-Critical considerations for clinical translation.}, journal = {Frontiers in bioengineering and biotechnology}, volume = {11}, number = {}, pages = {1138596}, pmid = {36873375}, issn = {2296-4185}, abstract = {CRISPR offers new hope for many patients and promises to transform the way we think of future therapies. Ensuring safety of CRISPR therapeutics is a top priority for clinical translation and specific recommendations have been recently released by the FDA. Rapid progress in the preclinical and clinical development of CRISPR therapeutics leverages years of experience with gene therapy successes and failures. Adverse events due to immunogenicity have been a major setback that has impacted the field of gene therapy. As several in vivo CRISPR clinical trials make progress, the challenge of immunogenicity remains a significant roadblock to the clinical availability and utility of CRISPR therapeutics. In this review, we examine what is currently known about the immunogenicity of CRISPR therapeutics and discuss several considerations to mitigate immunogenicity for the design of safe and clinically translatable CRISPR therapeutics.}, }
@article {pmid36869936, year = {2023}, author = {Zhou, S and Deng, L and Dong, J and Lu, P and Qi, N and Huang, Z and Yang, M and Huo, D and Hou, C}, title = {Electrochemical detection of the p53 gene using exponential amplification reaction (EXPAR) and CRISPR/Cas12a reactions.}, journal = {Mikrochimica acta}, volume = {190}, number = {4}, pages = {113}, pmid = {36869936}, issn = {1436-5073}, mesh = {Humans ; *CRISPR-Cas Systems ; *Genes, p53 ; DNA Primers ; Electrodes ; Fluorescence ; }, abstract = {An improved electrochemical sensor has been developed for sensitive detection of the p53 gene based on exponential amplification reaction (EXPAR) and CRISPR/Cas12a. Restriction endonuclease BstNI is introduced to specifically identify and cleave the p53 gene, generating primers to trigger the EXPAR cascade amplification. A large number of amplified products are then obtained to enable the lateral cleavage activity of CRISPR/Cas12a. For electrochemical detection, the amplified product activates Cas12a to digest the designed block probe, which allows the signal probe to be captured by the reduced graphene oxide-modified electrode (GCE/RGO), resulting in an enhanced electrochemical signal. Notably, the signal probe is labeled with large amounts of methylene blue (MB). Compared with traditional endpoint decoration, the special signal probe effectively amplifies the electrochemical signals by a factor of about 15. Experimental results show that the electrochemical sensor exhibits wide ranges from 500 aM to 10 pM and 10 pM to 1 nM, as well as a relatively low limit detection of 0.39 fM, which is about an order of magnitude lower than that of fluorescence detection. Moreover, the proposed sensor shows reliable application capability in real human serum, indicating that this work has great prospects for the construction of a CRISPR-based ultra-sensitive detection platform.}, }
@article {pmid36821893, year = {2023}, author = {Wang, Y and Peng, Y and Li, S and Han, D and Ren, S and Qin, K and Zhou, H and Han, T and Gao, Z}, title = {The development of a fluorescence/colorimetric biosensor based on the cleavage activity of CRISPR-Cas12a for the detection of non-nucleic acid targets.}, journal = {Journal of hazardous materials}, volume = {449}, number = {}, pages = {131044}, doi = {10.1016/j.jhazmat.2023.131044}, pmid = {36821893}, issn = {1873-3336}, mesh = {Male ; Humans ; CRISPR-Cas Systems ; Colorimetry ; Environmental Monitoring ; Hydrogels ; *Nucleic Acids ; Oligonucleotides ; DNA ; *Biosensing Techniques ; }, abstract = {Nano-biosensors are of great significance for the analysis and detection of important biological targets. Surprisingly, the CRISPR-Cas12a system not only provides us with excellent gene editing capabilities, it also plays an important role in biosensing due to its high base resolution and high levels of sensitivity. However, most CRISPR-Cas12a-based sensors are limited by their recognition and output modes, are therefore only utilized for the detection of nucleic acids using fluorescence as an output signal. In the present study, we further explored the potential application of CRISPR-Cas12a and developed a CRISPR-Cas12a-based fluorescence/colorimetric biosensor (UCNPs-Cas12a/hydrogel-MOF-Cas12a) that provides an efficient targeting system for small molecules and protein targets. These two sensors yield multiple types of signal outputs by converting the target molecule into a deoxyribonucleic acid (DNA) signal input system using aptamers, amplifying the DNA signal by catalyzed hairpin assembly (CHA), and then combining CRISPR-Cas12a with various nanomaterials. UCNPs-Cas12a/hydrogel-MOF-Cas12a exhibited prominent sensitivity and stability for the detection of estradiol (E2) and prostate-specific antigen (PSA), and was successfully applied for the detection of these targets in milk and serum samples. A major advantage of the hydrogel-MOF-Cas12a system is that the signal output can be observed directly. When combined with aptamers and nanomaterials, CRISPR-Cas12a can be used to target multiple targets, with a diverse array of signal outputs. Our findings create a foundation for the development of CRISPR-Cas12a-based technologies for application in the fields of food safety, environmental monitoring, and clinical diagnosis.}, }
@article {pmid36725926, year = {2023}, author = {Eccleston, A}, title = {CRISPR base editing protects the heart.}, journal = {Nature reviews. Drug discovery}, volume = {22}, number = {3}, pages = {179}, pmid = {36725926}, issn = {1474-1784}, mesh = {Humans ; *Gene Editing ; *Clustered Regularly Interspaced Short Palindromic Repeats ; CRISPR-Cas Systems ; }, }
@article {pmid36696824, year = {2023}, author = {Wang, W and Zhou, S and Cheng, Z and Ma, D and Liu, T}, title = {A glutathione-sensitive cationic polymer delivery of CRISPR-Cas9 RNA plasmid for targeting nasopharyngeal carcinoma gene therapy.}, journal = {Colloids and surfaces. B, Biointerfaces}, volume = {223}, number = {}, pages = {113146}, doi = {10.1016/j.colsurfb.2023.113146}, pmid = {36696824}, issn = {1873-4367}, mesh = {Humans ; Nasopharyngeal Carcinoma/genetics/therapy ; *CRISPR-Cas Systems ; Polymers ; Genetic Therapy/methods ; Plasmids ; Glutathione ; Oligopeptides/chemistry ; RNA, Small Interfering/genetics ; Amines/chemistry ; *Nasopharyngeal Neoplasms/genetics/therapy ; Tumor Microenvironment ; }, abstract = {CRISPR-Cas9 technology has been proven to be the most straightforward and accurate tool for gene therapy, but some limitations, such as the inefficient transfection or inability to precisely target, prevent the gene therapy from achieving the desired therapeutic effect. To overcome these, a kind of glutathione-sensitive cationic vectors, hyperbranched polyamide amine (HPAA) was designed for Delivery of CRISPR-Cas9 RNA plasmid, and the cyclic RGD (Arg-Gly-Asp) was conjugated for the targeted treatment of nasopharyngeal carcinoma (NPC). Disulfide bonds in HPAA segments can specifically respond to the high glutathione concentration in the tumor microenvironment. Meanwhile, RGD could especially interact to the integrin αvβ3 receptors which are highly expressed on the surface of NPC tumor cells. The results showed that more HPAA-RGD/SGK3-gRNA complexes could be uptaken by NPC HNE-1 cells after RGD was conjugated, and then the plasmid could be accumulated in the NPC tumor as well, which may assure the satisfied NPC therapy effect in vivo. In transfection assays, this complex showed the acceptable gene transfection efficiency in vitro and the obvious tumor inhibition effect in vivo, suggested a potential application in gene therapy to NPC.}, }
@article {pmid36669556, year = {2023}, author = {Yamada, K and Yamamoto, T and Uwasa, K and Osakabe, K and Takano, Y}, title = {The establishment of multiple knockout mutants of Colletotrichum orbiculare by CRISPR-Cas9 and Cre-loxP systems.}, journal = {Fungal genetics and biology : FG &amp; B}, volume = {165}, number = {}, pages = {103777}, doi = {10.1016/j.fgb.2023.103777}, pmid = {36669556}, issn = {1096-0937}, mesh = {*CRISPR-Cas Systems ; Quinic Acid ; Integrases/genetics/metabolism ; *Colletotrichum/genetics ; Gene Editing/methods ; }, abstract = {Colletotrichum orbiculare is employed as a model fungus to analyze molecular aspects of plant-fungus interactions. Although gene disruption via homologous recombination (HR) was established for C. orbiculare, this approach is laborious due to its low efficiency. Here we developed methods to generate multiple knockout mutants of C. orbiculare efficiently. We first found that CRISPR-Cas9 system massively promoted gene-targeting efficiency. By transiently introducing a CRISPR-Cas9 vector, more than 90% of obtained transformants were knockout mutants. Furthermore, we optimized a self-excision Cre-loxP marker recycling system for C. orbiculare because a limited availability of desired selective markers hampers sequential gene disruption. In this system, the integrated selective marker is removable from the genome via Cre recombinase driven by a xylose-inducible promoter, enabling the reuse of the same selective marker for the next transformation. Using our CRISPR-Cas9 and Cre-loxP systems, we attempted to identify functional sugar transporters involved in fungal virulence. Multiple disruptions of putative quinate transporter genes restricted fungal growth on media containing quinate as a sole carbon source, confirming their functionality as quinate transporters. However, our analyses showed that quinate acquisition was dispensable for infection to host plants. In addition, we successfully built mutations of 17 cellobiose transporter genes in a strain. From the data of knockout mutants that we established in this study, we inferred that repetitive rounds of gene disruption using CRISPR-Cas9 and Cre-loxP systems do not cause adverse effects on fungal virulence and growth. Therefore, these systems will be powerful tools to perform a systematic loss-of-function approach for C. orbiculare.}, }
@article {pmid36868398, year = {2023}, author = {Makarova, KS and I Wolf, Y and V Koonin, E}, title = {In silico approaches for prediction of anti-CRISPR proteins.}, journal = {Journal of molecular biology}, volume = {}, number = {}, pages = {168036}, doi = {10.1016/j.jmb.2023.168036}, pmid = {36868398}, issn = {1089-8638}, abstract = {Numerous viruses infecting bacteria and archaea encode CRISPR-Cas system inhibitors, known as anti-CRISPR proteins (Acr). The Acrs typically are highly specific for particular CRISPR variants, resulting in remarkable sequence and structural diversity and complicating accurate prediction and identification of Acrs. In addition to their intrinsic interest for understanding the coevolution of defense and counter-defense systems in prokaryotes, Acrs could be natural, potent on-off switches for CRISPR-based biotechnological tools, so their discovery, characterization and application are of major importance. Here we discuss the computational approaches for Acr prediction. Due to the enormous diversity and likely multiple origins of the Acrs, sequence similarity searches are of limited use. However, multiple features of protein and gene organization have been successfully harnessed to this end including small protein size and distinct amino acid compositions of the Acrs, association of acr genes in virus genomes with genes encoding helix-turn-helix proteins that regulate Acr expression (Acr-associated proteins, Aca), and presence of self-target