@article {pmid33626339, year = {2021}, author = {Gonçalves, OS and de Oliveira Souza, F and Bruckner, FP and Santana, MF and Alfenas-Zerbini, P}, title = {Widespread distribution of prophages signaling the potential for adaptability and pathogenicity evolution of Ralstonia solanacearum species complex.}, journal = {Genomics}, volume = {}, number = {}, pages = {}, doi = {10.1016/j.ygeno.2021.02.011}, pmid = {33626339}, issn = {1089-8646}, abstract = {Integrated bacteriophages (prophages) can impact host cells, affecting their lifestyle, genomic diversity, and fitness. However, many basic aspects of how these organisms affect the host cell remain poorly understood. Ralstonia solanacearum is a gram-negative plant pathogenic bacterium that encompasses a great diversity of ecotypes regarded as a species complex (R. solanacearum Species Complex - RSSC). RSSC genomes have a mosaic structure containing numerous elements, signaling the potential for its evolution through horizontal gene transfer. Here, we analyzed 120 Ralstonia spp. genomes from the public database to identify prophage sequences. In total, 379 prophage-like elements were found in the chromosome and megaplasmid of Ralstonia spp. These elements encode genes related to host fitness, virulence factors, antibiotic resistance, and niche adaptation, which might contribute to RSSC adaptability. Prophage-like elements are widespread into the complex in different species and geographic origins, suggesting that the RSSC phages are ancestrally acquired. Complete prophages belonging to the families Inoviridae, Myoviridae, and Siphoviridae were found, being the members of Inoviridae the most abundant. Analysis of CRISPR-Cas spacer sequences demonstrated the presence of prophages sequences that indicate successive infection events during bacterial evolution. Besides complete prophages, we also demonstrated 14 novel putative prophages integrated into Ralstonia spp. genomes. Altogether, our results provide insights into the diversity of prophages in RSSC genomes and suggest that these elements may deeply affect the virulence and host adaptation and shaping the genomes among the strains of this important pathogen.}, }
@article {pmid33626327, year = {2021}, author = {Turchiano, G and Andrieux, G and Klermund, J and Blattner, G and Pennucci, V and El Gaz, M and Monaco, G and Poddar, S and Mussolino, C and Cornu, TI and Boerries, M and Cathomen, T}, title = {Quantitative evaluation of chromosomal rearrangements in gene-edited human stem cells by CAST-Seq.}, journal = {Cell stem cell}, volume = {}, number = {}, pages = {}, doi = {10.1016/j.stem.2021.02.002}, pmid = {33626327}, issn = {1875-9777}, abstract = {Genome editing has shown great promise for clinical translation but also revealed the risk of genotoxicity caused by off-target effects of programmable nucleases. Here we describe chromosomal aberrations analysis by single targeted linker-mediated PCR sequencing (CAST-Seq), a preclinical assay to identify and quantify chromosomal aberrations derived from on-target and off-target activities of CRISPR-Cas nucleases or transcriptional activator-like effector nucleases (TALENs), respectively, in human hematopoietic stem cells (HSCs). Depending on the employed designer nuclease, CAST-Seq detected translocations in 0%-0.5% of gene-edited human CD34+ HSCs, and up to 20% of on-target loci harbored gross rearrangements. Moreover, CAST-Seq detected distinct types of chromosomal aberrations, such as homology-mediated translocations, that are mediated by homologous recombination and not off-target activity. CAST-Seq is a sensitive assay able to identify and quantify unintended chromosomal rearrangements in addition to the more typical mutations at off-target sites. CAST-Seq analyses may be particularly relevant for therapeutic genome editing to enable thorough risk assessment before clinical application of gene-edited products.}, }
@article {pmid33360156, year = {2021}, author = {Ghafouri-Fard, S and Shoorei, H and Abak, A and Abbas Raza, SH and Pichler, M and Taheri, M}, title = {Role of non-coding RNAs in modulating the response of cancer cells to paclitaxel treatment.}, journal = {Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie}, volume = {134}, number = {}, pages = {111172}, doi = {10.1016/j.biopha.2020.111172}, pmid = {33360156}, issn = {1950-6007}, mesh = {Animals ; Antineoplastic Agents/adverse effects/chemistry/*therapeutic use ; CRISPR-Cas Systems ; Drug Carriers ; Drug Compounding ; *Drug Resistance, Neoplasm/drug effects ; Gene Editing ; Gene Expression Regulation, Neoplastic ; Humans ; MicroRNAs/genetics/*metabolism ; Neoplasms/*drug therapy/genetics/metabolism/pathology ; Oligonucleotides/therapeutic use ; Paclitaxel/adverse effects/chemistry/*therapeutic use ; RNA, Long Noncoding/genetics/*metabolism ; Signal Transduction ; }, abstract = {Paclitaxel is a chemotherapeutic substance that is administered for treatment of an extensive spectrum of human malignancies. In spite of its potent short-term effects against tumor cells, resistance to paclitaxel occurs in a number of patients precluding its long-term application in these patients. Non-coding RNAs have been shown to influence response of cancer cells to this chemotherapeutic agent via different mechanisms. Mechanistically, these transcripts regulate expression of several genes particularly those being involved in the apoptotic processes. Lots of in vivo and in vitro assays have demonstrated the efficacy of oligonucleotide-mediated microRNAs (miRNA)/ long non-coding RNAs (lncRNA) silencing in enhancement of response of cancer cells to paclitaxel. Therefore, targeted therapies against non-coding RNAs have been suggested as applicable modalities for combatting resistance to this agent. In the present review, we provide a summary of studies which assessed the role of miRNAs and lncRNAs in conferring resistance to paclitaxel.}, }
@article {pmid33227699, year = {2021}, author = {Zhang, M and Eshraghian, EA and Jammal, OA and Zhang, Z and Zhu, X}, title = {CRISPR technology: The engine that drives cancer therapy.}, journal = {Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie}, volume = {133}, number = {}, pages = {111007}, doi = {10.1016/j.biopha.2020.111007}, pmid = {33227699}, issn = {1950-6007}, mesh = {Animals ; CRISPR-Associated Proteins/*genetics/metabolism ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Diffusion of Innovation ; *Gene Editing ; Gene Expression Regulation, Neoplastic ; Gene Transfer Techniques ; *Genetic Therapy/adverse effects ; Humans ; *Immunotherapy/adverse effects ; Molecular Diagnostic Techniques ; *Molecular Targeted Therapy/adverse effects ; Neoplasms/genetics/immunology/metabolism/*therapy ; Predictive Value of Tests ; }, abstract = {CRISPR gene editing technology belongs to the third generation of gene editing technology. Since its discovery, it has attracted the attention of a large number of researchers. Investigators have published a series of academic articles and obtained breakthrough research results through in-depth research. In recent years, this technology has developed rapidly and been widely applied in many fields, especially in medicine. This review focuses on concepts of CRISPR gene editing technology, its application in cancer treatments, its existing limitations, and the new progress in recent years for detailed analysis and sharing.}, }
@article {pmid32828318, year = {2020}, author = {Chen, W and Gao, D and Xie, L and Wang, A and Zhao, H and Guo, C and Sun, Y and Nie, Y and Hong, A and Xiong, S}, title = {SCF-FBXO24 regulates cell proliferation by mediating ubiquitination and degradation of PRMT6.}, journal = {Biochemical and biophysical research communications}, volume = {530}, number = {1}, pages = {75-81}, doi = {10.1016/j.bbrc.2020.06.007}, pmid = {32828318}, issn = {1090-2104}, mesh = {CRISPR-Cas Systems ; Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; F-Box Proteins/genetics/*metabolism ; Humans ; Mutation ; Neoplasm Invasiveness/genetics ; Neoplasms/genetics/metabolism ; Nuclear Proteins/genetics/*metabolism ; Protein-Arginine N-Methyltransferases/genetics/*metabolism ; *Proteolysis ; *Ubiquitination ; Up-Regulation ; }, abstract = {The protein arginine methyltransferase 6 (PRMT6) is a coregulator of gene expression by methylation of the histone H3 on arginine 2 (H3R2), H4R3 and H2AR3 [1,2]. PRMT6 is aberrantly expressed in various types of human cancer, and abnormal methylation in cancers caused by overexpression of PRMT6 is considered to correlate with poor recovery prognosis [3,4]. However, mechanisms that regulate PRMT6 protein stability in cells remain largely unknown. Here we identified that an orphan F-box protein, FBXO24, that binds to 270 to 275 amino acid residues of PRMT6 to cause polyubiquitination of lysine at position 369 of PRMT6, which mediates its degradation via the ubiquitin-proteasome pathway. Overexpression of FBXO24 or knockout of PRMT6 was found to inhibit cell proliferation, migration, and invasion in H1299 cells. PRMT6 K369R mutant became resistant to degradation. Overexpression of PRMT6 K369R caused cell cycle progression, resulting in cell proliferation. Thus, our data confirm that FBXO24 regulates cell proliferation by mediating ubiquitin-dependent proteasomal degradation of PRMT6.}, }
@article {pmid32642978, year = {2020}, author = {Afzal, S and Sirohi, P and Singh, NK}, title = {A review of CRISPR associated genome engineering: application, advances and future prospects of genome targeting tool for crop improvement.}, journal = {Biotechnology letters}, volume = {42}, number = {9}, pages = {1611-1632}, doi = {10.1007/s10529-020-02950-w}, pmid = {32642978}, issn = {1573-6776}, mesh = {*CRISPR-Cas Systems ; Crops, Agricultural ; Epigenomics ; *Gene Editing ; *Plants, Genetically Modified ; Transcription Activator-Like Effector Nucleases ; Zinc Finger Nucleases ; }, abstract = {The Cas9 nuclease initiates double-stranded breaks at the target position in DNA, which are repaired by the intracellular restoration pathways to eliminate or insert pieces of DNA. CRISPR-Cas9 is proficient and cost-effective since cutting is guided by a piece of RNA instead of protein. Emphasis on this technology, in contrast with two recognized genome editing platforms (i.e., ZFNs and TALENs), is provided. This review evaluates the benefits of chemically synthesized gRNAs as well as the integration of chemical amendments to improve gene editing efficiencies. CRISPR is an indispensable means in biological investigations and is now as well transforming varied fields of biotechnology and agriculture. Recent advancement in targetable epigenomic-editing tools allows researchers to dispense direct functional and transcriptional significance to locus-explicit chromatin adjustments encompassing gene regulation and editing. An account of diverse sgRNA design tools is provided, principally on their target competence prediction model, off-target recognition algorithm, and generation of instructive annotations. The modern systems that have been utilized to deliver CRISPR-Cas9 in vivo and in vitro for crop improvement viz. nutritional enhancement, production of drought-tolerant and disease-resistant plants, are also highlighted. The conclusion is focused on upcoming directions, biosafety concerns, and expansive prospects of CRISPR technologies.}, }
@article {pmid32314149, year = {2020}, author = {Yue, X and Xia, T and Wang, S and Dong, H and Li, Y}, title = {Highly efficient genome editing in N. gerenzanensis using an inducible CRISPR/Cas9-RecA system.}, journal = {Biotechnology letters}, volume = {42}, number = {9}, pages = {1699-1706}, doi = {10.1007/s10529-020-02893-2}, pmid = {32314149}, issn = {1573-6776}, support = {ZR2015CL001//Natural Science Foundation of Shandong Province/ ; }, mesh = {Actinobacteria/*genetics ; Actinomyces/genetics ; Bacterial Proteins/genetics ; CRISPR-Cas Systems/*genetics ; Escherichia coli/genetics ; Gene Editing/*methods ; Mutation/genetics ; Rec A Recombinases/*genetics ; }, abstract = {OBJECTIVE: To develop an inducible CRISPR/Cas9-Recombinase A (RecA) system to manipulate genes in Nonomuraea gerenzanensis effectively.<br><br>RESULTS: Compared with traditional homologous recombination, the inducible CRISPR/Cas9 system achieved 68.8% editing efficiency, whereas, with both the inducible Cas9 and the overexpressed RecA, the efficiency of the combined genome editing system reached 100%. The dbv23-deleted mutant obtained by the inducible CRISPR/Cas9-RecA system was confirmed to produce more A40926 with an approximate yield of 200 mg L-1 than that of around 150 mg L-1 produced by the wild-type strain.<br><br>CONCLUSIONS: This inducible CRISPR/Cas9-RecA system was successfully constructed and can be utilized as an efficient genome editing tool for Actinomyces able to shorten editing time simultaneously.}, }
@article {pmid32231122, year = {2020}, author = {Abbasi, F and Kodani, M and Emori, C and Kiyozumi, D and Mori, M and Fujihara, Y and Ikawa, M}, title = {CRISPR/Cas9-Mediated Genome Editing Reveals Oosp Family Genes are Dispensable for Female Fertility in Mice.}, journal = {Cells}, volume = {9}, number = {4}, pages = {}, pmid = {32231122}, issn = {2073-4409}, mesh = {Amino Acid Sequence ; Animals ; CRISPR-Cas Systems/*genetics ; Computer Simulation ; Conserved Sequence ; Female ; Fertility/*physiology ; Gene Deletion ; *Gene Editing ; Gene Expression Regulation ; HEK293 Cells ; Humans ; Mice, Knockout ; Mice, Mutant Strains ; *Multigene Family ; Ovary/metabolism ; Phenotype ; Pregnancy Proteins/chemistry/genetics/*metabolism ; }, abstract = {There are over 200 genes that are predicted to be solely expressed in the oocyte and ovary, and thousands more that have expression patterns in the female reproductive tract. Unfortunately, many of their physiological functions, such as their roles in oogenesis or fertilization, have yet to be elucidated. Previous knockout (KO) mice studies have proven that many of the genes that were once thought to be essential for fertility are dispensable in vivo. Therefore, it is extremely important to confirm the roles of all genes before spending immense time studying them in vitro. To do this, our laboratory analyzes the functions of ovary and oocyte-enriched genes in vivo through generating CRISPR/Cas9 KO mice and examining their fertility. In this study, we have knocked out three Oosp family genes (Oosp1, Oosp2, and Oosp3) that have expression patterns linked to the female reproductive system and found that the triple KO (TKO) mutant mice generated exhibited decreased prolificacy but were not infertile; thus, these genes may potentially be dispensable for fertility. We also generated Cd160 and Egfl6 KO mice and found these genes are individually dispensable for female fertility. KO mice with no phenotypic data are seldom published, but we believe that this information must be shared to prevent unnecessary experimentation by other laboratories.}, }
@article {pmid32038657, year = {2020}, author = {Cohen, LB and Lindsay, SA and Xu, Y and Lin, SJH and Wasserman, SA}, title = {The Daisho Peptides Mediate Drosophila Defense Against a Subset of Filamentous Fungi.}, journal = {Frontiers in immunology}, volume = {11}, number = {}, pages = {9}, pmid = {32038657}, issn = {1664-3224}, support = {R01 GM050545/GM/NIGMS NIH HHS/United States ; }, mesh = {Animals ; Animals, Genetically Modified ; Antimicrobial Cationic Peptides/genetics/*metabolism ; CRISPR-Cas Systems ; Candida glabrata/*immunology ; Drosophila Proteins/genetics/*metabolism ; Drosophila melanogaster/genetics/*immunology/metabolism/*microbiology ; Enterococcus faecalis/*immunology ; Fusarium/*immunology ; Gene Knockout Techniques ; Host-Pathogen Interactions/genetics/immunology ; Hyphae/immunology ; Immunity, Innate ; Signal Transduction/genetics/immunology ; }, abstract = {Fungal infections, widespread throughout the world, affect a broad range of life forms, including agriculturally relevant plants, humans, and insects. In defending against fungal infections, the fruit fly Drosophila melanogaster employs the Toll pathway to induce a large number of immune peptides. Some have been investigated, such as the antimicrobial peptides (AMPs) and Bomanins (Boms); many, however, remain uncharacterized. Here, we examine the role in innate immunity of two related peptides, Daisho1 and Daisho2 (formerly IM4 and IM14, respectively), found in hemolymph following Toll pathway activation. By generating a CRISPR/Cas9 knockout of both genes, Δdaisho, we find that the Daisho peptides are required for defense against a subset of filamentous fungi, including Fusarium oxysporum, but not other Toll-inducible pathogens, such as Enterococcus faecalis and Candida glabrata. Analysis of null alleles and transgenes revealed that the two daisho genes are each required for defense, although their functions partially overlap. Generating and assaying a genomic epitope-tagged Daisho2 construct, we detected interaction in vitro of Daisho2 peptide in hemolymph with the hyphae of F. oxysporum. Together, these results identify the Daisho peptides as a new class of innate immune effectors with humoral activity against a select set of filamentous fungi.}, }
@article {pmid33620655, year = {2021}, author = {Golz, JC and Stingl, K}, title = {Natural Competence and Horizontal Gene Transfer in Campylobacter.}, journal = {Current topics in microbiology and immunology}, volume = {431}, number = {}, pages = {265-292}, pmid = {33620655}, issn = {0070-217X}, abstract = {Thermophilic Campylobacter, in particular Campylobacter jejuni, C. coli and C. lari are the main relevant Campylobacter species for human infections. Due to their high capacity of genetic exchange by horizontal gene transfer (HGT), rapid adaptation to changing environmental and host conditions contribute to successful spreading and persistence of these foodborne pathogens. However, extensive HGT can exert dangerous side effects for the bacterium, such as the incorporation of gene fragments leading to disturbed gene functions. Here we discuss mechanisms of HGT, notably natural transformation, conjugation and bacteriophage transduction and limiting regulatory strategies of gene transfer. In particular, we summarize the current knowledge on how the DNA macromolecule is exchanged between single cells. Mechanisms to stimulate and to limit HGT obviously coevolved and maintained an optimal balance. Chromosomal rearrangements and incorporation of harmful mutations are risk factors for survival and can result in drastic loss of fitness. In Campylobacter, the restricted recognition and preferential uptake of free DNA from relatives are mediated by a short methylated DNA pattern and not by a classical DNA uptake sequence as found in other bacteria. A class two CRISPR-Cas system is present but also other DNases and restriction-modification systems appear to be important for Campylobacter genome integrity. Several lytic and integrated bacteriophages have been identified, which contribute to genome diversity. Furthermore, we focus on the impact of gene transfer on the spread of antibiotic resistance genes (resistome) and persistence factors. We discuss remaining open questions in the HGT field, supposed to be answered in the future by current technologies like whole-genome sequencing and single-cell approaches.}, }
@article {pmid33619799, year = {2021}, author = {Whitbread, AL and Dorn, A and Röhrig, S and Puchta, H}, title = {Different functional roles of RTR complex factors in DNA repair and meiosis in Arabidopsis and tomato.}, journal = {The Plant journal : for cell and molecular biology}, volume = {}, number = {}, pages = {}, doi = {10.1111/tpj.15211}, pmid = {33619799}, issn = {1365-313X}, abstract = {The RTR (RecQ/Top3/Rmi1) complex has been elucidated as essential for ensuring genome stability in eukaryotes. Fundamental for the dissolution of Holliday junction (HJ)-like recombination intermediates, the factors have been shown to play further, partly distinct roles in DNA repair and homologous recombination. Across all kingdoms, disruption of this complex results in characteristic phenotypes including hyperrecombination and sensitivity to genotoxins. The type IA topoisomerase TOP3α has been shown as essential for viability in various animals. In contrast, in the model plant species Arabidopsis, the top3α mutant is viable. rmi1 mutants are deficient in the repair of DNA damage. Moreover, as opposed to other eukaryotes, TOP3α and RMI1 were found to be indispensable for proper meiotic progression, with mutants showing severe meiotic defects and sterility. We now established mutants of both TOP3α and RMI1 in tomato using CRISPR/Cas technology. Surprisingly, we found phenotypes that differed dramatically from those of Arabidopsis: The top3α mutants proved to be embryo-lethal, implying an essential role of the topoisomerase in tomato. In contrast, no defect in somatic DNA repair or meiosis was detectable for rmi1 mutants in tomato. This points to a differentiation of function of RTR complex partners between plant species. Our results indicate that there are relevant differences in the roles of basic factors involved in DNA repair and meiosis within dicotyledons, and thus should be taken as a note of caution when generalising knowledge regarding basic biological processes obtained in the model plant Arabidopsis for the entire plant kingdom.}, }
@article {pmid33619565, year = {2021}, author = {Wu, C and Tang, D and Cheng, J and Hu, D and Yang, Z and Ma, X and He, H and Yao, S and Fu, TM and Yu, Y and Chen, Q}, title = {Mechanisms of spacer acquisition by sequential assembly of the adaptation module in Synechocystis.}, journal = {Nucleic acids research}, volume = {}, number = {}, pages = {}, doi = {10.1093/nar/gkab105}, pmid = {33619565}, issn = {1362-4962}, abstract = {CRISPR-Cas immune systems process and integrate short fragments of DNA from new invaders as spacers into the host CRISPR locus to establish molecular memory of prior infection, which is also known as adaptation in the field. Some CRISPR-Cas systems rely on Cas1 and Cas2 to complete the adaptation process, which has been characterized in a few systems. In contrast, many other CRISPR-Cas systems require an additional factor of Cas4 for efficient adaptation, the mechanism of which remains less understood. Here we present biochemical reconstitution of the Synechocystis sp. PCC6803 type I-D adaptation system, X-ray crystal structures of Cas1-Cas2-prespacer complexes, and negative stained electron microscopy structure of the Cas4-Cas1 complex. Cas4 and Cas2 compete with each other to interact with Cas1. In the absence of prespacer, Cas4 but not Cas2 assembles with Cas1 into a very stable complex for processing the prespacer. Strikingly, the Cas1-prespacer complex develops a higher binding affinity toward Cas2 to form the Cas1-Cas2-prespacer ternary complex for integration. Together, we show a two-step sequential assembly mechanism for the type I-D adaptation module of Synechocystis, in which Cas4-Cas1 and Cas1-Cas2 function as two exclusive complexes for prespacer processing, capture, and integration.}, }
@article {pmid33589617, year = {2021}, author = {Hsu, JY and Grünewald, J and Szalay, R and Shih, J and Anzalone, AV and Lam, KC and Shen, MW and Petri, K and Liu, DR and Keith Joung, J and Pinello, L}, title = {PrimeDesign software for rapid and simplified design of prime editing guide RNAs.}, journal = {Nature communications}, volume = {12}, number = {1}, pages = {1034}, pmid = {33589617}, issn = {2041-1723}, support = {R00 HG008399/HG/NHGRI NIH HHS/United States ; R35 HG010717/HG/NHGRI NIH HHS/United States ; RM1 HG009490/HG/NHGRI NIH HHS/United States ; R35 GM118158/GM/NIGMS NIH HHS/United States ; RM1 HG009490/HG/NHGRI NIH HHS/United States ; U01 AI142756/AI/NIAID NIH HHS/United States ; R01 EB022376/EB/NIBIB NIH HHS/United States ; R35 GM118062/GM/NIGMS NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; }, mesh = {Base Pairing ; Base Sequence ; CRISPR-Associated Protein 9/genetics/metabolism ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Databases, Genetic ; Fabry Disease/genetics/metabolism/pathology ; Gene Editing/*methods ; *Genome, Human ; Green Fluorescent Proteins/genetics/metabolism ; HEK293 Cells ; Hemophilia A/genetics/metabolism/pathology ; Humans ; Models, Biological ; Muscular Dystrophy, Duchenne/genetics/metabolism/pathology ; Mutation ; Nucleic Acid Conformation ; Plasmids/chemistry/metabolism ; RNA, Guide/*genetics/metabolism ; Recombinant Fusion Proteins/genetics/metabolism ; }, abstract = {Prime editing (PE) is a versatile genome editing technology, but design of the required guide RNAs is more complex than for standard CRISPR-based nucleases or base editors. Here we describe PrimeDesign, a user-friendly, end-to-end web application and command-line tool for the design of PE experiments. PrimeDesign can be used for single and combination editing applications, as well as genome-wide and saturation mutagenesis screens. Using PrimeDesign, we construct PrimeVar, a comprehensive and searchable database that includes candidate prime editing guide RNA (pegRNA) and nicking sgRNA (ngRNA) combinations for installing or correcting >68,500 pathogenic human genetic variants from the ClinVar database. Finally, we use PrimeDesign to design pegRNAs/ngRNAs to install a variety of human pathogenic variants in human cells.}, }
@article {pmid33579329, year = {2021}, author = {Zhou, T and Yuan, Z and Weng, J and Pei, D and Du, X and He, C and Lai, P}, title = {Challenges and advances in clinical applications of mesenchymal stromal cells.}, journal = {Journal of hematology &amp; oncology}, volume = {14}, number = {1}, pages = {24}, pmid = {33579329}, issn = {1756-8722}, support = {2017YFE0131600//National Key Research and Development Project of China/ ; 81870121//National Natural Science Foundation of China/ ; 81671585//National Natural Science Foundation of China/ ; 82070176//National Natural Science Foundation of China/ ; 81700825//National Natural Science Foundation of China/ ; 2019B151502006//Natural Science Foundation of Guangdong Province/ ; 2019B020236004//Natural Science Foundation of Guangdong Province/ ; 201906010076//Science and Technology Program of Guangzhou/ ; 201803040005//Science and Technology Program of Guangzhou/ ; 2017B020230004//Science and Technology Planning Project of Guangdong Province/ ; }, mesh = {Animals ; Artificial Intelligence ; COVID-19/therapy ; CRISPR-Cas Systems ; Cell Differentiation ; Cell Movement ; Clinical Trials as Topic ; Extracellular Vesicles/genetics/immunology/transplantation ; Genetic Engineering/methods ; Humans ; *Mesenchymal Stem Cell Transplantation/methods ; Mesenchymal Stem Cells/*cytology/immunology/metabolism ; }, abstract = {Mesenchymal stromal cells (MSCs), also known as mesenchymal stem cells, have been intensely investigated for clinical applications within the last decades. However, the majority of registered clinical trials applying MSC therapy for diverse human diseases have fallen short of expectations, despite the encouraging pre-clinical outcomes in varied animal disease models. This can be attributable to inconsistent criteria for MSCs identity across studies and their inherited heterogeneity. Nowadays, with the emergence of advanced biological techniques and substantial improvements in bio-engineered materials, strategies have been developed to overcome clinical challenges in MSC application. Here in this review, we will discuss the major challenges of MSC therapies in clinical application, the factors impacting the diversity of MSCs, the potential approaches that modify MSC products with the highest therapeutic potential, and finally the usage of MSCs for COVID-19 pandemic disease.}, }
@article {pmid33563994, year = {2021}, author = {Li, J and Mahata, B and Escobar, M and Goell, J and Wang, K and Khemka, P and Hilton, IB}, title = {Programmable human histone phosphorylation and gene activation using a CRISPR/Cas9-based chromatin kinase.}, journal = {Nature communications}, volume = {12}, number = {1}, pages = {896}, pmid = {33563994}, issn = {2041-1723}, mesh = {Acetylation ; CRISPR-Associated Protein 9/genetics/metabolism ; *CRISPR-Cas Systems ; Cell Line, Tumor ; Chromatin/genetics ; Drug Resistance, Neoplasm/genetics ; Epigenomics/*methods ; Histones/*metabolism ; Humans ; Indoles/pharmacology ; Phosphorylation ; Promoter Regions, Genetic/genetics ; Ribosomal Protein S6 Kinases, 90-kDa/genetics/*metabolism ; Sulfonamides/pharmacology ; Transcriptional Activation ; }, abstract = {Histone phosphorylation is a ubiquitous post-translational modification that allows eukaryotic cells to rapidly respond to environmental stimuli. Despite correlative evidence linking histone phosphorylation to changes in gene expression, establishing the causal role of this key epigenomic modification at diverse loci within native chromatin has been hampered by a lack of technologies enabling robust, locus-specific deposition of endogenous histone phosphorylation. To address this technological gap, here we build a programmable chromatin kinase, called dCas9-dMSK1, by directly fusing nuclease-null CRISPR/Cas9 to a hyperactive, truncated variant of the human MSK1 histone kinase. Targeting dCas9-dMSK1 to human promoters results in increased target histone phosphorylation and gene activation and demonstrates that hyperphosphorylation of histone H3 serine 28 (H3S28ph) in particular plays a causal role in the transactivation of human promoters. In addition, we uncover mediators of resistance to the BRAF V600E inhibitor PLX-4720 in human melanoma cells using genome-scale screening with dCas9-dMSK1. Collectively, our findings enable a facile way to reshape human chromatin using CRISPR/Cas9-based epigenome editing and further define the causal link between histone phosphorylation and human gene activation.}, }
@article {pmid33404234, year = {2021}, author = {Kachwala, MJ and Smith, CW and Nandu, N and Yigit, MV}, title = {Reprogrammable Gel Electrophoresis Detection Assay Using CRISPR-Cas12a and Hybridization Chain Reaction.}, journal = {Analytical chemistry}, volume = {93}, number = {4}, pages = {1934-1938}, doi = {10.1021/acs.analchem.0c04949}, pmid = {33404234}, issn = {1520-6882}, mesh = {Biosensing Techniques/methods ; *CRISPR-Cas Systems ; DNA/chemistry ; Electrophoresis, Gel, Two-Dimensional/*methods ; Nucleic Acid Amplification Techniques/methods ; Nucleic Acid Hybridization/methods ; }, abstract = {Hybridization chain reaction (HCR) is a DNA-based target-induced cascade reaction. Due to its unique enzyme-free amplification feature, HCR is often employed for sensing applications. Much like DNA nanostructures that have been designed to respond to a specific stimulus, HCR employs nucleic acids that reconfigure and assemble in the presence of a specific trigger. Despite its standalone capabilities, HCR is highly modular; therefore, it can be advanced and repurposed when coupled with latest discoveries. To this effect, we have developed a gel electrophoresis-based detection approach which combines the signal amplification feature of HCR with the programmability and sensitivity of the CRISPR-Cas12a system. By incorporating CRISPR-Cas12a, we have achieved greater sensitivity and reversed the signal output from TURN OFF to TURN ON. CRISPR-Cas12a also enabled us to rapidly reprogram the assay for the detection of both ssDNA and dsDNA target sequences by replacing a single reaction component in the detection kit. Detection of conserved, both ssDNA and dsDNA, regions of tobacco curly shoot virus (TCSV) and hepatitis B virus (HepBV) genomes is demonstrated with this methodology. This low-cost gel electrophoresis assay can detect as little as 1.5 fmol of the target without any additional target amplification steps and is about 100-fold more sensitive than HCR-alone approach.}, }
@article {pmid33375604, year = {2020}, author = {Désaulniers, K and Ortiz, L and Dufour, C and Claudel, A and Plourde, MB and Merindol, N and Berthoux, L}, title = {Editing of the TRIM5 Gene Decreases the Permissiveness of Human T Lymphocytic Cells to HIV-1.}, journal = {Viruses}, volume = {13}, number = {1}, pages = {}, pmid = {33375604}, issn = {1999-4915}, mesh = {CRISPR-Cas Systems ; *Gene Editing ; Gene Expression Regulation ; Gene Knockdown Techniques ; HIV Infections/*genetics/*virology ; HIV-1/*physiology ; Host-Pathogen Interactions/genetics ; Humans ; Jurkat Cells ; RNA, Guide ; T-Lymphocytes/immunology/*metabolism/*virology ; Tripartite Motif Proteins/*genetics ; Ubiquitin-Protein Ligases/*genetics ; Viral Tropism/*genetics ; }, abstract = {Tripartite-motif-containing protein 5 isoform α (TRIM5α) is a cytoplasmic antiretroviral effector upregulated by type I interferons (IFN-I). We previously showed that two points mutations, R332G/R335G, in the retroviral capsid-binding region confer human TRIM5α the capacity to target and strongly restrict HIV-1 upon overexpression of the mutated protein. Here, we used clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9-mediated homology-directed repair (HDR) to introduce these two mutations in the endogenous human TRIM5 gene. We found 6 out of 47 isolated cell clones containing at least one HDR-edited allele. One clone (clone 6) had both alleles containing R332G, but only one of the two alleles containing R335G. Upon challenge with an HIV-1 vector, clone 6 was significantly less permissive compared to unmodified cells, whereas the cell clones with monoallelic modifications were only slightly less permissive. Following interferon (IFN)-β treatment, inhibition of HIV-1 infection in clone 6 was significantly enhanced (~40-fold inhibition). TRIM5α knockdown confirmed that HIV-1 was inhibited by the edited TRIM5 gene products. Quantification of HIV-1 reverse transcription products showed that inhibition occurred through the expected mechanism. In conclusion, we demonstrate the feasibility of potently inhibiting a viral infection through the editing of innate effector genes. Our results also emphasize the importance of biallelic modification in order to reach significant levels of inhibition by TRIM5α.}, }
@article {pmid33053801, year = {2020}, author = {Olson, A and Basukala, B and Lee, S and Gagne, M and Wong, WW and Henderson, AJ}, title = {Targeted Chromatinization and Repression of HIV-1 Provirus Transcription with Repurposed CRISPR/Cas9.}, journal = {Viruses}, volume = {12}, number = {10}, pages = {}, pmid = {33053801}, issn = {1999-4915}, support = {R01 AI138960/NH/NIH HHS/United States ; P30 AI042853/AI/NIAID NIH HHS/United States ; }, mesh = {Acetylation ; CRISPR-Cas Systems ; Cell Line ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Epigenesis, Genetic/genetics ; HEK293 Cells ; HIV Infections/virology ; HIV Long Terminal Repeat/genetics ; HIV-1/*genetics ; Histones/metabolism ; Humans ; Jurkat Cells ; Methylation ; Proviruses/*genetics ; RNA, Guide/*genetics ; Repressor Proteins/metabolism ; Transcription, Genetic/genetics ; Virus Activation/*genetics ; Virus Latency/*genetics ; }, abstract = {The major barrier to HIV-1 cure is the persistence of latent provirus, which is not eradicated by antiretroviral therapy. The &quot;shock and kill&quot; approach entails stimulating viral production with latency-reversing agents followed by the killing of cells actively producing the virus by immune clearance. However, this approach does not induce all intact proviruses, leaving a residual reservoir. CRISPR/Cas9 has been utilized to excise integrated Human Immunodeficiency Virus (HIV) DNA from infected cells in an RNA-guided, sequence-specific manner. Here, we seek to epigenetically silence the proviral DNA by introducing nuclease-deficient disabled Cas9 (dCas9) coupled with a transcriptional repressor domain derived from Kruppel-associated box (KRAB). We show that specific guide RNAs (gRNAs) and dCas9-KRAB repress HIV-1 transcription and reactivation of latent HIV-1 provirus. This repression is correlated with chromatin changes, including decreased H3 histone acetylation and increased histone H3 lysine 9 trimethylation, histone marks that are associated with transcriptional repression. dCas9-KRAB-mediated inhibition of HIV-1 transcription suggests that CRISPR can be engineered as a tool for block-and-lock strategies.}, }
@article {pmid32781607, year = {2020}, author = {Szillat, KP and Koethe, S and Wernike, K and Höper, D and Beer, M}, title = {A CRISPR/Cas9 Generated Bovine CD46-knockout Cell Line-A Tool to Elucidate the Adaptability of Bovine Viral Diarrhea Viruses (BVDV).}, journal = {Viruses}, volume = {12}, number = {8}, pages = {}, pmid = {32781607}, issn = {1999-4915}, mesh = {Adaptation, Biological ; Amino Acid Substitution ; Animals ; CRISPR-Cas Systems ; Cattle ; Diarrhea Virus 1, Bovine Viral/genetics/*physiology ; Diarrhea Virus 2, Bovine Viral/genetics/*physiology ; Dogs ; Gene Knockout Techniques ; Host-Pathogen Interactions ; Madin Darby Canine Kidney Cells ; Membrane Cofactor Protein/*genetics/metabolism ; Protein Multimerization ; Receptors, Virus/*genetics/metabolism ; Ribonucleoproteins/metabolism ; Viral Envelope Proteins/chemistry/*genetics/metabolism ; Virus Internalization ; Virus Replication ; }, abstract = {Bovine viral diarrhea virus (BVDV) entry into a host cell is mediated by the interaction of the viral glycoprotein E2 with the cellular transmembrane CD46 receptor. In this study, we generated a stable Madin-Darby Bovine Kidney (MDBK) CD46-knockout cell line to study the ability of different pestivirus A and B species (BVDV-1 and -2) to escape CD46-dependent cell entry. Four different BVDV-1/2 isolates showed a clearly reduced infection rate after inoculation of the knockout cells. However, after further passaging starting from the remaining virus foci on the knockout cell line, all tested virus isolates were able to escape CD46-dependency and grew despite the lack of the entry receptor. Whole-genome sequencing of the escape-isolates suggests that the genetic basis for the observed shift in infectivity is an amino acid substitution of an uncharged (glycine/asparagine) for a charged amino acid (arginine/lysine) at position 479 in the ERNS in three of the four isolates tested. In the fourth isolate, the exchange of a cysteine at position 441 in the ERNS resulted in a loss of ERNS dimerization that is likely to influence viral cell-to-cell spread. In general, the CD46-knockout cell line is a useful tool to analyze the role of CD46 for pestivirus replication and the virus-receptor interaction.}, }
@article {pmid32768421, year = {2020}, author = {Kushawah, G and Hernandez-Huertas, L and Abugattas-Nuñez Del Prado, J and Martinez-Morales, JR and DeVore, ML and Hassan, H and Moreno-Sanchez, I and Tomas-Gallardo, L and Diaz-Moscoso, A and Monges, DE and Guelfo, JR and Theune, WC and Brannan, EO and Wang, W and Corbin, TJ and Moran, AM and Sánchez Alvarado, A and Málaga-Trillo, E and Takacs, CM and Bazzini, AA and Moreno-Mateos, MA}, title = {CRISPR-Cas13d Induces Efficient mRNA Knockdown in Animal Embryos.}, journal = {Developmental cell}, volume = {54}, number = {6}, pages = {805-817.e7}, doi = {10.1016/j.devcel.2020.07.013}, pmid = {32768421}, issn = {1878-1551}, mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; *Gene Editing/methods ; Gene Expression Regulation, Developmental/*genetics ; HEK293 Cells ; Humans ; RNA Interference/physiology ; RNA, Messenger/genetics ; }, abstract = {Early embryonic development is driven exclusively by maternal gene products deposited into the oocyte. Although critical in establishing early developmental programs, maternal gene functions have remained elusive due to a paucity of techniques for their systematic disruption and assessment. CRISPR-Cas13 systems have recently been employed to degrade RNA in yeast, plants, and mammalian cell lines. However, no systematic study of the potential of Cas13 has been carried out in an animal system. Here, we show that CRISPR-RfxCas13d (CasRx) is an effective and precise system to deplete specific mRNA transcripts in zebrafish embryos. We demonstrate that zygotically expressed and maternally provided transcripts are efficiently targeted, resulting in a 76% average decrease in transcript levels and recapitulation of well-known embryonic phenotypes. Moreover, we show that this system can be used in medaka, killifish, and mouse embryos. Altogether, our results demonstrate that CRISPR-RfxCas13d is an efficient knockdown platform to interrogate gene function in animal embryos.}, }
@article {pmid32759308, year = {2020}, author = {Prasai, A and Schmidt Cernohorska, M and Ruppova, K and Niederlova, V and Andelova, M and Draber, P and Stepanek, O and Huranova, M}, title = {The BBSome assembly is spatially controlled by BBS1 and BBS4 in human cells.}, journal = {The Journal of biological chemistry}, volume = {295}, number = {42}, pages = {14279-14290}, pmid = {32759308}, issn = {1083-351X}, mesh = {Bardet-Biedl Syndrome/genetics/metabolism/pathology ; CRISPR-Cas Systems/genetics ; Cell Line ; Cilia/metabolism ; Cytoplasm/metabolism ; Fluorescence Recovery After Photobleaching ; Gene Editing ; Humans ; Microscopy, Fluorescence ; Microtubule-Associated Proteins/deficiency/genetics/*metabolism ; Mutation ; Protein Subunits/genetics/metabolism ; }, abstract = {Bardet-Biedl syndrome (BBS) is a pleiotropic ciliopathy caused by dysfunction of primary cilia. More than half of BBS patients carry mutations in one of eight genes encoding for subunits of a protein complex, the BBSome, which mediates trafficking of ciliary cargoes. In this study, we elucidated the mechanisms of the BBSome assembly in living cells and how this process is spatially regulated. We generated a large library of human cell lines deficient in a particular BBSome subunit and expressing another subunit tagged with a fluorescent protein. We analyzed these cell lines utilizing biochemical assays, conventional and expansion microscopy, and quantitative fluorescence microscopy techniques: fluorescence recovery after photobleaching and fluorescence correlation spectroscopy. Our data revealed that the BBSome formation is a sequential process. We show that the pre-BBSome is nucleated by BBS4 and assembled at pericentriolar satellites, followed by the translocation of the BBSome into the ciliary base mediated by BBS1. Our results provide a framework for elucidating how BBS-causative mutations interfere with the biogenesis of the BBSome.}, }
@article {pmid32710848, year = {2020}, author = {Dabelsteen, S and Pallesen, EMH and Marinova, IN and Nielsen, MI and Adamopoulou, M and Rømer, TB and Levann, A and Andersen, MM and Ye, Z and Thein, D and Bennett, EP and Büll, C and Moons, SJ and Boltje, T and Clausen, H and Vakhrushev, SY and Bagdonaite, I and Wandall, HH}, title = {Essential Functions of Glycans in Human Epithelia Dissected by a CRISPR-Cas9-Engineered Human Organotypic Skin Model.}, journal = {Developmental cell}, volume = {54}, number = {5}, pages = {669-684.e7}, pmid = {32710848}, issn = {1878-1551}, mesh = {CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Epithelium/*physiology ; Gene Library ; Glycoproteins/genetics ; Glycosylation ; Humans ; Polysaccharides/*genetics ; Skin/metabolism/pathology ; }, abstract = {The glycome undergoes characteristic changes during histogenesis and organogenesis, but our understanding of the importance of select glycan structures for tissue formation and homeostasis is incomplete. Here, we present a human organotypic platform that allows genetic dissection of cellular glycosylation capacities and systematic interrogation of the roles of distinct glycan types in tissue formation. We used CRISPR-Cas9 gene targeting to generate a library of 3D organotypic skin tissues that selectively differ in their capacity to produce glycan structures on the main types of N- and O-linked glycoproteins and glycolipids. This tissue library revealed distinct changes in skin formation associated with a loss of features for all tested glycoconjugates. The organotypic skin model provides phenotypic cues for the distinct functions of glycoconjugates and serves as a unique resource for further genetic dissection and identification of the specific structural features involved. The strategy is also applicable to other organotypic tissue models.}, }
@article {pmid32644042, year = {2020}, author = {Zhang, W and Chen, Y and Yang, J and Zhang, J and Yu, J and Wang, M and Zhao, X and Wei, K and Wan, X and Xu, X and Jiang, Y and Chen, J and Gao, S and Mao, Z}, title = {A high-throughput small molecule screen identifies farrerol as a potentiator of CRISPR/Cas9-mediated genome editing.}, journal = {eLife}, volume = {9}, number = {}, pages = {}, pmid = {32644042}, issn = {2050-084X}, support = {2018YFC2000100//Chinese National Program on Key Basic Research Project/International ; 19JC1415300//the key project of the Science and Technology of Shanghai Municipality/International ; 19QA1409600//the Shanghai Rising-Star Program/International ; 2017ZZ02015//the Shanghai Municipal Medical and Health Discipline Construction Projects/International ; 2018QNRC001//the Young Elite Scientist Sponsorship Program by CAST/International ; 2017YFA010330//Chinese National Program on Key Basic Research Project/International ; 2016YFA0100400//Chinese National Program on Key Basic Research Project/International ; 31871438//the National Science Foundation of China/International ; 81972457//the National Science Foundation of China/International ; 31721003//the National Science Foundation of China/International ; 31871446//the National Science Foundation of China/International ; 19XD1403000//the Fundamental Research Funds for the Central Universities, Program of Shanghai Academic Research Leader/International ; 19SG18//Shuguang Program&quot; of Shanghai Education Development Foundation and Shanghai Municipal Education Commission&quot;/International ; 2017YFA0103300//Chinese National Program on Key Basic Research Project/International ; 31871438//National Science Foundation of China/International ; 81972457//National Science Foundation of China/International ; 31721003//National Science Foundation of China/International ; 31871446//National Science Foundation of China/International ; 19XD1403000//Program of Shanghai Academic Research Leader/International ; 19SG18//&quot;Shuguang Program&quot; of Shanghai Education Development Foundation and Shanghai Municipal Education Commission/International ; 19JC1415300//The key project of the Science and Technology of Shanghai Municipality/International ; 19QA1409600//Shanghai Rising-Star Program/International ; 2017ZZ02015//Shanghai Municipal Medical and Health Discipline Construction Projects/International ; 2018QNRC001//The Young Elite Scientist Sponsorship Program by CAST/International ; }, mesh = {Animals ; *CRISPR-Cas Systems ; Chromones/*analysis ; *Gene Editing ; HEK293 Cells ; *High-Throughput Screening Assays ; Humans ; Mice ; }, abstract = {Directly modulating the choice between homologous recombination (HR) and non-homologous end joining (NHEJ) - two independent pathways for repairing DNA double-strand breaks (DSBs) - has the potential to improve the efficiency of gene targeting by CRISPR/Cas9. Here, we have developed a rapid and easy-to-score screening approach for identifying small molecules that affect the choice between the two DSB repair pathways. Using this tool, we identified a small molecule, farrerol, that promotes HR but does not affect NHEJ. Further mechanistic studies indicate that farrerol functions through stimulating the recruitment of RAD51 to DSB sites. Importantly, we demonstrated that farrerol effectively promotes precise targeted integration in human cells, mouse cells and mouse embryos at multiple genomic loci. In addition, treating cells with farrerol did not have any obvious negative effect on genomic stability. Moreover, farrerol significantly improved the knock-in efficiency in blastocysts, and the subsequently generated knock-in mice retained the capacity for germline transmission.}, }
@article {pmid32479262, year = {2020}, author = {Wolf, G and de Iaco, A and Sun, MA and Bruno, M and Tinkham, M and Hoang, D and Mitra, A and Ralls, S and Trono, D and Macfarlan, TS}, title = {KRAB-zinc finger protein gene expansion in response to active retrotransposons in the murine lineage.}, journal = {eLife}, volume = {9}, number = {}, pages = {}, pmid = {32479262}, issn = {2050-084X}, support = {1ZIAHD008933//Eunice Kennedy Shriver National Institute of Child Health and Human Development/International ; 310030_152879/SNSF_/Swiss National Science Foundation/Switzerland ; 310030B_173337/SNSF_/Swiss National Science Foundation/Switzerland ; No. 268721/ERC_/European Research Council/International ; No 694658/ERC_/European Research Council/International ; }, mesh = {Animals ; CRISPR-Cas Systems ; *DNA/chemistry/genetics/metabolism ; DNA Transposable Elements/*genetics ; Evolution, Molecular ; Gene Editing ; Mice ; Mice, Knockout ; Mutation/genetics ; *Repressor Proteins/chemistry/genetics/metabolism ; Retroelements/*genetics ; Zinc Fingers/*genetics ; }, abstract = {The Krüppel-associated box zinc finger protein (KRAB-ZFP) family diversified in mammals. The majority of human KRAB-ZFPs bind transposable elements (TEs), however, since most TEs are inactive in humans it is unclear whether KRAB-ZFPs emerged to suppress TEs. We demonstrate that many recently emerged murine KRAB-ZFPs also bind to TEs, including the active ETn, IAP, and L1 families. Using a CRISPR/Cas9-based engineering approach, we genetically deleted five large clusters of KRAB-ZFPs and demonstrate that target TEs are de-repressed, unleashing TE-encoded enhancers. Homozygous knockout mice lacking one of two KRAB-ZFP gene clusters on chromosome 2 and chromosome 4 were nonetheless viable. In pedigrees of chromosome 4 cluster KRAB-ZFP mutants, we identified numerous novel ETn insertions with a modest increase in mutants. Our data strongly support the current model that recent waves of retrotransposon activity drove the expansion of KRAB-ZFP genes in mice and that many KRAB-ZFPs play a redundant role restricting TE activity.}, }
@article {pmid32290857, year = {2020}, author = {Jones, MG and Khodaverdian, A and Quinn, JJ and Chan, MM and Hussmann, JA and Wang, R and Xu, C and Weissman, JS and Yosef, N}, title = {Inference of single-cell phylogenies from lineage tracing data using Cassiopeia.}, journal = {Genome biology}, volume = {21}, number = {1}, pages = {92}, pmid = {32290857}, issn = {1474-760X}, support = {U19 AI090023/AI/NIAID NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; F32 GM125247/GM/NIGMS NIH HHS/United States ; T32 HG000047/HG/NHGRI NIH HHS/United States ; R01 DA036858/DA/NIDA NIH HHS/United States ; T32 GM067547/GM/NIGMS NIH HHS/United States ; RM1 HG009490/HG/NHGRI NIH HHS/United States ; }, mesh = {Algorithms ; CRISPR-Cas Systems ; *Cell Lineage ; Humans ; Mutation ; *Phylogeny ; *Single-Cell Analysis ; }, abstract = {The pairing of CRISPR/Cas9-based gene editing with massively parallel single-cell readouts now enables large-scale lineage tracing. However, the rapid growth in complexity of data from these assays has outpaced our ability to accurately infer phylogenetic relationships. First, we introduce Cassiopeia-a suite of scalable maximum parsimony approaches for tree reconstruction. Second, we provide a simulation framework for evaluating algorithms and exploring lineage tracer design principles. Finally, we generate the most complex experimental lineage tracing dataset to date, 34,557 human cells continuously traced over 15 generations, and use it for benchmarking phylogenetic inference approaches. We show that Cassiopeia outperforms traditional methods by several metrics and under a wide variety of parameter regimes, and provide insight into the principles for the design of improved Cas9-enabled recorders. Together, these should broadly enable large-scale mammalian lineage tracing efforts. Cassiopeia and its benchmarking resources are publicly available at www.github.com/YosefLab/Cassiopeia.}, }
@article {pmid32234056, year = {2020}, author = {Liu, SJ and Malatesta, M and Lien, BV and Saha, P and Thombare, SS and Hong, SJ and Pedraza, L and Koontz, M and Seo, K and Horlbeck, MA and He, D and Birk, HS and Jain, M and Olsen, HE and Akeson, M and Weissman, JS and Monje, M and Gupta, N and Raleigh, DR and Ullian, EM and Lim, DA}, title = {CRISPRi-based radiation modifier screen identifies long non-coding RNA therapeutic targets in glioma.}, journal = {Genome biology}, volume = {21}, number = {1}, pages = {83}, pmid = {32234056}, issn = {1474-760X}, support = {HG010053/NH/NIH HHS/United States ; R21 NS101395/NS/NINDS NIH HHS/United States ; 1R21 NS101395-01/NH/NIH HHS/United States ; T32 EB009383/EB/NIBIB NIH HHS/United States ; F30 NS092319-01/NH/NIH HHS/United States ; I01 BX000252/BX/BLRD VA/United States ; 1R01NS091544/NH/NIH HHS/United States ; R03AG063157/NH/NIH HHS/United States ; P30 EY002162/EY/NEI NIH HHS/United States ; R01 NS091544/NS/NINDS NIH HHS/United States ; T32 HD007470/HD/NICHD NIH HHS/United States ; }, mesh = {Adult ; Astrocytes ; Brain ; Brain Neoplasms/genetics/pathology/radiotherapy/*therapy ; *CRISPR-Cas Systems ; Cell Line, Tumor ; Combined Modality Therapy ; Glioblastoma/genetics/pathology/radiotherapy/*therapy ; Humans ; Oligonucleotides, Antisense ; Organoids ; RNA, Long Noncoding/*antagonists &amp; inhibitors ; Radiation Tolerance ; }, abstract = {BACKGROUND: Long non-coding RNAs (lncRNAs) exhibit highly cell type-specific expression and function, making this class of transcript attractive for targeted cancer therapy. However, the vast majority of lncRNAs have not been tested as potential therapeutic targets, particularly in the context of currently used cancer treatments. Malignant glioma is rapidly fatal, and ionizing radiation is part of the current standard-of-care used to slow tumor growth in both adult and pediatric patients.<br><br>RESULTS: We use CRISPR interference (CRISPRi) to screen 5689 lncRNA loci in human glioblastoma (GBM) cells, identifying 467 hits that modify cell growth in the presence of clinically relevant doses of fractionated radiation. Thirty-three of these lncRNA hits sensitize cells to radiation, and based on their expression in adult and pediatric gliomas, nine of these hits are prioritized as lncRNA Glioma Radiation Sensitizers (lncGRS). Knockdown of lncGRS-1, a primate-conserved, nuclear-enriched lncRNA, inhibits the growth and proliferation of primary adult and pediatric glioma cells, but not the viability of normal brain cells. Using human brain organoids comprised of mature neural cell types as a three-dimensional tissue substrate to model the invasive growth of glioma, we find that antisense oligonucleotides targeting lncGRS-1 selectively decrease tumor growth and sensitize glioma cells to radiation therapy.<br><br>CONCLUSIONS: These studies identify lncGRS-1 as a glioma-specific therapeutic target and establish a generalizable approach to rapidly identify novel therapeutic targets in the vast non-coding genome to enhance radiation therapy.}, }
@article {pmid32234052, year = {2020}, author = {Horii, T and Morita, S and Hino, S and Kimura, M and Hino, Y and Kogo, H and Nakao, M and Hatada, I}, title = {Successful generation of epigenetic disease model mice by targeted demethylation of the epigenome.}, journal = {Genome biology}, volume = {21}, number = {1}, pages = {77}, pmid = {32234052}, issn = {1474-760X}, mesh = {Animals ; CRISPR-Cas Systems ; DNA Methylation ; *Disease Models, Animal ; Embryonic Stem Cells/metabolism ; *Epigenesis, Genetic ; *Epigenome ; Epigenomics/methods ; Humans ; *Mice ; RNA, Long Noncoding/genetics ; Silver-Russell Syndrome/diagnostic imaging/*genetics ; Zygote/metabolism ; }, abstract = {BACKGROUND: Epigenetic modifications, including DNA methylation, play an important role in gene silencing and genome stability. Consequently, epigenetic dysregulation can cause several diseases, such as cancer, obesity, diabetes, autism, and imprinting disorders.<br><br>RESULTS: We validate three methods for the generation of epigenome-edited mice using the dCas9-SunTag and single-chain variable fragment-TET1 catalytic domain. We generate model mice for Silver-Russell syndrome (SRS), an imprinting disorder, by target-specific DNA demethylation in the H19 differentially methylated region. Like SRS patients, these mice show H19 upregulation and Igf2 downregulation, leading to severe intrauterine and postnatal growth retardation.<br><br>CONCLUSION: This is the first report of an imprinting disease model animal generated by targeted demethylation of specific loci of the epigenome in fertilized eggs. Epigenome-edited animals are also useful for exploring the causative epimutations in epigenetic diseases.}, }
@article {pmid32041354, year = {2020}, author = {Tromp, AT and Van Gent, M and Jansen, JP and Scheepmaker, LM and Velthuizen, A and De Haas, CJC and Van Kessel, KPM and Bardoel, BW and Boettcher, M and McManus, MT and Van Strijp, JAG and Lebbink, RJ and Haas, PA and Spaan, AN}, title = {Host-Receptor Post-Translational Modifications Refine Staphylococcal Leukocidin Cytotoxicity.}, journal = {Toxins}, volume = {12}, number = {2}, pages = {}, pmid = {32041354}, issn = {2072-6651}, support = {UU 2012-5667//KWF Kankerbestrijding/International ; }, mesh = {CRISPR-Cas Systems ; Cell Culture Techniques ; Cell Survival/genetics ; Drug Resistance, Bacterial/genetics ; Genetic Predisposition to Disease ; Genome-Wide Association Study ; HEK293 Cells ; Host Microbial Interactions/*genetics ; Humans ; Leukocidins/genetics/metabolism/*toxicity ; Phagocytes/microbiology/pathology ; Protein Binding ; *Protein Processing, Post-Translational ; Receptors, G-Protein-Coupled/genetics/*metabolism ; Staphylococcal Infections/microbiology/*pathology ; Staphylococcus aureus/genetics/metabolism/*pathogenicity ; U937 Cells ; }, abstract = {Staphylococcal bi-component pore-forming toxins, also known as leukocidins, target and lyse human phagocytes in a receptor-dependent manner. S-components of the leukocidins Panton-Valentine leukocidin (PVL), γ-haemolysin AB (HlgAB) and CB (HlgCB), and leukocidin ED (LukED) specifically employ receptors that belong to the class of G-protein coupled receptors (GPCRs). Although these receptors share a common structural architecture, little is known about the conserved characteristics of the interaction between leukocidins and GPCRs. In this study, we investigated host cellular pathways contributing to susceptibility towards S. aureus leukocidin cytotoxicity. We performed a genome-wide CRISPR/Cas9 library screen for toxin-resistance in U937 cells sensitized to leukocidins by ectopic expression of different GPCRs. Our screen identifies post-translational modification (PTM) pathways involved in the sulfation and sialylation of the leukocidin-receptors. Subsequent validation experiments show differences in the impact of PTM moieties on leukocidin toxicity, highlighting an additional layer of refinement and divergence in the staphylococcal host-pathogen interface. Leukocidin receptors may serve as targets for anti-staphylococcal interventions and understanding toxin-receptor interactions will facilitate the development of innovative therapeutics. Variations in the genes encoding PTM pathways could provide insight into observed differences in susceptibility of humans to infections with S. aureus.}, }
@article {pmid33617832, year = {2021}, author = {Spoelstra, WK and Jacques, JM and Gonzalez-Linares, R and Nobrega, FL and Haagsma, AC and Dogterom, M and Meijer, DH and Idema, T and Brouns, SJJ and Reese, L}, title = {CRISPR-based DNA and RNA detection with liquid-liquid phase separation.}, journal = {Biophysical journal}, volume = {}, number = {}, pages = {}, doi = {10.1016/j.bpj.2021.02.013}, pmid = {33617832}, issn = {1542-0086}, abstract = {The ability to detect specific nucleic acid sequences allows for a wide range of applications, such as the identification of pathogens, clinical diagnostics, and genotyping. CRISPR-Cas proteins Cas12a and Cas13a are RNA-guided endonucleases that bind and cleave specific DNA and RNA sequences, respectively. After recognition of a target sequence, both enzymes activate indiscriminate nucleic acid cleavage, which has been exploited for sequence specific molecular diagnostics of nucleic acids. Here, we present a label-free detection approach that uses a readout based on solution turbidity caused by liquid-liquid phase separation (LLPS). Our approach relies on the fact that the LLPS of oppositely charged polymers requires polymers to be longer than a critical length. This length dependence is predicted by the Voorn-Overbeek model, which we describe in detail and validate experimentally in mixtures of polynucleotides and polycations. We show that the turbidity resulting from LLPS can be used to detect the presence of specific nucleic acid sequences, by employing the programmable CRISPR-nucleases Cas12a and Cas13a. Because LLPS of polynucleotides and polycations causes solutions to become turbid, the detection of specific nucleic acid sequences can be observed with the naked eye. We also demonstrate that there is an optimal polynucleotide concentration for detection. Finally, we provide a theoretical prediction which hints towards possible improvements of a LLPS based detection assay. The deployment of LLPS complements CRISPR-based molecular diagnostic applications and facilitates easy and low-cost nucleotide sequence detection.}, }
@article {pmid33617628, year = {2021}, author = {Ishikawa, K and Soejima, S and Masuda, F and Saitoh, S}, title = {Implementation of dCas9-mediated CRISPRi in the fission yeast Schizosaccharomyces pombe.}, journal = {G3 (Bethesda, Md.)}, volume = {}, number = {}, pages = {}, doi = {10.1093/g3journal/jkab051}, pmid = {33617628}, issn = {2160-1836}, abstract = {Controllable and reversible transcriptional repression is an essential method to study gene functions. A systematic knock-down method using catalytically inactive Cas9 (dCas9) was originally established in bacteria. dCas9 forms a ribonucleoprotein with a small guide RNA and uses it to recognize a specific DNA sequence via Watson-Crick base-pairing. When specifically bound to a targeted DNA, dCas9 impairs RNA polymerase activity and represses transcription of that target gene. This technology, CRISPRi, has been implemented in several organisms, but not in Schizosaccharomyces pombe using dCas9. Here we provide a plasmid that expresses dCas9 and sgRNA in fission yeast. With this plasmid, CRISPRi repressed endogenous gene transcription by as much as 87%. This transcriptional repression method is controllable, reversible, and efficient enough to alter cellular phenotypes. Here we offer a CRISPRi method to choose proper targeting sequences for transcriptional repression in fission yeast. Implementation of CRISPRi will help to reveal gene functions and to develop tools based on dCas9 technology in S. pombe.}, }
@article {pmid33616113, year = {2021}, author = {Sun, R and Brogan, D and Buchman, A and Yang, T and Akbari, OS}, title = {Ubiquitous and Tissue-specific RNA Targeting in Drosophila Melanogaster using CRISPR/CasRx.}, journal = {Journal of visualized experiments : JoVE}, volume = {}, number = {168}, pages = {}, doi = {10.3791/62154}, pmid = {33616113}, issn = {1940-087X}, abstract = {CasRx, a member of the RNA-targeting Cas13 family, is a promising new addition of the CRISPR/Cas technologies in efficient gene transcript reduction with an attractive off-target profile at both cellular and organismal levels. It is recently reported that the CRISPR/CasRx system can be used to achieve ubiquitous and tissue-specific gene transcript reduction in Drosophila melanogaster. This paper details the methods from the recent work, consisting of three parts: 1) ubiquitous in vivo endogenous RNA targeting using a two-component CasRx system; 2) ubiquitous in vivo exogenous RNA targeting using a three-component CasRx system; and 3) tissue-specific in vivo RNA targeting using a three-component CasRx system. The effects of RNA targeting observed include targeted gene specific phenotypic changes, targeted RNA transcript reduction, and occasional lethality phenotypes associated with high expression of CasRx protein and collateral activity. Overall, these results showed that the CasRx system is capable of target RNA transcript reduction at the organismal level in a programmable and efficient manner, demonstrating that in vivo transcriptome targeting, and engineering is feasible and lays the foundation for future in vivo CRISPR-based RNA targeting technologies.}, }
@article {pmid33614630, year = {2020}, author = {Tong, B and Dong, H and Cui, Y and Jiang, P and Jin, Z and Zhang, D}, title = {The Versatile Type V CRISPR Effectors and Their Application Prospects.}, journal = {Frontiers in cell and developmental biology}, volume = {8}, number = {}, pages = {622103}, doi = {10.3389/fcell.2020.622103}, pmid = {33614630}, issn = {2296-634X}, abstract = {The class II clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems, characterized by a single effector protein, can be further subdivided into types II, V, and VI. The application of the type II CRISPR effector protein Cas9 as a sequence-specific nuclease in gene editing has revolutionized this field. Similarly, Cas13 as the effector protein of type VI provides a convenient tool for RNA manipulation. Additionally, the type V CRISPR-Cas system is another valuable resource with many subtypes and diverse functions. In this review, we summarize all the subtypes of the type V family that have been identified so far. According to the functions currently displayed by the type V family, we attempt to introduce the functional principle, current application status, and development prospects in biotechnology for all major members.}, }
@article {pmid33612399, year = {2021}, author = {Ma, W and Xu, YS and Sun, XM and Huang, H}, title = {Transposon-Associated CRISPR-Cas System: A Powerful DNA Insertion Tool.}, journal = {Trends in microbiology}, volume = {}, number = {}, pages = {}, doi = {10.1016/j.tim.2021.01.017}, pmid = {33612399}, issn = {1878-4380}, abstract = {The transposon-associated CRISPR-Cas system is emerging as a powerful tool for targeted genetic manipulation. Using this system, targeted genomic knockins and knockouts can be achieved, independently of the host-cell repair machinery, thereby significantly improving our ability to perform genome editing in a wider variety of cell types.}, }
@article {pmid33338046, year = {2020}, author = {O'Leary, S and Adelman, ZN}, title = {CRISPR/Cas9 knockout of female-biased genes AeAct-4 or myo-fem in Ae. aegypti results in a flightless phenotype in female, but not male mosquitoes.}, journal = {PLoS neglected tropical diseases}, volume = {14}, number = {12}, pages = {e0008971}, pmid = {33338046}, issn = {1935-2735}, mesh = {Aedes/*genetics/physiology/virology ; Animals ; *CRISPR-Cas Systems ; Female ; Flight, Animal ; Gene Knockout Techniques ; Humans ; Insecticides/*pharmacology ; Male ; *Mosquito Control ; Mosquito Vectors/*genetics/physiology/virology ; Phenotype ; Zika Virus/*physiology ; Zika Virus Infection/*prevention &amp; control/transmission/virology ; }, abstract = {Aedes aegypti is a vector of dengue, chikungunya, and Zika viruses. Current vector control strategies such as community engagement, source reduction, and insecticides have not been sufficient to prevent viral outbreaks. Thus, interest in novel strategies involving genetic engineering is growing. Female mosquitoes rely on flight to mate with males and obtain a bloodmeal from a host. We hypothesized that knockout of genes specifically expressed in female mosquitoes associated with the indirect flight muscles would result in a flightless female mosquito. Using CRISPR-Cas9 we generated loss-of-function mutations in several genes hypothesized to control flight in mosquitoes, including actin (AeAct-4) and myosin (myo-fem) genes expressed specifically in the female flight muscle. Genetic knockout of these genes resulted in 100% flightless females, with homozygous males able to fly, mate, and produce offspring, albeit at a reduced rate when compared to wild type males. Interestingly, we found that while AeAct-4 was haplosufficient, with most heterozygous individuals capable of flight, this was not the case for myo-fem, where about half of individuals carrying only one intact copy could not fly. These findings lay the groundwork for developing novel mechanisms of controlling Ae. aegypti populations, and our results suggest that this mechanism could be applicable to other vector species of mosquito.}, }
@article {pmid33270627, year = {2020}, author = {Navarro-Payá, D and Flis, I and Anderson, MAE and Hawes, P and Li, M and Akbari, OS and Basu, S and Alphey, L}, title = {Targeting female flight for genetic control of mosquitoes.}, journal = {PLoS neglected tropical diseases}, volume = {14}, number = {12}, pages = {e0008876}, pmid = {33270627}, issn = {1935-2735}, support = {/MRC_/Medical Research Council/United Kingdom ; 200171/Z/15/Z/WT_/Wellcome Trust/United Kingdom ; BBS/E/I/00007033/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BBS/E/I/00007038/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BBS/E/I/00007039/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; }, mesh = {Aedes/*genetics ; Animals ; CRISPR-Cas Systems ; Culex/*genetics/*physiology ; Female ; *Flight, Animal ; Gene Deletion ; Gene Expression Regulation ; Insect Proteins/genetics/*metabolism ; Male ; *Mosquito Control ; Mutation ; }, abstract = {Aedes aegypti Act4 is a paralog of the Drosophila melanogaster indirect flight muscle actin gene Act88F. Act88F has been shown to be haploinsufficient for flight in both males and females (amorphic mutants are dominant). Whereas Act88F is expressed in indirect flight muscles of both males and females, expression of Act4 is substantially female-specific. We therefore used CRISPR/Cas9 and homology directed repair to examine the phenotype of Act4 mutants in two Culicine mosquitoes, Aedes aegypti and Culex quinquefasciatus. A screen for dominant female-flightless mutants in Cx. quinquefasciatus identified one such mutant associated with a six base pair deletion in the CxAct4 coding region. A similar screen in Ae. aegypti identified no dominant mutants. Disruption of the AeAct4 gene by homology-dependent insertion of a fluorescent protein marker cassette gave a recessive female-flightless phenotype in Ae. aegypti. Reproducing the six-base deletion from Cx. quinquefasciatus in Ae. aegypti using oligo-directed mutagenesis generated dominant female-flightless mutants and identified additional dominant female-flightless mutants with other in-frame insertions or deletions. Our data indicate that loss of function mutations in the AeAct4 gene are recessive but that short in-frame deletions produce dominant-negative versions of the AeAct4 protein that interfere with flight muscle function. This makes Act4 an interesting candidate for genetic control methods, particularly population-suppression gene drives targeting female viability/fertility.}, }
@article {pmid33077937, year = {2020}, author = {Porto, EM and Komor, AC and Slaymaker, IM and Yeo, GW}, title = {Base editing: advances and therapeutic opportunities.}, journal = {Nature reviews. Drug discovery}, volume = {19}, number = {12}, pages = {839-859}, pmid = {33077937}, issn = {1474-1784}, support = {R01 EY029166/EY/NEI NIH HHS/United States ; R01 NS103172/NS/NINDS NIH HHS/United States ; R21 GM135736/GM/NIGMS NIH HHS/United States ; T32 GM008326/GM/NIGMS NIH HHS/United States ; }, mesh = {*CRISPR-Cas Systems ; Disease/*genetics ; *Gene Editing ; Hearing Loss/genetics/*therapy ; Humans ; Liver Diseases/genetics/*therapy ; Muscular Dystrophy, Duchenne/genetics/*therapy ; Neoplasms/genetics/*therapy ; Polymorphism, Single Nucleotide ; }, abstract = {Base editing - the introduction of single-nucleotide variants (SNVs) into DNA or RNA in living cells - is one of the most recent advances in the field of genome editing. As around half of known pathogenic genetic variants are due to SNVs, base editing holds great potential for the treatment of numerous genetic diseases, through either temporary RNA or permanent DNA base alterations. Recent advances in the specificity, efficiency, precision and delivery of DNA and RNA base editors are revealing exciting therapeutic opportunities for these technologies. We expect the correction of single point mutations will be a major focus of future precision medicine.}, }
@article {pmid32868807, year = {2020}, author = {Traenkle, B and Segan, S and Fagbadebo, FO and Kaiser, PD and Rothbauer, U}, title = {A novel epitope tagging system to visualize and monitor antigens in live cells with chromobodies.}, journal = {Scientific reports}, volume = {10}, number = {1}, pages = {14267}, pmid = {32868807}, issn = {2045-2322}, mesh = {Antigens/*immunology ; Blotting, Western ; CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Cells/immunology/ultrastructure ; Electrophoresis, Polyacrylamide Gel ; Epitopes/*immunology ; Fluorescent Antibody Technique ; Gene Editing ; HEK293 Cells ; Humans ; Immunoprecipitation ; Microscopy, Confocal ; Peptides/*immunology ; Recombinant Proteins ; Transfection ; }, abstract = {Epitope tagging is a versatile approach to study different proteins using a well-defined and established methodology. To date, most epitope tags such as myc, HA, V5 and FLAG tags are recognized by antibodies, which limits their use to fixed cells, tissues or protein samples. Here we introduce a broadly applicable tagging strategy utilizing a short peptide tag (PepTag) which is specifically recognized by a nanobody (PepNB). We demonstrated that the PepNB can be easily functionalized for immunoprecipitation or direct immunofluorescence staining of Pep-tagged proteins in vitro. For in cellulo studies we converted the PepNB into a fluorescently labeled Pep-chromobody (PepCB) which is functionally expressed in living cells. The addition of the small PepTag does not interfere with the examined structures in different cellular compartments and its detection with the PepCB enables optical antigen tracing in real time. By employing the phenomenon of antigen-mediated chromobody stabilization (AMCBS) using a turnover-accelerated PepCB we demonstrated that the system is suitable to visualize and quantify changes in Pep-tagged antigen concentration by quantitative live-cell imaging. We expect that this novel tagging strategy offers new opportunities to study the dynamic regulation of proteins, e.g. during cellular signaling, cell differentiation, or upon drug action.}, }
@article {pmid32859098, year = {2020}, author = {Nawaz, G and Usman, B and Zhao, N and Han, Y and Li, Z and Wang, X and Liu, Y and Li, R}, title = {CRISPR/Cas9 Directed Mutagenesis of OsGA20ox2 in High Yielding Basmati Rice (Oryza sativa L.) Line and Comparative Proteome Profiling of Unveiled Changes Triggered by Mutations.}, journal = {International journal of molecular sciences}, volume = {21}, number = {17}, pages = {}, pmid = {32859098}, issn = {1422-0067}, support = {SKLWSA-a201914//State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources/ ; }, mesh = {CRISPR-Cas Systems ; Dioxygenases/*genetics ; Gene Expression Regulation, Developmental ; Gene Expression Regulation, Plant ; Gibberellins/*metabolism ; Glycolysis ; Mutation ; Oryza/genetics/*growth &amp; development/metabolism ; Oxidative Phosphorylation ; Photosynthesis ; Plant Proteins/genetics/metabolism ; Protein Interaction Maps ; Proteomics/*methods ; }, abstract = {In rice, semi-dwarfism is among the most required characteristics, as it facilitates better yields and offers lodging resistance. Here, semi-dwarf rice lines lacking any residual transgene-DNA and off-target effects were generated through CRISPR/Cas9-guided mutagenesis of the OsGA20ox2 gene in a high yielding Basmati rice line, and the isobaric tags for relative and absolute quantification (iTRAQ) strategy was utilized to elucidate the proteomic changes in mutants. The results indicated the reduced gibberellins (GA1 and GA4) levels, plant height (28.72%), and flag leaf length, while all the other traits remained unchanged. The OsGA20ox2 expression was highly suppressed, and the mutants exhibited decreased cell length, width, and restored their plant height by exogenous GA3 treatment. Comparative proteomics of the wild-type and homozygous mutant line (GXU43_9) showed an altered level of 588 proteins, 273 upregulated and 315 downregulated, respectively. The identified differentially expressed proteins (DEPs) were mainly enriched in the carbon metabolism and fixation, glycolysis/gluconeogenesis, photosynthesis, and oxidative phosphorylation pathways. The proteins (Q6AWY7, Q6AWY2, Q9FRG8, Q6EPP9, Q6AWX8) associated with growth-regulating factors (GRF2, GRF7, GRF9, GRF10, and GRF11) and GA (Q8RZ73, Q9AS97, Q69VG1, Q8LNJ6, Q0JH50, and Q5MQ85) were downregulated, while the abscisic stress-ripening protein 5 (ASR5) and abscisic acid receptor (PYL5) were upregulated in mutant lines. We integrated CRISPR/Cas9 with proteomic screening as the most reliable strategy for rapid assessment of the CRISPR experiments outcomes.}, }
@article {pmid32752068, year = {2020}, author = {Jung, YJ and Kim, JH and Lee, HJ and Kim, DH and Yu, J and Bae, S and Cho, YG and Kang, KK}, title = {Generation and Transcriptome Profiling of Slr1-d7 and Slr1-d8 Mutant Lines with a New Semi-Dominant Dwarf Allele of SLR1 Using the CRISPR/Cas9 System in Rice.}, journal = {International journal of molecular sciences}, volume = {21}, number = {15}, pages = {}, pmid = {32752068}, issn = {1422-0067}, support = {PJ01477203//Rural Development Administration/ ; }, mesh = {Alleles ; Amino Acid Sequence/genetics ; CRISPR-Cas Systems/genetics ; Gene Expression Profiling ; Gene Expression Regulation, Plant/genetics ; Gibberellins/metabolism ; Mutation/genetics ; Oryza/*genetics/growth &amp; development ; Phenotype ; Plant Growth Regulators/*genetics/metabolism ; Plant Proteins/*genetics ; Plants, Genetically Modified/genetics ; Signal Transduction/genetics ; Transcriptome/*genetics ; }, abstract = {The rice SLR1 gene encodes the DELLA protein, and a loss-of-function mutation is dwarfed by inhibiting plant growth. We generate slr1-d mutants with a semi-dominant dwarf phenotype to target mutations of the DELLA/TVHYNP domain using CRISPR/Cas9 genome editing in rice. Sixteen genetic edited lines out of 31 transgenic plants were generated. Deep sequencing results showed that the mutants had six different mutation types at the target site of the TVHYNP domain of the SLR1 gene. The homo-edited plants selected individuals without DNA (T-DNA) transcribed by segregation in the T1 generation. The slr1-d7 and slr1-d8 plants caused a gibberellin (GA)-insensitive dwarf phenotype with shrunken leaves and shortened internodes. A genome-wide gene expression analysis by RNA-seq indicated that the expression levels of two GA-related genes, GA20OX2 (Gibberellin oxidase) and GA3OX2, were increased in the edited mutant plants, suggesting that GA20OX2 acts as a convert of GA12 signaling. These mutant plants are required by altering GA responses, at least partially by a defect in the phytohormone signaling system process and prevented cell elongation. The new mutants, namely, the slr1-d7 and slr1-d8 lines, are valuable semi-dominant dwarf alleles with potential application value for molecule breeding using the CRISPR/Cas9 system in rice.}, }
@article {pmid32408486, year = {2020}, author = {Dai, Y and Hu, G and Dupas, A and Medina, L and Blandels, N and Clemente, HS and Ladouce, N and Badawi, M and Hernandez-Raquet, G and Mounet, F and Grima-Pettenati, J and Cassan-Wang, H}, title = {Implementing the CRISPR/Cas9 Technology in Eucalyptus Hairy Roots Using Wood-Related Genes.}, journal = {International journal of molecular sciences}, volume = {21}, number = {10}, pages = {}, pmid = {32408486}, issn = {1422-0067}, support = {ANR-10-LABX-41; ANR-11-IDEX-0002-02//Agence Nationale de la Recherche/ ; 201708440282//China Scholarship Council/ ; }, mesh = {Aldehyde Oxidoreductases/genetics/metabolism ; Base Sequence ; *CRISPR-Cas Systems ; Eucalyptus/*genetics/metabolism ; Gene Editing/*methods ; Genes, Plant/*genetics ; Lignin/biosynthesis/genetics ; Multivariate Analysis ; Mutation ; Plant Roots/*genetics/metabolism ; Plants, Genetically Modified ; Spectroscopy, Fourier Transform Infrared ; Transcription Factors/genetics/metabolism ; Wood/*genetics/metabolism ; }, abstract = {Eucalypts are the most planted hardwoods worldwide. The availability of the Eucalyptus grandis genome highlighted many genes awaiting functional characterization, lagging behind because of the lack of efficient genetic transformation protocols. In order to efficiently generate knock-out mutants to study the function of eucalypts genes, we implemented the powerful CRISPR/Cas9 gene editing technology with the hairy roots transformation system. As proofs-of-concept, we targeted two wood-related genes: Cinnamoyl-CoA Reductase1 (CCR1), a key lignin biosynthetic gene and IAA9A an auxin dependent transcription factor of Aux/IAA family. Almost all transgenic hairy roots were edited but the allele-editing rates and spectra varied greatly depending on the gene targeted. Most edition events generated truncated proteins, the prevalent edition types were small deletions but large deletions were also quite frequent. By using a combination of FT-IR spectroscopy and multivariate analysis (partial least square analysis (PLS-DA)), we showed that the CCR1-edited lines, which were clearly separated from the controls. The most discriminant wave-numbers were attributed to lignin. Histochemical analyses further confirmed the decreased lignification and the presence of collapsed vessels in CCR1-edited lines, which are characteristics of CCR1 deficiency. Although the efficiency of editing could be improved, the method described here is already a powerful tool to functionally characterize eucalypts genes for both basic research and industry purposes.}, }
@article {pmid32380756, year = {2020}, author = {Murphy, BG and Wolf, T and Vogel, H and Castillo, D and Woolard, K}, title = {An RNA-Directed Gene Editing Strategy for Attenuating the Infectious Potential of Feline Immunodeficiency Virus-Infected Cells: A Proof of Concept.}, journal = {Viruses}, volume = {12}, number = {5}, pages = {}, pmid = {32380756}, issn = {1999-4915}, mesh = {Animals ; CRISPR-Cas Systems ; Cats ; Feline Acquired Immunodeficiency Syndrome/therapy/*virology ; Gene Editing ; HIV/genetics/physiology ; HIV Infections/therapy/virology ; Humans ; Immunodeficiency Virus, Feline/*genetics/physiology ; RNA, Viral/*genetics ; T-Lymphocytes/virology ; }, abstract = {Modern antiretroviral therapy for immunodeficiency viruses, although remarkably effective in controlling viral transcription, and overt virus-associated morbidity, has failed to absolutely eradicate retroviruses from their infected hosts as a result of proviral integration in long-lived reservoir cells. Immunodeficiency virus-infected patients are therefore consigned to lifelong antiviral therapy as a means to control viremia, viral transmission, and infection-associated morbidity. Unfortunately, lifelong antiviral therapies can be difficult for patients to continuously maintain and may be associated with therapy-specific morbidities. Patient advocates have argued for new methods to achieve retroviral eradication. As a proof-of-concept study, a lentivirus-delivered RNA-directed gene editing strategy was utilized in a series of in vitro experiments in an attempt to attenuate the feline immunodeficiency virus (FIV) proviral load, viral transcription, and production of infectious virions. We found that a feline T lymphocyte cell line (MCH5-4) treated with an FIV-specific clustered regularly interspersed short palindromic repeats (CRISPR)-associated protein 9 (Cas9) gene editing tool resulted in a reduction of cell-free viral RNA relative to control cells. Decreased infectious potential was demonstrated in a two-step FIV infection study-naïve MCH5-4 cells infected with cell-free FIV harvested from FIV-infected and CRISPR lentivirus-treated cells had less integrated proviral DNA than control cells. This study represents the initial steps towards the development of an effective method of proviral eradication in an immunodeficiency virus-infected host.}, }
@article {pmid32088728, year = {2021}, author = {Zhang, B and Chen, Y and Shi, X and Zhou, M and Bao, L and Hatanpaa, KJ and Patel, T and DeBerardinis, RJ and Wang, Y and Luo, W}, title = {Regulation of branched-chain amino acid metabolism by hypoxia-inducible factor in glioblastoma.}, journal = {Cellular and molecular life sciences : CMLS}, volume = {78}, number = {1}, pages = {195-206}, pmid = {32088728}, issn = {1420-9071}, support = {CA168746/CA/NCI NIH HHS/United States ; CA222393/CA/NCI NIH HHS/United States ; I-1939//Welch Foundation/ ; I-1903//Welch Foundation/ ; AG066166/AG/NIA NIH HHS/United States ; NS078049//National Institutes of Health (US)/ ; R35 GM124693/GM/NIGMS NIH HHS/United States ; R00 NS078049/NS/NINDS NIH HHS/United States ; GM124693/NH/NIH HHS/United States ; RP190358//Cancer Prevention and Research Institute of Texas/ ; RP170671//Cancer Prevention and Research Institute of Texas/ ; R01 AG066166/AG/NIA NIH HHS/United States ; RR140036//Cancer Prevention and Research Institute of Texas/ ; CA168746/CA/NCI NIH HHS/United States ; CA222393/CA/NCI NIH HHS/United States ; GM124693/NH/NIH HHS/United States ; AG066166/AG/NIA NIH HHS/United States ; }, mesh = {Amino Acids, Branched-Chain/*metabolism ; Basic Helix-Loop-Helix Transcription Factors/deficiency/genetics/*metabolism ; Brain Neoplasms/metabolism/pathology ; CRISPR-Cas Systems/genetics ; Cell Hypoxia ; Cell Proliferation ; Cells, Cultured ; Gene Expression Regulation, Neoplastic ; Gene Knockout Techniques ; Glioblastoma/metabolism/pathology ; Glutamic Acid/metabolism ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit/deficiency/genetics/*metabolism ; Large Neutral Amino Acid-Transporter 1/genetics/metabolism ; Protein Binding ; Transaminases/antagonists &amp; inhibitors/genetics/metabolism ; }, abstract = {Hypoxia-inducible factors (HIFs) mediate metabolic reprogramming in response to hypoxia. However, the role of HIFs in branched-chain amino acid (BCAA) metabolism remains unknown. Here we show that hypoxia upregulates mRNA and protein levels of the BCAA transporter LAT1 and the BCAA metabolic enzyme BCAT1, but not their paralogs LAT2-4 and BCAT2, in human glioblastoma (GBM) cell lines as well as primary GBM cells. Hypoxia-induced LAT1 protein upregulation is mediated by both HIF-1 and HIF-2 in GBM cells. Although both HIF-1α and HIF-2α directly bind to the hypoxia response element at the first intron of the human BCAT1 gene, HIF-1α is exclusively responsible for hypoxia-induced BCAT1 expression in GBM cells. Knockout of HIF-1α and HIF-2α significantly reduces glutamate labeling from BCAAs in GBM cells under hypoxia, which provides functional evidence for HIF-mediated reprogramming of BCAA metabolism. Genetic or pharmacological inhibition of BCAT1 inhibits GBM cell growth under hypoxia. Together, these findings uncover a previously unrecognized HIF-dependent metabolic pathway that increases GBM cell growth under conditions of hypoxic stress.}, }
@article {pmid32088047, year = {2020}, author = {Mukai, C and Nelson, JL and Cheong, SH and Diel de Amorim, M and Travis, AJ}, title = {Impacts of oocyte/zygote timing for in vitro fertilization and gene editing in the dog.}, journal = {Theriogenology}, volume = {150}, number = {}, pages = {347-352}, doi = {10.1016/j.theriogenology.2020.02.003}, pmid = {32088047}, issn = {1879-3231}, mesh = {Animals ; CRISPR-Cas Systems ; DNA End-Joining Repair/physiology ; Dogs/*physiology ; Embryo Transfer ; Fertilization in Vitro/*veterinary ; Fibroblast Growth Factor 5/genetics/metabolism ; Gene Editing/methods/*veterinary ; Gene Expression Regulation ; Genotype ; In Vitro Oocyte Maturation Techniques/*veterinary ; Keratins, Hair-Specific/genetics/metabolism ; Oocytes/*physiology ; Time Factors ; Zygote/*physiology ; }, abstract = {Previously, we reported the first live births of dogs using in vitro fertilization (IVF), embryo cryopreservation, and transfer. These techniques have potential applications in the conservation of endangered canids, and development of gene editing/repair technologies that could improve animal welfare by restoring normal gene function and removing predisposition to disease. Here, we used IVF as a springboard for initial attempts at genetic modification through gene editing/repair using the Clustered Regularly-Interspaced Short Palindromic Repeat (CRISPR)-CRISPR-associated endonuclease (Cas9) system. We showed previously that timing is critical for successful IVF in that the canine oocyte must be exposed to the oviductal environment beyond simply reaching metaphase II. Others have shown that timing of injection of CRISPR-Cas9 constructs is critical in gene editing, influencing the extent of genetic mosaicism. Therefore, we investigated whether timing of injection of the gene editing/repair constructs might influence the success of embryo production and gene editing in the dog. We achieved similar IVF success to our prior report in generating 2-cell control embryos, and found equally reduced embryo production whether injection was performed in oocytes prior to fertilization, or in presumptive single-cell zygotes already exposed to sperm. We had no success at generating offspring with precise single-nucleotide changes in KRT71 via homology-directed repair (HDR), but did identify mutation of FGF5 using non-homologous end joining (NHEJ). These findings underscore the difficulties inherent to gene repair, but represent important progress on reproducibility of canine IVF, improved techniques of oocyte/embryo handling, and impact of timing of injections on embryo development.}, }
@article {pmid32053368, year = {2020}, author = {Scheidler, CM and Vrabel, M and Schneider, S}, title = {Genetic Code Expansion, Protein Expression, and Protein Functionalization in Bacillus subtilis.}, journal = {ACS synthetic biology}, volume = {9}, number = {3}, pages = {486-493}, doi = {10.1021/acssynbio.9b00458}, pmid = {32053368}, issn = {2161-5063}, mesh = {Bacillus subtilis/*genetics/metabolism ; CRISPR-Cas Systems ; Click Chemistry ; Creatine Kinase, MM Form/metabolism ; Enzyme-Linked Immunosorbent Assay ; Gene Expression Regulation, Bacterial/drug effects ; Genetic Code ; Genetic Vectors ; Green Fluorescent Proteins/genetics/metabolism ; Humans ; Isopropyl Thiogalactoside/pharmacology ; Lysine/chemistry ; Norbornanes/chemistry ; Protein Engineering/*methods ; Recombinant Proteins/chemistry/genetics/isolation &amp; purification/*metabolism ; }, abstract = {The site-specific chemical modification of proteins through incorporation of noncanonical amino acids enables diverse applications, such as imaging, probing, and expanding protein functions, as well as to precisely engineer therapeutics. Here we report a general strategy that allows the incorporation of noncanonical amino acids into target proteins using the amber suppression method and their efficient secretion in the biotechnological relevant expression host Bacillus subtilis. This facilitates efficient purification of target proteins directly from the supernatant, followed by their functionalization using click chemistry. We used this strategy to site-specifically introduce norbornene lysine into a single chain antibody and functionalize it with fluorophores for the detection of human target proteins.}, }
@article {pmid33609360, year = {2021}, author = {Stamatiadis, P and Boel, A and Cosemans, G and Popovic, M and Bekaert, B and Guggilla, R and Tang, M and De Sutter, P and Van Nieuwerburgh, F and Menten, B and Stoop, D and Chuva de Sousa Lopes, SM and Coucke, P and Heindryckx, B}, title = {Comparative analysis of mouse and human preimplantation development following POU5F1 CRISPR/Cas9 targeting reveals interspecies differences.}, journal = {Human reproduction (Oxford, England)}, volume = {}, number = {}, pages = {}, doi = {10.1093/humrep/deab027}, pmid = {33609360}, issn = {1460-2350}, abstract = {STUDY QUESTION: What is the role of POU class 5 homeobox 1 (POU5F1) in human preimplantation development and how does it compare with the mouse model?<br><br>SUMMARY ANSWER: POU5F1 is required for successful development of mouse and human embryos to the blastocyst stage as knockout embryos exhibited a significantly lower blastocyst formation rate, accompanied by lack of inner cell mass (ICM) formation.<br><br>WHAT IS KNOWN ALREADY: Clustered regularly interspaced short palindromic repeats-CRISPR associated genes (CRISPR-Cas9) has previously been used to examine the role of POU5F1 during human preimplantation development. The reported POU5F1-targeted blastocysts always retained POU5F1 expression in at least one cell, because of incomplete CRISPR-Cas9 editing. The question remains of whether the inability to obtain fully edited POU5F1-targeted blastocysts in human results from incomplete editing or the actual inability of these embryos to reach the blastocyst stage.<br><br>STUDY DESIGN, SIZE, DURATION: The efficiency of CRISPR-Cas9 to induce targeted gene mutations was first optimized in the mouse model. Two CRISPR-Cas9 delivery methods were compared in the B6D2F1 strain: S-phase injection (zygote stage) (n = 135) versus metaphase II-phase (M-phase) injection (oocyte stage) (n = 23). Four control groups were included: non-injected media-control zygotes (n = 43)/oocytes (n = 48); sham-injected zygotes (n = 45)/oocytes (n = 47); Cas9-protein injected zygotes (n = 23); and Cas9 protein and scrambled guide RNA (gRNA)-injected zygotes (n = 27). Immunofluorescence analysis was performed in Pou5f1-targeted zygotes (n = 37), media control zygotes (n = 19), and sham-injected zygotes (n = 15). To assess the capacity of Pou5f1-null embryos to develop further in vitro, additional groups of Pou5f1-targeted zygotes (n = 29) and media control zygotes (n = 30) were cultured to postimplantation stages (8.5 dpf). Aiming to identify differences in developmental capacity of Pou5f1-null embryos attributed to strain variation, zygotes from a second mouse strain-B6CBA (n = 52) were targeted. Overall, the optimized methodology was applied in human oocytes following IVM (metaphase II stage) (n = 101). The control group consisted of intracytoplasmically sperm injected (ICSI) IVM oocytes (n = 33). Immunofluorescence analysis was performed in human CRISPR-injected (n = 10) and media control (n = 9) human embryos.<br><br>A gRNA-Cas9 protein mixture targeting exon 2 of Pou5f1/POU5F1 was microinjected in mouse oocytes/zygotes or human IVM oocytes. Reconstructed embryos were cultured for 4 days (mouse) or 6.5 days (human) in sequential culture media. An additional group of mouse-targeted zygotes was cultured to postimplantation stages. Embryonic development was assessed daily, with detailed scoring at late blastocyst stage. Genomic editing was assessed by immunofluorescence analysis and next-generation sequencing.<br><br>Genomic analysis in mouse revealed very high editing efficiencies with 95% of the S-Phase and 100% of the M-Phase embryos containing genetic modifications, of which 89.47% in the S-Phase and 84.21% in the M-Phase group were fully edited. The developmental capacity was significantly compromised as only 46.88% embryos in the S-Phase and 19.05% in the M-Phase group reached the blastocyst stage, compared to 86.36% in control M-Phase and 90.24% in control S-Phase groups, respectively. Immunofluorescence analysis confirmed the loss of Pou5f1 expression and downregulation of the primitive marker SRY-Box transcription factor (Sox17). Our experiments confirmed the requirement of Pou5f1 expression for blastocyst development in the second B6CBA strain. Altogether, our data obtained in mouse reveal that Pou5f1 expression is essential for development to the blastocyst stage. M-Phase injection in human IVM oocytes (n = 101) similarly resulted in 88.37% of the POU5F1-targeted embryos being successfully edited. The developmental capacity of generated embryos was compromised from the eight-cell stage onwards. Only 4.55% of the microinjected embryos reached the late blastocyst stage and the embryos exhibited complete absence of ICM and an irregular trophectoderm cell layer. Loss of POU5F1 expression resulted in absence of SOX17 expression, as in mouse. Interestingly, genetic mosaicism was eliminated in a subset of targeted human embryos (9 out of 38), three of which developed into blastocysts.<br><br>One of the major hurdles of CRISPR-Cas9 germline genome editing is the occurrence of mosaicism, which may complicate phenotypic analysis and interpretation of developmental behavior of the injected embryos. Furthermore, in this study, spare IVM human oocytes were used, which may not recapitulate the developmental behavior of in vivo matured oocytes.<br><br>Comparison of developmental competency following CRISPR-Cas-mediated gene targeting in mouse and human may be influenced by the selected mouse strain. Gene targeting by CRISPR-Cas9 is subject to variable targeting efficiencies. Therefore, striving to reduce mosaicism can provide novel molecular insights into mouse and human embryogenesis.<br><br>The research was funded by the Ghent University Hospital and Ghent University and supported by the FWO-Vlaanderen (Flemish fund for scientific research, Grant no. G051516N), and Hercules funding (FWO.HMZ.2016.00.02.01). The authors declare no competing interests.<br><br>TRIAL REGISTRATION NUMBER: N/A.}, }
@article {pmid33609253, year = {2021}, author = {Elliott, EK and Haupt, LM and Griffiths, LR}, title = {Mini review: genome and transcriptome editing using CRISPR-cas systems for haematological malignancy gene therapy.}, journal = {Transgenic research}, volume = {}, number = {}, pages = {}, pmid = {33609253}, issn = {1573-9368}, abstract = {The recent introduction of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR associated protein (Cas) systems, offer an array of genome and transcriptome editing tools for clinical repair strategies. These include Cas9, Cas12a, dCas9 and more recently Cas13 effectors. RNA targeting CRISPR-Cas13 complexes show unique characteristics with the capability to engineer transcriptomes and modify gene expression, providing a potential clinical cancer therapy tool across various tissue types. Cas13 effectors such as RNA base editing for A to I replacement allows for precise transcript modification. Further applications of Cas13a highlights its capability of producing rapid diagnostic results in a mobile platform. This review will focus on the adaptions of existing CRISPR-Cas systems, along with new Cas effectors for transcriptome or RNA modifications used in disease modelling and gene therapy for haematological malignancy. We also address the current diagnostic and therapeutic potential of CRISPR-Cas systems for personalised haematological malignancy.}, }
@article {pmid33607867, year = {2021}, author = {Liu, Y and Zhan, L and Qin, Z and Sackrison, J and Bischof, JC}, title = {Ultrasensitive and Highly Specific Lateral Flow Assays for Point-of-Care Diagnosis.}, journal = {ACS nano}, volume = {}, number = {}, pages = {}, doi = {10.1021/acsnano.0c10035}, pmid = {33607867}, issn = {1936-086X}, abstract = {Lateral flow assays (LFAs) are paper-based point-of-care (POC) diagnostic tools that are widely used because of their low cost, ease of use, and rapid format. Unfortunately, traditional commercial LFAs have significantly poorer sensitivities (μM) and specificities than standard laboratory tests (enzyme-linked immunosorbent assay, ELISA: pM-fM; polymerase chain reaction, PCR: aM), thus limiting their impact in disease control. In this Perspective, we review the evolving efforts to increase the sensitivity and specificity of LFAs. Recent work to improve the sensitivity through assay improvement includes optimization of the assay kinetics and signal amplification by either reader systems or additional reagents. Together, these efforts have produced LFAs with ELISA-level sensitivities (pM-fM). In addition, sample preamplification can be applied to both nucleic acids (direct amplification) and other analytes (indirect amplification) prior to LFA testing, which can lead to PCR-level (aM) sensitivity. However, these amplification strategies also increase the detection time and assay complexity, which inhibits the large-scale POC use of LFAs. Perspectives to achieve future rapid (<30 min), ultrasensitive (PCR-level), and &quot;sample-to-answer&quot; POC diagnostics are also provided. In the case of LFA specificity, recent research efforts have focused on high-affinity molecules and assay optimization to reduce nonspecific binding. Furthermore, novel highly specific molecules, such as CRISPR/Cas systems, can be integrated into diagnosis with LFAs to produce not only ultrasensitive but also highly specific POC diagnostics. In summary, with continuing improvements, LFAs may soon offer performance at the POC that is competitive with laboratory techniques while retaining a rapid format.}, }
@article {pmid33607266, year = {2021}, author = {Wang, Q and Chear, S and Wing, K and Stellon, D and Thuan Nguyen Tran, M and Talbot, J and Pébay, A and Hewitt, AW and Cook, AL}, title = {Use of CRISPR/Cas ribonucleoproteins for high throughput gene editing of induced pluripotent stemcells.}, journal = {Methods (San Diego, Calif.)}, volume = {}, number = {}, pages = {}, doi = {10.1016/j.ymeth.2021.02.009}, pmid = {33607266}, issn = {1095-9130}, abstract = {Induced pluripotent stem cells (iPSCs) have become widely used for disease modelling, particularly with regard to predisposing genetic risk factors and causal gene variants. Alongside this, technologies such as the CRISPR/Cas system have been adapted to enable programmable gene editing in human cells. When combined, CRISPR/Cas gene editing of donor-specific iPSC to generate isogenic cell lines that differ only at specific gene variants provides a powerful model with which to investigate genetic variants associated with diseases affecting many organs, including the brain and eye. Here we describe our optimized protocol for using CRISPR/Cas ribonucleoproteins to edit disease causing gene variants in human iPSCs. We discuss design of crRNAs and homology-directed repair templates, assembly of CRISPR/Cas ribonucleoproteins, optimization of delivery via nucleofection, and strategies for single cell cloning, efficient clone cryopreservation and genotyping for identifying iPSC clones for further characterization.}, }
@article {pmid33526930, year = {2021}, author = {Dunbar, CE}, title = {A plethora of gene therapies for hemoglobinopathies.}, journal = {Nature medicine}, volume = {27}, number = {2}, pages = {202-204}, pmid = {33526930}, issn = {1546-170X}, support = {NA see below//U.S. Department of Health &amp; Human Services | NIH | National Heart, Lung, and Blood Institute (NHLBI)/ ; }, mesh = {*Anemia, Sickle Cell/genetics/therapy ; CRISPR-Cas Systems ; Gene Editing ; *Hemoglobinopathies/genetics/therapy ; Humans ; *beta-Thalassemia ; }, }
@article {pmid33514753, year = {2021}, author = {Wang, Y and Cheng, H and Liu, Y and Liu, Y and Wen, X and Zhang, K and Ni, X and Gao, N and Fan, L and Zhang, Z and Liu, J and Chen, J and Wang, L and Guo, Y and Zheng, P and Wang, M and Sun, J and Ma, Y}, title = {In-situ generation of large numbers of genetic combinations for metabolic reprogramming via CRISPR-guided base editing.}, journal = {Nature communications}, volume = {12}, number = {1}, pages = {678}, pmid = {33514753}, issn = {2041-1723}, mesh = {Bacillus subtilis/genetics/metabolism ; Bacterial Proteins/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Corynebacterium glutamicum/genetics/metabolism ; DNA, Bacterial/genetics ; Escherichia coli/genetics ; Gene Editing/*methods ; Genes, Bacterial/genetics ; Glycerol/metabolism ; Industrial Microbiology/*methods ; Lycopene/metabolism ; Metabolic Engineering/*methods ; Metabolic Networks and Pathways/genetics ; Multigene Family/*genetics ; Transformation, Bacterial ; Xylose/metabolism ; }, abstract = {Reprogramming complex cellular metabolism requires simultaneous regulation of multigene expression. Ex-situ cloning-based methods are commonly used, but the target gene number and combinatorial library size are severely limited by cloning and transformation efficiencies. In-situ methods such as multiplex automated genome engineering (MAGE) depends on high-efficiency transformation and incorporation of heterologous DNA donors, which are limited to few microorganisms. Here, we describe a Base Editor-Targeted and Template-free Expression Regulation (BETTER) method for simultaneously diversifying multigene expression. BETTER repurposes CRISPR-guided base editors and in-situ generates large numbers of genetic combinations of diverse ribosome binding sites, 5' untranslated regions, or promoters, without library construction, transformation, and incorporation of DNA donors. We apply BETTER to simultaneously regulate expression of up to ten genes in industrial and model microorganisms Corynebacterium glutamicum and Bacillus subtilis. Variants with improved xylose catabolism, glycerol catabolism, or lycopene biosynthesis are respectively obtained. This technology will be useful for large-scale fine-tuning of multigene expression in both genetically tractable and intractable microorganisms.}, }
@article {pmid33514718, year = {2021}, author = {Wilkinson, AC and Dever, DP and Baik, R and Camarena, J and Hsu, I and Charlesworth, CT and Morita, C and Nakauchi, H and Porteus, MH}, title = {Cas9-AAV6 gene correction of beta-globin in autologous HSCs improves sickle cell disease erythropoiesis in mice.}, journal = {Nature communications}, volume = {12}, number = {1}, pages = {686}, pmid = {33514718}, issn = {2041-1723}, support = {K99 HL150218/HL/NHLBI NIH HHS/United States ; R01 HL147124/HL/NHLBI NIH HHS/United States ; R01 HL135607/HL/NHLBI NIH HHS/United States ; R01 DK116944/DK/NIDDK NIH HHS/United States ; R01 AI120766/AI/NIAID NIH HHS/United States ; R01 AI097320/AI/NIAID NIH HHS/United States ; }, mesh = {Anemia, Sickle Cell/blood/diagnosis/genetics/*therapy ; Animals ; CRISPR-Cas Systems/genetics ; Combined Modality Therapy/methods ; Disease Models, Animal ; Erythropoiesis/*genetics ; Female ; Gene Editing/methods ; Gene Knock-In Techniques ; Genetic Therapy/*methods ; Genetic Vectors/genetics ; Hematopoietic Stem Cell Transplantation/*methods ; Humans ; Mice ; Mice, Transgenic ; Parvovirinae/genetics ; Transplantation, Autologous/methods ; beta-Globins/*genetics ; }, abstract = {CRISPR/Cas9-mediated beta-globin (HBB) gene correction of sickle cell disease (SCD) patient-derived hematopoietic stem cells (HSCs) in combination with autologous transplantation represents a recent paradigm in gene therapy. Although several Cas9-based HBB-correction approaches have been proposed, functional correction of in vivo erythropoiesis has not been investigated previously. Here, we use a humanized globin-cluster SCD mouse model to study Cas9-AAV6-mediated HBB-correction in functional HSCs within the context of autologous transplantation. We discover that long-term multipotent HSCs can be gene corrected ex vivo and stable hemoglobin-A production can be achieved in vivo from HBB-corrected HSCs following autologous transplantation. We observe a direct correlation between increased HBB-corrected myeloid chimerism and normalized in vivo red blood cell (RBC) features, but even low levels of chimerism resulted in robust hemoglobin-A levels. Moreover, this study offers a platform for gene editing of mouse HSCs for both basic and translational research.}, }
@article {pmid33508235, year = {2021}, author = {Patrizi, C and Llado, M and Benati, D and Iodice, C and Marrocco, E and Guarascio, R and Surace, EM and Cheetham, ME and Auricchio, A and Recchia, A}, title = {Allele-specific editing ameliorates dominant retinitis pigmentosa in a transgenic mouse model.}, journal = {American journal of human genetics}, volume = {108}, number = {2}, pages = {295-308}, doi = {10.1016/j.ajhg.2021.01.006}, pmid = {33508235}, issn = {1537-6605}, support = {/WT_/Wellcome Trust/United Kingdom ; }, mesh = {Alleles ; Animals ; CRISPR-Cas Systems ; Cell Line ; Dependovirus/genetics ; Disease Models, Animal ; Electroretinography ; *Gene Editing ; Genetic Therapy ; Humans ; INDEL Mutation ; Mice ; Mice, Transgenic ; Mutation, Missense ; Photoreceptor Cells, Vertebrate/metabolism ; Retina/metabolism/physiopathology ; Retinitis Pigmentosa/*genetics/*therapy ; Rhodopsin/*genetics/metabolism ; }, abstract = {Retinitis pigmentosa (RP) is a group of progressive retinal degenerations of mostly monogenic inheritance, which cause blindness in about 1:3,500 individuals worldwide. Heterozygous variants in the rhodopsin (RHO) gene are the most common cause of autosomal dominant RP (adRP). Among these, missense variants at C-terminal proline 347, such as p.Pro347Ser, cause severe adRP recurrently in European affected individuals. Here, for the first time, we use CRISPR/Cas9 to selectively target the p.Pro347Ser variant while preserving the wild-type RHO allele in vitro and in a mouse model of adRP. Detailed in vitro, genomic, and biochemical characterization of the rhodopsin C-terminal editing demonstrates a safe downregulation of p.Pro347Ser expression leading to partial recovery of photoreceptor function in a transgenic mouse model treated with adeno-associated viral vectors. This study supports the safety and efficacy of CRISPR/Cas9-mediated allele-specific editing and paves the way for a permanent and precise correction of heterozygous variants in dominantly inherited retinal diseases.}, }
@article {pmid33482281, year = {2021}, author = {Wang, S and Xu, C and Shi, J and Wang, H and Wu, S and Bao, W}, title = {Regulatory effect and mechanism of APN gene on porcine epidemic diarrhea virus resistance.}, journal = {Gene}, volume = {775}, number = {}, pages = {145448}, doi = {10.1016/j.gene.2021.145448}, pmid = {33482281}, issn = {1879-0038}, mesh = {Animals ; CD13 Antigens/*genetics/metabolism ; CRISPR-Cas Systems ; Cell Cycle ; Cell Line ; Cell Proliferation ; DNA Methylation ; *Disease Resistance ; Gene Knockout Techniques ; *Polymorphism, Single Nucleotide ; Porcine epidemic diarrhea virus/genetics/*pathogenicity ; Promoter Regions, Genetic ; RNA, Viral/genetics ; Swine ; *Up-Regulation ; }, abstract = {PURPOSE: The expression level of aminopeptidase N (APN) is evidently correlated with porcine epidemic diarrhea virus (PEDV) infectivity. This study aims to examine the mechanisms regulating APN expression level in response to PEDV infection.<br><br>METHODS: Quantitative real time PCR was performed herein to detect gene expression dynamics at various timepoints after PEDV infection. Subsequently, CRISPR/Cas9 gene editing technology was used to generate a APN-knockout IPEC-J2 cell line, exploring the effects of APN on cell proliferation by propidium iodide staining and anti-PEDV activity by indirect immunofluorescence assay. Ultimately, the effects of single nucleotide polymorphisms (SNPs) and methylation in the APN promoter region on gene expression were analyzed by using bisulfite sequencing PCR and dual luciferase reporter gene assay.<br><br>RESULTS: APN expression was significantly upregulated within 4-24 h post-infection. The cytoactivity of the APN-knockout IPEC-J2 cell line was markedly suppressed at different timepoints. Further, cell cycle analyses indicated an increase in the number of G1-phase cells and a significant decrease in that of S-phase cells. Moreover, key cyclical factors regulating the G1 phase were highly expressed in APN-knockout cells. The RNA copies of viral particles and mRNA levels of antiviral genes and inflammatory cytokines in APN-knockout cells were markedly decreased within 24 h of PEDV infection. Similarly, indirect immunofluorescence assay confirmed that the number of PEDV particles was significantly decreased. Sequence analysis revealed two CpG islands in the APN promoter region. However, there was no evident correlation between the methylation status of APN promoter and mRNA levels. Dual luciferase reporter gene assay showed that the SNP rs326030589 (G/A) significantly increased the promoter activity of APN.<br><br>CONCLUSIONS: These results suggested that APN knockout enhanced the resistance of IPEC-J2 cells to PEDV. Moreover, rs326030589 in the APN promoter region participated in gene transcription regulation. Our results provide a reference for studying the mechanisms regulating APN and may contribute to the application of APN gene in resistance breeding of swine epidemic diarrhea.}, }
@article {pmid33262250, year = {2021}, author = {Miller, SR and Zhang, X and Hau, RK and Jilek, JL and Jennings, EQ and Galligan, JJ and Foil, DH and Zorn, KM and Ekins, S and Wright, SH and Cherrington, NJ}, title = {Predicting Drug Interactions with Human Equilibrative Nucleoside Transporters 1 and 2 Using Functional Knockout Cell Lines and Bayesian Modeling.}, journal = {Molecular pharmacology}, volume = {99}, number = {2}, pages = {147-162}, pmid = {33262250}, issn = {1521-0111}, support = {T32 ES007091/ES/NIEHS NIH HHS/United States ; R01 GM123643/GM/NIGMS NIH HHS/United States ; P30 ES006694/ES/NIEHS NIH HHS/United States ; R41 GM131433/GM/NIGMS NIH HHS/United States ; R44 GM122196/GM/NIGMS NIH HHS/United States ; }, mesh = {Acetates/*pharmacology ; Bayes Theorem ; Biological Transport ; CRISPR-Cas Systems ; Cell Line ; Dideoxynucleosides/*pharmacology ; Drug Interactions ; Equilibrative Nucleoside Transporter 1/*genetics/metabolism ; Equilibrative-Nucleoside Transporter 2/*genetics/metabolism ; Gene Knockout Techniques ; HeLa Cells ; Humans ; Machine Learning ; Nevirapine/*pharmacology ; Thioinosine/analogs &amp; derivatives/pharmacology ; Ticagrelor/*pharmacology ; Uridine/*analogs &amp; derivatives/*metabolism/pharmacology ; }, abstract = {Equilibrative nucleoside transporters (ENTs) 1 and 2 facilitate nucleoside transport across the blood-testis barrier (BTB). Improving drug entry into the testes with drugs that use endogenous transport pathways may lead to more effective treatments for diseases within the reproductive tract. In this study, CRISPR/CRISPR-associated protein 9 was used to generate HeLa cell lines in which ENT expression was limited to ENT1 or ENT2. We characterized uridine transport in these cell lines and generated Bayesian models to predict interactions with the ENTs. Quantification of [3H]uridine uptake in the presence of the ENT-specific inhibitor S-(4-nitrobenzyl)-6-thioinosine (NBMPR) demonstrated functional loss of each transporter. Nine nucleoside reverse-transcriptase inhibitors and 37 nucleoside/heterocycle analogs were evaluated to identify ENT interactions. Twenty-one compounds inhibited uridine uptake and abacavir, nevirapine, ticagrelor, and uridine triacetate had different IC50 values for ENT1 and ENT2. Total accumulation of four identified inhibitors was measured with and without NBMPR to determine whether there was ENT-mediated transport. Clofarabine and cladribine were ENT1 and ENT2 substrates, whereas nevirapine and lexibulin were ENT1 and ENT2 nontransported inhibitors. Bayesian models generated using Assay Central machine learning software yielded reasonably high internal validation performance (receiver operator characteristic > 0.7). ENT1 IC50-based models were generated from ChEMBL; subvalidations using this training data set correctly predicted 58% of inhibitors when analyzing activity by percent uptake and 63% when using estimated-IC50 values. Determining drug interactions with these transporters can be useful in identifying and predicting compounds that are ENT1 and ENT2 substrates and can thereby circumvent the BTB through this transepithelial transport pathway in Sertoli cells. SIGNIFICANCE STATEMENT: This study is the first to predict drug interactions with equilibrative nucleoside transporter (ENT) 1 and ENT2 using Bayesian modeling. Novel CRISPR/CRISPR-associated protein 9 functional knockouts of ENT1 and ENT2 in HeLa S3 cells were generated and characterized. Determining drug interactions with these transporters can be useful in identifying and predicting compounds that are ENT1 and ENT2 substrates and can circumvent the blood-testis barrier through this transepithelial transport pathway in Sertoli cells.}, }
@article {pmid32823735, year = {2020}, author = {Feng, M and Bai, Y and Chen, Y and Wang, K}, title = {Knockout of the Transducin-Like Enhancer of Split 6 Gene Affects the Proliferation and Cell Cycle Process of Mouse Spermatogonia.}, journal = {International journal of molecular sciences}, volume = {21}, number = {16}, pages = {}, pmid = {32823735}, issn = {1422-0067}, support = {2019012222//Foundation for Distinguished Young Talents in Higher Education of Guangdong/ ; }, mesh = {Animals ; Base Sequence ; CRISPR-Associated Protein 9/metabolism ; CRISPR-Cas Systems/genetics ; *Cell Cycle ; Cell Proliferation ; Co-Repressor Proteins/*genetics/metabolism ; Green Fluorescent Proteins/metabolism ; Male ; Mice, Knockout ; Spermatogonia/*cytology/*metabolism ; }, abstract = {Tle6 (Transducin-like enhancer of split 6) is a member of the Tle co-repressor superfamily, which is expressed in various tissues of invertebrates and vertebrates and participates in the developmental process. However, the current research has only found that the TLE6 mutation is related to infertility, and the key regulatory mechanism of TLE6 remains to be explored. In this study, we combined Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 and the Tet-on system to construct mouse spermatogonia cell lines that induced TLE6 protein knockout (KO), and studied the effect of Tle6 on mouse spermatogonia proliferation and the cell cycle. The results showed that, after drug induction, the Tle6 gene in mouse spermatogonia was successfully knocked out at the genome and protein levels, and the Tle6 gene knockout efficiency was confirmed to be 87.5% with gene-cloning technology. At the same time, we also found that the mouse spermatogonia proliferated slowly after the Tle6 knockout. Using flow cytometry, we found that the cells did not undergo significant apoptosis, and the number of cells in the S phase decreased. After real-time quantity PCR (qRT-PCR) analysis, we found that the expression of cell-proliferation-related genes, CCAAT enhancer-binding protein α(C/ebp α), granulocyte-colony stimulating factor(G-csf), cyclin-dependent kinases 4(Cdk 4), Cyclin E, proliferating cell nuclear antigen(Pcna), and S-phase kinase-associated protein 2 (Skp2) was significantly reduced, which further affected cell growth. In summary, Tle6 can regulate spermatogonia cell proliferation and the cell cycle and provide a scientific basis for studying the role of TLE6 on spermatogenesis.}, }
@article {pmid32784649, year = {2020}, author = {Ahmar, S and Saeed, S and Khan, MHU and Ullah Khan, S and Mora-Poblete, F and Kamran, M and Faheem, A and Maqsood, A and Rauf, M and Saleem, S and Hong, WJ and Jung, KH}, title = {A Revolution toward Gene-Editing Technology and Its Application to Crop Improvement.}, journal = {International journal of molecular sciences}, volume = {21}, number = {16}, pages = {}, pmid = {32784649}, issn = {1422-0067}, support = {PJ01492703//Rural Development Administration/ ; PJ01325901//Rural Development Administration/ ; PJ01366401//Rural Development Administration/ ; }, mesh = {CRISPR-Cas Systems ; Crops, Agricultural/*genetics ; Endonucleases/metabolism ; Gene Editing/*methods ; Genome, Plant ; Plant Breeding ; }, abstract = {Genome editing is a relevant, versatile, and preferred tool for crop improvement, as well as for functional genomics. In this review, we summarize the advances in gene-editing techniques, such as zinc-finger nucleases (ZFNs), transcription activator-like (TAL) effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR) associated with the Cas9 and Cpf1 proteins. These tools support great opportunities for the future development of plant science and rapid remodeling of crops. Furthermore, we discuss the brief history of each tool and provide their comparison and different applications. Among the various genome-editing tools, CRISPR has become the most popular; hence, it is discussed in the greatest detail. CRISPR has helped clarify the genomic structure and its role in plants: For example, the transcriptional control of Cas9 and Cpf1, genetic locus monitoring, the mechanism and control of promoter activity, and the alteration and detection of epigenetic behavior between single-nucleotide polymorphisms (SNPs) investigated based on genetic traits and related genome-wide studies. The present review describes how CRISPR/Cas9 systems can play a valuable role in the characterization of the genomic rearrangement and plant gene functions, as well as the improvement of the important traits of field crops with the greatest precision. In addition, the speed editing strategy of gene-family members was introduced to accelerate the applications of gene-editing systems to crop improvement. For this, the CRISPR technology has a valuable advantage that particularly holds the scientist's mind, as it allows genome editing in multiple biological systems.}, }
@article {pmid32744504, year = {2020}, author = {Gaertner, B and van Heesch, S and Schneider-Lunitz, V and Schulz, JF and Witte, F and Blachut, S and Nguyen, S and Wong, R and Matta, I and Hübner, N and Sander, M}, title = {A human ESC-based screen identifies a role for the translated lncRNA LINC00261 in pancreatic endocrine differentiation.}, journal = {eLife}, volume = {9}, number = {}, pages = {}, pmid = {32744504}, issn = {2050-084X}, support = {R01 DK068471/DK/NIDDK NIH HHS/United States ; P30 DK063491/DK/NIDDK NIH HHS/United States ; 2015-D-021-FEL//Larry L. Hillblom Foundation/International ; R01 DK078803/DK/NIDDK NIH HHS/United States ; AdG788970//Horizon 2020 Framework Programme/International ; ALTF 186-2015//European Molecular Biology Organization/International ; }, mesh = {CRISPR-Cas Systems ; Cell Differentiation/*physiology ; Cells, Cultured ; Gene Deletion ; Gene Expression Regulation, Developmental ; Gene Knockout Techniques ; HEK293 Cells ; Human Embryonic Stem Cells ; Humans ; Islets of Langerhans/*cytology/embryology ; Male ; Protein Biosynthesis ; RNA, Long Noncoding/genetics/*physiology ; Transcription Factors/metabolism ; }, abstract = {Long noncoding RNAs (lncRNAs) are a heterogenous group of RNAs, which can encode small proteins. The extent to which developmentally regulated lncRNAs are translated and whether the produced microproteins are relevant for human development is unknown. Using a human embryonic stem cell (hESC)-based pancreatic differentiation system, we show that many lncRNAs in direct vicinity of lineage-determining transcription factors (TFs) are dynamically regulated, predominantly cytosolic, and highly translated. We genetically ablated ten such lncRNAs, most of them translated, and found that nine are dispensable for pancreatic endocrine cell development. However, deletion of LINC00261 diminishes insulin+ cells, in a manner independent of the nearby TF FOXA2. One-by-one disruption of each of LINC00261's open reading frames suggests that the RNA, rather than the produced microproteins, is required for endocrine development. Our work highlights extensive translation of lncRNAs during hESC pancreatic differentiation and provides a blueprint for dissection of their coding and noncoding roles.}, }
@article {pmid32698485, year = {2020}, author = {Key, J and Harter, PN and Sen, NE and Gradhand, E and Auburger, G and Gispert, S}, title = {Mid-Gestation lethality of Atxn2l-Ablated Mice.}, journal = {International journal of molecular sciences}, volume = {21}, number = {14}, pages = {}, pmid = {32698485}, issn = {1422-0067}, mesh = {Animals ; CRISPR-Cas Systems ; Cell Line ; Embryo Loss/*genetics/pathology ; Female ; *Gene Deletion ; Gene Expression Regulation, Developmental ; Humans ; Mice/*embryology/genetics ; Pregnancy ; }, abstract = {Depletion of yeast/fly Ataxin-2 rescues TDP-43 overexpression toxicity. In mouse models of Amyotrophic Lateral Sclerosis via TDP-43 overexpression, depletion of its ortholog ATXN2 mitigated motor neuron degeneration and extended lifespan from 25 days to >300 days. There is another ortholog in mammals, named ATXN2L (Ataxin-2-like), which is almost uncharacterized but also functions in RNA surveillance at stress granules. We generated mice with Crispr/Cas9-mediated deletion of Atxn2l exons 5-8, studying homozygotes prenatally and heterozygotes during aging. Our novel findings indicate that ATXN2L absence triggers mid-gestational embryonic lethality, affecting female animals more strongly. Weight and development stages of homozygous mutants were reduced. Placenta phenotypes were not apparent, but brain histology showed lamination defects and apoptosis. Aged heterozygotes showed no locomotor deficits or weight loss over 12 months. Null mutants in vivo displayed compensatory efforts to maximize Atxn2l expression, which were prevented upon nutrient abundance in vitro. Mouse embryonal fibroblast cells revealed more multinucleated giant cells upon ATXN2L deficiency. In addition, in human neural cells, transcript levels of ATXN2L were induced upon starvation and glucose and amino acids exposure, but this induction was partially prevented by serum or low cholesterol administration. Neither ATXN2L depletion triggered dysregulation of ATXN2, nor a converse effect was observed. Overall, this essential role of ATXN2L for embryogenesis raises questions about its role in neurodegenerative diseases and neuroprotective therapies.}, }
@article {pmid32698333, year = {2020}, author = {Zhang, B}, title = {CRISPR/Cas9: A Robust Genome-Editing Tool with Versatile Functions and Endless Application.}, journal = {International journal of molecular sciences}, volume = {21}, number = {14}, pages = {}, pmid = {32698333}, issn = {1422-0067}, mesh = {*CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing/methods ; Genome, Plant ; Plants/*genetics ; Plants, Genetically Modified/*genetics ; }, abstract = {Since a potential genome editing tool was first recognized in 2012 [...].}, }
@article {pmid32696132, year = {2020}, author = {Tang, R and Xu, Z}, title = {Gene therapy: a double-edged sword with great powers.}, journal = {Molecular and cellular biochemistry}, volume = {474}, number = {1-2}, pages = {73-81}, doi = {10.1007/s11010-020-03834-3}, pmid = {32696132}, issn = {1573-4919}, support = {81771001//National Natural Science Foundation of China (CN)/ ; }, mesh = {*CRISPR-Cas Systems ; Gene Editing/*methods ; Gene Transfer Techniques/*standards ; *Genetic Predisposition to Disease ; Genetic Therapy/methods/*trends ; Humans ; Immunotherapy, Adoptive/*methods ; }, abstract = {Gene therapy is the treatment of a disease through transferring genetic material into cells of the patients. In the recent several years, gene therapy has experienced rapid progress and achieved huge success. Over two dozens of gene therapies have been approved for clinical use by the drug regulatory agencies from different countries. However, concerns about its efficacy and safety have accompanied gene therapy since its birth. In the present manuscript, we first introduce various strategies employed in gene therapy, which includes ex vivo gene delivery v.s. in vivo gene delivery; gene addition v.s. genome editing; inherited disease v.s. acquired disease; and somatic gene therapy v.s. germline gene therapy. Then we discuss the clinical outcomes of some approved gene therapies. We finish our discussion with the safety issues related to gene therapy. We will see that with the technology improvement, somatic gene therapy has been proved to be efficient and safe enough for clinical practice. However, germline gene therapy has important efficiency and safety issues at present, and should not be put into clinical practice before these issues are solved.}, }
@article {pmid32325942, year = {2020}, author = {Luo, J and Teng, M and Zai, X and Tang, N and Zhang, Y and Mandviwala, A and Reddy, VRAP and Baigent, S and Yao, Y and Nair, V}, title = {Efficient Mutagenesis of Marek's Disease Virus-Encoded microRNAs Using a CRISPR/Cas9-Based Gene Editing System.}, journal = {Viruses}, volume = {12}, number = {4}, pages = {}, pmid = {32325942}, issn = {1999-4915}, support = {BBS/E/I/00007039/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BBS/E/I/00007032, BB/R007896/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; }, mesh = {Animals ; Base Sequence ; *CRISPR-Cas Systems ; Cells, Cultured ; Chick Embryo ; Cloning, Molecular ; *Gene Editing ; Gene Knockdown Techniques ; Genome, Viral ; Marek Disease/*genetics ; MicroRNAs/*genetics ; *Mutagenesis ; Open Reading Frames ; RNA, Guide ; RNA, Messenger/genetics ; RNA, Viral/*genetics ; Sequence Analysis, DNA ; Virus Replication ; }, abstract = {The virus-encoded microRNAs (miRNAs) have been demonstrated to have important regulatory roles in herpesvirus biology, including virus replication, latency, pathogenesis and/or tumorigenesis. As an emerging efficient tool for gene editing, the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system has been successfully applied in manipulating the genomes of large DNA viruses. Herein, utilizing the CRISPR/Cas9 system with a double-guide RNAs transfection/virus infection strategy, we have established a new platform for mutagenesis of viral miRNAs encoded by the Marek's disease virus serotype 1 (MDV-1), an oncogenic alphaherpesvirus that can induce rapid-onset T-cell lymphomas in chickens. A series of miRNA-knocked out (miR-KO) mutants with deletions of the Meq- or the mid-clustered miRNAs, namely RB-1B∆Meq-miRs, RB-1B∆M9-M2, RB-1B∆M4, RB-1B∆M9 and RB-1B∆M11, were generated from vvMDV strain RB-1B virus. Interestingly, mutagenesis of the targeted miRNAs showed changes in the in vitro virus growth kinetics, which is consistent with that of the in vivo proliferation curves of our previously reported GX0101 mutants produced by the bacterial artificial chromosome (BAC) clone and Rec E/T homologous recombination techniques. Our data demonstrate that the CRISPR/Cas9-based gene editing is a simple, efficient and relatively nondisruptive approach for manipulating the small non-coding genes from the genome of herpesvirus and will undoubtedly contribute significantly to the future progress in herpesvirus biology.}, }
@article {pmid32276275, year = {2020}, author = {Svaton, M and Skvarova Kramarzova, K and Kanderova, V and Mancikova, A and Smisek, P and Jesina, P and Krijt, J and Stiburkova, B and Dobrovolny, R and Sokolova, J and Bakardjieva-Mihaylova, V and Vodickova, E and Rackova, M and Stuchly, J and Kalina, T and Stary, J and Trka, J and Fronkova, E and Kozich, V}, title = {A homozygous deletion in the SLC19A1 gene as a cause of folate-dependent recurrent megaloblastic anemia.}, journal = {Blood}, volume = {135}, number = {26}, pages = {2427-2431}, pmid = {32276275}, issn = {1528-0020}, mesh = {Adolescent ; Anemia, Megaloblastic/drug therapy/*genetics ; CRISPR-Cas Systems ; Cells, Cultured ; Clone Cells ; Folic Acid/*therapeutic use ; Frameshift Mutation ; Gene Knockout Techniques ; Homozygote ; Humans ; Hyperhomocysteinemia/drug therapy/*genetics ; K562 Cells ; Male ; Recurrence ; Sequence Deletion ; Sodium-Hydrogen Exchanger 1/*deficiency/genetics ; Vitamin B 12/therapeutic use ; Whole Exome Sequencing ; }, }
@article {pmid32159213, year = {2020}, author = {Gao, B and Sabnis, R and Costantini, T and Jinkerson, R and Sun, Q}, title = {A peek in the micro-sized world: a review of design principles, engineering tools, and applications of engineered microbial community.}, journal = {Biochemical Society transactions}, volume = {48}, number = {2}, pages = {399-409}, doi = {10.1042/BST20190172}, pmid = {32159213}, issn = {1470-8752}, mesh = {Animals ; Biodegradation, Environmental ; Biotechnology ; CRISPR-Cas Systems ; Gene Editing ; Gene Transfer Techniques ; Genetic Engineering/*methods ; Humans ; *Microbial Consortia ; *Microbial Interactions ; Synthetic Biology/*methods ; Waste Disposal, Fluid ; }, abstract = {Microbial communities drive diverse processes that impact nearly everything on this planet, from global biogeochemical cycles to human health. Harnessing the power of these microorganisms could provide solutions to many of the challenges that face society. However, naturally occurring microbial communities are not optimized for anthropogenic use. An emerging area of research is focusing on engineering synthetic microbial communities to carry out predefined functions. Microbial community engineers are applying design principles like top-down and bottom-up approaches to create synthetic microbial communities having a myriad of real-life applications in health care, disease prevention, and environmental remediation. Multiple genetic engineering tools and delivery approaches can be used to 'knock-in' new gene functions into microbial communities. A systematic study of the microbial interactions, community assembling principles, and engineering tools are necessary for us to understand the microbial community and to better utilize them. Continued analysis and effort are required to further the current and potential applications of synthetic microbial communities.}, }
@article {pmid32147798, year = {2020}, author = {Lambert, M and Leijonhufvud, C and Segerberg, F and Melenhorst, JJ and Carlsten, M}, title = {CRISPR/Cas9-Based Gene Engineering of Human Natural Killer Cells: Protocols for Knockout and Readouts to Evaluate Their Efficacy.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2121}, number = {}, pages = {213-239}, doi = {10.1007/978-1-0716-0338-3_18}, pmid = {32147798}, issn = {1940-6029}, mesh = {*CRISPR-Cas Systems ; Cell Movement/immunology ; Flow Cytometry/*methods ; Gene Editing/history/*methods ; Gene Knockout Techniques/history/*methods ; History, 20th Century ; History, 21st Century ; Humans ; Killer Cells, Natural/*metabolism ; Real-Time Polymerase Chain Reaction/*methods ; Sequence Analysis, DNA/methods ; }, abstract = {Natural killer (NK) cells are cytotoxic lymphocytes of our immune system with the ability to identify and kill certain virally infected and tumor-transformed cells. During the past 15 years, it has become increasingly clear that NK cells are involved in tumor immune surveillance and that they can be utilized to treat cancer patients. However, their ability to induce durable responses in settings of adoptive cell therapy needs to be further improved. One possible approach is to genetically engineer NK cells to augment their cytotoxicity per se, but also their ability to persist in vivo and home to the tumor-bearing tissue. In recent years, investigators have explored the potential of viral transduction and mRNA electroporation to modify NK cells. Although these methods have generated promising data, they are associated with certain limitations. With the increasing advances in the CRISPR/Cas9 technology, investigators have now turned their attention toward using this technology with NK cells as an alternative method. In this book chapter, we introduce NK cells and provide an historical overview of techniques to genetically engineer lymphocytes. Further, we elucidate protocols for inducing double-strand breaks in NK cells via CRISPR/Cas9 together with readouts to address its efficacy and functional outcome. We also discuss the pros and cons of the described readouts. The overall aim of this book chapter is to help introduce the CRISPR/Cas9 technology to the broader audience of NK cell researchers.}, }
@article {pmid32112380, year = {2020}, author = {Jin, Y and Shen, Y and Weintraub, NL and Tang, Y}, title = {Using iRFP Genetic Labeling Technology to Track Tumorogenesis of Transplanted CRISPR/Cas9-Edited iPSC in Skeletal Muscle.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2126}, number = {}, pages = {73-83}, doi = {10.1007/978-1-0716-0364-2_7}, pmid = {32112380}, issn = {1940-6029}, mesh = {Animals ; CRISPR-Cas Systems ; Carcinogenesis/genetics/metabolism/*pathology ; Cell Tracking/methods ; Dystrophin/deficiency/*genetics ; Gene Editing ; Humans ; Induced Pluripotent Stem Cells/*cytology/metabolism/transplantation ; Luminescent Proteins/genetics/*metabolism ; Mice ; Molecular Imaging ; Muscle, Skeletal/*cytology/metabolism ; Muscular Dystrophy, Animal/genetics/*physiopathology/therapy ; Spectroscopy, Near-Infrared ; Stem Cell Transplantation/*adverse effects ; }, abstract = {Tumorigenesis and attendant safety risks are significant concerns of induced pluripotent stem cell (iPSC)-based therapies. Thus, it is crucial to evaluate iPSC proliferation, differentiation, and tumor formation after transplantation. Several approaches have been employed for tracking the donor cells, including fluorescent protein and luciferase, but both have limitations. Here, we introduce a protocol using iRFP genetic labeling technology to track tumor formation of iPSCs in skeletal muscle after CRISPR/Cas9 gene editing.}, }
@article {pmid32110989, year = {2020}, author = {Sato, M and Miyagasako, R and Takabayashi, S and Ohtsuka, M and Hatada, I and Horii, T}, title = {Sequential i-GONAD: An Improved In Vivo Technique for CRISPR/Cas9-Based Genetic Manipulations in Mice.}, journal = {Cells}, volume = {9}, number = {3}, pages = {}, pmid = {32110989}, issn = {2073-4409}, mesh = {Animals ; Base Sequence ; CRISPR-Associated Protein 9/*metabolism ; CRISPR-Cas Systems/*genetics ; Dextrans/chemistry ; Exons/genetics ; Female ; Fluorescence ; Gene Editing ; *Gene Transfer Techniques ; INDEL Mutation/genetics ; Introns/genetics ; Methyl-CpG-Binding Protein 2/genetics ; Mice, Inbred C57BL ; Mice, Transgenic ; Nucleic Acids/*metabolism ; Oviducts/*metabolism ; }, abstract = {Improved genome-editing via oviductal nucleic acid delivery (i-GONAD) is a technique capable of inducing genomic changes in preimplantation embryos (zygotes) present within the oviduct of a pregnant female. i-GONAD involves intraoviductal injection of a solution containing genome-editing components via a glass micropipette under a dissecting microscope, followed by in vivo electroporation using tweezer-type electrodes. i-GONAD does not involve ex vivo handling of embryos (isolation of zygotes, microinjection or electroporation of zygotes, and egg transfer of the treated embryos to the oviducts of a recipient female), which is required for in vitro genome-editing of zygotes. i-GONAD enables the generation of indels, knock-in (KI) of ~ 1 kb sequence of interest, and large deletion at a target locus. i-GONAD is usually performed on Day 0.7 of pregnancy, which corresponds to the late zygote stage. During the initial development of this technique, we performed i-GONAD on Days 1.4-1.5 (corresponding to the 2-cell stage). Theoretically, this means that at least two GONAD steps (on Day 0.7 and Day 1.4-1.5) must be performed. If this is practically demonstrated, it provides additional options for various clustered regularly interspaced palindrome repeats (CRISPR)/Caspase 9 (Cas9)-based genetic manipulations. For example, it is usually difficult to induce two independent indels at the target sites, which are located very close to each other, by simultaneous transfection of two guide RNAs and Cas9 protein. However, the sequential induction of indels at a target site may be possible when repeated i-GONAD is performed on different days. Furthermore, simultaneous introduction of two mutated lox sites (to which Cre recombinase bind) for making a floxed allele is reported to be difficult, as it often causes deletion of a sequence between the two gRNA target sites. However, differential KI of lox sites may be possible when repeated i-GONAD is performed on different days. In this study, we performed proof-of-principle experiments to demonstrate the feasibility of the proposed approach called &quot;sequential i-GONAD (si-GONAD).&quot;}, }
@article {pmid33595147, year = {2021}, author = {Che, P and Chang, S and Simon, MK and Zhang, Z and Shaharyar, A and Ourada, J and O'Neill, D and Torres-Mendoza, M and Guo, Y and Marasigan, KM and Vielle-Calzada, JP and Ozias-Akins, P and Albertsen, MC and Jones, TJ}, title = {Developing a rapid and highly efficient cowpea regeneration, transformation and genome editing system using embryonic axis explants.}, journal = {The Plant journal : for cell and molecular biology}, volume = {}, number = {}, pages = {}, doi = {10.1111/tpj.15202}, pmid = {33595147}, issn = {1365-313X}, abstract = {Cowpea is one of the most important legume crops planted worldwide, but despite decades of effort, cowpea transformation is still challenging due to inefficient Agrobacterium-mediated T-DNA delivery, transgenic selection and in vitro shoot regeneration. Here, we report a highly efficient transformation system using embryonic axis explants isolated from imbibed mature seeds. We found that removal of the shoot apical meristem from the explants stimulated direct multiple shoot organogenesis from the cotyledonary node tissue. The application of a previously reported ternary transformation vector system provided efficient Agrobacterium-mediated gene delivery, while the utilization of spcN as selectable marker enabled more robust transgenic selection, plant recovery and generated transgenic plants without escapes and chimera formation. Transgenic cowpea plantlets developed exclusively from the cotyledonary nodes at high frequencies of 4 to 37% across a wide range of cowpea genotypes. CRISPR/Cas-mediated gene editing was successfully demonstrated. The transformation principles established here could also be applied to other legumes to increase transformation efficiencies. Supporting information.}, }
@article {pmid33593370, year = {2021}, author = {Sun, Y and Yu, L and Liu, C and Ye, S and Chen, W and Li, D and Huang, W}, title = {One-tube SARS-CoV-2 detection platform based on RT-RPA and CRISPR/Cas12a.}, journal = {Journal of translational medicine}, volume = {19}, number = {1}, pages = {74}, pmid = {33593370}, issn = {1479-5876}, support = {2019YFA0906000//National Key R&amp;D Program of China/ ; 2020A1515110032//Natural Science Foundation of Guangdong Province/ ; 2017B030301015//Natural Science Foundation of Guangdong Province/ ; 2020A1515010235//Natural Science Foundation of Guangdong Province/ ; 81772737//National Natural Science Foundation of China/ ; 31670757//National Natural Science Foundation of China/ ; JCYJ20170413161749433//Shenzhen Municipal Government of China/ ; SZSM202011017//Sanming Project of Medicine in Shenzhen/ ; SZXK020//Shenzhen Key Medical Discipline Construction Fund/ ; JCYJ20200109120016553//Shenzhen Basic Research Program/ ; }, abstract = {BACKGROUND: COVID-19 has spread rapidly around the world, affecting a large percentage of the population. When lifting certain mandatory measures for an economic restart, robust surveillance must be established and implemented, with nucleic acid detection for SARS-CoV-2 as an essential component.<br><br>METHODS: We tried to develop a one-tube detection platform based on RT-RPA (Reverse Transcription and Recombinase Polymerase Isothermal Amplification) and DNA Endonuclease-Targeted CRISPR Trans Reporter (DETECTR) technology, termed OR-DETECTR, to detect SARS-CoV-2. We designed RT-RPA primers of the RdRp and N genes following the SARS-CoV-2 gene sequence. We optimized reaction components so that the detection process could be carried out in one tube. Specificity was demonstrated by detecting nucleic acid samples from pseudoviruses from seven human coronaviruses and Influenza A (H1N1). Clinical samples were used to validate the platform and all results were compared to rRT-PCR. RNA standards and pseudoviruses diluted by different gradients were used to demonstrate the detection limit. Additionally, we have developed a lateral flow assay based on OR-DETECTR for detecting COVID-19.<br><br>RESULTS: The OR-DETECTR detection process can be completed in one tube, which takes approximately 50 min. This method can specifically detect SARS-CoV-2 from seven human coronaviruses and Influenza A (H1N1), with a low detection limit of 2.5 copies/µl input (RNA standard) and 1 copy/µl input (pseudovirus). Results of six samples from SARS-CoV-2 patients, eight samples from patients with fever but no SARS-CoV-2 infection, and one mixed sample from 40 negative controls showed that OR-DETECTR is 100% consistent with rRT-PCR. The lateral flow assay based on OR-DETECTR can be used for the detection of COVID-19, and the detection limit is 2.5 copies/µl input.<br><br>CONCLUSIONS: The OR-DETECTR platform for the detection of COVID-19 is rapid, accurate, tube closed, easy-to-operate, and free of large instruments.}, }
@article {pmid33574281, year = {2021}, author = {Zhu, Y and Feng, F and Hu, G and Wang, Y and Yu, Y and Zhu, Y and Xu, W and Cai, X and Sun, Z and Han, W and Ye, R and Qu, D and Ding, Q and Huang, X and Chen, H and Xu, W and Xie, Y and Cai, Q and Yuan, Z and Zhang, R}, title = {A genome-wide CRISPR screen identifies host factors that regulate SARS-CoV-2 entry.}, journal = {Nature communications}, volume = {12}, number = {1}, pages = {961}, pmid = {33574281}, issn = {2041-1723}, mesh = {A549 Cells ; Angiotensin-Converting Enzyme 2/genetics/metabolism ; Animals ; COVID-19/genetics/*virology ; *CRISPR-Cas Systems ; Chlorocebus aethiops ; Disease Models, Animal ; Endosomes/virology ; *Genome-Wide Association Study ; HeLa Cells ; *Host-Pathogen Interactions ; Humans ; Mesocricetus ; SARS-CoV-2/*physiology ; Serine Endopeptidases ; Spike Glycoprotein, Coronavirus/metabolism ; Vero Cells ; *Virus Internalization ; }, abstract = {The global spread of SARS-CoV-2 is posing major public health challenges. One feature of SARS-CoV-2 spike protein is the insertion of multi-basic residues at the S1/S2 subunit cleavage site. Here, we find that the virus with intact spike (Sfull) preferentially enters cells via fusion at the plasma membrane, whereas a clone (Sdel) with deletion disrupting the multi-basic S1/S2 site utilizes an endosomal entry pathway. Using Sdel as model, we perform a genome-wide CRISPR screen and identify several endosomal entry-specific regulators. Experimental validation of hits from the CRISPR screen shows that host factors regulating the surface expression of angiotensin-converting enzyme 2 (ACE2) affect entry of Sfull virus. Animal-to-animal transmission with the Sdel virus is reduced compared to Sfull in the hamster model. These findings highlight the critical role of the S1/S2 boundary of SARS-CoV-2 spike protein in modulating virus entry and transmission and provide insights into entry of coronaviruses.}, }
@article {pmid33349703, year = {2021}, author = {Jin, S and Gao, Q and Gao, C}, title = {An unbiased method for evaluating the genome-wide specificity of base editors in rice.}, journal = {Nature protocols}, volume = {16}, number = {1}, pages = {431-457}, pmid = {33349703}, issn = {1750-2799}, mesh = {Agrobacterium tumefaciens/genetics ; CRISPR-Cas Systems ; Gene Editing/*methods ; Genome, Plant ; Oryza/*genetics ; Polymorphism, Single Nucleotide ; Transformation, Genetic ; Whole Genome Sequencing ; }, abstract = {Base editors can achieve targeted genomic base conversion. However, the off-target issue is one of the major concerns in their application. Whole-genome sequencing (WGS) at the individual level can provide direct information on genome-wide specificity, but it is difficult to distinguish true off-target single-nucleotide variants (SNVs) induced by base editors from background variation. Here we describe an unbiased WGS method for evaluating the specificity of base editors in rice. In this protocol, we describe the experimental design and provide details of vector construction, rice transformation and tissue culture, as well as a comprehensive WGS data analysis pipeline for overcoming two related core problems in various plant species: high background mutation rates and the heterogeneity of examined populations. Using this protocol, researchers can straightforwardly and accurately assess the genome-wide specificity of base editors and other genome editing tools in 12-15 weeks.}, }
@article {pmid33288953, year = {2021}, author = {Bao, XR and Pan, Y and Lee, CM and Davis, TH and Bao, G}, title = {Tools for experimental and computational analyses of off-target editing by programmable nucleases.}, journal = {Nature protocols}, volume = {16}, number = {1}, pages = {10-26}, pmid = {33288953}, issn = {1750-2799}, support = {R01 HL152314/HL/NHLBI NIH HHS/United States ; UG3 HL151545/HL/NHLBI NIH HHS/United States ; }, mesh = {Animals ; *CRISPR-Cas Systems ; Endonucleases/genetics/metabolism ; Gene Editing/*methods ; Genomics/methods ; High-Throughput Nucleotide Sequencing/methods ; Humans ; }, abstract = {Genome editing using programmable nucleases is revolutionizing life science and medicine. Off-target editing by these nucleases remains a considerable concern, especially in therapeutic applications. Here we review tools developed for identifying potential off-target editing sites and compare the ability of these tools to properly analyze off-target effects. Recent advances in both in silico and experimental tools for off-target analysis have generated remarkably concordant results for sites with high off-target editing activity. However, no single tool is able to accurately predict low-frequency off-target editing, presenting a bottleneck in therapeutic genome editing, because even a small number of cells with off-target editing can be detrimental. Therefore, we recommend that at least one in silico tool and one experimental tool should be used together to identify potential off-target sites, and amplicon-based next-generation sequencing (NGS) should be used as the gold standard assay for assessing the true off-target effects at these candidate sites. Future work to improve off-target analysis includes expanding the true off-target editing dataset to evaluate new experimental techniques and to train machine learning algorithms; performing analysis using the particular genome of the cells in question rather than the reference genome; and applying novel NGS techniques to improve the sensitivity of amplicon-based off-target editing quantification.}, }
@article {pmid33247284, year = {2021}, author = {Hendriks, D and Artegiani, B and Hu, H and Chuva de Sousa Lopes, S and Clevers, H}, title = {Establishment of human fetal hepatocyte organoids and CRISPR-Cas9-based gene knockin and knockout in organoid cultures from human liver.}, journal = {Nature protocols}, volume = {16}, number = {1}, pages = {182-217}, pmid = {33247284}, issn = {1750-2799}, mesh = {*CRISPR-Cas Systems ; Cell Culture Techniques/methods ; Cells, Cultured ; Fetus/cytology/metabolism ; Gene Editing/methods ; Gene Knock-In Techniques/methods ; Gene Knockout Techniques/methods ; Hepatocytes/*cytology/metabolism ; Humans ; Liver/*cytology/metabolism ; Organoids/*cytology/metabolism ; }, abstract = {The liver is composed of two epithelial cell types: hepatocytes and liver ductal cells. Culture conditions for expansion of human liver ductal cells in vitro as organoids were previously described in a protocol; however, primary human hepatocytes remained hard to expand, until recently. In this protocol, we provide full details of how we overcame this limitation, establishing culture conditions that facilitate long-term expansion of human fetal hepatocytes as organoids. In addition, we describe how to generate (multi) gene knockouts using CRISPR-Cas9 in both human fetal hepatocyte and adult liver ductal organoid systems. Using a CRISPR-Cas9 and homology-independent organoid transgenesis (CRISPR-HOT) approach, efficient gene knockin can be achieved in these systems. These gene knockin and knockout approaches, and their multiplexing, should be useful for a variety of applications, such as disease modeling, investigating gene functions and studying processes, such as cellular differentiation and cell division. The protocol to establish human fetal hepatocyte organoid cultures takes ~1-2 months. The protocols to genome engineer human liver ductal organoids and human fetal hepatocyte organoids take 2-3 months.}, }
@article {pmid33130330, year = {2020}, author = {Kauffman, MR and Nazemidashtarjandi, S and Ghazanfari, D and Allen, AE and Reynolds, NM and Faik, A and Burdick, MM and McCall, KD and Goetz, DJ}, title = {Evidence that knock down of GSK-3β in Chronic Myelogenous Leukemia cells augments IFN-γ-induced apoptosis.}, journal = {Leukemia research}, volume = {99}, number = {}, pages = {106464}, pmid = {33130330}, issn = {1873-5835}, support = {R15 GM110602/GM/NIGMS NIH HHS/United States ; }, mesh = {Antineoplastic Agents/pharmacology ; Apoptosis/*drug effects ; CRISPR-Cas Systems ; Cell Adhesion/drug effects ; Cell Line, Tumor/drug effects ; Codon, Nonsense ; Drug Interactions ; Flow Cytometry ; Frameshift Mutation ; Fusion Proteins, bcr-abl/antagonists &amp; inhibitors ; Gene Knockdown Techniques ; Glycogen Synthase Kinase 3 beta/*antagonists &amp; inhibitors/genetics ; Humans ; Interferon-gamma/*pharmacology/therapeutic use ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive/*drug therapy/enzymology/pathology ; Male ; Neoplasm Proteins/*antagonists &amp; inhibitors ; Protein Kinase Inhibitors/*pharmacology ; RNA, Guide/genetics/pharmacology ; Spectrometry, Fluorescence ; }, abstract = {The role of interferon-gamma (IFN-γ) in Chronic Myelogenous/Myeloid Leukemia (CML) and in the treatment of CML remains unclear; specifically, the effect of IFN-γ on apoptosis. There is reported interplay between IFN-γ and glycogen synthase kinase-3 (GSK-3), a kinase which has been implicated in both cell death and, conversely, cell survival. Thus, we utilized the CML-derived HAP1 cell line and a mutant HAP1 GSK-3β knocked-down cell line (GSK-3β 31bp) to investigate whether GSK-3 modulates IFN-γ's action on CML cells. Significantly less GSK-3β 31bp cells, relative to HAP1 cells, were present after 48 h treatment with IFN-γ. IFN-γ treatment significantly decreased GSK-3β 31bp substrate adhesiveness (relative to HAP1 cells); an observation often correlated with cell death. Fluorescence microscopy revealed that IFN-γ induces a modest level of apoptosis in the HAP1 cells and that IFN-γ induced apoptosis is significantly enhanced in GSK-3β 31bp cells. Utilizing a complementary GSK-3β knocked-down cell line (8bp) we found, via flow cytometric analysis, that IFN-γ induced apoptosis is significantly enhanced in GSK-3β 8bp cells relative to HAP1 cells. Combined, our findings suggest that IFN-γ induces apoptosis of CML cells and that loss of GSK-3β significantly augments IFN-γ-induced apoptosis.}, }
@article {pmid33116147, year = {2020}, author = {Schmidt, JK and Strelchenko, N and Park, MA and Kim, YH and Mean, KD and Schotzko, ML and Kang, HJ and Golos, TG and Slukvin, II}, title = {Genome editing of CCR5 by CRISPR-Cas9 in Mauritian cynomolgus macaque embryos.}, journal = {Scientific reports}, volume = {10}, number = {1}, pages = {18457}, pmid = {33116147}, issn = {2045-2322}, support = {HD099154-01/HD/NICHD NIH HHS/United States ; R24OD021322/NH/NIH HHS/United States ; P51 OD011106/OD/NIH HHS/United States ; }, mesh = {Animals ; *Animals, Genetically Modified/embryology/genetics ; *CRISPR-Cas Systems ; Embryo, Mammalian/*metabolism ; *Gene Editing ; Macaca fascicularis ; *Receptors, CCR5/genetics/metabolism ; }, abstract = {The discovery that CCR5 serves as an R5-HIV-1 co-receptor, coupled with findings of protection from HIV infection in individuals lacking CCR5, led to the exploration of novel therapeutic strategies for HIV infection based on genome editing of CCR5. Advancing translation of CCR5-mutant-based cellular therapies for HIV requires development of novel physiologically relevant animal models. Mauritian cynomolgus macaques (MCMs), with high degree of MHC allele sharing, are valuable models for HIV-1 research and stem cell therapies. To facilitate the generation of a CCR5-mutant MHC-defined MCM model, we explored editing the CCR5 gene in MCM embryos via CRISPR-Cas9. We refined ovarian stimulation and in vitro fertilization (IVF) methods established for Chinese cynomolgus macaques to generate in vitro MCM embryos. Time-lapse embryo imaging was performed to assess the timing of MCM embryonic developmental events in control and CRISPR-Cas9 microinjected embryos. Using a dual-guide gene targeting approach, biallelic deletions in the CCR5 gene were introduced into ~ 23-37% of MCM embryos. In addition, single blastomere PCR analysis revealed mosaicism in CCR5 editing within the same embryo. Successful development of IVF and CCR5 editing protocols in MCM embryos lays a foundation for the creation of CCR5-mutant MCMs to assess novel stem cell-based HIV therapeutics.}, }
@article {pmid32764465, year = {2020}, author = {Son, J and Jang, SH and Cha, JW and Jeong, KJ}, title = {Development of CRISPR Interference (CRISPRi) Platform for Metabolic Engineering of Leuconostoc citreum and Its Application for Engineering Riboflavin Biosynthesis.}, journal = {International journal of molecular sciences}, volume = {21}, number = {16}, pages = {}, pmid = {32764465}, issn = {1422-0067}, support = {NRF-2014M3A6A8066443//Intelligent Synthetic Biology Center of the Global Frontier Project/ ; NRF-2020R1A2C2012537//National Research Foundation of Korea/ ; }, mesh = {CRISPR-Cas Systems/*genetics ; Humans ; Lactic Acid/metabolism ; Leuconostoc/*genetics/metabolism ; *Metabolic Engineering ; Plasmids/genetics ; Probiotics/metabolism ; RNA, Guide/genetics ; Riboflavin/biosynthesis/*genetics ; Streptococcus pyogenes/genetics ; }, abstract = {Leuconostoccitreum, a hetero-fermentative type of lactic acid bacteria, is a crucial probiotic candidate because of its ability to promote human health. However, inefficient gene manipulation tools limit its utilization in bioindustries. We report, for the first time, the development of a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) interference (CRISPRi) system for engineering L. citreum. For reliable expression, the expression system of synthetic single guide RNA (sgRNA) and the deactivated Cas9 of Streptococcus pyogenes (SpdCas9) were constructed in a bicistronic design (BCD) platform using a high-copy-number plasmid. The expression of SpdCas9 and sgRNA was optimized by examining the combination of two synthetic promoters and Shine-Dalgarno sequences; the strong expression of sgRNA and the weak expression of SpdCas9 exhibited the most significant downregulation (20-fold decrease) of the target gene (sfGFP), without cell growth retardation caused by SpdCas9 overexpression. The feasibility of the optimized CRISPRi system was demonstrated by modulating the biosynthesis of riboflavin. Using the CRISPRi system, the expression of ribF and folE genes was downregulated (3.3-fold and 5.6-fold decreases, respectively), thereby improving riboflavin production. In addition, the co-expression of the rib operon was introduced and the production of riboflavin was further increased up to 1.7 mg/L, which was 1.53 times higher than that of the wild-type strain.}, }
@article {pmid32726977, year = {2020}, author = {Chokhachi Baradaran, P and Kozovska, Z and Furdova, A and Smolkova, B}, title = {Targeting Epigenetic Modifications in Uveal Melanoma.}, journal = {International journal of molecular sciences}, volume = {21}, number = {15}, pages = {}, pmid = {32726977}, issn = {1422-0067}, support = {APVV-17-0369//Agentúra na Podporu Výskumu a Vývoja/ ; KEGA 016UK-4/2018//Kultúrna a Edukacná Grantová Agentúra MŠVVaŠ SR/ ; }, mesh = {CRISPR-Cas Systems ; *DNA Methylation ; *DNA, Neoplasm/genetics/metabolism ; *Epigenesis, Genetic ; *Gene Expression Regulation, Neoplastic ; *Genetic Therapy ; Histones/genetics/metabolism ; Humans ; *Melanoma/genetics/metabolism/therapy ; Neoplasm Proteins/genetics/metabolism ; Protein Processing, Post-Translational ; *Uveal Neoplasms/genetics/metabolism/therapy ; }, abstract = {Uveal melanoma (UM), the most common intraocular malignancy in adults, is a rare subset of melanoma. Despite effective primary therapy, around 50% of patients will develop the metastatic disease. Several clinical trials have been evaluated for patients with advanced UM, though outcomes remain dismal due to the lack of efficient therapies. Epigenetic dysregulation consisting of aberrant DNA methylation, histone modifications, and small non-coding RNA expression, silencing tumor suppressor genes, or activating oncogenes, have been shown to play a significant role in UM initiation and progression. Given that there is no evidence any approach improves results so far, adopting combination therapies, incorporating a new generation of epigenetic drugs targeting these alterations, may pave the way for novel promising therapeutic options. Furthermore, the fusion of effector enzymes with nuclease-deficient Cas9 (dCas9) in clustered regularly interspaced short palindromic repeats (CRISPR) associated protein 9 (Cas9) system equips a potent tool for locus-specific erasure or establishment of DNA methylation as well as histone modifications and, therefore, transcriptional regulation of specific genes. Both, CRISPR-dCas9 potential for driver epigenetic alterations discovery, and possibilities for their targeting in UM are highlighted in this review.}, }
@article {pmid32705536, year = {2020}, author = {Park, CW and Bae, JS and Ryu, KY}, title = {Simultaneous Disruption of Both Polyubiquitin Genes Affects Proteasome Function and Decreases Cellular Proliferation.}, journal = {Cell biochemistry and biophysics}, volume = {78}, number = {3}, pages = {321-329}, doi = {10.1007/s12013-020-00933-2}, pmid = {32705536}, issn = {1559-0283}, support = {2019R1F1A1061484, 2020R1F1A1070847//National Research Foundation of Korea (KR)/ ; }, mesh = {CRISPR-Cas Systems ; Cell Proliferation ; Gene Knockout Techniques ; HEK293 Cells ; HeLa Cells ; Humans ; Phosphorylation ; Polyubiquitin/*chemistry ; Proteasome Endopeptidase Complex/*chemistry ; Transfection ; Ubiquitin/*chemistry ; Ubiquitin C/chemistry/metabolism ; }, abstract = {The ubiquitin (Ub) proteasome system is important for maintaining protein homeostasis and has various roles in cell signaling, proliferation, and cell cycle regulation. In mammals, Ub is encoded by two monoubiquitin and two polyubiquitin genes. Although reduced levels of Ub due to the disruption of one polyubiquitin gene are known to decrease cell proliferation, the effect of disrupting both polyubiquitin genes remains elusive. Polyubiquitin gene Ubc knockout mice are embryonically lethal and polyubiquitin gene Ubb knockout mice are infertile. Thus, it is difficult to study the effects of double knockouts (DKOs). In the present study, the CRISPR/Cas9 system was used to simultaneously knockout both polyubiquitin genes, UBB and UBC, in HEK293T and HeLa cells. In DKO cells, growth decreased significantly compared to the control cells. We observed reduced proteasome function and reduced levels of free Ub in DKO cells. However, the levels of purified proteasome were not different between control and DKO cells, although the mRNA levels of proteasomal subunits were significantly increased in latter. We propose that the reduction of Ub levels, by disruption of both polyubiquitin genes, resulted in an altered proteasomal status, leading to the reduced proteasome activity, and decreased cellular proliferation.}, }
@article {pmid32549410, year = {2020}, author = {Sarrou, E and Richmond, L and Carmody, RJ and Gibson, B and Keeshan, K}, title = {CRISPR Gene Editing of Murine Blood Stem and Progenitor Cells Induces MLL-AF9 Chromosomal Translocation and MLL-AF9 Leukaemogenesis.}, journal = {International journal of molecular sciences}, volume = {21}, number = {12}, pages = {}, pmid = {32549410}, issn = {1422-0067}, support = {NA//Kay Kendall Leukaemia Fund/ ; NA//The Howat Foundation/ ; C596/A18076//Cancer Research UK Glasgow Centre/ ; C596/A17196//Cancer Research UK Beatson Institute/ ; NA//Yorkhill Children's Leukaemia Research Fund/ ; NA//Children with Cancer UK/ ; }, mesh = {Animals ; CRISPR-Cas Systems ; Cells, Cultured ; Chromosome Breakpoints ; Disease Models, Animal ; Gene Editing/*methods ; HEK293 Cells ; Hematopoietic Stem Cells/*cytology/metabolism ; Histone-Lysine N-Methyltransferase/*genetics ; Humans ; Leukemia/*genetics ; Mice ; Models, Biological ; Myeloid-Lymphoid Leukemia Protein/*genetics ; NIH 3T3 Cells ; Nuclear Proteins/*genetics ; Proto-Oncogene Proteins c-kit/*genetics ; }, abstract = {Chromosomal rearrangements of the mixed lineage leukaemia (MLL, also known as KMT2A) gene on chromosome 11q23 are amongst the most common genetic abnormalities observed in human acute leukaemias. MLL rearrangements (MLLr) are the most common cytogenetic abnormalities in infant and childhood acute myeloid leukaemia (AML) and acute lymphocytic leukaemia (ALL) and do not normally acquire secondary mutations compared to other leukaemias. To model these leukaemias, we have used clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing to induce MLL-AF9 (MA9) chromosomal rearrangements in murine hematopoietic stem and progenitor cell lines and primary cells. By utilizing a dual-single guide RNA (sgRNA) approach targeting the breakpoint cluster region of murine Mll and Af9 equivalent to that in human MA9 rearrangements, we show efficient de novo generation of MA9 fusion product at the DNA and RNA levels in the bulk population. The leukaemic features of MA9-induced disease were observed including increased clonogenicity, enrichment of c-Kit-positive leukaemic stem cells and increased MA9 target gene expression. This approach provided a rapid and reliable means of de novo generation of Mll-Af9 genetic rearrangements in murine haematopoietic stem and progenitor cells (HSPCs), using CRISPR/Cas9 technology to produce a cellular model of MA9 leukaemias which faithfully reproduces many features of the human disease in vitro.}, }
@article {pmid32466287, year = {2020}, author = {Ibragimova, S and Szebenyi, C and Sinka, R and Alzyoud, EI and Homa, M and Vágvölgyi, C and Nagy, G and Papp, T}, title = {CRISPR-Cas9-Based Mutagenesis of the Mucormycosis-Causing Fungus Lichtheimia corymbifera.}, journal = {International journal of molecular sciences}, volume = {21}, number = {10}, pages = {}, pmid = {32466287}, issn = {1422-0067}, support = {LP2016-8/2016//Magyar Tudományos Akadémia/ ; 460050//Magyar Tudományos Akadémia/ ; K131796//Nemzeti Kutatási Fejlesztési és Innovációs Hivatal/ ; TUDFO/4738-1/2019 ITM//Emberi Eroforrások Minisztériuma/ ; }, mesh = {*CRISPR-Cas Systems ; Fungal Proteins/genetics ; Mucorales/*genetics ; *Mutagenesis ; Orotidine-5'-Phosphate Decarboxylase/genetics ; }, abstract = {Lichtheimia corymbifera is considered as one of the most frequent agents of mucormycosis. The lack of efficient genetic manipulation tools hampers the characterization of the pathomechanisms and virulence factors of this opportunistic pathogenic fungus. Although such techniques have been described for certain species, the performance of targeted mutagenesis and the construction of stable transformants have remained a great challenge in Mucorales fungi. In the present study, a plasmid-free CRISPR-Cas9 system was applied to carry out a targeted gene disruption in L. corymbifera. The described method is based on the non-homologous end-joining repair of the double-strand break caused by the Cas9 enzyme. Using this method, short, one-to-five nucleotide long-targeted deletions could be induced in the orotidine 5'-phosphate decarboxylase gene (pyrG) and, as a result, uracil auxotrophic strains were constructed. These strains are applicable as recipient strains in future gene manipulation studies. As we know, this is the first genetic modification of this clinically relevant fungus.}, }
@article {pmid32299104, year = {2020}, author = {Khan, DH and Mullokandov, M and Wu, Y and Voisin, V and Gronda, M and Hurren, R and Wang, X and MacLean, N and Jeyaraju, DV and Jitkova, Y and Xu, GW and Laister, R and Seneviratne, A and Blatman, ZM and Ketela, T and Bader, GD and Marhon, SA and De Carvalho, DD and Minden, MD and Gross, A and Schimmer, AD}, title = {Mitochondrial carrier homolog 2 is necessary for AML survival.}, journal = {Blood}, volume = {136}, number = {1}, pages = {81-92}, doi = {10.1182/blood.2019000106}, pmid = {32299104}, issn = {1528-0020}, support = {/CAPMC/CIHR/Canada ; }, mesh = {Acetylation ; Animals ; CRISPR-Cas Systems ; Cell Differentiation ; Cell Line, Tumor ; Cell Nucleus/metabolism ; Fetal Blood/cytology ; Gene Expression Regulation, Leukemic/genetics ; Gene Knockdown Techniques ; Histones/metabolism ; Humans ; Leukemia, Myeloid, Acute/genetics/*metabolism/pathology ; Mice ; Mice, Inbred C57BL ; Mitochondria/*metabolism ; Mitochondrial Membrane Transport Proteins/*physiology ; Myeloid-Lymphoid Leukemia Protein/physiology ; Neoplasm Proteins/*physiology ; Oncogene Proteins, Fusion/physiology ; Protein Processing, Post-Translational ; Pyruvic Acid/metabolism ; RNA Interference ; RNA, Small Interfering/genetics/pharmacology ; }, abstract = {Through a clustered regularly insterspaced short palindromic repeats (CRISPR) screen to identify mitochondrial genes necessary for the growth of acute myeloid leukemia (AML) cells, we identified the mitochondrial outer membrane protein mitochondrial carrier homolog 2 (MTCH2). In AML, knockdown of MTCH2 decreased growth, reduced engraftment potential of stem cells, and induced differentiation. Inhibiting MTCH2 in AML cells increased nuclear pyruvate and pyruvate dehydrogenase (PDH), which induced histone acetylation and subsequently promoted the differentiation of AML cells. Thus, we have defined a new mechanism by which mitochondria and metabolism regulate AML stem cells and gene expression.}, }
@article {pmid33592378, year = {2021}, author = {Yin, JH and Elumalai, P and Kim, SY and Zhang, SZ and Shin, S and Lee, M and Chung, YJ}, title = {TNNC1 knockout reverses metastatic potential of ovarian cancer cells by inactivating epithelial-mesenchymal transition and suppressing F-actin polymerization.}, journal = {Biochemical and biophysical research communications}, volume = {547}, number = {}, pages = {44-51}, doi = {10.1016/j.bbrc.2021.02.021}, pmid = {33592378}, issn = {1090-2104}, abstract = {Troponin C type 1 (TNNC1) is commonly overexpressed in ovarian cancer. However, the biological implications of TNNC1 overexpression on ovarian cancer malignization and its related mechanism remain unknown. To elucidate these implications, we knocked out the TNNC1 gene in TNNC1-overexpressing SKOV-3-13 ovarian cancer cells using CRISPR/Cas-9 technology and observed the changes in metastatic phenotypes and related molecular pathways. TNNC1-knockout (KO) cells showed significantly reduced proliferation and colony formation when compared with TNNC1 wild-type cells (P < 0.01). In TNNC1-KO cells, wound healing, migration, and invasive phenotypes decreased. Upon observation of upstream regulators of epithelial-mesenchymal transition (EMT), levels of phosphorylated AKT (Ser-473 and Thr-308) and GSK-3β (inactive form) were found to be decreased in TNNC1-KO cells. Accordingly, SNAIL and SLUG expression decreased and were almost completely localized in the cytoplasm following TNNC1 silencing. Regarding downstream EMT markers, N-cadherin and vimentin expression decreased, but E-cadherin expression increased. Related matrix metalloproteinase and chemokine expression generally decreased. TNNC1 deficiency also suppressed F-actin polymerization. In conclusion, TNNC1 overexpression contributes to the metastatic behavior of ovarian cancer by perturbation of EMT and actin microfilaments. Our results provide a better understanding of the detailed molecular mechanism of ovarian cancer metastasis associated with TNNC1 overexpression.}, }
@article {pmid33414202, year = {2021}, author = {Kaiser, J}, title = {Gene therapy beats premature-aging syndrome in mice.}, journal = {Science (New York, N.Y.)}, volume = {371}, number = {6525}, pages = {114}, doi = {10.1126/science.371.6525.114}, pmid = {33414202}, issn = {1095-9203}, mesh = {Adolescent ; Animals ; CRISPR-Cas Systems ; Child ; Child, Preschool ; Disease Models, Animal ; Gene Editing/*methods ; Genetic Therapy/*methods ; Humans ; Mice ; Progeria/*therapy ; }, }
@article {pmid33310733, year = {2021}, author = {Ning, B and Yu, T and Zhang, S and Huang, Z and Tian, D and Lin, Z and Niu, A and Golden, N and Hensley, K and Threeton, B and Lyon, CJ and Yin, XM and Roy, CJ and Saba, NS and Rappaport, J and Wei, Q and Hu, TY}, title = {A smartphone-read ultrasensitive and quantitative saliva test for COVID-19.}, journal = {Science advances}, volume = {7}, number = {2}, pages = {}, doi = {10.1126/sciadv.abe3703}, pmid = {33310733}, issn = {2375-2548}, support = {P51 OD011104/OD/NIH HHS/United States ; R01 AI113725/AI/NIAID NIH HHS/United States ; R01 AI122932/AI/NIAID NIH HHS/United States ; R01 HD090927/HD/NICHD NIH HHS/United States ; R21 AI126361/AI/NIAID NIH HHS/United States ; }, mesh = {Animals ; COVID-19/*diagnosis ; *COVID-19 Nucleic Acid Testing ; CRISPR-Cas Systems ; Chlorocebus aethiops ; Computer Simulation ; Female ; Humans ; Limit of Detection ; Macaca mulatta ; Male ; Molecular Diagnostic Techniques/instrumentation ; *Point-of-Care Testing ; Reverse Transcriptase Polymerase Chain Reaction ; Saliva/*virology ; Sensitivity and Specificity ; *Smartphone ; Vero Cells ; Viral Load ; }, abstract = {Point-of-care COVID-19 assays that are more sensitive than the current RT-PCR (reverse transcription polymerase chain reaction) gold standard assay are needed to improve disease control efforts. We describe the development of a portable, ultrasensitive saliva-based COVID-19 assay with a 15-min sample-to-answer time that does not require RNA isolation or laboratory equipment. This assay uses CRISPR-Cas12a activity to enhance viral amplicon signal, which is stimulated by the laser diode of a smartphone-based fluorescence microscope device. This device robustly quantified viral load over a broad linear range (1 to 105 copies/μl) and exhibited a limit of detection (0.38 copies/μl) below that of the RT-PCR reference assay. CRISPR-read SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) RNA levels were similar in patient saliva and nasal swabs, and viral loads measured by RT-PCR and the smartphone-read CRISPR assay demonstrated good correlation, supporting the potential use of this portable assay for saliva-based point-of-care COVID-19 diagnosis.}, }
@article {pmid33305456, year = {2021}, author = {Mohamadian, M and Chiti, H and Shoghli, A and Biglari, S and Parsamanesh, N and Esmaeilzadeh, A}, title = {COVID-19: Virology, biology and novel laboratory diagnosis.}, journal = {The journal of gene medicine}, volume = {23}, number = {2}, pages = {e3303}, doi = {10.1002/jgm.3303}, pmid = {33305456}, issn = {1521-2254}, mesh = {COVID-19/*diagnosis/physiopathology/prevention &amp; control/virology ; COVID-19 Nucleic Acid Testing/methods ; CRISPR-Cas Systems/genetics ; High-Throughput Nucleotide Sequencing/methods ; Humans ; Microarray Analysis ; Molecular Diagnostic Techniques/methods ; Nucleic Acid Amplification Techniques/methods ; Real-Time Polymerase Chain Reaction/methods ; SARS-CoV-2/genetics/metabolism/*pathogenicity ; }, abstract = {BACKGROUND: At the end of December 2019, a novel coronavirus tentatively named SARS-CoV-2 in Wuhan, a central city in China, was announced by the World Health Organization. SARS-CoV-2 is an RNA virus that has become a major public health concern after the outbreak of the Middle East Respiratory Syndrome-CoV (MERS-CoV) and Severe Acute Respiratory Syndrome-CoV (SARS-CoV) in 2002 and 2012, respectively. As of 29 October 2020, the total number of COVID-19 cases had reached over 44 million worldwide, with more than 1.17 million confirmed deaths.<br><br>DISCUSSION: SARS-CoV-2 infected patients usually present with severe viral pneumonia. Similar to SARS-CoV, the virus enters respiratory tract cells via the angiotensin-converting enzyme receptor 2. The structural proteins play an essential role in budding the virus particles released from different host cells. To date, an approved vaccine or treatment option of a preventive character to avoid severe courses of COVID-19 is still not available.<br><br>CONCLUSIONS: In the present study, we provide a brief review of the general biological features of CoVs and explain the pathogenesis, clinical symptoms and diagnostic approaches regarding monitoring future infectivity and prevent emerging COVID-19 infections.}, }
@article {pmid32809800, year = {2020}, author = {Chen, M and Luo, R and Li, S and Li, H and Qin, Y and Zhou, D and Liu, H and Gong, X and Chang, J}, title = {Paper-Based Strip for Ultrasensitive Detection of OSCC-Associated Salivary MicroRNA via CRISPR/Cas12a Coupling with IS-Primer Amplification Reaction.}, journal = {Analytical chemistry}, volume = {92}, number = {19}, pages = {13336-13342}, doi = {10.1021/acs.analchem.0c02642}, pmid = {32809800}, issn = {1520-6882}, mesh = {Bacterial Proteins/genetics ; CRISPR-Associated Proteins/genetics ; CRISPR-Cas Systems/genetics ; Endodeoxyribonucleases/genetics ; Head and Neck Neoplasms/*diagnosis ; Humans ; MicroRNAs/*analysis/genetics ; *Nucleic Acid Amplification Techniques ; *Paper ; Reagent Strips/*chemistry ; Saliva/*chemistry ; Squamous Cell Carcinoma of Head and Neck/*diagnosis ; }, abstract = {As the most common malignancy in humans, oral squamous cell carcinoma (OSCC) not only harms the people's health but also undermines their confidence after facial surgery. Early detection and treatment can effectively reduce these damages. The unique collateral trans-cleavage nuclease activity of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a system was utilized to realize the detection of nucleic acid with high sensitivity. So, in this work, we designed a point-of-care testing (POCT) platform for the detection of OSCC-associated salivary hsa-miRNA 31-5p (miR-31) via the cascade signal amplification of &quot;invading stacking primer&quot; (IS-primer) amplification reaction (ISAR), CRISPR/Cas12a, and dual-mode paper-based strip (dm-Strip). To amplify the detection signal of trace miR-31, the cascade signal amplification of CRISPR/Cas12a system coupling with ISAR was designed in a one-pot reaction at a constant temperature. The target miR-31 could activate the ISAR to generate numerous DNAs, which would further trigger the trans-cleavage effect of Cas12a to catalyze the nonspecific single-stranded DNA (ssDNA) cleavage. This ssDNA was labeled with digoxin and biotin at the 5' and 3' termini (digoxin/ssDNA/biotin), respectively, which led to generate the naked-eye signal and fluorescent signal of the designed dm-Strip. The whole detection time was 90 min with limit-of-detection (LOD) down to aM level. This ISAR/Cas12a-based dm-Strip (ISAR/Cas12a-dmStrip) allowed for the portable and ultrasensitive detection of miRNA, an important step in early diagnosis of OSCC and biomedical research.}, }
@article {pmid32783942, year = {2020}, author = {Zhou, P and Chan, BKC and Wan, YK and Yuen, CTL and Choi, GCG and Li, X and Tong, CSW and Zhong, SSW and Sun, J and Bao, Y and Mak, SYL and Chow, MZY and Khaw, JV and Leung, SY and Zheng, Z and Cheung, LWT and Tan, K and Wong, KH and Chan, HYE and Wong, ASL}, title = {A Three-Way Combinatorial CRISPR Screen for Analyzing Interactions among Druggable Targets.}, journal = {Cell reports}, volume = {32}, number = {6}, pages = {108020}, doi = {10.1016/j.celrep.2020.108020}, pmid = {32783942}, issn = {2211-1247}, mesh = {Animals ; Antineoplastic Agents/pharmacology ; *CRISPR-Cas Systems ; Drosophila melanogaster ; *Drug Combinations ; Drug Delivery Systems/*methods ; Gene Knockout Techniques ; HEK293 Cells ; High-Throughput Screening Assays/*methods ; Humans ; Mice ; Neoplasms/drug therapy ; Parkinson Disease/drug therapy ; RNA, Guide ; }, abstract = {We present a CRISPR-based multi-gene knockout screening system and toolkits for extensible assembly of barcoded high-order combinatorial guide RNA libraries en masse. We apply this system for systematically identifying not only pairwise but also three-way synergistic therapeutic target combinations and successfully validate double- and triple-combination regimens for suppression of cancer cell growth and protection against Parkinson's disease-associated toxicity. This system overcomes the practical challenges of experimenting on a large number of high-order genetic and drug combinations and can be applied to uncover the rare synergistic interactions between druggable targets.}, }
@article {pmid32751693, year = {2020}, author = {Mazumdar, A and Urdinez, J and Boro, A and Migliavacca, J and Arlt, MJE and Muff, R and Fuchs, B and Snedeker, JG and Gvozdenovic, A}, title = {Osteosarcoma-Derived Extracellular Vesicles Induce Lung Fibroblast Reprogramming.}, journal = {International journal of molecular sciences}, volume = {21}, number = {15}, pages = {}, pmid = {32751693}, issn = {1422-0067}, mesh = {Actins/*genetics ; CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; Cellular Reprogramming/*genetics ; Extracellular Vesicles/metabolism/pathology ; Fibroblasts/metabolism/pathology ; Gene Expression Regulation, Neoplastic/genetics ; Humans ; Lung/metabolism ; Neoplasm Invasiveness/genetics/pathology ; Neoplasm Metastasis ; Osteosarcoma/*genetics/pathology ; Receptor, Transforming Growth Factor-beta Type I/antagonists &amp; inhibitors/*genetics ; }, abstract = {Tumor-secreted extracellular vesicles (EVs) have been identified as mediators of cancer-host intercellular communication and shown to support pre-metastatic niche formation by modulating stromal cells at future metastatic sites. While osteosarcoma, the most common primary malignant bone tumor in children and adolescents, has a high propensity for pulmonary metastases, the interaction of osteosarcoma cells with resident lung cells remains poorly understood. Here, we deliver foundational in vitro evidence that osteosarcoma cell-derived EVs drive myofibroblast/cancer-associated fibroblast differentiation. Human lung fibroblasts displayed increased invasive competence, in addition to increased α-smooth muscle actin expression and fibronectin production upon EV treatment. Furthermore, we demonstrate, through the use of transforming growth factor beta receptor 1 (TGFBR1) inhibitors and CRISPR-Cas9-mediated knockouts, that TGFβ1 present in osteosarcoma cell-derived EVs is responsible for lung fibroblast differentiation. Overall, our study highlights osteosarcoma-derived EVs as novel regulators of lung fibroblast activation and provides mechanistic insight into how osteosarcoma cells can modulate distant cells to potentially support metastatic progression.}, }
@article {pmid32686823, year = {2020}, author = {Okada, K and Takase, R and Hamaoka, Y and Honda, A and Ikeda, A and Hokazono, Y and Maeda, Y and Hayasaka, O and Kotani, T and Komatsu, M and Shiozaki, K}, title = {Establishment and characterization of Neu1-knockout zebrafish and its abnormal clinical phenotypes.}, journal = {The Biochemical journal}, volume = {477}, number = {15}, pages = {2841-2857}, doi = {10.1042/BCJ20200348}, pmid = {32686823}, issn = {1470-8728}, mesh = {Animals ; Animals, Genetically Modified ; Body Weight/genetics ; CRISPR-Cas Systems ; Disease Models, Animal ; Embryo, Nonmammalian ; Female ; Gene Expression Regulation, Developmental ; Gene Knockout Techniques ; Glycoproteins/genetics/metabolism ; HEK293 Cells ; Humans ; Hydrogen-Ion Concentration ; Male ; Mucolipidoses/etiology/genetics ; N-Acetylneuraminic Acid/metabolism ; Neuraminidase/*genetics/*metabolism ; Osteogenesis/genetics ; Phenotype ; Zebrafish/embryology/*genetics ; Zebrafish Proteins/*genetics/metabolism ; }, abstract = {Mammalian sialidase Neu1 is involved in various physiological functions, including cell adhesion, differentiation, cancer metastasis, and diabetes through lysosomal catabolism and desialylation of glycoproteins at the plasma membrane. Various animal models have been established to further explore the functions of vertebrate Neu1. The present study focused on zebrafish (Danio rerio) belonging to Cypriniformes as an experimental animal model with neu1 gene deficiency. The results revealed that the zebrafish Neu1 desialyzed both α2-3 and α2-6 sialic acid linkages from oligosaccharides and glycoproteins at pH 4.5, and it is highly conserved with other fish species and mammalian Neu1. Furthermore, Neu1-knockout zebrafish (Neu1-KO) was established through CRISPR/Cas9 genome editing. Neu1-KO fish exhibited slight abnormal embryogenesis with the accumulation of pleural effusion; however, no embryonic lethality was observed. Although Neu1-KO fish were able to be maintained as homozygous, they showed smaller body length and weight than the wild-type (WT) fish, and muscle atrophy and curvature of the vertebra were observed in adult Neu1-KO fish (8 months). The expression patterns of myod and myog transcription factors regulating muscle differentiation varied between Neu1-KO and WT fish embryo. Expression of lysosomal-related genes, including ctsa, lamp1a, and tfeb were up-regulated in adult Neu1-KO muscle as compared with WT. Furthermore, the expression pattern of genes involved in bone remodeling (runx2a, runx2b, and mmp9) was decreased in Neu1-KO fish. These phenotypes were quite similar to those of Neu1-KO mice and human sialidosis patients, indicating the effectiveness of the established Neu1-KO zebrafish for the study of vertebrate Neu1 sialidase.}, }
@article {pmid32650483, year = {2020}, author = {Delos Santos, K and Kwon, E and Moon, NS}, title = {PRPS-Associated Disorders and the Drosophila Model of Arts Syndrome.}, journal = {International journal of molecular sciences}, volume = {21}, number = {14}, pages = {}, pmid = {32650483}, issn = {1422-0067}, support = {162337/CAPMC/CIHR/Canada ; }, mesh = {Animals ; Ataxia/*genetics ; CRISPR-Cas Systems/genetics ; Deaf-Blind Disorders/*genetics ; Disease Models, Animal ; Drosophila melanogaster/*genetics ; Gene Editing/methods ; Genetic Diseases, X-Linked/*genetics ; Humans ; Mutation/genetics ; Ribose-Phosphate Pyrophosphokinase/*genetics ; }, abstract = {While a plethora of genetic techniques have been developed over the past century, modifying specific sequences of the fruit fly genome has been a difficult, if not impossible task. clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 truly redefined molecular genetics and provided new tools to model human diseases in Drosophila melanogaster. This is particularly true for genes whose protein sequences are highly conserved. Phosphoribosyl pyrophosphate synthetase (PRPS) is a rate-limiting enzyme in nucleotide metabolism whose missense mutations are found in several neurological disorders, including Arts syndrome. In addition, PRPS is deregulated in cancer, particularly those that become resistant to cancer therapy. Notably, DrosophilaPRPS shares about 90% protein sequence identity with its human orthologs, making it an ideal gene to study via CRISPR/Cas9. In this review, we will summarize recent findings on PRPS mutations in human diseases including cancer and on the molecular mechanisms by which PRPS activity is regulated. We will also discuss potential applications of Drosophila CRISPR/Cas9 to model PRPS-dependent disorders and other metabolic diseases that are associated with nucleotide metabolism.}, }
@article {pmid32580383, year = {2020}, author = {Hao, W and Wang, L and Li, S}, title = {FKBP5 Regulates RIG-I-Mediated NF-κB Activation and Influenza A Virus Infection.}, journal = {Viruses}, volume = {12}, number = {6}, pages = {}, pmid = {32580383}, issn = {1999-4915}, support = {R15 AI126360/AI/NIAID NIH HHS/United States ; R01 AI141399/AI/NIAID NIH HHS/United States ; R21 AI137750/AI/NIAID NIH HHS/United States ; }, mesh = {A549 Cells ; CRISPR-Cas Systems/genetics ; Cell Line ; DEAD Box Protein 58/*metabolism ; Gene Knockout Techniques ; HEK293 Cells ; Humans ; Immunity, Innate/immunology ; Influenza A virus/*immunology ; Influenza, Human/*immunology/pathology ; NF-KappaB Inhibitor alpha/*metabolism ; NF-kappa B/metabolism ; Tacrolimus Binding Proteins/genetics/*metabolism ; }, abstract = {Influenza A virus (IAV) is a highly transmissible respiratory pathogen and is a constant threat to global health with considerable economic and social impact. Influenza viral RNA is sensed by host pattern recognition receptors (PRRs), such as the Toll-like receptor 7 (TLR7) and retinoic acid-inducible gene I (RIG-I). The activation of these PRRs instigates the interferon regulatory factor (IRF) and nuclear factor kappa B (NF-κB) signaling pathways that induce the expression of interferon-stimulated genes (ISGs) and inflammatory genes. FK506-binding protein 5 (FKBP5) has been implied in the IκBα kinase (IKK) complex. However, the role of FKBP5 in the RIG-I signaling and IAV infection is not well elucidated. Here, we demonstrate that the knockout of FKBP5 increases IAV infection. Furthermore, FKBP5 binds IKKα, which is critical for RIG-I-induced innate immune responses and ISG expression. Taken together, FKBP5 is a novel anti-influenza host factor that restricts IAV infection by the activation of RIG-I-mediated NF-κB signaling.}, }
@article {pmid32516948, year = {2020}, author = {Ansari, WA and Chandanshive, SU and Bhatt, V and Nadaf, AB and Vats, S and Katara, JL and Sonah, H and Deshmukh, R}, title = {Genome Editing in Cereals: Approaches, Applications and Challenges.}, journal = {International journal of molecular sciences}, volume = {21}, number = {11}, pages = {}, pmid = {32516948}, issn = {1422-0067}, support = {BL/17-18/0577//University Grants Commission/ ; 2017//Department of Biotechnology , Ministry of Science and Technology/ ; }, mesh = {CRISPR-Cas Systems ; Crops, Agricultural/genetics ; Edible Grain/*genetics ; *Gene Editing ; *Genome, Plant ; *Genomics/methods ; Plants, Genetically Modified ; Stress, Physiological ; Transformation, Genetic ; }, abstract = {Over the past decades, numerous efforts were made towards the improvement of cereal crops mostly employing traditional or molecular breeding approaches. The current scenario made it possible to efficiently explore molecular understanding by targeting different genes to achieve desirable plants. To provide guaranteed food security for the rising world population particularly under vulnerable climatic condition, development of high yielding stress tolerant crops is needed. In this regard, technologies upgradation in the field of genome editing looks promising. Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 is a rapidly growing genome editing technique being effectively applied in different organisms, that includes both model and crop plants. In recent times CRISPR/Cas9 is being considered as a technology which revolutionized fundamental as well as applied research in plant breeding. Genome editing using CRISPR/Cas9 system has been successfully demonstrated in many cereal crops including rice, wheat, maize, and barley. Availability of whole genome sequence information for number of crops along with the advancement in genome-editing techniques provides several possibilities to achieve desirable traits. In this review, the options available for crop improvement by implementing CRISPR/Cas9 based genome-editing techniques with special emphasis on cereal crops have been summarized. Recent advances providing opportunities to simultaneously edit many target genes were also discussed. The review also addressed recent advancements enabling precise base editing and gene expression modifications. In addition, the article also highlighted limitations such as transformation efficiency, specific promoters and most importantly the ethical and regulatory issues related to commercial release of novel crop varieties developed through genome editing.}, }
@article {pmid32472264, year = {2020}, author = {Li, Q and Lu, L and Liu, H and Bai, X and Zhou, X and Wu, B and Yuan, M and Yang, L and Xing, Y}, title = {A minor QTL, SG3, encoding an R2R3-MYB protein, negatively controls grain length in rice.}, journal = {TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik}, volume = {133}, number = {8}, pages = {2387-2399}, doi = {10.1007/s00122-020-03606-z}, pmid = {32472264}, issn = {1432-2242}, support = {91935302//National Natural Science Foundation of China/ ; 2016YFD0100403//National Basic Research Program of China (973 Program) (CN)/ ; }, mesh = {Alleles ; CRISPR-Cas Systems ; Cell Nucleus/metabolism ; Chromosome Mapping/*methods ; Codon, Nonsense ; Edible Grain/*genetics ; Exons ; Gene Expression Regulation, Plant/genetics ; Gene Knockout Techniques ; *Genes, Plant ; INDEL Mutation ; Oryza/*genetics/growth &amp; development ; Phylogeny ; Plant Breeding/*methods ; Plants, Genetically Modified ; Polymorphism, Single Nucleotide ; Quantitative Trait Loci ; Seeds/*genetics/growth &amp; development ; Transcription Factors/*genetics/metabolism ; }, abstract = {KEY MESSAGE: SG3, an R2R3 MYB protein coding gene that tightly linked to a major QTLGS3, negatively regulates grain length while dependent on the status ofGS3in rice. It is often very difficult to isolate a minor QTL that is closely linked to a major QTL in rice. In this study, we focused on the isolation of a minor grain length QTL, small grain 3 (SG3), which is closely linked to the major QTL grain size 3 (GS3). The genetic effect of SG3 on grain length was dependent on GS3 status. Its genetic effect was larger in the presence of nonfunctional sg3 than functional SG3. A large number of near-isogenic F2 plants in which GS3 was fixed with nonfunctional alleles were developed to clone SG3. A gene encoding an R2R3 MYB domain transcriptional regulator was identified as the candidate gene for SG3. SG3 overexpression and knockdown plants showed shortened and elongated grains, respectively, which demonstrated that SG3 acts as a negative regulator of grain length. SG3 was preferentially expressed in panicles after flowering, and SG3 acted as a transcription activator. Comparative sequencing analysis identified a 12-bp insertion in the third exon of NYZ that led to a frameshift and resulted in a premature stop codon. The insertion/deletion was associated with grain length in the presence of functional GS3 in the indica subspecies. SG3 and GS3 were frequently in coupling phase in indica rice, making them good targets for the breeding of cultivars with short or long grains. The isolation of the SG3 MYB gene provides new gene resource and contributes to the regulatory network of grain length in rice.}, }
@article {pmid32471232, year = {2020}, author = {Yun, Y and Ha, Y}, title = {CRISPR/Cas9-Mediated Gene Correction to Understand ALS.}, journal = {International journal of molecular sciences}, volume = {21}, number = {11}, pages = {}, pmid = {32471232}, issn = {1422-0067}, mesh = {Amyotrophic Lateral Sclerosis/genetics/*therapy ; Animals ; C9orf72 Protein/genetics ; *CRISPR-Cas Systems ; DNA-Binding Proteins/genetics ; Humans ; RNA-Binding Protein FUS/genetics ; Superoxide Dismutase-1/genetics ; Targeted Gene Repair/*methods ; }, abstract = {Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease caused by the death of motor neurons in the spinal cord and brainstem. ALS has a diverse genetic origin; at least 20 genes have been shown to be related to ALS. Most familial and sporadic cases of ALS are caused by variants of the SOD1, C9orf72,FUS, and TARDBP genes. Genome editing using clustered regularly interspaced short palindromic repeats/CRISPR-associated system 9 (CRISPR/Cas9) can provide insights into the underlying genetics and pathophysiology of ALS. By correcting common mutations associated with ALS in animal models and patient-derived induced pluripotent stem cells (iPSCs), CRISPR/Cas9 has been used to verify the effects of ALS-associated mutations and observe phenotype differences between patient-derived and gene-corrected iPSCs. This technology has also been used to create mutations to investigate the pathophysiology of ALS. Here, we review recent studies that have used CRISPR/Cas9 to understand the genetic underpinnings of ALS.}, }
@article {pmid32466470, year = {2020}, author = {Han, JP and Chang, YJ and Song, DW and Choi, BS and Koo, OJ and Yi, SY and Park, TS and Yeom, SC}, title = {High Homology-Directed Repair Using Mitosis Phase and Nucleus Localizing Signal.}, journal = {International journal of molecular sciences}, volume = {21}, number = {11}, pages = {}, pmid = {32466470}, issn = {1422-0067}, support = {NRF-2017M3A9B4061409//National Research Foundation of Korea/ ; 2018R1A1A1A05078158//National Research Foundation of Korea/ ; }, mesh = {Animals ; CRISPR-Associated Protein 9/*genetics/metabolism ; CRISPR-Cas Systems ; Gene Editing/*methods ; Gene Knock-In Techniques/*methods ; Mice ; Mice, Inbred C57BL ; *Mitosis ; NIH 3T3 Cells ; *Nuclear Localization Signals ; Recombinant Proteins/genetics/metabolism ; Recombinational DNA Repair ; }, abstract = {In homology-directed repair, mediated knock-in single-stranded oligodeoxynucleotides (ssODNs) can be used as a homologous template and present high efficiency, but there is still a need to improve efficiency. Previous studies have mainly focused on controlling double-stranded break size, ssODN stability, and the DNA repair cycle. Nevertheless, there is a lack of research on the correlation between the cell cycle and single-strand template repair (SSTR) efficiency. Here, we investigated the relationship between cell cycle and SSTR efficiency. We found higher SSTR efficiency during mitosis, especially in the metaphase and anaphase. A Cas9 protein with a nuclear localization signal (NLS) readily migrated to the nucleus; however, the nuclear envelope inhibited the nuclear import of many nucleotide templates. This seemed to result in non-homologous end joining (NHEJ) before the arrival of the homologous template. Thus, we assessed whether NLS-tagged ssODNs and free NLS peptides could circumvent problems posed by the nuclear envelope. NLS-tagging ssODNs enhanced SSTR and indel efficiency by 4-fold compared to the control. Our results suggest the following: (1) mitosis is the optimal phase for SSTR, (2) the donor template needs to be delivered to the nucleus before nuclease delivery, and (3) NLS-tagging ssODNs improve SSTR efficiency, especially high in mitosis.}, }
@article {pmid32448919, year = {2020}, author = {Huang, H and Cui, T and Zhang, L and Yang, Q and Yang, Y and Xie, K and Fan, C and Zhou, Y}, title = {Modifications of fatty acid profile through targeted mutation at BnaFAD2 gene with CRISPR/Cas9-mediated gene editing in Brassica napus.}, journal = {TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik}, volume = {133}, number = {8}, pages = {2401-2411}, doi = {10.1007/s00122-020-03607-y}, pmid = {32448919}, issn = {1432-2242}, support = {2017YFE0104800//National Key Research and Development Program/ ; 2016YFD0100506-3//National Key Research and Development Program/ ; 31671725//National Natural Science Foundation of China/ ; }, mesh = {Alleles ; Brassica napus/*genetics/metabolism ; CRISPR-Cas Systems ; Chromatography, Gas ; Chromatography, Liquid ; Fatty Acid Desaturases/*genetics/metabolism ; Fatty Acids/*metabolism ; Frameshift Mutation ; Gene Editing/*methods ; *Genes, Plant ; Genotype ; Hypocotyl/genetics/metabolism ; Linoleic Acid/analysis ; Mutation ; Oleic Acid/analysis ; Plant Leaves/genetics/growth &amp; development/metabolism ; Plant Proteins/*genetics/metabolism ; Plant Roots/genetics/metabolism ; Plants, Genetically Modified/genetics ; Polyploidy ; RNA-Seq ; Seedlings/genetics/growth &amp; development/metabolism ; Seeds/chemistry/*genetics/growth &amp; development/metabolism ; alpha-Linolenic Acid/analysis ; }, abstract = {KEY MESSAGE: Genomic editing with CRISPR/Cas9 system can simultaneously modify multiple copies of theBnaFAD2 gene to develop novel variations in fatty acids profiles in polyploidy rapeseed. Fatty acid composition affects edible and processing quality of vegetable oil and has been one of the primary targets for genetic modification in oilseed crops including rapeseed (Brassica napus). Fatty acid desaturase 2 gene, FAD2, is a key player that affects three major fatty acids, namely oleic, linoleic and linolenic acid, in oilseed plants. Previously, we showed that there are four copies of BnaFAD2 in allotetraploid rapeseed. In this study, we further established spatiotemporal expression pattern of each copy of BnaFAD2 using published RNA-seq data. Genomic editing technology based on CRISPR/Cas9 system was used to mutate all the copies of BnaFAD2 to create novel allelic variations in oleic acid and other fatty acid levels. A number of mutants at two targeting sites were identified, and the phenotypic variation in the mutants was systematically evaluated. The oleic acid content in the seed of the mutants increased significantly with the highest exceeding 80% compared with wild type of 66.43%, while linoleic and linolenic acid contents decreased accordingly. Mutations on BnaFAD2.A5 caused more dramatic changes of fatty acid profile than the mutations on BnaFAD2.C5 alleles that were identified with gene editing technique for the first time. Moreover, combining different mutated alleles of BnaFAD2 can even broaden the variation more dramatically. It was found that effects of different mutation types at BnaFAD2 alleles on oleic levels varied, indicating a possibility to manipulate fatty acid levels by precise mutation at specific region of a gene.}, }
@article {pmid32443745, year = {2020}, author = {Korablev, A and Lukyanchikova, V and Serova, I and Battulin, N}, title = {On-Target CRISPR/Cas9 Activity Can Cause Undesigned Large Deletion in Mouse Zygotes.}, journal = {International journal of molecular sciences}, volume = {21}, number = {10}, pages = {}, pmid = {32443745}, issn = {1422-0067}, support = {18-29-07022//Russian Foundation for Basic Research/ ; 0324-2019-0041-C-01//Budget project of the Institute of Cytology and Genetics/ ; }, mesh = {Animals ; *CRISPR-Cas Systems ; Female ; *Gene Deletion ; Gene Knock-In Techniques/*adverse effects/methods ; Male ; Mice ; Mice, Inbred C57BL ; Recombinational DNA Repair ; Zygote/*metabolism ; }, abstract = {Genome engineering has been tremendously affected by the appearance of the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9)-based approach. Initially discovered as an adaptive immune system for prokaryotes, the method has rapidly evolved over the last decade, overtaking multiple technical challenges and scientific tasks and becoming one of the most effective, reliable, and easy-to-use technologies for precise genomic manipulations. Despite its undoubtable advantages, CRISPR/Cas9 technology cannot ensure absolute accuracy and predictability of genomic editing results. One of the major concerns, especially for clinical applications, is mutations resulting from error-prone repairs of CRISPR/Cas9-induced double-strand DNA breaks. In some cases, such error-prone repairs can cause unpredicted and unplanned large genomic modifications within the CRISPR/Cas9 on-target site. Here we describe the largest, to the best of our knowledge, undesigned on-target deletion with a size of ~293 kb that occurred after the cytoplasmic injection of CRISPR/Cas9 system components into mouse zygotes and speculate about its origin. We suppose that deletion occurred as a result of the truncation of one of the ends of a double-strand break during the repair.}, }
@article {pmid32405769, year = {2020}, author = {Biswas, S and Li, R and Hong, J and Zhao, X and Yuan, Z and Zhang, D and Shi, J}, title = {Effective identification of CRISPR/Cas9-induced and naturally occurred mutations in rice using a multiplex ligation-dependent probe amplification-based method.}, journal = {TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik}, volume = {133}, number = {8}, pages = {2323-2334}, doi = {10.1007/s00122-020-03600-5}, pmid = {32405769}, issn = {1432-2242}, support = {2016ZX08012-002//Agro-Industry Research and Development Special Fund of China/ ; 2017ZX08013001-001//Agro-Industry Research and Development Special Fund of China/ ; 2016ZX08009-003-007//Agro-Industry Research and Development Special Fund of China/ ; B14016//Overseas Expertise Introduction Project for Discipline Innovation/ ; }, mesh = {*CRISPR-Cas Systems ; Electrophoresis, Capillary ; Gene Editing/*methods ; Genetic Variation ; Genome, Plant ; INDEL Mutation ; Multiplex Polymerase Chain Reaction/*methods ; *Mutagenesis ; Mutation ; Oryza/*genetics ; Plants, Genetically Modified/*genetics ; Sensitivity and Specificity ; }, abstract = {KEY MESSAGE: A multiplex ligation-dependent probe amplification (MLPA)-based method was developed and successfully utilized to efficiently detect both CRISPR/Cas9-induced and naturally occurred mutations in rice. The site-specific nuclease-based CRISPR/Cas9 system has emerged as one of the most efficient genome editing tools to modify multiple genomic targets simultaneously in various organisms, including plants for both fundamental and applied researches. Screening for both on-target and off-target mutations in CRISPR/Cas9-generated mutants at the early stages is an indispensable step for functional analysis and subsequent application. Various methods have been developed to detect CRISPR/Cas9-induced mutations in plants. Still, very few have focused on the detection of both on- and off-targets simultaneously, let alone the detection of natural mutations. Here, we report a multiplex capable method that allows to detect CRISPR/Cas9 induced on- and off-target mutations as well as naturally occurred mutation based on a multiplex ligation-dependent probe amplification (MLPA) method. We demonstrated that unlike other methods, the modified target-specific MLPA method can accurately identify any INDELs generated naturally or by the CRISPR/Cas9 system and that it can detect natural variation and zygosity of the CRISPR/Cas9-generated mutants in rice as well. Furthermore, its high sensitivity allowed to define INDELs down to 1 bp and substitutions to a single nucleotide. Therefore, this sensitive, reliable, and cheap method would further accelerate functional analysis and marker-assisted breeding in plants, including rice.}, }
@article {pmid32357367, year = {2020}, author = {Freund, EC and Lock, JY and Oh, J and Maculins, T and Delamarre, L and Bohlen, CJ and Haley, B and Murthy, A}, title = {Efficient gene knockout in primary human and murine myeloid cells by non-viral delivery of CRISPR-Cas9.}, journal = {The Journal of experimental medicine}, volume = {217}, number = {7}, pages = {}, pmid = {32357367}, issn = {1540-9538}, mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Cells, Cultured ; Dendritic Cells/metabolism ; Gene Deletion ; Gene Editing ; *Gene Knockout Techniques ; *Gene Transfer Techniques ; Genetic Engineering ; Genome ; Humans ; Macrophages/metabolism ; Mice ; Monocytes/metabolism ; Myeloid Cells/*metabolism ; Phagocytosis ; Phenotype ; RNA, Guide/genetics ; Ribonucleoproteins/metabolism ; Viruses/metabolism ; }, abstract = {Myeloid cells play critical and diverse roles in mammalian physiology, including tissue development and repair, innate defense against pathogens, and generation of adaptive immunity. As cells that show prolonged recruitment to sites of injury or pathology, myeloid cells represent therapeutic targets for a broad range of diseases. However, few approaches have been developed for gene editing of these cell types, likely owing to their sensitivity to foreign genetic material or virus-based manipulation. Here we describe optimized strategies for gene disruption in primary myeloid cells of human and murine origin. Using nucleofection-based delivery of Cas9-ribonuclear proteins (RNPs), we achieved near population-level genetic knockout of single and multiple targets in a range of cell types without selection or enrichment. Importantly, we show that cellular fitness and response to immunological stimuli is not significantly impacted by the gene editing process. This provides a significant advance in the study of myeloid cell biology, thus enabling pathway discovery and drug target validation across species in the field of innate immunity.}, }
@article {pmid32230737, year = {2020}, author = {Li, J and Fang, K and Rong, Z and Li, X and Ren, X and Ma, H and Chen, H and Li, X and Qian, P}, title = {Comparison of gE/gI- and TK/gE/gI-Gene-Deleted Pseudorabies Virus Vaccines Mediated by CRISPR/Cas9 and Cre/Lox Systems.}, journal = {Viruses}, volume = {12}, number = {4}, pages = {}, pmid = {32230737}, issn = {1999-4915}, mesh = {Amino Acid Sequence ; Animals ; Antibodies, Viral/immunology ; *CRISPR-Cas Systems ; Female ; *Gene Deletion ; Gene Targeting ; Genetic Engineering ; Genome, Viral ; HEK293 Cells ; Herpesvirus 1, Suid/*genetics/*immunology/isolation &amp; purification ; *Homologous Recombination ; Humans ; Integrases/*metabolism ; Mice ; Pseudorabies Vaccines/*genetics/*immunology ; Swine ; T-Lymphocyte Subsets/immunology/metabolism ; Virulence ; }, abstract = {Pseudorabies (PR), caused by pseudorabies virus (PRV), is an acute and febrile infectious disease in swine. To eradicate PR, a more efficacious vaccine needs to be developed. Here, the gE/gI- and TK/gE/gI-gene-deleted recombinant PRV (rGXΔgE/gI and rGXΔTK/gE/gI) are constructed through CRISPR/Cas9 and Cre/Lox systems. We found that the rGXΔTK/gE/gI was safer than rGXΔgE/gI in mice. Additionally, the effects of rGXΔgE/gI and rGXΔTK/gE/gI were further evaluated in swine. The rGXΔgE/gI and rGXΔTK/gE/gI significantly increased numbers of IFN-γ-producing CD4+ and CD8+ T-cells in swine, whereas there was no difference between rGXΔgE/gI and rGXΔTK/gE/gI. Moreover, rGXΔgE/gI and rGXΔTK/gE/gI promoted a PRV-specific humoral immune response. The PRV-specific humoral immune response induced by rGXΔgE/gI was consistent with that caused by rGXΔTK/gE/gI. After the challenge, swine vaccinated with rGXΔgE/gI and rGXΔTK/gE/gI showed no clinical signs and viral shedding. However, histopathological detection revealed that rGXΔgE/gI, not rGXΔTK/gE/gI, caused pathological lesions in brain and lung tissues. In summary, these results demonstrate that the TK/gE/gI-gene-deleted recombinant PRV was safer compared with rGXΔgE/gI in swine. The data imply that the TK/gE/gI-gene-deleted recombinant PRV may be a more efficacious vaccine candidate for the prevention of PR.}, }
@article {pmid32207772, year = {2020}, author = {Auckland, P and Roscioli, E and Coker, HLE and McAinsh, AD}, title = {CENP-F stabilizes kinetochore-microtubule attachments and limits dynein stripping of corona cargoes.}, journal = {The Journal of cell biology}, volume = {219}, number = {5}, pages = {}, pmid = {32207772}, issn = {1540-8140}, support = {208384/Z/17/Z/WT_/Wellcome Trust/United Kingdom ; 106151/Z/14/Z/WT_/Wellcome Trust/United Kingdom ; }, mesh = {CRISPR-Cas Systems/genetics ; Chromosomal Proteins, Non-Histone/*genetics ; Chromosome Segregation/*genetics ; Chromosomes/genetics ; Dyneins/genetics ; HeLa Cells ; Humans ; *Kinetochores ; Microfilament Proteins/*genetics ; Microtubule-Associated Proteins/*genetics ; Microtubules/genetics ; Mutant Proteins/genetics ; Protein Binding/genetics ; Spindle Apparatus/genetics ; }, abstract = {Accurate chromosome segregation demands efficient capture of microtubules by kinetochores and their conversion to stable bioriented attachments that can congress and then segregate chromosomes. An early event is the shedding of the outermost fibrous corona layer of the kinetochore following microtubule attachment. Centromere protein F (CENP-F) is part of the corona, contains two microtubule-binding domains, and physically associates with dynein motor regulators. Here, we have combined CRISPR gene editing and engineered separation-of-function mutants to define how CENP-F contributes to kinetochore function. We show that the two microtubule-binding domains make distinct contributions to attachment stability and force transduction but are dispensable for chromosome congression. We further identify a specialized domain that functions to limit the dynein-mediated stripping of corona cargoes through a direct interaction with Nde1. This antagonistic activity is crucial for maintaining the required corona composition and ensuring efficient kinetochore biorientation.}, }
@article {pmid33586253, year = {2021}, author = {Stojkovic, M and Han, D and Jeong, M and Stojkovic, P and Stankovic, KM}, title = {Human Induced Pluripotent Stem Cells and CRISPR/Cas-Mediated Targeted Genome Editing: Platforms to Tackle Sensorineural Hearing Loss.}, journal = {Stem cells (Dayton, Ohio)}, volume = {}, number = {}, pages = {}, doi = {10.1002/stem.3353}, pmid = {33586253}, issn = {1549-4918}, abstract = {Hearing loss (HL) is a major global health problem of pandemic proportions. The most common type of HL is sensorineural hearing loss (SNHL) which typically occurs when cells within the inner ear are damaged. Human induced pluripotent stem cells (hiPSCs) can be generated from any individual including those who suffer from different types of HL. The development of new differentiation protocols to obtain cells of the inner ear including hair cells (HCs) and spiral ganglion neurons (SGNs) promises to expedite cell-based therapy and screening of potential pharmacologic and genetic therapies using human models. Considering age-related, acoustic, ototoxic and genetic insults which are the most frequent causes of irreversible damage of HCs and SGNs, new methods of genome editing (GE), especially the CRISPR/Cas9 technology, could bring additional opportunities to understand the pathogenesis of human SNHL and identify novel therapies. However, important challenges associated with both hiPSCs and GE need to be overcome before scientific discoveries are correctly translated to effective and patient-safe applications. The purpose of the present review is a) to summarize the findings from published reports utilizing hiPSCs for studies of SNHL, hence complementing recent reviews focused on animal studies, and b) to outline promising future directions for deciphering SNHL using disruptive molecular and genomic technologies. © AlphaMed Press 2021 SIGNIFICANCE STATEMENT: Hearing loss (HL) is the most common sensory deficit in the world and current therapies for it are severely limited. According to the WHO, 466 million people worldwide have disabling HL and this number is expected to increase to 900 million people by 2050. Here, we highlight the roles of two platforms, human induced pluripotent stem cells and genome editing in accelerating basic and translational research in neurobiology. We illustrate the potentially transformative impact of these platforms in deciphering early human development and the pathogenesis of neurodegenerative or inherited diseases using sensorineural hearing loss as a model.}, }
@article {pmid33585286, year = {2020}, author = {Newsom, S and Parameshwaran, HP and Martin, L and Rajan, R}, title = {The CRISPR-Cas Mechanism for Adaptive Immunity and Alternate Bacterial Functions Fuels Diverse Biotechnologies.}, journal = {Frontiers in cellular and infection microbiology}, volume = {10}, number = {}, pages = {619763}, doi = {10.3389/fcimb.2020.619763}, pmid = {33585286}, issn = {2235-2988}, abstract = {Bacterial and archaeal CRISPR-Cas systems offer adaptive immune protection against foreign mobile genetic elements (MGEs). This function is regulated by sequence specific binding of CRISPR RNA (crRNA) to target DNA/RNA, with an additional requirement of a flanking DNA motif called the protospacer adjacent motif (PAM) in certain CRISPR systems. In this review, we discuss how the same fundamental mechanism of RNA-DNA and/or RNA-RNA complementarity is utilized by bacteria to regulate two distinct functions: to ward off intruding genetic materials and to modulate diverse physiological functions. The best documented examples of alternate functions are bacterial virulence, biofilm formation, adherence, programmed cell death, and quorum sensing. While extensive complementarity between the crRNA and the targeted DNA and/or RNA seems to constitute an efficient phage protection system, partial complementarity seems to be the key for several of the characterized alternate functions. Cas proteins are also involved in sequence-specific and non-specific RNA cleavage and control of transcriptional regulator expression, the mechanisms of which are still elusive. Over the past decade, the mechanisms of RNA-guided targeting and auxiliary functions of several Cas proteins have been transformed into powerful gene editing and biotechnological tools. We provide a synopsis of CRISPR technologies in this review. Even with the abundant mechanistic insights and biotechnology tools that are currently available, the discovery of new and diverse CRISPR types holds promise for future technological innovations, which will pave the way for precision genome medicine.}, }
@article {pmid33584573, year = {2020}, author = {Tripathi, L and Ntui, VO and Tripathi, JN and Kumar, PL}, title = {Application of CRISPR/Cas for Diagnosis and Management of Viral Diseases of Banana.}, journal = {Frontiers in microbiology}, volume = {11}, number = {}, pages = {609784}, doi = {10.3389/fmicb.2020.609784}, pmid = {33584573}, issn = {1664-302X}, abstract = {Viral diseases are significant biotic constraints for banana (Musa spp.) production as they affect the yield and limit the international movement of germplasm. Among all the viruses known to infect banana, the banana bunchy top virus and banana streak viruses are widespread and economically damaging. The use of virus-resistant bananas is the most cost-effective option to minimize the negative impacts of viral-diseases on banana production. CRISPR/Cas-based genome editing is emerging as the most powerful tool for developing virus-resistant crop varieties in several crops, including the banana. The availability of a vigorous genetic transformation and regeneration system and a well-annotated whole-genome sequence of banana makes it a compelling candidate for genome editing. A robust CRISPR/Cas9-based genome editing of the banana has recently been established, which can be applied in developing disease-resistant varieties. Recently, the CRISPR system was exploited to detect target gene sequences using Cas9, Cas12, Cas13, and Cas14 enzymes, thereby unveiling the use of this technology for virus diagnosis. This article presents a synopsis of recent advancements and perspectives on the application of CRISPR/Cas-based genome editing for diagnosing and developing resistance against banana viruses and challenges in genome-editing of banana.}, }
@article {pmid33584572, year = {2020}, author = {Shahid, MS and Sattar, MN and Iqbal, Z and Raza, A and Al-Sadi, AM}, title = {Next-Generation Sequencing and the CRISPR-Cas Nexus: A Molecular Plant Virology Perspective.}, journal = {Frontiers in microbiology}, volume = {11}, number = {}, pages = {609376}, doi = {10.3389/fmicb.2020.609376}, pmid = {33584572}, issn = {1664-302X}, abstract = {In recent years, next-generation sequencing (NGS) and contemporary Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated (Cas) technologies have revolutionized the life sciences and the field of plant virology. Both these technologies offer an unparalleled platform for sequencing and deciphering viral metagenomes promptly. Over the past two decades, NGS technologies have improved enormously and have impacted plant virology. NGS has enabled the detection of plant viruses that were previously undetectable by conventional approaches, such as quarantine and archeological plant samples, and has helped to track the evolutionary footprints of viral pathogens. The CRISPR-Cas-based genome editing (GE) and detection techniques have enabled the development of effective approaches to virus resistance. Different versions of CRISPR-Cas have been employed to successfully confer resistance against diverse plant viruses by directly targeting the virus genome or indirectly editing certain host susceptibility factors. Applications of CRISPR-Cas systems include targeted insertion and/or deletion, site-directed mutagenesis, induction/expression/repression of the gene(s), epigenome re-modeling, and SNPs detection. The CRISPR-Cas toolbox has been equipped with precision GE tools to engineer the target genome with and without double-stranded (ds) breaks or donor templates. This technique has also enabled the generation of transgene-free genetically engineered plants, DNA repair, base substitution, prime editing, detection of small molecules, and biosensing in plant virology. This review discusses the utilities, advantages, applications, bottlenecks of NGS, and CRISPR-Cas in plant virology.}, }
@article {pmid33582559, year = {2021}, author = {Zhou, CM and Wu, Q and Wang, B and Lin, P and Wu, M and Yu, XJ}, title = {Calcium-responsive kinase LadS modulates type I-F CRISPR-Cas adaptive immunity.}, journal = {Biochemical and biophysical research communications}, volume = {546}, number = {}, pages = {155-161}, doi = {10.1016/j.bbrc.2021.01.100}, pmid = {33582559}, issn = {1090-2104}, abstract = {The CRISPR-Cas systems are recently discovered adaptive immune strategies in bacteria and archaea against foreign genetic elements. Although gene-editing enabled by CRISPR-Cas9 has shown great promise for clinical application, little is known about potential mechanisms of CRISPR-Cas systems for regulating their own gene expression and altering the virulence within bacteria. Here, Gram-negative bacterium Pseudomonas aeruginosa PA14 that contains a Type I-F CRISPR-Cas system was used to study the mechanism endogenous CRISPR-Cas of regulation mechanism. We delineated the role of calcium as a positive regulator of the transcription of cas/csy complex and CRISPR-Cas immunity through the two-component system (TCS) protein kinase LadS. Furthermore, we identified a LadS downstream post-transcriptional regulator, RsmA, which targeted translation region of cas mRNA via A(N)GGA motif. Importantly, calcium-mediated influencing of CRISPR-Cas system was dependent on LadS and RsmA. Altogether, our findings uncover the previously unrecognized role of LadS/RsmA in modulating Type I-F CRISPR-Cas system via sensing calcium.}, }
@article {pmid33580373, year = {2021}, author = {Wan, ZY and Lin, VCL and Hua, YG}, title = {Pomc Plays an Important Role in Sexual Size Dimorphism in Tilapia.}, journal = {Marine biotechnology (New York, N.Y.)}, volume = {}, number = {}, pages = {}, pmid = {33580373}, issn = {1436-2236}, support = {9200//Temasek Life Sciences Laboratory/ ; }, abstract = {Sexual dimorphism is common across the animal kingdom. Knowledge of the mechanisms of sexual size dimorphism is limited although it is important in biology and aquaculture. Tilapia is the common name for ~ 100 species of cichlid fish. Some are important aquaculture species and males outgrow females. To gain novel insights into the mechanisms underlying sexual size dimorphism, we analyzed the differences of brain transcriptomes between males and females in Mozambique tilapia and studied the function of the pro-opiomelanocortin (Pomc) gene in tilapia and zebrafish. The transcriptome analysis identified 123, 55, and 2706 sex-biased genes at 5, 30, and 90 dph (days post-hatch), respectively, indicating sexual dimorphism of gene expressions in the brain. The expression of Pomc in the tilapia brain was a female-biased at 30, 90, and 120 dph. An analysis of the DNA sequence located upstream of the tilapia Pomc transcriptional start site identified two estrogenic response elements. In vitro luciferase assay of the two elements revealed that β-estradiol significantly enhanced the expression of luciferase activity, suggesting that the expression of Pomc is mediated by estrogen. We knocked out Pomc in zebrafish using Crispr/Cas-9. The Pomc-knockout zebrafish showed faster growth and higher sensitivity to feeding as compared to the wild-type fish. Taken together, our results indicate that Pomc contributes to sexual size dimorphism and suggest that the high estrogen level in females promotes the expression of Pomc and suppresses feeding in female tilapias, which leads to the slower growth of female tilapias.}, }
@article {pmid33560305, year = {2021}, author = {Abbasi, J}, title = {CRISPR-Based COVID-19 Smartphone Test in Development.}, journal = {JAMA}, volume = {325}, number = {6}, pages = {522}, doi = {10.1001/jama.2021.0493}, pmid = {33560305}, issn = {1538-3598}, mesh = {COVID-19/*diagnosis ; COVID-19 Nucleic Acid Testing/*methods ; *CRISPR-Cas Systems ; Humans ; Microscopy, Fluorescence ; Nose/virology ; RNA, Viral/isolation &amp; purification ; SARS-CoV-2/*genetics/isolation &amp; purification ; *Smartphone ; Viral Load ; }, }
@article {pmid32617796, year = {2021}, author = {Yoshimi, K and Oka, Y and Miyasaka, Y and Kotani, Y and Yasumura, M and Uno, Y and Hattori, K and Tanigawa, A and Sato, M and Oya, M and Nakamura, K and Matsushita, N and Kobayashi, K and Mashimo, T}, title = {Combi-CRISPR: combination of NHEJ and HDR provides efficient and precise plasmid-based knock-ins in mice and rats.}, journal = {Human genetics}, volume = {140}, number = {2}, pages = {277-287}, pmid = {32617796}, issn = {1432-1203}, mesh = {Animals ; CRISPR-Cas Systems/*genetics ; DNA/genetics ; DNA End-Joining Repair/*genetics ; Exons/genetics ; Female ; Gene Editing/methods ; Gene Knock-In Techniques/*methods ; Genome/genetics ; Introns/genetics ; Mice ; Mice, Inbred C57BL ; Mutation/genetics ; Plasmids/*genetics ; Rats ; Rats, Long-Evans ; Rats, Wistar ; Recombinational DNA Repair/*genetics ; }, abstract = {CRISPR-Cas9 are widely used for gene targeting in mice and rats. The non-homologous end-joining (NHEJ) repair pathway, which is dominant in zygotes, efficiently induces insertion or deletion (indel) mutations as gene knockouts at targeted sites, whereas gene knock-ins (KIs) via homology-directed repair (HDR) are difficult to generate. In this study, we used a double-stranded DNA (dsDNA) donor template with Cas9 and two single guide RNAs, one designed to cut the targeted genome sequences and the other to cut both the flanked genomic region and one homology arm of the dsDNA plasmid, which resulted in 20-33% KI efficiency among G0 pups. G0 KI mice carried NHEJ-dependent indel mutations at one targeting site that was designed at the intron region, and HDR-dependent precise KIs of the various donor cassettes spanning from 1 to 5 kbp, such as EGFP, mCherry, Cre, and genes of interest, at the other exon site. These findings indicate that this combinatorial method of NHEJ and HDR mediated by the CRISPR-Cas9 system facilitates the efficient and precise KIs of plasmid DNA cassettes in mice and rats.}, }
@article {pmid33581428, year = {2021}, author = {Li, F and Ye, Q and Chen, M and Zhou, B and Zhang, J and Pang, R and Xue, L and Wang, J and Zeng, H and Wu, S and Zhang, Y and Ding, Y and Wu, Q}, title = {An ultrasensitive CRISPR/Cas12a based electrochemical biosensor for Listeria monocytogenes detection.}, journal = {Biosensors &amp; bioelectronics}, volume = {179}, number = {}, pages = {113073}, doi = {10.1016/j.bios.2021.113073}, pmid = {33581428}, issn = {1873-4235}, abstract = {Listeria monocytogenes is an important foodborne pathogen that can cause listeriosis with high patient mortality. Accordingly, it is necessary to develop a L. monocytogenes detection platform with high specificity, sensitivity, and exploitability. CRISPR/Cas systems have shown great potential in the development of next-generation biosensors for nucleic acid detection, owing to the trans-cleavage capabilities of the Cas effector proteins. Herein, we introduce the trans-cleavage activity of CRISPR/Cas12a into an electrochemical biosensor (E-CRISPR), combined with recombinase-assisted amplification (RAA), to establish a cost-effective, specific and ultrasensitive method; namely RAA-based E-CRISPR. The concept behind this approach is that the target will induce the number change of the surface signaling probe (containing an electrochemical tag), which leads to a variation in the electron transfer of the electrochemical tag. The introduction of an RAA-based Cas12a system into the E-CRISPR sensor achieves a more prominent signal change between the presence and absence of the target. Under optimized conditions, RAA-based E-CRISPR can detect as low as 0.68 aM of genomic DNA and 26 cfu/mL of L. monocytogenes in pure cultures. More importantly, the RAA-based E-CRISPR enables rapid and ultrasensitive detection of L. monocytogenes in spiked and natural Flammulina velutipes samples. Moreover, no cross-reactivity with other non-target bacteria was observed. This system thus demonstrates to be a simple, high-sensitivity, and high-accuracy platform for L. monocytogenes detection.}, }
@article {pmid33577797, year = {2021}, author = {Jia, R and Bonifacino, JS}, title = {The ubiquitin isopeptidase USP10 deubiquitinates LC3B to increase LC3B levels and autophagic activity.}, journal = {The Journal of biological chemistry}, volume = {}, number = {}, pages = {100405}, doi = {10.1016/j.jbc.2021.100405}, pmid = {33577797}, issn = {1083-351X}, abstract = {Components of the autophagy machinery are subject to regulation by various post-translational modifications. Previous studies have shown that monoubiquitination of LC3B catalyzed by the ubiquitin-activating enzyme UBA6 and ubiquitin-conjugating enzyme/ubiquitin ligase BIRC6 targets LC3B for proteasomal degradation, thus reducing LC3B levels and autophagic activity under conditions of stress. However, mechanisms capable of counteracting this process are not known. Herein, we report that LC3B ubiquitination is reversed by the action of the deubiquitinating enzyme USP10. We identified USP10 in a CRISPR-Cas9 knockout screen for ubiquitination-related genes that regulate LC3B levels. Biochemical analyses showed that silencing of USP10 reduces the levels of both the LC3B-I and LC3B-II forms of LC3B through increased ubiquitination and proteasomal degradation. In turn, the reduced LC3B levels result in slower degradation of the autophagy receptors SQSTM1 and NBR1, and an increased accumulation of puromycin-induced aggresome-like structures. Taken together, these findings indicate that the levels of LC3B and autophagic activity are controlled through cycles of LC3B ubiquitination and deubiquitination.}, }
@article {pmid33576013, year = {2021}, author = {Bhat, MA and Mir, RA and Kumar, V and Shah, AA and Zargar, SM and Rahman, S and Jan, AT}, title = {Mechanistic insights of CRISPR/Cas mediated genome editing towards enhancing abiotic stress tolerance in plants.}, journal = {Physiologia plantarum}, volume = {}, number = {}, pages = {}, doi = {10.1111/ppl.13359}, pmid = {33576013}, issn = {1399-3054}, abstract = {Abiotic stresses such as temperature (high/low), drought, salinity and others, make the environment hostile to plants. Abiotic stressors adversely affect plant growth and development; and thereby makes a direct impact on overall plant productivity. Plants confront stress by developing an internal defense system orchestrated by compatible solutes, reactive oxygen species (ROS) scavengers and phytohormones. However, routine exposure to unpredictable environmental stressors makes it essential to equip plants with a system that contributes to sustainable agricultural productivity, besides imparting multi-stress tolerance. The sustainable approach against abiotic stress is accomplished through breeding of tolerant cultivars. Though eco-friendly, tedious screening and crossing protocols limits its usage to overcome stress and in attaining the goal of global food security. Advancement on the technological front has enabled adoption of genomic engineering approaches to perform site specific modification in plant genome for improving adaptability, increasing the yield and in attributing resilience against different stressors. Of the different genome editing approaches, CRISPR/Cas has revolutionized biological research with wider applicability to crop plants. CRISPR/Cas emerged as a versatile tool in editing genomes for desired traits in highly accurate and precise manner. The present study summarizes advancement of the CRISPR/Cas genome editing tool in its adoption to manipulate plant genomes for novel traits for developing high yielding and climate resilient crop varieties.}, }
@article {pmid33575613, year = {2020}, author = {Podlevsky, JD and Hudson, CM and Timlin, JA and Williams, KP}, title = {CasCollect: targeted assembly of CRISPR-associated operons from high-throughput sequencing data.}, journal = {NAR genomics and bioinformatics}, volume = {2}, number = {3}, pages = {lqaa063}, doi = {10.1093/nargab/lqaa063}, pmid = {33575613}, issn = {2631-9268}, abstract = {CRISPR arrays and CRISPR-associated (Cas) proteins comprise a widespread adaptive immune system in bacteria and archaea. These systems function as a defense against exogenous parasitic mobile genetic elements that include bacteriophages, plasmids and foreign nucleic acids. With the continuous spread of antibiotic resistance, knowledge of pathogen susceptibility to bacteriophage therapy is becoming more critical. Additionally, gene-editing applications would benefit from the discovery of new cas genes with favorable properties. While next-generation sequencing has produced staggering quantities of data, transitioning from raw sequencing reads to the identification of CRISPR/Cas systems has remained challenging. This is especially true for metagenomic data, which has the highest potential for identifying novel cas genes. We report a comprehensive computational pipeline, CasCollect, for the targeted assembly and annotation of cas genes and CRISPR arrays-even isolated arrays-from raw sequencing reads. Benchmarking our targeted assembly pipeline demonstrates significantly improved timing by almost two orders of magnitude compared with conventional assembly and annotation, while retaining the ability to detect CRISPR arrays and cas genes. CasCollect is a highly versatile pipeline and can be used for targeted assembly of any specialty gene set, reconfigurable for user provided Hidden Markov Models and/or reference nucleotide sequences.}, }
@article {pmid33575551, year = {2020}, author = {Pan, M and Hidalgo-Cantabrana, C and Barrangou, R}, title = {Host and body site-specific adaptation of Lactobacillus crispatus genomes.}, journal = {NAR genomics and bioinformatics}, volume = {2}, number = {1}, pages = {lqaa001}, doi = {10.1093/nargab/lqaa001}, pmid = {33575551}, issn = {2631-9268}, abstract = {Lactobacillus crispatus is a common inhabitant of both healthy poultry gut and human vaginal tract, and the absence of this species has been associated with a higher risk of developing infectious diseases. In this study, we analyzed 105 L. crispatus genomes isolated from a variety of ecological niches, including the human vaginal tract, human gut, chicken gut and turkey gut, to shed light on the genetic and functional features that drive evolution and adaptation of this important species. We performed in silico analyses to identify the pan and core genomes of L. crispatus, and to reveal the genomic differences and similarities associated with their origins of isolation. Our results demonstrated that, although a significant portion of the genomic content is conserved, human and poultry L. crispatus isolates evolved to encompass different genomic features (e.g. carbohydrate usage, CRISPR-Cas immune systems, prophage occurrence) in order to thrive in different environmental niches. We also observed that chicken and turkey L. crispatus isolates can be differentiated based on their genomic information, suggesting significant differences may exist between these two poultry gut niches. These results provide insights into host and niche-specific adaptation patterns in species of human and animal importance.}, }
@article {pmid33573639, year = {2021}, author = {Abdulrachman, D and Eurwilaichitr, L and Champreda, V and Chantasingh, D and Pootanakit, K}, title = {Development of a CRISPR/Cpf1 system for targeted gene disruption in Aspergillus aculeatus TBRC 277.}, journal = {BMC biotechnology}, volume = {21}, number = {1}, pages = {15}, pmid = {33573639}, issn = {1472-6750}, abstract = {BACKGROUND: CRISPR-Cas genome editing technologies have revolutionized biotechnological research particularly in functional genomics and synthetic biology. As an alternative to the most studied and well-developed CRISPR/Cas9, a new class 2 (type V) CRISPR-Cas system called Cpf1 has emerged as another versatile platform for precision genome modification in a wide range of organisms including filamentous fungi.<br><br>RESULTS: In this study, we developed AMA1-based single CRISPR/Cpf1 expression vector that targets pyrG gene in Aspergillus aculeatus TBRC 277, a wild type filamentous fungus and potential enzyme-producing cell factory. The results showed that the Cpf1 codon optimized from Francisella tularensis subsp. novicida U112, FnCpf1, works efficiently to facilitate RNA-guided site-specific DNA cleavage. Specifically, we set up three different guide crRNAs targeting pyrG gene and demonstrated that FnCpf1 was able to induce site-specific double-strand breaks (DSBs) followed by an endogenous non-homologous end-joining (NHEJ) DNA repair pathway which caused insertions or deletions (indels) at these site-specific loci.<br><br>CONCLUSIONS: The use of FnCpf1 as an alternative class II (type V) nuclease was reported for the first time in A. aculeatus TBRC 277 species. The CRISPR/Cpf1 system developed in this study highlights the feasibility of CRISPR/Cpf1 technology and could be envisioned to further increase the utility of the CRISPR/Cpf1 in facilitating strain improvements as well as functional genomics of filamentous fungi.}, }
@article {pmid33566028, year = {2021}, author = {Wang, Y and Xie, Y and Dong, ZC and Jiang, XJ and Gong, P and Lu, J and Wan, F}, title = {[Levels of sgRNA as a Major Factor Affecting CRISPRi Knockdown Efficiency in K562 Cells].}, journal = {Molekuliarnaia biologiia}, volume = {55}, number = {1}, pages = {86-95}, doi = {10.31857/S0026898421010146}, pmid = {33566028}, issn = {0026-8984}, mesh = {CRISPR-Associated Protein 9 ; *CRISPR-Cas Systems/genetics ; DNA-Binding Proteins ; Humans ; K562 Cells ; Promoter Regions, Genetic ; *RNA, Guide ; Transcription Factors ; }, abstract = {To determine how nuclease deactivated Cas9 (dCas9) or single-guide RNA (sgRNA) expression levels affect the knockdown efficiency of CRISPRi, we created K562 cell clones expressing KRAB-dCas9 protein either with the inducible Tet-on system or with the constitutive SFFV promotor. Single clones were selected by fluorescence-activated cell sorting (FACS) for further study. Six genes with various expression levels were targeted using lentiviral sgRNA from two libraries in four cell clones with various KRAB-dCas9 expression levels. The expression level of dCas9 protein/sgRNA levels and the knockdown efficiency were determined by flow cytometry. The cell clone with the highest KRAB-dCas9 expression level achieved effective CRISPRi knockdown. The data describing this clone were statistically different from that on other clones, indicating the strong KRAB-dCas9 expression might be a prerequisite for CRISPRi. By adopting different multiplicity of infection (MOI) in lentiviral transduction of this clone, we modified the expression level of sgRNA and found that the knockdown efficiency was neither affected by the target gene expression level nor correlated with KRAB-dCas9 levels, which remained relatively constant across all knockdown experiments (coefficient of variation = 2.2%). As an example, the following levels of the knockdowns: 74.72, 72.28 and 39.08% for mmadhc, rpia and znf148 genes, respectively, were achieved. These knockdown efficiencies correlated well with the respective sgRNA expression levels. Linear regression models built using this data indicate that the knockdown efficiency may be significantly affected by the levels of both KRAB-dCas9 and sgRNA. Notably, the sgRNA levels have greater impact, being a major factor affecting CRISPRi efficiency.}, }
@article {pmid33493182, year = {2021}, author = {Papa, G and Mallery, DL and Albecka, A and Welch, LG and Cattin-Ortolá, J and Luptak, J and Paul, D and McMahon, HT and Goodfellow, IG and Carter, A and Munro, S and James, LC}, title = {Furin cleavage of SARS-CoV-2 Spike promotes but is not essential for infection and cell-cell fusion.}, journal = {PLoS pathogens}, volume = {17}, number = {1}, pages = {e1009246}, doi = {10.1371/journal.ppat.1009246}, pmid = {33493182}, issn = {1553-7374}, support = {/MRC_/Medical Research Council/United Kingdom ; }, mesh = {Animals ; COVID-19 ; CRISPR-Cas Systems ; *Cell Fusion ; Chlorocebus aethiops ; Furin/*genetics ; Gene Knockout Techniques ; HEK293 Cells ; Humans ; Protein Structure, Tertiary ; SARS-CoV-2 ; Serine Endopeptidases ; Spike Glycoprotein, Coronavirus/*chemistry ; Vero Cells ; *Virus Internalization ; }, abstract = {Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) infects cells by binding to the host cell receptor ACE2 and undergoing virus-host membrane fusion. Fusion is triggered by the protease TMPRSS2, which processes the viral Spike (S) protein to reveal the fusion peptide. SARS-CoV-2 has evolved a multibasic site at the S1-S2 boundary, which is thought to be cleaved by furin in order to prime S protein for TMPRSS2 processing. Here we show that CRISPR-Cas9 knockout of furin reduces, but does not prevent, the production of infectious SARS-CoV-2 virus. Comparing S processing in furin knockout cells to multibasic site mutants reveals that while loss of furin substantially reduces S1-S2 cleavage it does not prevent it. SARS-CoV-2 S protein also mediates cell-cell fusion, potentially allowing virus to spread virion-independently. We show that loss of furin in either donor or acceptor cells reduces, but does not prevent, TMPRSS2-dependent cell-cell fusion, unlike mutation of the multibasic site that completely prevents syncytia formation. Our results show that while furin promotes both SARS-CoV-2 infectivity and cell-cell spread it is not essential, suggesting furin inhibitors may reduce but not abolish viral spread.}, }
@article {pmid32768274, year = {2020}, author = {Pham, QT and Raad, S and Mangahas, CL and M'Callum, MA and Raggi, C and Paganelli, M}, title = {High-throughput assessment of mutations generated by genome editing in induced pluripotent stem cells by high-resolution melting analysis.}, journal = {Cytotherapy}, volume = {22}, number = {10}, pages = {536-542}, doi = {10.1016/j.jcyt.2020.06.008}, pmid = {32768274}, issn = {1477-2566}, support = {//CIHR/Canada ; }, mesh = {Animals ; CRISPR-Cas Systems/genetics ; Cell Line ; DNA/genetics ; *Gene Editing ; High-Throughput Screening Assays/*methods ; Humans ; Induced Pluripotent Stem Cells/*metabolism ; Mice ; Mutation/*genetics ; *Nucleic Acid Denaturation ; Polymorphism, Single Nucleotide/genetics ; }, abstract = {BACKGROUND AND AIMS: Genome editing of induced pluripotent stem cells (iPSCs) holds great potential for both disease modeling and regenerative medicine. Although clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 provides an efficient and precise genome editing tool, iPSCs are especially difficult to transfect, resulting in a small percentage of cells carrying the desired correction. A high-throughput method to identify edited clones is required to reduce the time and costs of such an approach.<br><br>METHODS: Here we assess high-resolution melting analysis (HRMA), a simple and efficient real-time polymerase chain reaction-based method, and compare it with more commonly used assays.<br><br>RESULTS AND CONCLUSIONS: Our data show that HRMA is a robust and highly sensitive method, allowing the cost-effective and time-saving screening of genome-edited iPSCs. Samples can be prepared directly from 96-well microtiter plates for high-throughput analysis, and amplicons can be further analyzed with downstream techniques for further confirmation, if needed.}, }
@article {pmid32703430, year = {2020}, author = {Gonzalez-Salinas, F and Rojo, R and Martinez-Amador, C and Herrera-Gamboa, J and Trevino, V}, title = {Transcriptomic and cellular analyses of CRISPR/Cas9-mediated edition of FASN show inhibition of aggressive characteristics in breast cancer cells.}, journal = {Biochemical and biophysical research communications}, volume = {529}, number = {2}, pages = {321-327}, doi = {10.1016/j.bbrc.2020.05.172}, pmid = {32703430}, issn = {1090-2104}, mesh = {Breast Neoplasms/*genetics/pathology ; CRISPR-Cas Systems ; Cell Movement ; Cell Proliferation ; Fatty Acid Synthase, Type I/*genetics ; Female ; Humans ; MCF-7 Cells ; Mutation ; *Transcriptome ; }, abstract = {Several genes are significantly mutated in breast cancer but only a small percentage of mutations are well-known to contribute to cancer development. FASN is involved in de novo lipogenesis and the regulation of ERα signaling. However, the effect of genetic mutations affecting FASN in breast cancer has not thoroughly studied. Therefore, we used the CRISPR/Cas9 system to edit the FASN locus in MCF-7 cells and evaluated its biological effect. We obtained four clones carrying mutations and frameshifts in the acyl-transferase domain of FASN. We found that clones had reduced proliferation, migration, viability, and showed alterations in cell cycle profiles. RNA-Seq analysis demonstrates that a lack of fully functional FASN may have a more significant role in proliferation-related genes than in lipid metabolism. We conclude that functional knockouts in FASN contributes to decrease the proliferation and migration of breast cancer cells contrary to point mutations in breast cancer patients.}, }
@article {pmid32156541, year = {2020}, author = {Ge, L and Dong, X and Gong, X and Kang, J and Zhang, Y and Quan, F}, title = {Mutation in myostatin 3'UTR promotes C2C12 myoblast proliferation and differentiation by blocking the translation of MSTN.}, journal = {International journal of biological macromolecules}, volume = {154}, number = {}, pages = {634-643}, doi = {10.1016/j.ijbiomac.2020.03.043}, pmid = {32156541}, issn = {1879-0003}, mesh = {Animals ; CRISPR-Cas Systems ; Cell Differentiation ; Cell Proliferation ; *Gene Editing ; HEK293 Cells ; Humans ; Mice ; MicroRNAs/metabolism ; Mutation ; Myoblasts/*cytology ; Myostatin/*genetics/physiology ; Phenotype ; }, abstract = {The point mutation in myostatin (MSTN) can produce the Texel sheep double muscle phenotype. However, whether other species have the same mode of action as MSTN and whether breeding materials can be obtained through cross-species genetic editing remain unclear. The mutation in the mouse MSTN 3'UTR could create a target site for mmu-miR-1/206, as verified by the dual luciferase reporter system. A C2C12 cell model with the mutation in MSTN 3'UTR was constructed using CRISPR/Cas9 gene editing. Then, the mRNA and protein expression of MSTN was analyzed in the mutant C2C12 cell model. Results revealed that the mutation blocked the translational level of MSTN. By inhibiting mmu-mir-206, low expression of MSTN protein in mutant C2C12 cell can be rescued. Furthermore, the proliferation and differentiation abilities of the mutant C2C12 cell model were tested by RT-PCR, CCK8 analysis, EDU (5-ethynyl-2'-deoxyuridine) proliferation analysis, immunofluorescence analysis, Western blot, and myotube fusion statistics. This study may serve as a reference for elucidating the function and molecular mechanism of MSTN and as a foundation for accurate breeding improvement.}, }
@article {pmid31959210, year = {2020}, author = {Watanabe, S and Oiwa, K and Murata, Y and Komine, O and Sobue, A and Endo, F and Takahashi, E and Yamanaka, K}, title = {ALS-linked TDP-43M337V knock-in mice exhibit splicing deregulation without neurodegeneration.}, journal = {Molecular brain}, volume = {13}, number = {1}, pages = {8}, pmid = {31959210}, issn = {1756-6606}, mesh = {Alternative Splicing/genetics/*physiology ; Amyotrophic Lateral Sclerosis/genetics ; Animals ; Base Sequence ; Brain/metabolism ; CRISPR-Cas Systems ; DNA-Binding Proteins/*genetics/physiology ; Exons/genetics ; Gene Knock-In Techniques ; Humans ; Mice ; *Mutation, Missense ; *Point Mutation ; RNA, Messenger/metabolism ; Spinal Cord/metabolism ; }, abstract = {Abnormal accumulation of TAR DNA-binding protein 43 (TDP-43), a DNA/RNA binding protein, is a pathological signature of amyotrophic lateral sclerosis (ALS). Missense mutations in the TARDBP gene are also found in inherited and sporadic ALS, indicating that dysfunction in TDP-43 is causative for ALS. To model TDP-43-linked ALS in rodents, we generated TDP-43 knock-in mice with inherited ALS patient-derived TDP-43M337V mutation. Homozygous TDP-43M337V mice developed normally without exhibiting detectable motor dysfunction and neurodegeneration. However, splicing of mRNAs regulated by TDP-43 was deregulated in the spinal cords of TDP-43M337V mice. Together with the recently reported TDP-43 knock-in mice with ALS-linked mutations, our finding indicates that ALS patient-derived mutations in the TARDBP gene at a carboxyl-terminal domain of TDP-43 may cause a gain of splicing function by TDP-43, however, were insufficient to induce robust neurodegeneration in mice.}, }
@article {pmid31803983, year = {2020}, author = {Ortega, MS and Kelleher, AM and O'Neil, E and Benne, J and Cecil, R and Spencer, TE}, title = {NANOG is required to form the epiblast and maintain pluripotency in the bovine embryo.}, journal = {Molecular reproduction and development}, volume = {87}, number = {1}, pages = {152-160}, pmid = {31803983}, issn = {1098-2795}, support = {R01 HD072898/HD/NICHD NIH HHS/United States ; }, mesh = {Animals ; CRISPR-Cas Systems ; Cattle ; Cell Lineage/*genetics ; Embryo, Mammalian/*metabolism ; Exons ; Female ; Fertilization in Vitro/methods ; GATA6 Transcription Factor/metabolism ; Gene Expression Regulation, Developmental ; Genotype ; Germ Layers/*metabolism ; Nanog Homeobox Protein/genetics/*metabolism ; RNA, Guide ; Zygote/metabolism ; }, abstract = {During preimplantation development, the embryo undergoes two consecutive lineages specifications. The first cell fate decision determines which cells give rise to the trophectoderm (TE) and the inner cell mass (ICM). Subsequently, the ICM differentiates into hypoblast and epiblast, the latter giving rise to the embryo proper. The transcription factors that govern these cell fate decisions have been extensively studied in the mouse, but are still poorly understood in other mammalian species. In the present study, the role of NANOG in the formation of the epiblast and maintenance of pluripotency in the bovine embryo was investigated. Using a CRISPR-Cas9 approach, guide RNAs were designed to target exon 2, resulting in a functional deletion of bovine NANOG at the zygote stage. Disruption of NANOG resulted in the embryos that form a blastocoel and an ICM composed of hypoblast cells. Furthermore, NANOG-null embryos showed lower expression of epiblast cell markers SOX2 and HA2AFZ, and hypoblast marker GATA6; without affecting the expression of TE markers CDX2 and KRT8. Results indicate that NANOG, has no apparent role in segregation or maintenance of the TE, but it is required to derive and maintain the pluripotent epiblast and during the second lineage commitment in the bovine embryo.}, }
@article {pmid33571114, year = {2021}, author = {Shi, K and Xie, S and Tian, R and Wang, S and Lu, Q and Gao, D and Lei, C and Zhu, H and Nie, Z}, title = {A CRISPR-Cas autocatalysis-driven feedback amplification network for supersensitive DNA diagnostics.}, journal = {Science advances}, volume = {7}, number = {5}, pages = {}, doi = {10.1126/sciadv.abc7802}, pmid = {33571114}, issn = {2375-2548}, abstract = {Artificial nucleic acid circuits with precisely controllable dynamic and function have shown great promise in biosensing, but their utility in molecular diagnostics is still restrained by the inability to process genomic DNA directly and moderate sensitivity. To address this limitation, we present a CRISPR-Cas-powered catalytic nucleic acid circuit, namely, CRISPR-Cas-only amplification network (CONAN), for isothermally amplified detection of genomic DNA. By integrating the stringent target recognition, helicase activity, and trans-cleavage activity of Cas12a, a Cas12a autocatalysis-driven artificial reaction network is programmed to construct a positive feedback circuit with exponential dynamic in CONAN. Consequently, CONAN achieves one-enzyme, one-step, real-time detection of genomic DNA with attomolar sensitivity. Moreover, CONAN increases the intrinsic single-base specificity of Cas12a, and enables the effective detection of hepatitis B virus infection and human bladder cancer-associated single-nucleotide mutation in clinical samples, highlighting its potential as a powerful tool for disease diagnostics.}, }
@article {pmid33571043, year = {2021}, author = {Bloh, K and Kanchana, R and Bialk, P and Banas, K and Zhang, Z and Yoo, BC and Kmiec, EB}, title = {Deconvolution of Complex DNA Repair (DECODR): Establishing a Novel Deconvolution Algorithm for Comprehensive Analysis of CRISPR-Edited Sanger Sequencing Data.}, journal = {The CRISPR journal}, volume = {}, number = {}, pages = {}, doi = {10.1089/crispr.2020.0022}, pmid = {33571043}, issn = {2573-1602}, abstract = {During CRISPR-directed gene editing, multiple gene repair mechanisms interact to produce a wide and largely unpredictable variety of sequence changes across an edited population of cells. Shortcomings inherent to previously available proposal-based insertion and deletion (indel) analysis software necessitated the development of a more comprehensive tool that could detect a larger range and variety of indels while maintaining the ease of use of tools currently available. To that end, we developed Deconvolution of Complex DNA Repair (DECODR). DECODR can detect indels formed from single or multi-guide CRISPR experiments without a limit on indel size. The software is accurate in determining the identities and positions of inserted and deleted bases in DNA extracts from both clonally expanded and bulk cell populations. The accurate identification and output of any potential indel allows for DECODR analysis to be executed in experiments utilizing potentially any configuration of donor DNA sequences, CRISPR-Cas, and endogenous DNA repair pathways.}, }
@article {pmid33569817, year = {2021}, author = {Escalona-Noguero, C and López-Valls, M and Sot, B}, title = {CRISPR/Cas technology as a promising weapon to combat viral infections.}, journal = {BioEssays : news and reviews in molecular, cellular and developmental biology}, volume = {}, number = {}, pages = {e2000315}, doi = {10.1002/bies.202000315}, pmid = {33569817}, issn = {1521-1878}, support = {SAF2017-87305-R (B.S.)//Ministerio de Ciencia, Innovación y Universidades/ ; PEJD-2017-PRE/BMD-3730 (C.E-N.)//Madrid Regional Government/ ; PEJD-2018-PRE/IND-9584 (M.L-V)//Madrid Regional Government/ ; }, abstract = {The versatile clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system has emerged as a promising technology for therapy and molecular diagnosis. It is especially suited for overcoming viral infections outbreaks, since their effective control relies on an efficient treatment, but also on a fast diagnosis to prevent disease dissemination. The CRISPR toolbox offers DNA- and RNA-targeting nucleases that constitute dual weapons against viruses. They allow both the manipulation of viral and host genomes for therapeutic purposes and the detection of viral nucleic acids in &quot;Point of Care&quot; sensor devices. Here, we thoroughly review recent advances in the use of the CRISPR/Cas system for the treatment and diagnosis of viral deleterious infections such as HIV or SARS-CoV-2, examining their strengths and limitations. We describe the main points to consider when designing CRISPR antiviral strategies and the scientific efforts to develop more sensitive CRISPR-based viral detectors. Finally, we discuss future prospects to improve both applications.}, }
@article {pmid33347431, year = {2020}, author = {Lu, L and Hu, J and Chao, T and Chen, Z and Liu, Z and Luo, X and Liang, Y and He, P and Zhang, L}, title = {Loss of natural resistance to schistosome in T cell deficient rat.}, journal = {PLoS neglected tropical diseases}, volume = {14}, number = {12}, pages = {e0008909}, pmid = {33347431}, issn = {1935-2735}, mesh = {Animals ; CD3 Complex/genetics/metabolism ; CRISPR-Cas Systems ; Gene Deletion ; Gene Expression Regulation ; Lymphocyte Specific Protein Tyrosine Kinase p56(lck)/genetics/metabolism ; Mice, Inbred C57BL ; Mice, Knockout ; Rats ; Rats, Sprague-Dawley ; Schistosoma japonicum/physiology ; Schistosomiasis japonica/*immunology ; T-Lymphocytes/*physiology ; }, abstract = {Schistosomiasis is among the major neglected tropical diseases and effective prevention by boosting the immune system is still not available. T cells are key cellular components governing adaptive immune response to various infections. While common laboratory mice, such as C57BL/6, are highly susceptible to schistosomiasis, the SD rats are extremely resistant. However, whether adaptive immunity is necessary for such natural resistance to schistosomiasis in rats remains to be determined. Therefore, it is necessary to establish genetic model deficient in T cells and adaptive immunity on the resistant SD background, and to characterize liver pathology during schistosomiasis. In this study we compared experimental schistosomiasis in highly susceptible C57BL/6 (B6) mice and in resistant SD rats, using cercariae of Schistosoma japonicum. We observed a marked T cell expansion in the spleen of infected B6 mice, but not resistant SD rats. Interestingly, CD3e-/- B6 mice in which T cells are completely absent, the infectious burden of adult worms was significantly higher than that in WT mice, suggesting an anti-parasitic role for T cells in B6 mice during schistosome infection. In further experiments, we established Lck deficient SD rats by using CRISPR/Cas9 in which T cell development was completely abolished. Strikingly, we found that such Lck deficiency in SD rats severely impaired their natural resistance to schistosome infection, and fostered parasite growth. Together with an additional genetic model deficient in T cells, the CD3e-/- SD rats, we confirmed the absence of T cell resulted in loss of natural resistance to schistosome infection, but also mitigated liver immunopathology. Our further experiments showed that regulatory T cell differentiation in infected SD rats was significantly decreased during schistosomiasis, in contrast to significant increase of regulatory T cells in infected B6 mice. These data suggest that T cell mediated immune tolerance facilitates persistent infection in mice but not in SD rats. The demonstration of an important role for T cells in natural resistance of SD rats to schistosomiasis provides experimental evidences supporting the rationale to boost T cell responses in humans to prevent and treat schistosomiasis.}, }
@article {pmid33106643, year = {2020}, author = {Crunkhorn, S}, title = {Restoring vision loss with base editing.}, journal = {Nature reviews. Drug discovery}, volume = {19}, number = {12}, pages = {835}, doi = {10.1038/d41573-020-00186-x}, pmid = {33106643}, issn = {1474-1784}, mesh = {*Adenine ; Animals ; CRISPR-Cas Systems ; Gene Editing ; Mice ; *Retinal Diseases ; }, }
@article {pmid33087932, year = {2020}, author = {Wolter, JM and Mao, H and Fragola, G and Simon, JM and Krantz, JL and Bazick, HO and Oztemiz, B and Stein, JL and Zylka, MJ}, title = {Cas9 gene therapy for Angelman syndrome traps Ube3a-ATS long non-coding RNA.}, journal = {Nature}, volume = {587}, number = {7833}, pages = {281-284}, pmid = {33087932}, issn = {1476-4687}, support = {1R01NS109304-01A1/NH/NIH HHS/United States ; T32 HD040127/HD/NICHD NIH HHS/United States ; R01 MH118349/MH/NIMH NIH HHS/United States ; R00 MH102357/MH/NIMH NIH HHS/United States ; R01 MH120125/MH/NIMH NIH HHS/United States ; P50 HD103573/HD/NICHD NIH HHS/United States ; P30 NS045892/NS/NINDS NIH HHS/United States ; }, mesh = {Angelman Syndrome/*genetics/*therapy ; Animals ; CRISPR-Associated Protein 9/genetics/*metabolism ; CRISPR-Cas Systems/*genetics ; Dependovirus/genetics ; Disease Models, Animal ; Female ; *Gene Editing ; Gene Silencing ; Genetic Therapy/*methods ; Genetic Vectors/genetics ; Humans ; Male ; Mice ; Mice, Inbred C57BL ; Nervous System/metabolism ; Paternal Inheritance/genetics ; Phenotype ; RNA, Guide/genetics ; RNA, Long Noncoding/*genetics ; Ubiquitin-Protein Ligases/*genetics ; }, abstract = {Angelman syndrome (AS) is a severe neurodevelopmental disorder caused by a mutation or deletion of the maternally inherited UBE3A allele. In neurons, the paternally inherited UBE3A allele is silenced in cis by a long non-coding RNA called UBE3A-ATS. Here, as part of a systematic screen, we found that Cas9 can be used to activate ('unsilence') paternal Ube3a in cultured mouse and human neurons when targeted to Snord115 genes, which are small nucleolar RNAs that are clustered in the 3' region of Ube3a-ATS. A short Cas9 variant and guide RNA that target about 75 Snord115 genes were packaged into an adeno-associated virus and administered to a mouse model of AS during the embryonic and early postnatal stages, when the therapeutic benefit of restoring Ube3a is predicted to be greatest1,2. This early treatment unsilenced paternal Ube3a throughout the brain for at least 17 months and rescued anatomical and behavioural phenotypes in AS mice. Genomic integration of the adeno-associated virus vector into Cas9 target sites caused premature termination of Ube3a-ATS at the vector-derived polyA cassette, or when integrated in the reverse orientation, by transcriptional collision with the vector-derived Cas9 transcript. Our study shows that targeted genomic integration of a gene therapy vector can restore the function of paternally inherited UBE3A throughout life, providing a path towards a disease-modifying treatment for a syndromic neurodevelopmental disorder.}, }
@article {pmid32930578, year = {2020}, author = {Zou, D and Maina, SW and Zhang, F and Yan, Z and Ding, L and Shao, Y and Xin, Z}, title = {Mining New Plipastatins and Increasing the Total Yield Using CRISPR/Cas9 in Genome-Modified Bacillus subtilis 1A751.}, journal = {Journal of agricultural and food chemistry}, volume = {68}, number = {41}, pages = {11358-11367}, doi = {10.1021/acs.jafc.0c03694}, pmid = {32930578}, issn = {1520-5118}, mesh = {Bacillus subtilis/chemistry/*genetics/*metabolism ; *CRISPR-Cas Systems ; Chromatography, High Pressure Liquid ; Fatty Acids/*biosynthesis/chemistry/pharmacology ; Fungi/drug effects/growth &amp; development ; Gas Chromatography-Mass Spectrometry ; Genome, Bacterial ; Multigene Family ; Oligopeptides/*biosynthesis/chemistry/pharmacology ; Operon ; Peptides, Cyclic/*biosynthesis/chemistry/pharmacology ; }, abstract = {CRISPR/Cas9 is one of the robust and effective gene manipulation tools which has been widely applied in various organisms. In this study, the plipastatin gene cluster was successfully expressed in genome-modified Bacillus subtilis 1A751 by disrupting the surfactin operon (srf) through CRISPR/Cas9 technology. The presumed plipastatin biosynthetic pathway was proposed based on the analysis of its biosynthetic gene cluster. Two new plipastatins were identified by a combination of ultra-high performance liquid chromatography-coupled electron spray ionization-tandem mass spectrometry and gas chromatography-mass spectrometry analyses, together with nine known plipastatins or their derivatives. The yield of plipastatin was as high as 1600 mg/L which is the highest reported to date. Antimicrobial experiments revealed that its methanolic extracts exhibited powerful inhibitory effects on the growth of the tested pathogens and fungi. The results from this investigation highlight the remarkable utility of CRISPR/Cas9 in mining new plipastatins and increasing the total plipastatin yield, providing a new pipeline for the industrial application of plipastatin.}, }
@article {pmid32895548, year = {2021}, author = {Jones, SK and Hawkins, JA and Johnson, NV and Jung, C and Hu, K and Rybarski, JR and Chen, JS and Doudna, JA and Press, WH and Finkelstein, IJ}, title = {Massively parallel kinetic profiling of natural and engineered CRISPR nucleases.}, journal = {Nature biotechnology}, volume = {39}, number = {1}, pages = {84-93}, pmid = {32895548}, issn = {1546-1696}, support = {F32 AG053051/AG/NIA NIH HHS/United States ; R01 GM124141/GM/NIGMS NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; }, mesh = {*Bacterial Proteins/chemistry/genetics/metabolism ; *CRISPR-Associated Protein 9/chemistry/genetics/metabolism ; *CRISPR-Associated Proteins/chemistry/genetics/metabolism ; CRISPR-Cas Systems ; *Endodeoxyribonucleases/chemistry/genetics/metabolism ; Gene Editing ; High-Throughput Nucleotide Sequencing/*methods ; Kinetics ; Protein Binding/genetics ; Protein Engineering ; RNA, Guide/chemistry/genetics/metabolism ; Substrate Specificity/genetics ; }, abstract = {Engineered SpCas9s and AsCas12a cleave fewer off-target genomic sites than wild-type (wt) Cas9. However, understanding their fidelity, mechanisms and cleavage outcomes requires systematic profiling across mispaired target DNAs. Here we describe NucleaSeq-nuclease digestion and deep sequencing-a massively parallel platform that measures the cleavage kinetics and time-resolved cleavage products for over 10,000 targets containing mismatches, insertions and deletions relative to the guide RNA. Combining cleavage rates and binding specificities on the same target libraries, we benchmarked five SpCas9 variants and AsCas12a. A biophysical model built from these data sets revealed mechanistic insights into off-target cleavage. Engineered Cas9s, especially Cas9-HF1, dramatically increased cleavage specificity but not binding specificity compared to wtCas9. Surprisingly, AsCas12a cleavage specificity differed little from that of wtCas9. Initial DNA cleavage sites and end trimming varied by nuclease, guide RNA and the positions of mispaired nucleotides. More broadly, NucleaSeq enables rapid, quantitative and systematic comparisons of specificity and cleavage outcomes across engineered and natural nucleases.}, }
@article {pmid32690971, year = {2021}, author = {Kurt, IC and Zhou, R and Iyer, S and Garcia, SP and Miller, BR and Langner, LM and Grünewald, J and Joung, JK}, title = {CRISPR C-to-G base editors for inducing targeted DNA transversions in human cells.}, journal = {Nature biotechnology}, volume = {39}, number = {1}, pages = {41-46}, pmid = {32690971}, issn = {1546-1696}, support = {R35 GM118158/GM/NIGMS NIH HHS/United States ; RM1 HG009490/HG/NHGRI NIH HHS/United States ; }, mesh = {CRISPR-Cas Systems/*genetics ; Cytidine Deaminase/metabolism ; Cytosine/*metabolism ; DNA/genetics/metabolism ; Gene Editing/*methods ; Guanine/metabolism ; HEK293 Cells ; Humans ; }, abstract = {CRISPR-guided DNA cytosine and adenine base editors are widely used for many applications1-4 but primarily create DNA base transitions (that is, pyrimidine-to-pyrimidine or purine-to-purine). Here we describe the engineering of two base editor architectures that can efficiently induce targeted C-to-G base transversions, with reduced levels of unwanted C-to-W (W = A or T) and indel mutations. One of these C-to-G base editors (CGBE1), consists of an RNA-guided Cas9 nickase, an Escherichia coli-derived uracil DNA N-glycosylase (eUNG) and a rat APOBEC1 cytidine deaminase variant (R33A) previously shown to have reduced off-target RNA and DNA editing activities5,6. We show that CGBE1 can efficiently induce C-to-G edits, particularly in AT-rich sequence contexts in human cells. We also removed the eUNG domain to yield miniCGBE1, which reduced indel frequencies but only modestly decreased editing efficiency. CGBE1 and miniCGBE1 enable C-to-G edits and will serve as a basis for optimizing C-to-G base editors for research and therapeutic applications.}, }
@article {pmid32690970, year = {2021}, author = {Zhao, D and Li, J and Li, S and Xin, X and Hu, M and Price, MA and Rosser, SJ and Bi, C and Zhang, X}, title = {Glycosylase base editors enable C-to-A and C-to-G base changes.}, journal = {Nature biotechnology}, volume = {39}, number = {1}, pages = {35-40}, pmid = {32690970}, issn = {1546-1696}, mesh = {APOBEC-1 Deaminase/genetics/metabolism ; Adenine/metabolism ; Animals ; Base Pairing/genetics ; CRISPR-Associated Protein 9/genetics/metabolism ; CRISPR-Cas Systems/*genetics ; Cytidine Deaminase ; Cytosine/*metabolism ; *DNA Glycosylases ; DNA Repair/genetics ; Deoxyribonuclease I/genetics/metabolism ; Escherichia coli/genetics ; Gene Editing/*methods ; Guanine/metabolism ; Rats ; Uracil-DNA Glycosidase ; }, abstract = {Current base editors (BEs) catalyze only base transitions (C to T and A to G) and cannot produce base transversions. Here we present BEs that cause C-to-A transversions in Escherichia coli and C-to-G transversions in mammalian cells. These glycosylase base editors (GBEs) consist of a Cas9 nickase, a cytidine deaminase and a uracil-DNA glycosylase (Ung). Ung excises the U base created by the deaminase, forming an apurinic/apyrimidinic (AP) site that initiates the DNA repair process. In E. coli, we used activation-induced cytidine deaminase (AID) to construct AID-nCas9-Ung and found that it converts C to A with an average editing specificity of 93.8% ± 4.8% and editing efficiency of 87.2% ± 6.9%. For use in mammalian cells, we replaced AID with rat APOBEC1 (APOBEC-nCas9-Ung). We tested APOBEC-nCas9-Ung at 30 endogenous sites, and we observed C-to-G conversions with a high editing specificity at the sixth position of the protospacer between 29.7% and 92.2% and an editing efficiency between 5.3% and 53.0%. APOBEC-nCas9-Ung supplements the current adenine and cytidine BEs (ABE and CBE, respectively) and could be used to target G/C disease-causing mutations.}, }
@article {pmid32661438, year = {2021}, author = {DeWeirdt, PC and Sanson, KR and Sangree, AK and Hegde, M and Hanna, RE and Feeley, MN and Griffith, AL and Teng, T and Borys, SM and Strand, C and Joung, JK and Kleinstiver, BP and Pan, X and Huang, A and Doench, JG}, title = {Optimization of AsCas12a for combinatorial genetic screens in human cells.}, journal = {Nature biotechnology}, volume = {39}, number = {1}, pages = {94-104}, pmid = {32661438}, issn = {1546-1696}, support = {P01 HL142494/HL/NHLBI NIH HHS/United States ; R00 CA218870/CA/NCI NIH HHS/United States ; U19 AI133524/AI/NIAID NIH HHS/United States ; }, mesh = {Acidaminococcus/genetics ; Apoptosis/genetics ; *Bacterial Proteins/genetics/metabolism ; CRISPR-Associated Protein 9 ; *CRISPR-Associated Proteins/genetics/metabolism ; CRISPR-Cas Systems/*genetics ; Cell Line, Tumor ; *Endodeoxyribonucleases/genetics/metabolism ; Gene Editing/*methods ; Gene Library ; HEK293 Cells ; Humans ; *RNA, Guide/genetics/metabolism ; }, abstract = {Cas12a RNA-guided endonucleases are promising tools for multiplexed genetic perturbations because they can process multiple guide RNAs expressed as a single transcript, and subsequently cleave target DNA. However, their widespread adoption has lagged behind Cas9-based strategies due to low activity and the lack of a well-validated pooled screening toolkit. In the present study, we describe the optimization of enhanced Cas12a from Acidaminococcus (enAsCas12a) for pooled, combinatorial genetic screens in human cells. By assaying the activity of thousands of guides, we refine on-target design rules and develop a comprehensive set of off-target rules to predict and exclude promiscuous guides. We also identify 38 direct repeat variants that can substitute for the wild-type sequence. We validate our optimized AsCas12a toolkit by screening for synthetic lethalities in OVCAR8 and A375 cancer cells, discovering an interaction between MARCH5 and WSB2. Finally, we show that enAsCas12a delivers similar performance to Cas9 in genome-wide dropout screens but at greatly reduced library size, which will facilitate screens in challenging models.}, }
@article {pmid33509781, year = {2020}, author = {Cao, JX and Wang, YL and Wang, ZX}, title = {Advances in precise regulation of CRISPR/Cas9 gene editing technology.}, journal = {Yi chuan = Hereditas}, volume = {42}, number = {12}, pages = {1168-1177}, doi = {10.16288/j.yczz.20-069}, pmid = {33509781}, issn = {0253-9772}, mesh = {Animals ; Biotechnology ; *CRISPR-Cas Systems ; Crops, Agricultural ; *Gene Editing ; Humans ; Livestock ; RNA, Guide/*genetics ; }, abstract = {Gene editing is a genetic engineering technology that can modify, delete, or insert a small piece of DNA at a specific point in the genome of cells and organisms. Gene editing technology holds great promises in the fields of disease treatment, gene function regulation, gene detection, drug research and development, and crop breeding. However, side effects, such as off-target editing, genotoxicity and other issues, have gradually emerged in the application. In the CRISPR (clustered regularly interspaced short palindromic repeats) system, the Cas9 nuclease can specifically recognize the target DNA by the base pairing of a guide RNA (gRNA) with the target DNA. Upon target recognition, the two DNA strands are cleaved by distinct domains of the Cas9 nuclease. Since both Cas9 nuclease and gRNA possess different characteristics in their own activities, recognition sites and binding ability to specific target, it is essential to precisely regulate the activity of Cas9 nuclease and gRNA in both time and space manners, thus preventing the risk of side effects and enhancing the precise regulation of the CRISPR/Cas9 gene editing technology. In this review, we summarize the advances in the precise control of gene editing, especially CRISPR/cas9 over several dimensions using fusion Cas9 proteins regulated by light, temperature and drugs, exploiting and screening anti-CRISPRs proteins, synthesizing and identifying small molecules- inhibitors, and developing other therapeutic agents, thereby providing a reference and research ideas for human disease treatment, crop and livestock improvement and prevention of biotechnology misuse.}, }
@article {pmid33446553, year = {2021}, author = {Harding, AT and Goff, MA and Froggatt, HM and Lim, JK and Heaton, NS}, title = {GPER1 is required to protect fetal health from maternal inflammation.}, journal = {Science (New York, N.Y.)}, volume = {371}, number = {6526}, pages = {271-276}, doi = {10.1126/science.aba9001}, pmid = {33446553}, issn = {1095-9203}, support = {R21 AI139593/AI/NIAID NIH HHS/United States ; R21 AI144844/AI/NIAID NIH HHS/United States ; R01 AI150837/AI/NIAID NIH HHS/United States ; R01 HL142985/HL/NHLBI NIH HHS/United States ; }, mesh = {Animals ; Benzodioxoles/pharmacology ; CRISPR-Cas Systems ; Female ; Fetal Diseases/*immunology/virology ; Fetus/immunology/virology ; Humans ; Inflammation/*immunology ; Influenza A virus/immunology ; Influenza, Human/immunology ; Interferon Type I/immunology ; Maternal-Fetal Exchange/*immunology ; Mice ; Mice, Inbred C57BL ; Placenta/immunology/virology ; Pregnancy ; Pregnancy Complications, Infectious/*immunology ; Quinolines/pharmacology ; Receptors, Estrogen/antagonists &amp; inhibitors/*metabolism ; Receptors, G-Protein-Coupled/antagonists &amp; inhibitors/*metabolism ; }, abstract = {Type I interferon (IFN) signaling in fetal tissues causes developmental abnormalities and fetal demise. Although pathogens that infect fetal tissues can induce birth defects through the local production of type I IFN, it remains unknown why systemic IFN generated during maternal infections only rarely causes fetal developmental defects. Here, we report that activation of the guanine nucleotide-binding protein-coupled estrogen receptor 1 (GPER1) during pregnancy is both necessary and sufficient to suppress IFN signaling and does so disproportionately in reproductive and fetal tissues. Inactivation of GPER1 in mice halted fetal development and promoted fetal demise, but only in the context of maternal inflammation. Thus, GPER1 is a central regulator of IFN signaling during pregnancy that allows dynamic antiviral responses in maternal tissues while also preserving fetal health.}, }
@article {pmid33398196, year = {2021}, author = {Torre, EA and Arai, E and Bayatpour, S and Jiang, CL and Beck, LE and Emert, BL and Shaffer, SM and Mellis, IA and Fane, ME and Alicea, GM and Budinich, KA and Weeraratna, AT and Shi, J and Raj, A}, title = {Genetic screening for single-cell variability modulators driving therapy resistance.}, journal = {Nature genetics}, volume = {53}, number = {1}, pages = {76-85}, pmid = {33398196}, issn = {1546-1718}, support = {T32 DK007780/DK/NIDDK NIH HHS/United States ; DP5 OD028144/OD/NIH HHS/United States ; P30 CA006973/CA/NCI NIH HHS/United States ; R01 CA238237/CA/NCI NIH HHS/United States ; P30 CA010815/CA/NCI NIH HHS/United States ; F30 CA236129/CA/NCI NIH HHS/United States ; P01 CA114046/CA/NCI NIH HHS/United States ; T32 GM007170/GM/NIGMS NIH HHS/United States ; P50 CA174523/CA/NCI NIH HHS/United States ; R01 CA207935/CA/NCI NIH HHS/United States ; U01 CA227550/CA/NCI NIH HHS/United States ; F30 HG010822/HG/NHGRI NIH HHS/United States ; R01 CA174746/CA/NCI NIH HHS/United States ; R01 CA232256/CA/NCI NIH HHS/United States ; U54 CA193417/CA/NCI NIH HHS/United States ; RM1 HG007743/HG/NHGRI NIH HHS/United States ; P30 CA016520/CA/NCI NIH HHS/United States ; U01 DK127405/DK/NIDDK NIH HHS/United States ; R01 GM137425/GM/NIGMS NIH HHS/United States ; F30 NS100595/NS/NINDS NIH HHS/United States ; U01 HL129998/HL/NHLBI NIH HHS/United States ; T32 HG000046/HG/NHGRI NIH HHS/United States ; }, mesh = {Animals ; CRISPR-Cas Systems/genetics ; Cell Differentiation/genetics ; Cell Line, Tumor ; Cell Plasticity/*genetics ; Cell Proliferation/genetics ; Drug Resistance, Neoplasm/*drug effects/*genetics ; *Genetic Testing ; Histone-Lysine N-Methyltransferase/genetics ; Humans ; Melanoma/drug therapy/genetics/pathology ; Mice, Inbred NOD ; Mice, SCID ; Models, Biological ; Molecular Targeted Therapy ; Neoplasms/drug therapy/*genetics/*pathology ; Proto-Oncogene Proteins B-raf/genetics ; Transcription, Genetic ; }, abstract = {Cellular plasticity describes the ability of cells to transition from one set of phenotypes to another. In melanoma, transient fluctuations in the molecular state of tumor cells mark the formation of rare cells primed to survive BRAF inhibition and reprogram into a stably drug-resistant fate. However, the biological processes governing cellular priming remain unknown. We used CRISPR-Cas9 genetic screens to identify genes that affect cell fate decisions by altering cellular plasticity. We found that many factors can independently affect cellular priming and fate decisions. We discovered a new plasticity-based mode of increasing resistance to BRAF inhibition that pushes cells towards a more differentiated state. Manipulating cellular plasticity through inhibition of DOT1L before the addition of the BRAF inhibitor resulted in more therapy resistance than concurrent administration. Our results indicate that modulating cellular plasticity can alter cell fate decisions and may prove useful for treating drug resistance in other cancers.}, }
@article {pmid33376220, year = {2020}, author = {Wang, R and Lenoir, WF and Wang, C and Su, D and McLaughlin, M and Hu, Q and Shen, X and Tian, Y and Klages-Mundt, N and Lynn, E and Wood, RD and Chen, J and Hart, T and Li, L}, title = {DNA polymerase ι compensates for Fanconi anemia pathway deficiency by countering DNA replication stress.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {117}, number = {52}, pages = {33436-33445}, doi = {10.1073/pnas.2008821117}, pmid = {33376220}, issn = {1091-6490}, support = {P01 CA193124/CA/NCI NIH HHS/United States ; P30 CA016672/CA/NCI NIH HHS/United States ; R01 CA190635/CA/NCI NIH HHS/United States ; R35 GM130119/GM/NIGMS NIH HHS/United States ; }, mesh = {CRISPR-Cas Systems/genetics ; Cyclin-Dependent Kinase 4 ; DNA Damage ; *DNA Replication ; DNA-Directed DNA Polymerase/*metabolism ; Fanconi Anemia/*enzymology ; Genome, Human ; HCT116 Cells ; Humans ; Mutation/genetics ; *Stress, Physiological ; Synthetic Lethal Mutations/genetics ; }, abstract = {Fanconi anemia (FA) is caused by defects in cellular responses to DNA crosslinking damage and replication stress. Given the constant occurrence of endogenous DNA damage and replication fork stress, it is unclear why complete deletion of FA genes does not have a major impact on cell proliferation and germ-line FA patients are able to progress through development well into their adulthood. To identify potential cellular mechanisms that compensate for the FA deficiency, we performed dropout screens in FA mutant cells with a whole genome guide RNA library. This uncovered a comprehensive genome-wide profile of FA pathway synthetic lethality, including POLI and CDK4 As little is known of the cellular function of DNA polymerase iota (Pol ι), we focused on its role in the loss-of-function FA knockout mutants. Loss of both FA pathway function and Pol ι leads to synthetic defects in cell proliferation and cell survival, and an increase in DNA damage accumulation. Furthermore, FA-deficient cells depend on the function of Pol ι to resume replication upon replication fork stalling. Our results reveal a critical role for Pol ι in DNA repair and replication fork restart and suggest Pol ι as a target for therapeutic intervention in malignancies carrying an FA gene mutation.}, }
@article {pmid33376219, year = {2020}, author = {Marshall, JL and Doughty, BR and Subramanian, V and Guckelberger, P and Wang, Q and Chen, LM and Rodriques, SG and Zhang, K and Fulco, CP and Nasser, J and Grinkevich, EJ and Noel, T and Mangiameli, S and Bergman, DT and Greka, A and Lander, ES and Chen, F and Engreitz, JM}, title = {HyPR-seq: Single-cell quantification of chosen RNAs via hybridization and sequencing of DNA probes.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {117}, number = {52}, pages = {33404-33413}, doi = {10.1073/pnas.2010738117}, pmid = {33376219}, issn = {1091-6490}, support = {DP5 OD024583/OD/NIH HHS/United States ; K99 HG009917/HG/NHGRI NIH HHS/United States ; R00 HG009917/HG/NHGRI NIH HHS/United States ; }, mesh = {Animals ; CRISPR-Cas Systems/genetics ; DNA Probes/*genetics ; Gene Expression ; High-Throughput Nucleotide Sequencing/*methods ; Humans ; Introns/genetics ; K562 Cells ; Kidney/cytology ; Mice ; *Nucleic Acid Hybridization ; Polyadenylation ; RNA/*metabolism ; RNA, Messenger/genetics/metabolism ; *Single-Cell Analysis ; THP-1 Cells ; Time Factors ; }, abstract = {Single-cell quantification of RNAs is important for understanding cellular heterogeneity and gene regulation, yet current approaches suffer from low sensitivity for individual transcripts, limiting their utility for many applications. Here we present Hybridization of Probes to RNA for sequencing (HyPR-seq), a method to sensitively quantify the expression of hundreds of chosen genes in single cells. HyPR-seq involves hybridizing DNA probes to RNA, distributing cells into nanoliter droplets, amplifying the probes with PCR, and sequencing the amplicons to quantify the expression of chosen genes. HyPR-seq achieves high sensitivity for individual transcripts, detects nonpolyadenylated and low-abundance transcripts, and can profile more than 100,000 single cells. We demonstrate how HyPR-seq can profile the effects of CRISPR perturbations in pooled screens, detect time-resolved changes in gene expression via measurements of gene introns, and detect rare transcripts and quantify cell-type frequencies in tissue using low-abundance marker genes. By directing sequencing power to genes of interest and sensitively quantifying individual transcripts, HyPR-seq reduces costs by up to 100-fold compared to whole-transcriptome single-cell RNA-sequencing, making HyPR-seq a powerful method for targeted RNA profiling in single cells.}, }
@article {pmid33318207, year = {2020}, author = {Patel, AK and Broyer, RM and Lee, CD and Lu, T and Louie, MJ and La Torre, A and Al-Ali, H and Vu, MT and Mitchell, KL and Wahlin, KJ and Berlinicke, CA and Jaskula-Ranga, V and Hu, Y and Duan, X and Vilar, S and Bixby, JL and Weinreb, RN and Lemmon, VP and Zack, DJ and Welsbie, DS}, title = {Inhibition of GCK-IV kinases dissociates cell death and axon regeneration in CNS neurons.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {117}, number = {52}, pages = {33597-33607}, pmid = {33318207}, issn = {1091-6490}, support = {R01 EY023295/EY/NEI NIH HHS/United States ; P30 EY022589/EY/NEI NIH HHS/United States ; R01 EY029342/EY/NEI NIH HHS/United States ; R01 EY024932/EY/NEI NIH HHS/United States ; R01 EY028106/EY/NEI NIH HHS/United States ; }, mesh = {Animals ; Axons/*enzymology/*pathology ; Base Sequence ; CRISPR-Cas Systems/genetics ; Cell Death/drug effects ; Cell Survival/drug effects ; Central Nervous System/*pathology ; Dependovirus/metabolism ; Disease Models, Animal ; Germinal Center Kinases/*metabolism ; Humans ; Mice, Inbred C57BL ; *Nerve Regeneration/drug effects ; Neuronal Outgrowth/drug effects ; Optic Nerve Injuries/metabolism/pathology ; Organoids/metabolism ; Protein Kinase Inhibitors/pharmacology ; Retinal Ganglion Cells/drug effects/metabolism ; Signal Transduction/drug effects ; }, abstract = {Axon injury is a hallmark of many neurodegenerative diseases, often resulting in neuronal cell death and functional impairment. Dual leucine zipper kinase (DLK) has emerged as a key mediator of this process. However, while DLK inhibition is robustly protective in a wide range of neurodegenerative disease models, it also inhibits axonal regeneration. Indeed, there are no genetic perturbations that are known to both improve long-term survival and promote regeneration. To identify such a neuroprotective target, we conducted a set of complementary high-throughput screens using a protein kinase inhibitor library in human stem cell-derived retinal ganglion cells (hRGCs). Overlapping compounds that promoted both neuroprotection and neurite outgrowth were bioinformatically deconvoluted to identify specific kinases that regulated neuronal death and axon regeneration. This work identified the role of germinal cell kinase four (GCK-IV) kinases in cell death and additionally revealed their unexpected activity in suppressing axon regeneration. Using an adeno-associated virus (AAV) approach, coupled with genome editing, we validated that GCK-IV kinase knockout improves neuronal survival, comparable to that of DLK knockout, while simultaneously promoting axon regeneration. Finally, we also found that GCK-IV kinase inhibition also prevented the attrition of RGCs in developing retinal organoid cultures without compromising axon outgrowth, addressing a major issue in the field of stem cell-derived retinas. Together, these results demonstrate a role for the GCK-IV kinases in dissociating the cell death and axonal outgrowth in neurons and their druggability provides for therapeutic options for neurodegenerative diseases.}, }
@article {pmid32703416, year = {2020}, author = {Antonson, P and Apolinário, LM and Shamekh, MM and Humire, P and Poutanen, M and Ohlsson, C and Nalvarte, I and Gustafsson, JÅ}, title = {Generation of an all-exon Esr2 deleted mouse line: Effects on fertility.}, journal = {Biochemical and biophysical research communications}, volume = {529}, number = {2}, pages = {231-237}, doi = {10.1016/j.bbrc.2020.06.063}, pmid = {32703416}, issn = {1090-2104}, mesh = {Animals ; CRISPR-Cas Systems ; Estrogen Receptor beta/*genetics ; Exons ; Female ; *Fertility ; Gene Deletion ; Male ; Mice ; Ovary/physiology ; Ovulation ; }, abstract = {Estrogen receptor beta (ERβ), encoded by the Esr2 gene, is one of two nuclear receptors that mediate the functions of the steroid hormone estradiol. The binding of estradiol to the receptor results in enhanced transcription of many genes that have estrogen response elements in promoter or enhancer regions. Several genetically modified mouse lines with mutations or deletions of exons in the Esr2 gene have been developed and results from analysis of these are not completely consistent, especially regarding ERβ's role in fertility. To address these controversies, we have used the CRISPR/Cas9 genome editing system to make a deletion of the entire Esr2 gene in the mouse genome and determined the effect of this mutation on fertility. We show that female Esr2 deleted mice, Esr2ΔE1-10, are subfertile at young age, with fewer litters and smaller litter size, and that they become infertile/have severely reduced fertility at around six months of age, while the male Esr2ΔE1-10 mice are fertile. Ovaries from Esr2ΔE1-10 mice are smaller than those from wild-type littermates and the morphology of the ovary displays very few corpora lutea, indicating a defect in ovulation. We also show that the estradiol levels are reduced at diestrus, the phase in the estrous cycle when levels are expected to start to increase before ovulation. Our results verify that ERβ has an important function in female reproduction, likely as a regulator of serum estradiol levels, and that its loss does not affect male reproductive function.}, }
@article {pmid32214056, year = {2020}, author = {Libetti, D and Bernardini, A and Sertic, S and Messina, G and Dolfini, D and Mantovani, R}, title = {The Switch from NF-YAl to NF-YAs Isoform Impairs Myotubes Formation.}, journal = {Cells}, volume = {9}, number = {3}, pages = {}, pmid = {32214056}, issn = {2073-4409}, mesh = {Animals ; Base Sequence ; CCAAT-Binding Factor/*metabolism ; CRISPR-Cas Systems/genetics ; Cell Differentiation ; Cell Fusion ; Cell Line ; Clone Cells ; Exons/genetics ; Gene Expression Regulation ; Mice ; Muscle Fibers, Skeletal/cytology/*metabolism ; MyoD Protein/metabolism ; Myogenin/metabolism ; Protein Isoforms/genetics/metabolism ; Transcription Factors/metabolism ; }, abstract = {NF-YA, the regulatory subunit of the trimeric transcription factor (TF) NF-Y, is regulated by alternative splicing (AS) generating two major isoforms, &quot;long&quot; (NF-YAl) and &quot;short&quot; (NF-YAs). Muscle cells express NF-YAl. We ablated exon 3 in mouse C2C12 cells by a four-guide CRISPR/Cas9n strategy, obtaining clones expressing exclusively NF-YAs (C2-YAl-KO). C2-YAl-KO cells grow normally, but are unable to differentiate. Myogenin and-to a lesser extent, MyoD- levels are substantially lower in C2-YAl-KO, before and after differentiation. Expression of the fusogenic Myomaker and Myomixer genes, crucial for the early phases of the process, is not induced. Myomaker and Myomixer promoters are bound by MyoD and Myogenin, and Myogenin overexpression induces their expression in C2-YAl-KO. NF-Y inactivation reduces MyoD and Myogenin, but not directly: the Myogenin promoter is CCAAT-less, and the canonical CCAAT of the MyoD promoter is not bound by NF-Y in vivo. We propose that NF-YAl, but not NF-YAs, maintains muscle commitment by indirectly regulating Myogenin and MyoD expression in C2C12 cells. These experiments are the first genetic evidence that the two NF-YA isoforms have functionally distinct roles.}, }
@article {pmid32088034, year = {2020}, author = {Menchaca, A and Dos Santos-Neto, PC and Mulet, AP and Crispo, M}, title = {CRISPR in livestock: From editing to printing.}, journal = {Theriogenology}, volume = {150}, number = {}, pages = {247-254}, doi = {10.1016/j.theriogenology.2020.01.063}, pmid = {32088034}, issn = {1879-3231}, mesh = {Animals ; *Animals, Genetically Modified ; Biotechnology ; *CRISPR-Cas Systems ; *Gene Editing ; Livestock/*genetics ; Printing, Three-Dimensional ; }, abstract = {Precise genome editing of large animals applied to livestock and biomedicine is nowadays possible since the CRISPR revolution. This review summarizes the latest advances and the main technical issues that determine the success of this technology. The pathway from editing to printing, from engineering the genome to achieving the desired animals, does not always imply an easy, fast and safe journey. When applied in large animals, CRISPR involves time- and cost-consuming projects, and it is mandatory not only to choose the best approach for genome editing, but also for embryo production, zygote microinjection or electroporation, cryopreservation and embryo transfer. The main technical refinements and most frequent questions to improve this disruptive biotechnology in large animals are presented. In addition, we discuss some CRISPR applications to enhance livestock production in the context of a growing global demand of food, in terms of increasing efficiency, reducing the impact of farming on the environment, enhancing pest control, animal welfare and health. The challenge is no longer technical. Controversies and consensus, opportunities and threats, benefits and risks, ethics and science should be reconsidered to enter into the CRISPR era.}, }
@article {pmid32000993, year = {2020}, author = {Lee, K and Uh, K and Farrell, K}, title = {Current progress of genome editing in livestock.}, journal = {Theriogenology}, volume = {150}, number = {}, pages = {229-235}, doi = {10.1016/j.theriogenology.2020.01.036}, pmid = {32000993}, issn = {1879-3231}, support = {R21 OD027062/OD/NIH HHS/United States ; }, mesh = {Animals ; *Animals, Genetically Modified ; CRISPR-Cas Systems ; Gene Editing/*veterinary ; Genetic Engineering/*veterinary ; Livestock/*genetics ; }, abstract = {Historically, genetic engineering in livestock proved to be challenging. Without stable embryonic stem cell lines to utilize, somatic cell nuclear transfer (SCNT) had to be employed to produce many of the genetically engineered (GE) livestock models. Through the genetic engineering of somatic cells followed by SCNT, GE livestock models could be generated carrying site-specific modifications. Although successful, only a few GE livestock models were generated because of low efficiency and associated birth defects. Recently, there have been major strides in the development of genome editing tools: Zinc-Finger Nucleases (ZFNs), Transcription activator-like effector nucleases (TALENS), and Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated 9 (Cas9) system. These tools rely on the generation of a double strand DNA break, followed by one of two repair pathways: non-homologous end joining (NHEJ) or homology directed repair (HDR). Compared to the traditional approaches, these tools dramatically reduce time and effort needed to establish a GE animal. Another benefit of utilizing genome editing tools is the application of direct injection into developing embryos to induce targeted mutations, therefore, eliminating side effects associated with SCNT. Emerging technological advancements of genome editing systems have dramatically improved efficiency to generate GE livestock models for both biomedical and agricultural purposes. Although the efficiency of genome editing tools has revolutionized GE livestock production, improvements for safe and consistent application are desired. This review will provide an overview of genome editing techniques, as well as examples of GE livestock models for agricultural and biomedical purposes.}, }
@article {pmid33559722, year = {2021}, author = {Huang, X and Hilscher, J and Stoger, E and Christou, P and Zhu, C}, title = {Modification of cereal plant architecture by genome editing to improve yields.}, journal = {Plant cell reports}, volume = {}, number = {}, pages = {}, pmid = {33559722}, issn = {1432-203X}, support = {RTI2018-097613-B-I00//the Spanish Ministry of Economy and Competitiveness (MINECO)/ ; PGC2018-097655-B-I00//the Spanish Ministry of Economy and Competitiveness (MINECO)/ ; Grant N°771134//PROSTRIG, ERA-NET Cofund SusCrop/ ; I2823-B25//the Austrian Science Fund FWF/ ; }, abstract = {KEY MESSAGE: We summarize recent genome editing studies that have focused on the examination (or reexamination) of plant architectural phenotypes in cereals and the modification of these traits for crop improvement. Plant architecture is defined as the three-dimensional organization of the entire plant. Shoot architecture refers to the structure and organization of the aboveground components of a plant, reflecting the developmental patterning of stems, branches, leaves and inflorescences/flowers. Root system architecture is essentially determined by four major shape parameters-growth, branching, surface area and angle. Interest in plant architecture has arisen from the profound impact of many architectural traits on agronomic performance, and the genetic and hormonal regulation of these traits which makes them sensitive to both selective breeding and agronomic practices. This is particularly important in staple crops, and a large body of literature has, therefore, accumulated on the control of architectural phenotypes in cereals, particularly rice due to its twin role as one of the world's most important food crops as well as a model organism in plant biology and biotechnology. These studies have revealed many of the molecular mechanisms involved in the regulation of tiller/axillary branching, stem height, leaf and flower development, root architecture and the grain characteristics that ultimately help to determine yield. The advent of genome editing has made it possible, for the first time, to introduce precise mutations into cereal crops to optimize their architecture and close in on the concept of the ideotype. In this review, we consider recent genome editing studies that have focused on the examination (or reexamination) of plant architectural phenotypes in cereals and the modification of these traits for crop improvement.}, }
@article {pmid33558716, year = {2021}, author = {Wang, H and Han, M and Qi, LS}, title = {Engineering 3D genome organization.}, journal = {Nature reviews. Genetics}, volume = {}, number = {}, pages = {}, pmid = {33558716}, issn = {1471-0064}, abstract = {Cancers and developmental disorders are associated with alterations in the 3D genome architecture in space and time (the fourth dimension). Mammalian 3D genome organization is complex and dynamic and plays an essential role in regulating gene expression and cellular function. To study the causal relationship between genome function and its spatio-temporal organization in the nucleus, new technologies for engineering and manipulating the 3D organization of the genome have been developed. In particular, CRISPR-Cas technologies allow programmable manipulation at specific genomic loci, enabling unparalleled opportunities in this emerging field of 3D genome engineering. We review advances in mammalian 3D genome engineering with a focus on recent manipulative technologies using CRISPR-Cas and related technologies.}, }
@article {pmid33558704, year = {2021}, author = {Lakshmanan, VK and Jindal, S and Packirisamy, G and Ojha, S and Lian, S and Kaushik, A and Alzarooni, AIMA and Metwally, YAF and Thyagarajan, SP and Do Jung, Y and Chouaib, S}, title = {Nanomedicine-based cancer immunotherapy: recent trends and future perspectives.}, journal = {Cancer gene therapy}, volume = {}, number = {}, pages = {}, pmid = {33558704}, issn = {1476-5500}, support = {BT/PR 25095/NER/95/1011/2017//Department of Biotechnology, Ministry of Science and Technology (DBT)/ ; }, abstract = {The combination of cancer immunotherapy with efficient functionalized nanosystems has emerged as a beneficial treatment strategy and its use has increased rapidly. The roles of stimuli-responsive nanosystems and nanomedicine-based cancer immunotherapy, a subsidiary discipline in the field of immunology, are pivotal. The present era is witnessing rapid advancements in the use of nanomedicine as a platform for investigating novel therapeutic applications and modern intelligent healthcare management strategies. The development of cancer nanomedicine has posthaste ratified the outcomes of immunotherapy to the subsequent stage in the current era of medical research. This review focuses on key findings with respect to the effectiveness of nanomedicine-based cancer immunotherapies and their applications, which include i) immune checkpoint inhibitors and nanomedicine, ii) CRISPR-Cas nanoparticles (NPs) in cancer immunotherapy, iii) combination cancer immunotherapy with core-shell nanoparticles, iv) biomimetic NPs for cancer immunotherapy, and v) CAR-T cells and cancer nanoimmunotherapy. By evaluating the state-of-the-art tools and taking the challenges involved into consideration, various aspects of the proposed nano-enabled therapeutic approaches have been discussed in this review.}, }
@article {pmid33558692, year = {2021}, author = {Hana, S and Peterson, M and McLaughlin, H and Marshall, E and Fabian, AJ and McKissick, O and Koszka, K and Marsh, G and Craft, M and Xu, S and Sorets, A and Torregrosa, T and Sun, C and Henderson, CE and Lo, SC}, title = {Highly efficient neuronal gene knockout in vivo by CRISPR-Cas9 via neonatal intracerebroventricular injection of AAV in mice.}, journal = {Gene therapy}, volume = {}, number = {}, pages = {}, pmid = {33558692}, issn = {1476-5462}, abstract = {CRISPR-Cas systems have emerged as a powerful tool to generate genetic models for studying normal and diseased central nervous system (CNS). Targeted gene disruption at specific loci has been demonstrated successfully in non-dividing neurons. Despite its simplicity, high specificity and low cost, the efficiency of CRISPR-mediated knockout in vivo can be substantially impacted by many parameters. Here, we used CRISPR-Cas9 to disrupt the neuronal-specific gene, NeuN, and optimized key parameters to achieve effective gene knockout broadly in the CNS in postnatal mice. Three cell lines and two primary neuron cultures were used to validate the disruption of NeuN by single-guide RNAs (sgRNA) harboring distinct spacers and scaffold sequences. This triage identified an optimal sgRNA design with the highest NeuN disruption in in vitro and in vivo systems. To enhance CRISPR efficiency, AAV-PHP.B, a vector with superior neuronal transduction, was used to deliver this sgRNA in Cas9 mice via neonatal intracerebroventricular (ICV) injection. This approach resulted in 99.4% biallelic indels rate in the transduced cells, leading to greater than 70% reduction of total NeuN proteins in the cortex, hippocampus and spinal cord. This work contributes to the optimization of CRISPR-mediated knockout and will be beneficial for fundamental and preclinical research.}, }
@article {pmid33557342, year = {2021}, author = {Deutsch, M and Günther, A and Lerchundi, R and Rose, CR and Balfanz, S and Baumann, A}, title = {AAV-Mediated CRISPRi and RNAi Based Gene Silencing in Mouse Hippocampal Neurons.}, journal = {Cells}, volume = {10}, number = {2}, pages = {}, doi = {10.3390/cells10020324}, pmid = {33557342}, issn = {2073-4409}, abstract = {Uncovering the physiological role of individual proteins that are part of the intricate process of cellular signaling is often a complex and challenging task. A straightforward strategy of studying a protein's function is by manipulating the expression rate of its gene. In recent years, the Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/Cas9-based technology was established as a powerful gene-editing tool for generating sequence specific changes in proliferating cells. However, obtaining homogeneous populations of transgenic post-mitotic neurons by CRISPR/Cas9 turned out to be challenging. These constraints can be partially overcome by CRISPR interference (CRISPRi), which mediates the inhibition of gene expression by competing with the transcription machinery for promoter binding and, thus, transcription initiation. Notably, CRISPR/Cas is only one of several described approaches for the manipulation of gene expression. Here, we targeted neurons with recombinant Adeno-associated viruses to induce either CRISPRi or RNA interference (RNAi), a well-established method for impairing de novo protein biosynthesis by using cellular regulatory mechanisms that induce the degradation of pre-existing mRNA. We specifically targeted hyperpolarization-activated and cyclic nucleotide-gated (HCN) channels, which are widely expressed in neuronal tissues and play essential physiological roles in maintaining biophysical characteristics in neurons. Both of the strategies reduced the expression levels of three HCN isoforms (HCN1, 2, and 4) with high specificity. Furthermore, detailed analysis revealed that the knock-down of just a single HCN isoform (HCN4) in hippocampal neurons did not affect basic electrical parameters of transduced neurons, whereas substantial changes emerged in HCN-current specific properties.}, }
@article {pmid33463756, year = {2020}, author = {Rautela, J and Surgenor, E and Huntington, ND}, title = {Drug target validation in primary human natural killer cells using CRISPR RNP.}, journal = {Journal of leukocyte biology}, volume = {108}, number = {4}, pages = {1397-1408}, doi = {10.1002/JLB.2MA0620-074R}, pmid = {33463756}, issn = {1938-3673}, mesh = {Animals ; *CRISPR-Cas Systems ; *Drug Discovery ; *Electroporation ; Humans ; Killer Cells, Natural/cytology/*immunology ; Mice ; }, abstract = {The ability to genetically modify CD8 T cells using viral gene delivery has facilitated the development of next generation of cancer immunotherapies such as chimeric Ag receptor (CAR) T cells engineered to specifically kill tumor cells. Development of immunotherapies targeting NK cells have stalled in part by their resistance to traditional viral gene delivery systems. Here, an efficient approach is described to genetically edit human NK cells by electroporation and CRISPR-Cas9 ribonucleoprotein (RNP) complexes. Electroporation pulse codes and buffer optimization for protein uptake by human NK cells and viability, and the efficiency of this approach over other methods are detailed. To highlight the transformative step this technique will have for NK cell immunotherapy drug discovery, NCR1 and CISH are deleted in primary human NK cells and murine findings are validated on their key roles in regulating NK cell antitumor function.}, }
@article {pmid32807825, year = {2020}, author = {Slattery, SS and Wang, H and Giguere, DJ and Kocsis, C and Urquhart, BL and Karas, BJ and Edgell, DR}, title = {Plasmid-based complementation of large deletions in Phaeodactylum tricornutum biosynthetic genes generated by Cas9 editing.}, journal = {Scientific reports}, volume = {10}, number = {1}, pages = {13879}, pmid = {32807825}, issn = {2045-2322}, mesh = {Ascomycota/*genetics/*metabolism ; *CRISPR-Cas Systems ; Complement System Proteins/*genetics ; Drug Resistance, Bacterial/genetics ; *Gene Deletion ; Gene Editing/*methods ; Genetic Engineering ; Histidine/biosynthesis ; Orotate Phosphoribosyltransferase/metabolism ; Plasmids/*genetics ; Tryptophan/biosynthesis ; Uracil/biosynthesis ; }, abstract = {The model diatom Phaeodactylum tricornutum is an attractive candidate for synthetic biology applications. Development of auxotrophic strains of P. tricornutum would provide alternative selective markers to commonly used antibiotic resistance genes. Here, using CRISPR/Cas9, we show successful editing of genes in the uracil, histidine, and tryptophan biosynthetic pathways. Nanopore long-read sequencing indicates that editing events are characterized by the occurrence of large deletions of up to ~ 2.7 kb centered on the editing site. The uracil and histidine-requiring phenotypes can be complemented by plasmid-based copies of the intact genes after curing of the Cas9-editing plasmid. Growth of uracil auxotrophs on media supplemented with 5-fluoroorotic acid and uracil results in loss of the complementing plasmid, providing a facile method for plasmid curing with potential applications in strain engineering and CRISPR editing. Metabolomic characterization of uracil auxotrophs revealed changes in cellular orotate concentrations consistent with partial or complete loss of orotate phosphoribosyltransferase activity. Our results expand the range of P. tricornutum auxotrophic strains and demonstrate that auxotrophic complementation markers provide a viable alternative to traditionally used antibiotic selection markers. Plasmid-based auxotrophic markers should expand the range of genome engineering applications and provide a means for biocontainment of engineered P. tricornutum strains.}, }
@article {pmid32762648, year = {2020}, author = {Tasaki, K and Yoshida, M and Nakajima, M and Higuchi, A and Watanabe, A and Nishihara, M}, title = {Molecular characterization of an anthocyanin-related glutathione S-transferase gene in Japanese gentian with the CRISPR/Cas9 system.}, journal = {BMC plant biology}, volume = {20}, number = {1}, pages = {370}, pmid = {32762648}, issn = {1471-2229}, support = {16K18654//Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science/ ; 18K14462//Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science/ ; 19190722_//Ministry of Agriculture, Forestry and Fisheries/ ; 15653424//Ministry of Agriculture, Forestry and Fisheries/ ; }, mesh = {Anthocyanins/chemistry/*metabolism ; Biological Transport ; *CRISPR-Cas Systems/genetics ; Cloning, Molecular ; Flavonoids/biosynthesis/genetics ; Flowers/metabolism ; Gene Editing ; Genes, Plant ; Genetic Complementation Test ; Gentiana/*enzymology/*genetics ; Glutathione Transferase/genetics/*metabolism ; High-Throughput Nucleotide Sequencing ; Phenotype ; Plant Leaves/metabolism ; Plant Proteins/genetics/*metabolism ; }, abstract = {BACKGROUND: The blue pigmentation of Japanese gentian flowers is due to a polyacylated anthocyanin, gentiodelphin, and all associated biosynthesis genes and several regulatory genes have been cloned and characterized. However, the final step involving the accumulation of anthocyanins in petal vacuoles remains unclear. We cloned and analyzed the glutathione S-transferases (GSTs) in Japanese gentian that are known to be involved in anthocyanin transport in other plant species.<br><br>RESULTS: We cloned GST1, which is expressed in gentian flower petals. Additionally, this gene belongs to the Phi-type GST clade related to anthocyanin biosynthesis. We used the CRISPR/Cas9-mediated genome editing system to generate loss-of-function GST1 alleles. The edited alleles were confirmed by Sanger and next-generation sequencing analyses. The GST1 genome-edited lines exhibited two types of mutant flower phenotypes, severe (almost white) and mild (pale blue). The phenotypes were associated with decreased anthocyanin accumulation in flower petals. In the GST1 genome-edited lines, sugar-induced stress conditions inhibited the accumulation of anthocyanins in stems and leaves, suggestvhing that GST1 is necessary for stress-related anthocyanin accumulation in organs other than flowers. These observations clearly demonstrate that GST1 is the gene responsible for anthocyanin transport in Japanese gentian, and is necessary for the accumulation of gentiodelphin in flowers.<br><br>CONCLUSIONS: In this study, an anthocyanin-related GST gene in Japanese gentian was functionally characterized. Unlike other biosynthesis genes, the functions of GST genes are difficult to examine in in vitro studies. Thus, the genome-editing strategy described herein may be useful for in vivo investigations of the roles of transport-related genes in gentian plants.}, }
@article {pmid32531864, year = {2020}, author = {Chow, SH and Deo, P and Yeung, ATY and Kostoulias, XP and Jeon, Y and Gao, ML and Seidi, A and Olivier, FAB and Sridhar, S and Nethercott, C and Cameron, D and Robertson, AAB and Robert, R and Mackay, CR and Traven, A and Jin, ZB and Hale, C and Dougan, G and Peleg, AY and Naderer, T}, title = {Targeting NLRP3 and Staphylococcal pore-forming toxin receptors in human-induced pluripotent stem cell-derived macrophages.}, journal = {Journal of leukocyte biology}, volume = {108}, number = {3}, pages = {967-981}, doi = {10.1002/JLB.4MA0420-497R}, pmid = {32531864}, issn = {1938-3673}, mesh = {Animals ; Bacterial Proteins/*antagonists &amp; inhibitors ; Bacterial Toxins/*antagonists &amp; inhibitors ; CD11b Antigen/immunology ; CRISPR-Cas Systems ; Cell Differentiation ; Cells, Cultured ; Exotoxins/*antagonists &amp; inhibitors/deficiency ; Gene Knock-In Techniques ; Humans ; Induced Pluripotent Stem Cells/*cytology ; Interleukin-1beta/metabolism ; Leukocidins/*antagonists &amp; inhibitors ; Leukocyte Common Antigens/physiology ; Lung/immunology/microbiology ; Macrophages/cytology/*drug effects/immunology ; Mice ; Mice, Inbred C57BL ; Monocytes/cytology ; NLR Family, Pyrin Domain-Containing 3 Protein/*antagonists &amp; inhibitors ; Peptide Fragments/immunology ; Pneumonia, Staphylococcal/immunology ; Protein Subunits ; Receptor, Anaphylatoxin C5a/deficiency/*drug effects/genetics/physiology ; Recombinant Proteins/metabolism ; *Staphylococcus aureus/physiology ; }, abstract = {Staphylococcus aureus causes necrotizing pneumonia by secreting toxins such as leukocidins that target front-line immune cells. The mechanism by which leukocidins kill innate immune cells and trigger inflammation during S. aureus lung infection, however, remains unresolved. Here, we explored human-induced pluripotent stem cell-derived macrophages (hiPSC-dMs) to study the interaction of the leukocidins Panton-Valentine leukocidin (PVL) and LukAB with lung macrophages, which are the initial leukocidin targets during S. aureus lung invasion. hiPSC-dMs were susceptible to the leukocidins PVL and LukAB and both leukocidins triggered NLPR3 inflammasome activation resulting in IL-1β secretion. hiPSC-dM cell death after LukAB exposure, however, was only temporarily dependent of NLRP3, although NLRP3 triggered marked cell death after PVL treatment. CRISPR/Cas9-mediated deletion of the PVL receptor, C5aR1, protected hiPSC-dMs from PVL cytotoxicity, despite the expression of other leukocidin receptors, such as CD45. PVL-deficient S. aureus had reduced ability to induce lung IL-1β levels in human C5aR1 knock-in mice. Unexpectedly, inhibiting NLRP3 activity resulted in increased wild-type S. aureus lung burdens. Our findings suggest that NLRP3 induces macrophage death and IL-1β secretion after PVL exposure and controls S. aureus lung burdens.}, }
@article {pmid32415294, year = {2020}, author = {Zhong, Y and Chen, B and Li, M and Wang, D and Jiao, Y and Qi, X and Wang, M and Liu, Z and Chen, C and Wang, Y and Chen, M and Li, J and Xiao, Z and Cheng, D and Liu, W and Boutilier, K and Liu, C and Chen, S}, title = {A DMP-triggered in vivo maternal haploid induction system in the dicotyledonous Arabidopsis.}, journal = {Nature plants}, volume = {6}, number = {5}, pages = {466-472}, doi = {10.1038/s41477-020-0658-7}, pmid = {32415294}, issn = {2055-0278}, mesh = {Arabidopsis/*genetics/physiology ; Arabidopsis Proteins/*genetics/physiology ; CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Gene Editing ; Genes, Plant/genetics/physiology ; *Haploidy ; Loss of Function Mutation/genetics ; Membrane Proteins/*genetics/physiology ; Plants, Genetically Modified ; }, abstract = {Doubled haploid technology using inducer lines carrying mutations in ZmPLA1/MTL/NLD and ZmDMP1-4 has revolutionized traditional maize breeding. ZmPLA1/MTL/NLD is conserved in monocots and has been used to extend the system from maize to other monocots5-7, but no functional orthologue has been identified in dicots, while ZmDMP-like genes exist in both monocots and dicots4,8,9. Here, we report that loss-of-function mutations in the Arabidopsis thaliana ZmDMP-like genes AtDMP8 and AtDMP9 induce maternal haploids, with an average haploid induction rate of 2.1 ± 1.1%. In addition, to facilitate haploid seed identification in dicots, we established an efficient FAST-Red fluorescent marker-based haploid identification system that enables the identification of haploid seeds with >90% accuracy. These results show that mutations in DMP genes also trigger haploid induction in dicots. The conserved expression patterns and amino acid sequences of ZmDMP-like genes in dicots suggest that DMP mutations could be used to develop in vivo haploid induction systems in dicots.}, }
@article {pmid32393878, year = {2020}, author = {Xiao, Y and Offringa, R}, title = {PDK1 regulates auxin transport and Arabidopsis vascular development through AGC1 kinase PAX.}, journal = {Nature plants}, volume = {6}, number = {5}, pages = {544-555}, doi = {10.1038/s41477-020-0650-2}, pmid = {32393878}, issn = {2055-0278}, mesh = {3-Phosphoinositide-Dependent Protein Kinases/metabolism/*physiology ; Arabidopsis/*growth &amp; development/physiology ; Arabidopsis Proteins/metabolism/*physiology ; CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Gene Editing ; Indoleacetic Acids/*metabolism ; Phloem/*growth &amp; development ; Phosphorylation ; Plant Growth Regulators/*metabolism ; }, abstract = {The 3-phosphoinositide-dependent protein kinase 1 (PDK1) is a conserved master regulator of AGC kinases in eukaryotic organisms. pdk1 loss of function causes a lethal phenotype in animals and yeasts, but only mild phenotypic defects in Arabidopsis thaliana (Arabidopsis). The Arabidopsis genome contains two PDK1-encoding genes, PDK1 and PDK2. Here, we used clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) to generate true loss-of-function pdk1 alleles, which, when combined with pdk2 alleles, showed severe developmental defects including fused cotyledons, a short primary root, dwarf stature and defects in male fertility. We obtained evidence that PDK1 is responsible for AGC1 kinase PROTEIN KINASE ASSOCIATED WITH BRX (PAX) activation by phosphorylation during vascular development, and that the PDK1 phospholipid-binding Pleckstrin Homology domain is not required for this process. Our data indicate that PDK1 regulates polar auxin transport by activating AGC1 clade kinases, resulting in PIN phosphorylation.}, }
@article {pmid32097467, year = {2020}, author = {Pauli, C and Liu, Y and Rohde, C and Cui, C and Fijalkowska, D and Gerloff, D and Walter, C and Krijgsveld, J and Dugas, M and Edemir, B and Pabst, C and Müller, LP and Zhou, F and Müller-Tidow, C}, title = {Site-specific methylation of 18S ribosomal RNA by SNORD42A is required for acute myeloid leukemia cell proliferation.}, journal = {Blood}, volume = {135}, number = {23}, pages = {2059-2070}, doi = {10.1182/blood.2019004121}, pmid = {32097467}, issn = {1528-0020}, mesh = {CRISPR-Cas Systems ; *Cell Proliferation ; *DNA Methylation ; Humans ; Leukemia, Myeloid, Acute/genetics/metabolism/*pathology ; RNA, Ribosomal, 18S/chemistry/*genetics ; RNA, Small Nucleolar/genetics/*metabolism ; Ribosomal Proteins/antagonists &amp; inhibitors/genetics/*metabolism ; Tumor Cells, Cultured ; }, abstract = {Noncoding RNAs, including small nucleolar RNAs (snoRNAs), play important roles in leukemogenesis, but the relevant mechanisms remain incompletely understood. We performed snoRNA-focused CRISPR-Cas9 knockout library screenings that targeted the entire snoRNAnome and corresponding host genes. The C/D box containing SNORD42A was identified as an essential modulator for acute myeloid leukemia (AML) cell survival and proliferation in multiple human leukemia cell lines. In line, SNORD42A was consistently expressed at higher levels in primary AML patient samples than in CD34+ progenitors, monocytes, and granulocytes. Functionally, knockout of SNORD42A reduced colony formation capability and inhibited proliferation. The SNORD42A acts as a C/D box snoRNA and directs 2'-O-methylation at uridine 116 of 18S ribosomal RNA (rRNA). Deletion of SNORD42A decreased 18S-U116 2'-O-methylation, which was associated with a specific decrease in the translation of ribosomal proteins. In line, the cell size of SNORD42A deletion carrying leukemia cells was decreased. Taken together, these findings establish that high-level expression of SNORD42A with concomitant U116 18S rRNA 2'-O-methylation is essential for leukemia cell growth and survival.}, }
@article {pmid31904924, year = {2020}, author = {Gamboa, L and Phung, EV and Li, H and Meyers, JP and Hart, AC and Miller, IC and Kwong, GA}, title = {Heat-Triggered Remote Control of CRISPR-dCas9 for Tunable Transcriptional Modulation.}, journal = {ACS chemical biology}, volume = {15}, number = {2}, pages = {533-542}, pmid = {31904924}, issn = {1554-8937}, support = {DP2 HD091793/HD/NICHD NIH HHS/United States ; T32 EB006343/EB/NIBIB NIH HHS/United States ; UL1 TR000454/TR/NCATS NIH HHS/United States ; }, mesh = {Animals ; CRISPR-Associated Protein 9/*genetics ; *CRISPR-Cas Systems ; Chemokine CCL21/metabolism ; Genes, Switch ; Granzymes/metabolism ; Green Fluorescent Proteins/metabolism ; HEK293 Cells ; HSP70 Heat-Shock Proteins/genetics ; *Heating ; Herpes Simplex Virus Protein Vmw65/genetics ; Humans ; Kruppel-Like Transcription Factors/genetics ; Mice, Nude ; Protein Domains ; Recombinant Fusion Proteins/genetics ; Repressor Proteins/genetics ; Simplexvirus/chemistry ; Transcription, Genetic/physiology ; Transcriptional Activation/*physiology ; }, abstract = {CRISPR-associated proteins (Cas) are enabling powerful new approaches to control mammalian cell functions, yet the lack of spatially defined, noninvasive modalities limits their use as biological tools. Here, we integrate thermal gene switches with dCas9 complexes to confer remote control of gene activation and suppression with short pulses of heat. Using a thermal switch constructed from the heat shock protein A6 (HSPA6) locus, we show that a single heat pulse 3-5 °C above basal temperature is sufficient to trigger expression of dCas9 complexes. We demonstrate that dCas9 fused to the transcriptional activator VP64 is functional after heat activation, and, depending on the number of heat pulses, drives transcription of endogenous genes GzmB and CCL21 to levels equivalent to that achieved by a constitutive viral promoter. Across a range of input temperatures, we find that downstream protein expression of GzmB closely correlates with transcript levels (R2 = 0.99). Using dCas9 fused with the transcriptional suppressor KRAB, we show that longitudinal suppression of the reporter d2GFP depends on key thermal input parameters including pulse magnitude, number of pulses, and dose fractionation. In living mice, we extend our study using photothermal heating to spatially target implanted cells to suppress d2GFP in vivo. Our study establishes a noninvasive and targeted approach to harness Cas-based proteins for modulation of gene expression to complement current methods for remote control of cell function.}, }
@article {pmid33555973, year = {2020}, author = {Makarova, KS and Wolf, YI and Shmakov, SA and Liu, Y and Li, M and Koonin, EV}, title = {Unprecedented Diversity of Unique CRISPR-Cas-Related Systems and Cas1 Homologs in Asgard Archaea.}, journal = {The CRISPR journal}, volume = {3}, number = {3}, pages = {156-163}, doi = {10.1089/crispr.2020.0012}, pmid = {33555973}, issn = {2573-1602}, abstract = {The principal function of archaeal and bacterial CRISPR-Cas systems is antivirus adaptive immunity. However, recent genome analyses identified a variety of derived CRISPR-Cas variants at least some of which appear to perform different functions. Here, we describe a unique repertoire of CRISPR-Cas-related systems that we discovered by searching archaeal metagenome-assemble genomes of the Asgard superphylum. Several of these variants contain extremely diverged homologs of Cas1, the integrase involved in CRISPR adaptation as well as casposon transposition. Strikingly, the diversity of Cas1 in Asgard archaea alone is greater than that detected so far among the rest of archaea and bacteria. The Asgard CRISPR-Cas derivatives also encode distinct forms of Cas4, Cas5, and Cas7 proteins, and/or additional nucleases. Some of these systems are predicted to perform defense functions, but possibly not programmable ones, whereas others are likely to represent previously unknown mobile genetic elements.}, }
@article {pmid33554142, year = {2021}, author = {Qiu, Y and Ding, Q}, title = {Optimized protocol for gene editing in adipocytes using CRISPR-Cas9 technology.}, journal = {STAR protocols}, volume = {2}, number = {1}, pages = {100307}, doi = {10.1016/j.xpro.2021.100307}, pmid = {33554142}, issn = {2666-1667}, abstract = {We present a detailed protocol for gene editing in adipocytes using the CRISPR-Cas technology. This protocol describes sgRNA design, preparation of lentiCRISPR-sgRNA vectors, functional validation of sgRNAs, preparation of lentiviruses, and lentiviruses transduction in adipocytes. Moreover, an optimized method of gene editing using the lentiCRISPRv2 vector expressing two sgRNAs targeting two different genes has also been described. For complete details on the use and execution of this protocol, please refer to Qiu et al. (2020).}, }
@article {pmid33552016, year = {2020}, author = {Huang, F and Zhu, B}, title = {The Cyclic Oligoadenylate Signaling Pathway of Type III CRISPR-Cas Systems.}, journal = {Frontiers in microbiology}, volume = {11}, number = {}, pages = {602789}, doi = {10.3389/fmicb.2020.602789}, pmid = {33552016}, issn = {1664-302X}, abstract = {Type III CRISPR-Cas systems, which are widespread in both bacteria and archaea, provide immunity against DNA viruses and plasmids in a transcription-dependent manner. Since an unprecedented cyclic oligoadenylate (cOA) signaling pathway was discovered in type III systems in 2017, the cOA signaling has been extensively studied in recent 3 years, which has expanded our understanding of type III systems immune defense and also its counteraction by viruses. In this review, we summarized recent advances in cOA synthesis, cOA-activated effector protein, cOA signaling-mediated immunoprotection, and cOA signaling inhibition, and highlighted the crosstalk between cOA signaling and other cyclic oligonucleotide-mediated immunity discovered very recently.}, }
@article {pmid33498106, year = {2021}, author = {Tian, T and Shu, B and Jiang, Y and Ye, M and Liu, L and Guo, Z and Han, Z and Wang, Z and Zhou, X}, title = {An Ultralocalized Cas13a Assay Enables Universal and Nucleic Acid Amplification-Free Single-Molecule RNA Diagnostics.}, journal = {ACS nano}, volume = {15}, number = {1}, pages = {1167-1178}, doi = {10.1021/acsnano.0c08165}, pmid = {33498106}, issn = {1936-086X}, mesh = {*CRISPR-Cas Systems ; Cell Line, Tumor ; Enterococcus faecalis ; Escherichia coli ; Humans ; Klebsiella pneumoniae ; MCF-7 Cells ; MicroRNAs/analysis ; *Microfluidics ; Pseudomonas aeruginosa ; RNA/*analysis ; RNA, Ribosomal, 16S/analysis ; Reverse Transcriptase Polymerase Chain Reaction ; SARS-CoV-2/genetics ; Staphylococcus aureus ; }, abstract = {Existing methods for RNA diagnostics, such as reverse transcription PCR (RT-PCR), mainly rely on nucleic acid amplification (NAA) and RT processes, which are known to introduce substantial issues, including amplification bias, cross-contamination, and sample loss. To address these problems, we introduce a confinement effect-inspired Cas13a assay for single-molecule RNA diagnostics, eliminating the need for NAA and RT. This assay involves confining the RNA-triggered Cas13a catalysis system in cell-like-sized reactors to enhance local concentrations of target and reporter simultaneously, via droplet microfluidics. It achieves >10 000-fold enhancement in sensitivity when compared to the bulk Cas13a assay and enables absolute digital single-molecule RNA quantitation. We experimentally demonstrate its broad applicability for precisely counting microRNAs, 16S rRNAs, and SARS-CoV-2 RNA from synthetic sequences to clinical samples with excellent accuracy. Notably, this direct RNA diagnostic technology enables detecting a wide range of RNA molecules at the single-molecule level. Moreover, its simplicity, universality, and excellent quantification capability might render it to be a dominant rival to RT-qPCR.}, }
@article {pmid33479218, year = {2021}, author = {Ward, CM and Aumann, RA and Whitehead, MA and Nikolouli, K and Leveque, G and Gouvi, G and Fung, E and Reiling, SJ and Djambazian, H and Hughes, MA and Whiteford, S and Caceres-Barrios, C and Nguyen, TNM and Choo, A and Crisp, P and Sim, SB and Geib, SM and Marec, F and Häcker, I and Ragoussis, J and Darby, AC and Bourtzis, K and Baxter, SW and Schetelig, MF}, title = {White pupae phenotype of tephritids is caused by parallel mutations of a MFS transporter.}, journal = {Nature communications}, volume = {12}, number = {1}, pages = {491}, pmid = {33479218}, issn = {2041-1723}, mesh = {Amino Acid Sequence ; Animals ; Base Sequence ; CRISPR-Cas Systems ; Ceratitis capitata/genetics ; Female ; Fertility/genetics ; Genome, Insect/genetics ; Insect Proteins/*genetics ; Male ; *Mutation ; Pest Control, Biological/*methods ; Phenotype ; Pupa/*genetics ; Reproduction/genetics ; Tephritidae/classification/*genetics ; }, abstract = {Mass releases of sterilized male insects, in the frame of sterile insect technique programs, have helped suppress insect pest populations since the 1950s. In the major horticultural pests Bactrocera dorsalis, Ceratitis capitata, and Zeugodacus cucurbitae, a key phenotype white pupae (wp) has been used for decades to selectively remove females before releases, yet the gene responsible remained unknown. Here, we use classical and modern genetic approaches to identify and functionally characterize causal wp- mutations in these distantly related fruit fly species. We find that the wp phenotype is produced by parallel mutations in a single, conserved gene. CRISPR/Cas9-mediated knockout of the wp gene leads to the rapid generation of white pupae strains in C. capitata and B. tryoni. The conserved phenotype and independent nature of wp- mutations suggest this technique can provide a generic approach to produce sexing strains in other major medical and agricultural insect pests.}, }
@article {pmid33367825, year = {2021}, author = {Cha, S and Hong, CP and Kang, HA and Hahn, JS}, title = {Differential activation mechanisms of two isoforms of Gcr1 transcription factor generated from spliced and un-spliced transcripts in Saccharomyces cerevisiae.}, journal = {Nucleic acids research}, volume = {49}, number = {2}, pages = {745-759}, pmid = {33367825}, issn = {1362-4962}, mesh = {CRISPR-Cas Systems ; Cell Respiration ; Chromatin Immunoprecipitation ; DNA-Binding Proteins/chemistry/*genetics ; Enzyme Activation ; Ethanol/metabolism ; Glycerol/metabolism ; Glycolysis ; Protein Binding ; Protein Domains ; Protein Isoforms/chemistry/genetics ; RNA-Seq ; Reverse Transcriptase Polymerase Chain Reaction ; Saccharomyces cerevisiae/*enzymology/genetics/growth &amp; development ; Saccharomyces cerevisiae Proteins/chemistry/*genetics ; Transcription Factors/chemistry/deficiency/*genetics ; }, abstract = {Gcr1, an important transcription factor for glycolytic genes in Saccharomyces cerevisiae, was recently revealed to have two isoforms, Gcr1U and Gcr1S, produced from un-spliced and spliced transcripts, respectively. In this study, by generating strains expressing only Gcr1U or Gcr1S using the CRISPR/Cas9 system, we elucidate differential activation mechanisms of these two isoforms. The Gcr1U monomer forms an active complex with its coactivator Gcr2 homodimer, whereas Gcr1S acts as a homodimer without Gcr2. The USS domain, 55 residues at the N-terminus existing only in Gcr1U, inhibits dimerization of Gcr1U and even acts in trans to inhibit Gcr1S dimerization. The Gcr1S monomer inhibits the metabolic switch from fermentation to respiration by directly binding to the ALD4 promoter, which can be restored by overexpression of the ALD4 gene, encoding a mitochondrial aldehyde dehydrogenase required for ethanol utilization. Gcr1U and Gcr1S regulate almost the same target genes, but show unique activities depending on growth phase, suggesting that these isoforms play differential roles through separate activation mechanisms depending on environmental conditions.}, }
@article {pmid33283409, year = {2021}, author = {Kim, HM and Lee, SH and Lim, J and Yoo, J and Hwang, DY}, title = {The epidermal growth factor receptor variant type III mutation frequently found in gliomas induces astrogenesis in human cerebral organoids.}, journal = {Cell proliferation}, volume = {54}, number = {2}, pages = {e12965}, pmid = {33283409}, issn = {1365-2184}, support = {//Korea National Institute of Health/ ; HI6C1559 and HI18C0096//Korea Ministry of Health and Welfare/ ; }, mesh = {Apoptosis/drug effects ; Astrocytes/*cytology/metabolism ; Brain/cytology/drug effects/metabolism/pathology ; Brain Neoplasms/genetics/*pathology ; CRISPR-Cas Systems/genetics ; Cell Differentiation ; Cell Line ; Cell Proliferation ; ErbB Receptors/genetics/*metabolism ; Gene Editing ; Glioblastoma/genetics/*pathology ; Human Embryonic Stem Cells/cytology/metabolism ; Humans ; Karyotype ; Models, Biological ; Mutation ; Organoids/cytology/drug effects/metabolism/*pathology ; Temozolomide/pharmacology ; }, abstract = {OBJECTIVES: The epidermal growth factor receptor variant type III (EGFRvIII) is the most common mutation of EGFR in glioblastoma multiforme (GBM) and is found in approximately 25% of all GBMs. Intriguingly, EGFRvIII is mostly found in GFAP+ astrocytic tumour cells in the brain, suggesting connection of EGFRvIII to astrogenesis. In this study, we explored whether EGFRvIII mutation facilitates astrogenesis in human development setting.<br><br>MATERIALS AND METHODS: Using CRISPR-Cas9, we generated EGFRvIII mutations in H9-hESCs. Wild type (wt) H9-hESCs were used as an isogenic control. Next, we generated cerebral organoids using the wt and EGFRvIII-hESCs and examined the astrogenic differentiation of the brain organoids.<br><br>RESULTS: EGFRvIII-organoids showed abundant astrocytes (GFAP+ , S100β+), while no astrocytes were detected in wt hESC-derived organoids at day 49. On the contrary, TUJ1+ neurons were more abundant in the wt-organoids than the EGFRvIII-organoids. This result suggested that constitutively active EGFRvIII promoted astrogenesis at the expense of neurogenesis. In addition, the EGFRvIII-organoids were larger in size and retained more Ki67+ cells than wt-organoids, indicating enhanced cell proliferation by the mutation. The EGFRvIII-organoids displayed massive apoptotic cell death after treatment with temozolomide and hence, could be used for evaluation of anti-GBM drugs.<br><br>CONCLUSIONS: EGFRvIII mutation-induced astrogenesis and massive cell proliferation in a human brain development model. These results provide us new insights into the mechanisms relating EGFRvIII mutation-mediated gliogenesis and gliomagenesis.}, }
@article {pmid33270888, year = {2021}, author = {Wang, J and Sui, X and Ding, Y and Fu, Y and Feng, X and Liu, M and Zhang, Y and Xian, M and Zhao, G}, title = {A fast and robust iterative genome-editing method based on a Rock-Paper-Scissors strategy.}, journal = {Nucleic acids research}, volume = {49}, number = {2}, pages = {e12}, pmid = {33270888}, issn = {1362-4962}, mesh = {CRISPR-Cas Systems ; Chloramphenicol/pharmacology ; Clone Cells ; Drug Resistance, Microbial/*genetics ; Escherichia coli/drug effects/genetics/metabolism ; Gene Editing/*methods ; Gene Expression Regulation, Bacterial ; Gene Knockout Techniques ; Genes, Bacterial ; Kanamycin/pharmacology ; Kanamycin Resistance/genetics ; Klebsiella pneumoniae/drug effects/genetics/metabolism ; Lactates/metabolism ; Mutation ; Nucleotide Motifs ; Plasmids/*genetics ; Promoter Regions, Genetic/genetics ; Pyruvic Acid/metabolism ; RNA, Guide/*genetics ; Selection, Genetic ; Tetracycline/pharmacology ; Tetracycline Resistance/genetics ; Time Factors ; Transformation, Bacterial ; }, abstract = {The production of optimized strains of a specific phenotype requires the construction and testing of a large number of genome modifications and combinations thereof. Most bacterial iterative genome-editing methods include essential steps to eliminate selection markers, or to cure plasmids. Additionally, the presence of escapers leads to time-consuming separate single clone picking and subsequent cultivation steps. Herein, we report a genome-editing method based on a Rock-Paper-Scissors (RPS) strategy. Each of three constructed sgRNA plasmids can cure, or be cured by, the other two plasmids in the system; plasmids from a previous round of editing can be cured while the current round of editing takes place. Due to the enhanced curing efficiency and embedded double check mechanism, separate steps for plasmid curing or confirmation are not necessary, and only two times of cultivation are needed per genome-editing round. This method was successfully demonstrated in Escherichia coli and Klebsiella pneumoniae with both gene deletions and replacements. To the best of our knowledge, this is the fastest and most robust iterative genome-editing method, with the least times of cultivation decreasing the possibilities of spontaneous genome mutations.}, }
@article {pmid33231685, year = {2021}, author = {Abid, HZ and Young, E and McCaffrey, J and Raseley, K and Varapula, D and Wang, HY and Piazza, D and Mell, J and Xiao, M}, title = {Customized optical mapping by CRISPR-Cas9 mediated DNA labeling with multiple sgRNAs.}, journal = {Nucleic acids research}, volume = {49}, number = {2}, pages = {e8}, pmid = {33231685}, issn = {1362-4962}, mesh = {Alleles ; Base Sequence ; Benzoxazoles/analysis ; *CRISPR-Cas Systems ; Chromosome Mapping/*methods ; Chromosomes, Bacterial/*genetics ; Computer Simulation ; Conserved Sequence/genetics ; DNA-Directed RNA Polymerases ; Drug Resistance, Bacterial/genetics ; Fluorescent Dyes/analysis ; Gene Editing/methods ; Genome, Bacterial ; Genome, Human ; Haemophilus influenzae/drug effects/*genetics ; Haplotypes/genetics ; Humans ; Lab-On-A-Chip Devices ; Nalidixic Acid/pharmacology ; Novobiocin/pharmacology ; Nucleotide Motifs/genetics ; Polymorphism, Single Nucleotide ; Quinolinium Compounds/analysis ; RNA, Guide/chemical synthesis/*genetics ; Repetitive Sequences, Nucleic Acid/genetics ; Sequence Alignment ; Staining and Labeling/methods ; Viral Proteins ; }, abstract = {Whole-genome mapping technologies have been developed as a complementary tool to provide scaffolds for genome assembly and structural variation analysis (1,2). We recently introduced a novel DNA labeling strategy based on a CRISPR-Cas9 genome editing system, which can target any 20bp sequences. The labeling strategy is specifically useful in targeting repetitive sequences, and sequences not accessible to other labeling methods. In this report, we present customized mapping strategies that extend the applications of CRISPR-Cas9 DNA labeling. We first design a CRISPR-Cas9 labeling strategy to interrogate and differentiate the single allele differences in NGG protospacer adjacent motifs (PAM sequence). Combined with sequence motif labeling, we can pinpoint the single-base differences in highly conserved sequences. In the second strategy, we design mapping patterns across a genome by selecting sets of specific single-guide RNAs (sgRNAs) for labeling multiple loci of a genomic region or a whole genome. By developing and optimizing a single tube synthesis of multiple sgRNAs, we demonstrate the utility of CRISPR-Cas9 mapping with 162 sgRNAs targeting the 2Mb Haemophilus influenzae chromosome. These CRISPR-Cas9 mapping approaches could be particularly useful for applications in defining long-distance haplotypes and pinpointing the breakpoints in large structural variants in complex genomes and microbial mixtures.}, }
@article {pmid33226787, year = {2021}, author = {Shokr, A and Pacheco, LGC and Thirumalaraju, P and Kanakasabapathy, MK and Gandhi, J and Kartik, D and Silva, FSR and Erdogmus, E and Kandula, H and Luo, S and Yu, XG and Chung, RT and Li, JZ and Kuritzkes, DR and Shafiee, H}, title = {Mobile Health (mHealth) Viral Diagnostics Enabled with Adaptive Adversarial Learning.}, journal = {ACS nano}, volume = {15}, number = {1}, pages = {665-673}, doi = {10.1021/acsnano.0c06807}, pmid = {33226787}, issn = {1936-086X}, support = {R01 AI118502/AI/NIAID NIH HHS/United States ; R01 AI138800/AI/NIAID NIH HHS/United States ; R61 AI140489/AI/NIAID NIH HHS/United States ; }, mesh = {Antigens, Viral/isolation &amp; purification ; COVID-19/*diagnosis ; COVID-19 Testing/*instrumentation/*methods ; CRISPR-Cas Systems ; Communicable Disease Control ; *Deep Learning ; Disaster Planning ; Humans ; Image Processing, Computer-Assisted/methods ; Metal Nanoparticles/chemistry ; Neural Networks, Computer ; Platinum ; Point-of-Care Testing ; Public Health ; Reproducibility of Results ; *Signal Processing, Computer-Assisted ; Smartphone ; Telemedicine/*methods ; }, abstract = {Deep-learning (DL)-based image processing has potential to revolutionize the use of smartphones in mobile health (mHealth) diagnostics of infectious diseases. However, the high variability in cellphone image data acquisition and the common need for large amounts of specialist-annotated images for traditional DL model training may preclude generalizability of smartphone-based diagnostics. Here, we employed adversarial neural networks with conditioning to develop an easily reconfigurable virus diagnostic platform that leverages a dataset of smartphone-taken microfluidic chip photos to rapidly generate image classifiers for different target pathogens on-demand. Adversarial learning was also used to augment this real image dataset by generating 16,000 realistic synthetic microchip images, through style generative adversarial networks (StyleGAN). We used this platform, termed smartphone-based pathogen detection resource multiplier using adversarial networks (SPyDERMAN), to accurately detect different intact viruses in clinical samples and to detect viral nucleic acids through integration with CRISPR diagnostics. We evaluated the performance of the system in detecting five different virus targets using 179 patient samples. The generalizability of the system was confirmed by rapid reconfiguration to detect SARS-CoV-2 antigens in nasal swab samples (n = 62) with 100% accuracy. Overall, the SPyDERMAN system may contribute to epidemic preparedness strategies by providing a platform for smartphone-based diagnostics that can be adapted to a given emerging viral agent within days of work.}, }
@article {pmid33154568, year = {2020}, author = {Marx, V}, title = {Guide RNAs: it's good to be choosy.}, journal = {Nature methods}, volume = {17}, number = {12}, pages = {1179-1182}, pmid = {33154568}, issn = {1548-7105}, mesh = {CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Editing/*methods ; Gene Knockout Techniques/methods ; Humans ; RNA, Guide/*genetics ; }, }
@article {pmid32682409, year = {2020}, author = {Zhang, F and Liu, R and Zhang, H and Liu, C and Liu, C and Lu, Y}, title = {Suppressing Dazl modulates tumorigenicity and stemness in human glioblastoma cells.}, journal = {BMC cancer}, volume = {20}, number = {1}, pages = {673}, pmid = {32682409}, issn = {1471-2407}, support = {shslczdzk03303//Shanghai Municipal Key Clinical Specialty of China/ ; 81372141//National Natural Science Foundation of China/ ; 81372351//National Natural Science Foundation of China/ ; 82600202//National Natural Science Foundation of China/ ; }, mesh = {Animals ; Antineoplastic Agents/pharmacology/therapeutic use ; Brain Neoplasms/drug therapy/*genetics/pathology ; CRISPR-Cas Systems/genetics ; Carcinogenesis/genetics ; Cell Line, Tumor ; Cell Movement/genetics ; Cell Proliferation/genetics ; Doxorubicin/pharmacology/therapeutic use ; Drug Resistance, Neoplasm/genetics ; Female ; *Gene Expression Regulation, Neoplastic ; Gene Knockdown Techniques ; Glioblastoma/drug therapy/*genetics/pathology ; Humans ; Mice ; Neoplasm Grading ; Neoplastic Stem Cells/*pathology ; Oncogenes/genetics ; RNA-Binding Proteins/*genetics ; Temozolomide/pharmacology/therapeutic use ; Up-Regulation ; Xenograft Model Antitumor Assays ; }, abstract = {BACKGROUND: Glioblastoma is devastating cancer with a high frequency of occurrence and poor survival rate and it is urgent to discover novel glioblastoma-specific antigens for the therapy. Cancer-germline genes are known to be related to the formation and progression of several cancer types by promoting tumor transformation. Dazl is one such germline gene and is up-regulated in a few germ cell cancers. In this study, we analyzed the expression of Dazl in human glioblastoma tissues and cells, and investigated its significance in proliferation, migration, invasion and chemoresistance of the glioblastoma cell lines.<br><br>METHODS: We evaluated the expression of Dazl in different pathologic grades of glioblastoma tissues by immunohistochemistry. We assessed the expression of Dazl in glioblastoma cells and normal human astrocytes (NHA) cells by western blotting and RT-qPCR. Then we generated Dazl knockout glioblastoma cell lines using the CRISPR/Cas9 gene-editing technology to explore the cellular function of Dazl. We detected the proliferation and germline traits via CCK-8 assays and alkaline phosphatase staining, respectively. Boyden chamber assays were performed to measure glioblastoma cell migration and invasion. Crystal violet staining was used to determine the number of viable cells after the treatment of Doxorubicin and Temozolomide. Finally, we used subcutaneous xenograft studies to measure the growth of tumors in vivo.<br><br>RESULTS: We found that Dazl was upregulated in glioblastoma tissues and glioblastoma cell lines. Dazl knockdown glioblastoma cells showed decreased cellular proliferation, migration, invasion, and resistance in vitro, and inhibited the initiation of glioblastoma in vivo. The glioblastoma cell lines A172, U251, and LN229 were found to express stem cell markers CD133, Oct4, Nanog, and Sox2. The expression of these markers was downregulated in Dazl-deficient cells.<br><br>CONCLUSIONS: Our results indicated that Dazl contributes to the tumorigenicity of glioblastoma via reducing cell stemness. Therefore, cancer-germline genes might represent a new paradigm of glioblastoma-initiating cells in the treatment of malignant tumors.}, }
@article {pmid32525845, year = {2020}, author = {Xiao, S and Shimura, D and Baum, R and Hernandez, DM and Agvanian, S and Nagaoka, Y and Katsumata, M and Lampe, PD and Kleber, AG and Hong, T and Shaw, RM}, title = {Auxiliary trafficking subunit GJA1-20k protects connexin-43 from degradation and limits ventricular arrhythmias.}, journal = {The Journal of clinical investigation}, volume = {130}, number = {9}, pages = {4858-4870}, pmid = {32525845}, issn = {1558-8238}, support = {R01 HL133286/HL/NHLBI NIH HHS/United States ; R01 HL136463/HL/NHLBI NIH HHS/United States ; R01 HL138577/HL/NHLBI NIH HHS/United States ; R01 HL152691/HL/NHLBI NIH HHS/United States ; }, mesh = {Animals ; Arrhythmias, Cardiac/genetics/*metabolism/pathology ; CRISPR-Cas Systems ; Connexin 43/genetics/*metabolism ; Gap Junctions/genetics/*metabolism/pathology ; Heart Ventricles/*metabolism/pathology ; Mice ; Mice, Mutant Strains ; Protein Transport ; *Proteolysis ; }, abstract = {Connexin-43 (Cx43) gap junctions provide intercellular coupling, which ensures rapid action potential propagation and synchronized heart contraction. Alterations in Cx43 localization and reductions in gap junction coupling occur in failing hearts, contributing to ventricular arrhythmias and sudden cardiac death. Recent reports have found that an internally translated Cx43 isoform, GJA1-20k, is an auxiliary subunit for the trafficking of Cx43 in heterologous expression systems. Here, we have created a mouse model by using CRISPR technology to mutate a single internal translation initiation site in Cx43 (M213L mutation), which generates full-length Cx43, but not GJA1-20k. We found that GJA1M213L/M213L mice had severely abnormal electrocardiograms despite preserved contractile function, reduced total Cx43, and reduced gap junctions, and they died suddenly at 2 to 4 weeks of age. Heterozygous GJA1M213L/WT mice survived to adulthood with increased ventricular ectopy. Biochemical experiments indicated that cytoplasmic Cx43 had a half-life that was 50% shorter than membrane-associated Cx43. Without GJA1-20k, poorly trafficked Cx43 was degraded. The data support that GJA1-20k, an endogenous entity translated independently of Cx43, is critical for Cx43 gap junction trafficking, maintenance of Cx43 protein, and normal electrical function of the mammalian heart.}, }
@article {pmid32439759, year = {2020}, author = {Mereu, L and Morf, MK and Spiri, S and Gutierrez, P and Escobar-Restrepo, JM and Daube, M and Walser, M and Hajnal, A}, title = {Polarized epidermal growth factor secretion ensures robust vulval cell fate specification in Caenorhabditis elegans.}, journal = {Development (Cambridge, England)}, volume = {147}, number = {11}, pages = {}, pmid = {32439759}, issn = {1477-9129}, mesh = {Animals ; Animals, Genetically Modified/growth &amp; development/metabolism ; CRISPR-Cas Systems/genetics ; Caenorhabditis elegans/growth &amp; development/*metabolism ; Caenorhabditis elegans Proteins/antagonists &amp; inhibitors/genetics/*metabolism ; Epidermal Growth Factor/antagonists &amp; inhibitors/genetics/*metabolism ; ErbB Receptors/metabolism ; Female ; Gene Editing ; Larva/metabolism ; Mitogen-Activated Protein Kinases/metabolism ; Mutagenesis ; Netrins/genetics/metabolism ; RNA Interference ; RNA-Dependent RNA Polymerase/genetics/metabolism ; Signal Transduction ; Stem Cells/cytology/metabolism ; Vulva/cytology/growth &amp; development/*metabolism ; ras GTPase-Activating Proteins/antagonists &amp; inhibitors/genetics/metabolism ; }, abstract = {The anchor cell (AC) in C. elegans secretes an epidermal growth factor (EGF) homolog that induces adjacent vulval precursor cells (VPCs) to differentiate. The EGF receptor in the nearest VPC sequesters the limiting EGF amounts released by the AC to prevent EGF from spreading to distal VPCs. Here, we show that not only EGFR localization in the VPCs but also EGF polarity in the AC is necessary for robust fate specification. The AC secretes EGF in a directional manner towards the nearest VPC. Loss of AC polarity causes signal spreading and, when combined with MAPK pathway hyperactivation, the ectopic induction of distal VPCs. In a screen for genes preventing distal VPC induction, we identified sra-9 and nlp-26 as genes specifically required for polarized EGF secretion. sra-9(lf) and nlp-26(lf) mutants exhibit errors in vulval fate specification, reduced precision in VPC to AC alignment and increased variability in MAPK activation. sra-9 encodes a seven-pass transmembrane receptor acting in the AC and nlp-26 a neuropeptide-like protein expressed in the VPCs. SRA-9 and NLP-26 may transduce a feedback signal to channel EGF secretion towards the nearest VPC.}, }
@article {pmid32439756, year = {2020}, author = {Kesavan, G and Machate, A and Hans, S and Brand, M}, title = {Cell-fate plasticity, adhesion and cell sorting complementarily establish a sharp midbrain-hindbrain boundary.}, journal = {Development (Cambridge, England)}, volume = {147}, number = {11}, pages = {}, doi = {10.1242/dev.186882}, pmid = {32439756}, issn = {1477-9129}, mesh = {Animals ; Animals, Genetically Modified/growth &amp; development/metabolism ; CRISPR-Cas Systems/genetics ; Cadherins/genetics/metabolism ; Cell Adhesion/*physiology ; Cell Lineage ; Embryo, Nonmammalian/metabolism ; Ephrins/antagonists &amp; inhibitors/genetics/metabolism ; Gastrulation ; Gene Editing ; Mesencephalon/*metabolism/pathology ; Microscopy, Atomic Force ; Microscopy, Fluorescence ; Morpholinos/metabolism ; Otx Transcription Factors/genetics/metabolism ; Rhombencephalon/*metabolism/pathology ; Signal Transduction ; Time-Lapse Imaging ; Zebrafish/growth &amp; development/*metabolism ; Zebrafish Proteins/genetics/metabolism ; }, abstract = {The formation and maintenance of sharp boundaries between groups of cells play a vital role during embryonic development as they serve to compartmentalize cells with similar fates. Some of these boundaries also act as organizers, with the ability to induce specific cell fates and morphogenesis in the surrounding cells. The midbrain-hindbrain boundary (MHB) is such an organizer: it acts as a lineage restriction boundary to prevent the intermingling of cells with different developmental fates. However, the mechanisms underlying the lineage restriction process remain unclear. Here, using novel fluorescent knock-in reporters, live imaging, Cre/lox-mediated lineage tracing, atomic force microscopy-based cell adhesion assays and mutant analysis, we analyze the process of lineage restriction at the MHB and provide mechanistic details. Specifically, we show that lineage restriction occurs by the end of gastrulation, and that the subsequent formation of sharp gene expression boundaries in the developing MHB occur through complementary mechanisms, i.e. cell-fate plasticity and cell sorting. Furthermore, we show that cell sorting at the MHB involves differential adhesion among midbrain and hindbrain cells that is mediated by N-cadherin and Eph-ephrin signaling.}, }
@article {pmid32380730, year = {2020}, author = {Pavan, E and Ormazabal, M and Peruzzo, P and Vaena, E and Rozenfeld, P and Dardis, A}, title = {CRISPR/Cas9 Editing for Gaucher Disease Modelling.}, journal = {International journal of molecular sciences}, volume = {21}, number = {9}, pages = {}, pmid = {32380730}, issn = {1422-0067}, mesh = {Biomarkers ; *CRISPR-Cas Systems ; Cell Line ; Disease Susceptibility ; Endoplasmic Reticulum Stress ; Endoplasmic Reticulum-Associated Degradation ; Gaucher Disease/*genetics/metabolism/pathology ; *Gene Editing ; Gene Expression ; Glucosylceramidase/genetics ; Humans ; Inflammation Mediators/metabolism ; *Models, Biological ; Monocytes/metabolism ; Mutation ; Unfolded Protein Response ; }, abstract = {Gaucher disease (GD) is an autosomal recessive lysosomal storage disorder caused by mutations in the acid β-glucosidase gene (GBA1). Besides causing GD, GBA1 mutations constitute the main genetic risk factor for developing Parkinson's disease. The molecular basis of neurological manifestations in GD remain elusive. However, neuroinflammation has been proposed as a key player in this process. We exploited CRISPR/Cas9 technology to edit GBA1 in the human monocytic THP-1 cell line to develop an isogenic GD model of monocytes and in glioblastoma U87 cell lines to generate an isogenic GD model of glial cells. Both edited (GBA1 mutant) cell lines presented low levels of mutant acid β-glucosidase expression, less than 1% of residual activity and massive accumulation of substrate. Moreover, U87 GBA1 mutant cells showed that the mutant enzyme was retained in the ER and subjected to proteasomal degradation, triggering unfolded protein response (UPR). U87 GBA1 mutant cells displayed an increased production of interleukin-1β, both with and without inflammosome activation, α-syn accumulation and a higher rate of cell death in comparison with wild-type cells. In conclusion, we developed reliable, isogenic, and easy-to-handle cellular models of GD obtained from commercially accessible cells to be employed in GD pathophysiology studies and high-throughput drug screenings.}, }
@article {pmid32326617, year = {2020}, author = {Mikolajewicz, N and Komarova, SV}, title = {Role of UDP-Sugar Receptor P2Y14 in Murine Osteoblasts.}, journal = {International journal of molecular sciences}, volume = {21}, number = {8}, pages = {}, pmid = {32326617}, issn = {1422-0067}, support = {PJT-165939/CAPMC/CIHR/Canada ; RGPIN-288253//Natural Sciences and Engineering Research Council of Canada/ ; }, mesh = {Adenosine Diphosphate/pharmacology ; Adenosine Triphosphate/pharmacology ; Animals ; Bone Density/genetics ; CRISPR-Cas Systems ; Calcium/metabolism ; Cell Line ; Cell Proliferation/drug effects/*genetics ; Cells, Cultured ; Cyclic AMP/metabolism ; Gene Knockout Techniques ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Mitogen-Activated Protein Kinase 1/metabolism ; Mitogen-Activated Protein Kinase 3/metabolism ; Osteoblasts/*metabolism ; Osteogenesis/drug effects/*genetics ; Phosphorylation ; Purinergic Antagonists/metabolism/*pharmacology ; Receptors, Purinergic P2Y/genetics/*metabolism ; Signal Transduction/drug effects/*genetics ; Uridine Diphosphate Glucose/metabolism/pharmacology ; Uridine Diphosphate Sugars/*metabolism/pharmacology ; }, abstract = {The purinergic (P2) receptor P2Y14 is the only P2 receptor that is stimulated by uridine diphosphate (UDP)-sugars and its role in bone formation is unknown. We confirmed P2Y14 expression in primary murine osteoblasts (CB-Ob) and the C2C12-BMP2 osteoblastic cell line (C2-Ob). UDP-glucose (UDPG) had undiscernible effects on cAMP levels, however, induced dose-dependent elevations in the cytosolic free calcium concentration ([Ca2+]i) in CB-Ob, but not C2-Ob cells. To antagonize the P2Y14 function, we used the P2Y14 inhibitor PPTN or generated CRISPR-Cas9-mediated P2Y14 knockout C2-Ob clones (Y14KO). P2Y14 inhibition facilitated calcium signalling and altered basal cAMP levels in both models of osteoblasts. Importantly, P2Y14 inhibition augmented Ca2+ signalling in response to ATP, ADP and mechanical stimulation. P2Y14 knockout or inhibition reduced osteoblast proliferation and decreased ERK1/2 phosphorylation and increased AMPKα phosphorylation. During in vitro osteogenic differentiation, P2Y14 inhibition modulated the timing of osteogenic gene expression, collagen deposition, and mineralization, but did not significantly affect differentiation status by day 28. Of interest, while P2ry14-/- mice from the International Mouse Phenotyping Consortium were similar to wild-type controls in bone mineral density, their tibia length was significantly increased. We conclude that P2Y14 in osteoblasts reduces cell responsiveness to mechanical stimulation and mechanotransductive signalling and modulates osteoblast differentiation.}, }
@article {pmid32326615, year = {2020}, author = {Malek, N and Mrówczyńska, E and Michrowska, A and Mazurkiewicz, E and Pavlyk, I and Mazur, AJ}, title = {Knockout of ACTB and ACTG1 with CRISPR/Cas9(D10A) Technique Shows that Non-Muscle β and γ Actin Are Not Equal in Relation to Human Melanoma Cells' Motility and Focal Adhesion Formation.}, journal = {International journal of molecular sciences}, volume = {21}, number = {8}, pages = {}, pmid = {32326615}, issn = {1422-0067}, support = {2015/17/B/NZ3/03604//National Science Center, Poland/ ; }, mesh = {Actins/analysis/genetics/*metabolism ; *CRISPR-Cas Systems ; Cell Line, Tumor ; Cell Movement/drug effects/genetics ; Focal Adhesions/drug effects/genetics/*metabolism ; Gene Editing/*methods ; Gene Knockout Techniques/*methods ; Humans ; Lysophospholipids/pharmacology ; Melanoma/genetics/*metabolism ; Neoplasm Invasiveness/genetics ; Protein Isoforms/metabolism ; Signal Transduction/drug effects/genetics ; Stress Fibers/genetics/metabolism ; Tetradecanoylphorbol Acetate/analogs &amp; derivatives/pharmacology ; }, abstract = {Non-muscle actins have been studied for many decades; however, the reason for the existence of both isoforms is still unclear. Here we show, for the first time, a successful inactivation of the ACTB (CRISPR clones with inactivated ACTB, CR-ACTB) and ACTG1 (CRISPR clones with inactivated ACTG1, CR-ACTG1) genes in human melanoma cells (A375) via the RNA-guided D10A mutated Cas9 nuclease gene editing [CRISPR/Cas9(D10A)] technique. This approach allowed us to evaluate how melanoma cell motility was impacted by the lack of either β actin coded by ACTB or γ actin coded by ACTG1. First, we observed different distributions of β and γ actin in the cells, and the absence of one actin isoform was compensated for via increased expression of the other isoform. Moreover, we noted that γ actin knockout had more severe consequences on cell migration and invasion than β actin knockout. Next, we observed that the formation rate of bundled stress fibers in CR-ACTG1 cells was increased, but lamellipodial activity in these cells was impaired, compared to controls. Finally, we discovered that the formation rate of focal adhesions (FAs) and, subsequently, FA-dependent signaling were altered in both the CR-ACTB and CR-ACTG1 clones; however, a more detrimental effect was observed for γ actin-deficient cells. Our research shows that both non-muscle actins play distinctive roles in melanoma cells' FA formation and motility.}, }
@article {pmid32299090, year = {2020}, author = {Huang, P and Peslak, SA and Lan, X and Khandros, E and Yano, JA and Sharma, M and Keller, CA and Giardine, B and Qin, K and Abdulmalik, O and Hardison, RC and Shi, J and Blobel, GA}, title = {The HRI-regulated transcription factor ATF4 activates BCL11A transcription to silence fetal hemoglobin expression.}, journal = {Blood}, volume = {135}, number = {24}, pages = {2121-2132}, pmid = {32299090}, issn = {1528-0020}, support = {R56 DK065806/DK/NIDDK NIH HHS/United States ; R01 HL119479/HL/NHLBI NIH HHS/United States ; R01 DK065806/DK/NIDDK NIH HHS/United States ; R01 DK054937/DK/NIDDK NIH HHS/United States ; U54 HG006998/HG/NHGRI NIH HHS/United States ; }, mesh = {Activating Transcription Factor 4/*genetics ; Anemia, Sickle Cell/blood/genetics/therapy ; Animals ; CRISPR-Cas Systems ; Cells, Cultured ; Enhancer Elements, Genetic ; Erythroblasts/metabolism ; Fetal Hemoglobin/*genetics ; Gene Expression Regulation ; Gene Silencing ; Humans ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Protein-Serine-Threonine Kinases/deficiency/genetics ; Repressor Proteins/*genetics ; Species Specificity ; beta-Thalassemia/blood/genetics/therapy ; eIF-2 Kinase/*genetics ; gamma-Globins/biosynthesis/genetics ; }, abstract = {Reactivation of fetal hemoglobin remains a critical goal in the treatment of patients with sickle cell disease and β-thalassemia. Previously, we discovered that silencing of the fetal γ-globin gene requires the erythroid-specific eIF2α kinase heme-regulated inhibitor (HRI), suggesting that HRI might present a pharmacologic target for raising fetal hemoglobin levels. Here, via a CRISPR-Cas9-guided loss-of-function screen in human erythroblasts, we identify transcription factor ATF4, a known HRI-regulated protein, as a novel γ-globin regulator. ATF4 directly stimulates transcription of BCL11A, a repressor of γ-globin transcription, by binding to its enhancer and fostering enhancer-promoter contacts. Notably, HRI-deficient mice display normal Bcl11a levels, suggesting species-selective regulation, which we explain here by demonstrating that the analogous ATF4 motif at the murine Bcl11a enhancer is largely dispensable. Our studies uncover a linear signaling pathway from HRI to ATF4 to BCL11A to γ-globin and illustrate potential limits of murine models of globin gene regulation.}, }
@article {pmid32251518, year = {2020}, author = {Watanabe, Y and Okuya, K and Takada, Y and Kinoshita, M and Yokoi, S and Chisada, S and Kamei, Y and Tatsukawa, H and Yamamoto, N and Abe, H and Hashimoto, H and Hitomi, K}, title = {Gene disruption of medaka (Oryzias latipes) orthologue for mammalian tissue-type transglutaminase (TG2) causes movement retardation.}, journal = {Journal of biochemistry}, volume = {168}, number = {3}, pages = {213-222}, doi = {10.1093/jb/mvaa038}, pmid = {32251518}, issn = {1756-2651}, mesh = {Animals ; Animals, Genetically Modified ; Behavior, Animal ; Brain/metabolism ; CRISPR-Cas Systems ; Fish Proteins/*genetics ; GTP-Binding Proteins/*genetics/*metabolism ; Gene Editing ; Humans ; *Movement ; Oryzias/*genetics/*metabolism ; Phenotype ; Transglutaminases/*genetics/*metabolism ; }, abstract = {Transglutaminases are an enzyme family that catalyses protein cross-linking essential for several biological functions. In the previous studies, we characterized the orthologues of the mammalian transglutaminase family in medaka (Oryzias latipes), an established fish model. Among the human isozymes, tissue-type transglutaminase (TG2) has multiple functions that are involved in several biological phenomena. In this study, we established medaka mutants deficient for the orthologue of human TG2 using the CRISPR/Cas9 and transcription activator-like effector nucleases systems. Although apparent morphological changes in the phenotype were not observed, movement retardation was found in the mutant fish when evaluated by a tank-diving test. Furthermore, comparative immunohistochemistry analysis using in this fish model revealed that orthologue of human TG2 was expressed at the periventricular layer of the optic tectum. Our findings provide novel insight for the relationship between tissue-type transglutaminase and the nervous system and the associated behaviour.}, }
@article {pmid32185620, year = {2020}, author = {Chang, Y and Shao, J and Gao, Y and Liu, W and Gao, Z and Hu, Y and Chang, H}, title = {Reporter gene knock-in into Marc-145 cells using CRISPR/Cas9-mediated homologous recombination.}, journal = {Biotechnology letters}, volume = {42}, number = {8}, pages = {1317-1325}, doi = {10.1007/s10529-020-02860-x}, pmid = {32185620}, issn = {1573-6776}, support = {1504NKCA057//Key Science and Technology Foundation of Gansu Province/ ; 2017YFD0500902//National Key Research and Development Program of China/ ; 2016YFE0204100//National Key Research and Development Program of China/ ; 2017RCZX-24//Science and Technology Innovation Funds of Gansu Agricultural University/ ; }, mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Cell Line ; Chlorocebus aethiops ; Gene Knock-In Techniques/*methods ; Genes, Reporter/*genetics ; Green Fluorescent Proteins/genetics/metabolism ; Homologous Recombination/*genetics ; Polymerase Chain Reaction ; Recombinant Proteins/genetics/metabolism ; }, abstract = {OBJECTIVES: Marc-145 cells (monkey embryonic kidney epithelial cells) play a critical role in the biotechnology industry as certain virus host cells. To investigate the expression of enhanced green fluorescent protein (eGFP) gene as a foreign gene in Marc-145 cells, which we developed an approach of foreign gene site-specific knock-in into Marc-145 cells by clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) and putatively explored appropriate genomic recombination sites in Marc-145 cells.<br><br>RESULTS: Our study demonstrated that the specific homologous recombination (HR) site between the Rac GTPase activating protein 1 (RACGAP1) and the acid-sensing ion channel subunit 1 (ASIC1) genes of the 11th chromosome could be used as the target site of Cas9 for the generation of target gene knock-in into Marc-145 cells, by the insertion of the eGFP cassette into the specific HR site and subsequent expression.<br><br>CONCLUSIONS: Junction PCR, sequencing, Southern blot and fluorescence assay determined eGFP gene-specific knock-in HR site between the RACGAP1 and ASIC1 genes of the 11th chromosome, which was identified by the genomic safe harbours in Marc-145 cells. Our study encouraged a broader range of applications, such as Marc-145 cells development and engineering for virus adaption and yield increase in the vaccine biotechnology industry.}, }
@article {pmid32150344, year = {2020}, author = {Johnson, CG and Chen, T and Furey, N and Hemmingsen, MG and Bissig, KD}, title = {Somatic Liver Knockout (SLiK): A Quick and Efficient Way to Generate Liver-Specific Knockout Mice Using Multiplex CRISPR/Cas9 Gene Editing.}, journal = {Current protocols in molecular biology}, volume = {130}, number = {1}, pages = {e117}, pmid = {32150344}, issn = {1934-3647}, support = {R56 DK115461/DK/NIDDK NIH HHS/United States ; T32 GM088129/GM/NIGMS NIH HHS/United States ; R01 HL132840/HL/NHLBI NIH HHS/United States ; P30 CA014236/CA/NCI NIH HHS/United States ; R01 HL134510/HL/NHLBI NIH HHS/United States ; R01 DK115461/DK/NIDDK NIH HHS/United States ; }, mesh = {Animals ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Editing/*methods ; Hepatocytes/*metabolism ; Liver/*metabolism ; Mice ; Mice, Knockout ; RNA, Guide/genetics ; Transfection/methods ; }, abstract = {Somatic liver knockout (SLiK) is a method developed to rapidly generate a liver-specific knockout of one or several genes. This technique combines the strengths of CRISPR/Cas9 gene editing and hydrodynamic tail-vein injection, a simple in vivo method for transfection of hepatocytes, to harness the powerful selection pressure of tyrosinemic livers to replace host hepatocytes with any desired gene deletion. In this protocol, we will describe sgRNA design and cloning, hydrodynamic tail-vein injection of targeting constructs, and screening and validation methods for efficient in vivo gene editing. © 2020 by John Wiley &amp; Sons, Inc. Support Protocol 1: sgRNA design Support Protocol 2: sgRNA construction: daisy chaining multiple sgRNAs Basic Protocol: Delivery of DNA by hydrodynamic tail-vein injection and liver repopulation of edited hepatocytes Support Protocol 3: Validation of CRISPR/Cas9 cutting in vivo.}, }
@article {pmid32130564, year = {2020}, author = {Liu, H and Hou, G and Wang, P and Guo, G and Wang, Y and Yang, N and Rehman, MNU and Li, C and Li, Q and Zheng, J and Zeng, J and Li, S}, title = {A double-locus scarless genome editing system in Escherichia coli.}, journal = {Biotechnology letters}, volume = {42}, number = {8}, pages = {1457-1465}, doi = {10.1007/s10529-020-02856-7}, pmid = {32130564}, issn = {1573-6776}, support = {2017CXTD005//Science Foundation of Hainan Province for Creative Research group/ ; 31460699//National Natural Science Foundation of China/ ; 2019RC084//Scinece Foundation of Hainan for high-level talents/ ; 2019NHMJ030//Talent Program of the South China Sea/ ; }, mesh = {CRISPR-Cas Systems/genetics ; DNA, Bacterial/genetics ; Escherichia coli/*genetics ; Gene Editing/*methods ; Genome, Bacterial/*genetics ; Plasmids/genetics ; Recombination, Genetic/genetics ; }, abstract = {OBJECTIVE: To develop a convenient double-locus scarless genome editing system in Escherichia coli, based on the type II Streptococcus pyogenes CRISPR/Cas9 and λ Red recombination cassette.<br><br>RESULTS: A two-plasmid genome editing system was constructed. The large-sized plasmid harbors the cas9 and λ Red recombination genes (gam, bet, and exo), while the small-molecular plasmid can simultaneously express two different gRNAs (targeting genome RNAs). The recombination efficiency was tested by targeting the galK, lacZ, and dbpA genes in E. coli with ssDNA or dsDNA. Resulting concurrent double-locus recombination efficiencies were 88 ± 5.5% (point mutation), 39.7 ± 4.3% (deletion/insertion), and 57.8 ± 3.4%-58.5 ± 4.1% (mixed point and deletion/insertion mutation), depending on 30 (ssDNA) or 40 bp (dsDNA) homologous side arms employed. In addition, the curing efficiency of the guide plasmid expressing gRNAs for negative selection was higher (96 ± 3% in 4 h) than the help plasmid carrying cas9 and λ Red (92 ± 2% in 9 h).<br><br>CONCLUSIONS: The new editing system is convenient and efficient for simultaneous double-locus recombination in the genome and should be favorable for high-throughput multiplex genome editing in synthetic biology and metabolic engineering.}, }
@article {pmid31960370, year = {2020}, author = {Meadows, SK and Brandsmeier, LA and Newberry, KM and Betti, MJ and Nesmith, AS and Mackiewicz, M and Partridge, EC and Mendenhall, EM and Myers, RM}, title = {Epitope Tagging ChIP-Seq of DNA Binding Proteins Using CETCh-Seq.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2117}, number = {}, pages = {3-34}, doi = {10.1007/978-1-0716-0301-7_1}, pmid = {31960370}, issn = {1940-6029}, support = {U54 HG006998/HG/NHGRI NIH HHS/United States ; }, mesh = {Binding Sites ; CRISPR-Cas Systems ; Cell Adhesion ; Chromatin Immunoprecipitation Sequencing ; Epitopes/*metabolism ; Gene Editing ; Hep G2 Cells ; Humans ; Protein Binding ; Transcription Factors/*analysis/*genetics ; }, abstract = {Chromatin immunoprecipitation followed by next-generation DNA sequencing (ChIP-seq) has been used to identify transcription factor (TF) binding proteins throughout the genome. Unfortunately, this approach traditionally requires commercially available, ChIP-seq grade antibodies that frequently fail to generate acceptable datasets. To obtain data for the many TFs for which there is no appropriate antibody, we recently developed a new method for performing ChIP-seq by epitope tagging endogenous TFs using CRISPR/Cas9 genome editing technology (CETCh-seq). Here, we describe our general protocol of CETCh-seq for both adherent and nonadherent cell lines using a commercially available FLAG antibody.}, }
@article {pmid31923454, year = {2020}, author = {McCloskey, AG and Miskelly, MG and Moore, CBT and Nesbit, MA and Christie, KA and Owolabi, AI and Flatt, PR and McKillop, AM}, title = {CRISPR/Cas9 gene editing demonstrates metabolic importance of GPR55 in the modulation of GIP release and pancreatic beta cell function.}, journal = {Peptides}, volume = {125}, number = {}, pages = {170251}, doi = {10.1016/j.peptides.2019.170251}, pmid = {31923454}, issn = {1873-5169}, mesh = {Animals ; CRISPR-Cas Systems ; Cell Line ; Diabetes Mellitus, Experimental/genetics/*metabolism/physiopathology ; Disease Models, Animal ; Gastric Inhibitory Polypeptide/*metabolism ; Gene Editing/*methods ; Glucagon-Like Peptide 1/*metabolism ; Humans ; Insulin-Secreting Cells/*metabolism ; Male ; Mice ; Receptors, Cannabinoid/chemistry/genetics/*metabolism ; }, abstract = {G-protein coupled receptor-55 (GPR55), an endocannabinoid receptor, is a novel anti-diabetic target. This study aimed to assess the metabolic functionality of GPR55 ligands using CRISPR/Cas9 gene editing to determine their regulatory role in beta cell function and incretin-secreting enteroendocrine cells. A clonal Gpr55 knockout beta cell line was generated by CRISPR/Cas9 gene editing to investigate insulin secretion and Gpr55 signalling. Acute effects of GPR55 agonists were investigated in high fat fed (HFD) diabetic HsdOla:TO (Swiss TO) mice. Atypical and endogenous endocannabinoid ligands (10-7-10-4M) stimulated insulin secretion (p < 0.05-0.001) in rodent (BRIN-BD11) and human (1.1B4) beta cells, with 2-2.7-fold (p < 0.001) increase demonstrated in BRIN-BD11 cells (10-4M). The insulinotropic effect of Abn-CBD (42 %), AM251 (30 %) and PEA (53 %) were impaired (p < 0.05) in Gpr55 knockout BRIN-BD11 cells, with the secretory effect of O-1602 completely abolished (p < 0.001). Gpr55 ablation abolished the release of intracellular Ca2+ upon treatment with O-1602, Abn-CBD and PEA. Upregulation of insulin mRNA by Abn-CBD and AM251 (1.7-3-fold; p < 0.01) was greatly diminished (p < 0.001) in Gpr55 null cells. Orally administered Abn-CBD and AM251 (0.1 μmol/kgBW) improved GIP (p < 0.05-p < 0.01), GLP-1 (p < 0.05-p < 0.001), glucose tolerance (p < 0.001) and circulating insulin (p < 0.05-p < 0.001) in HFD diabetic mice. Abn-CBD in combination therapy with DPP-IV inhibitor (Sitagliptin) resulted in greater improvement in glucose tolerance (p < 0.05) and insulin release (p < 0.05). Antagonism of Gpr55 in-vivo attenuated the glucoregulatory effects of Abn-CBD (p < 0.05). Conclusively, GPR55 agonists enhance insulin, GIP and GLP-1 release, thereby promoting GPR55 agonist monotherapy and combinational therapy as a novel approach for the treatment of type-2-diabetes.}, }
@article {pmid31869524, year = {2020}, author = {Bosch, JA and Knight, S and Kanca, O and Zirin, J and Yang-Zhou, D and Hu, Y and Rodiger, J and Amador, G and Bellen, HJ and Perrimon, N and Mohr, SE}, title = {Use of the CRISPR-Cas9 System in Drosophila Cultured Cells to Introduce Fluorescent Tags into Endogenous Genes.}, journal = {Current protocols in molecular biology}, volume = {130}, number = {1}, pages = {e112}, pmid = {31869524}, issn = {1934-3647}, support = {P41 GM132087/GM/NIGMS NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; R01 GM067858/GM/NIGMS NIH HHS/United States ; P30 CA006516/CA/NCI NIH HHS/United States ; R24 OD019847/OD/NIH HHS/United States ; R01 GM067761/GM/NIGMS NIH HHS/United States ; }, mesh = {Animals ; *CRISPR-Cas Systems ; Cells, Cultured ; Clustered Regularly Interspaced Short Palindromic Repeats ; DNA, Single-Stranded/genetics ; Drosophila/*cytology/*genetics ; Gene Editing/methods ; Gene Knock-In Techniques/*methods ; *Genes, Insect ; Green Fluorescent Proteins/*genetics ; *Open Reading Frames ; Plasmids/genetics ; RNA, Guide/genetics ; Transfection ; }, abstract = {The CRISPR-Cas9 system makes it possible to cause double-strand breaks in specific regions, inducing repair. In the presence of a donor construct, repair can involve insertion or 'knock-in' of an exogenous cassette. One common application of knock-in technology is to generate cell lines expressing fluorescently tagged endogenous proteins. The standard approach relies on production of a donor plasmid with ∼500 to 1000 bp of homology on either side of an insertion cassette that contains the fluorescent protein open reading frame (ORF). We present two alternative methods for knock-in of fluorescent protein ORFs into Cas9-expressing Drosophila S2R+ cultured cells, the single-stranded DNA (ssDNA) Drop-In method and the CRISPaint universal donor method. Both methods eliminate the need to clone a large plasmid donor for each target. We discuss the advantages and limitations of the standard, ssDNA Drop-In, and CRISPaint methods for fluorescent protein tagging in Drosophila cultured cells. © 2019 by John Wiley &amp; Sons, Inc. Basic Protocol 1: Knock-in into Cas9-positive S2R+ cells using the ssDNA Drop-In approach Basic Protocol 2: Knock-in into Cas9-positive S2R+ cells by homology-independent insertion of universal donor plasmids that provide mNeonGreen (CRISPaint method) Support Protocol 1: sgRNA design and cloning Support Protocol 2: ssDNA donor synthesis Support Protocol 3: Transfection using Effectene Support Protocol 4: Electroporation of S2R+-MT::Cas9 Drosophila cells Support Protocol 5: Single-cell isolation of fluorescent cells using FACS.}, }
@article {pmid33547443, year = {2021}, author = {Walton, RT and Hsu, JY and Joung, JK and Kleinstiver, BP}, title = {Scalable characterization of the PAM requirements of CRISPR-Cas enzymes using HT-PAMDA.}, journal = {Nature protocols}, volume = {}, number = {}, pages = {}, pmid = {33547443}, issn = {1750-2799}, support = {R00-CA218870//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; P01-HL142494//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; R35 GM118158/GM/NIGMS NIH HHS/United States ; RM1 HG009490/HG/NHGRI NIH HHS/United States ; P01-HL142494//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; R35 GM118158/GM/NIGMS NIH HHS/United States ; RM1 HG009490/HG/NHGRI NIH HHS/United States ; R35 GM118158/GM/NIGMS NIH HHS/United States ; }, abstract = {The continued expansion of the genome-editing toolbox necessitates methods to characterize important properties of CRISPR-Cas enzymes. One such property is the requirement for Cas proteins to recognize a protospacer-adjacent motif (PAM) in DNA target sites. The high-throughput PAM determination assay (HT-PAMDA) is a method that enables scalable characterization of the PAM preferences of different Cas proteins. Here, we provide a step-by-step protocol for the method, discuss experimental design considerations, and highlight how the method can be used to profile naturally occurring CRISPR-Cas9 enzymes, engineered derivatives with improved properties, orthologs of different classes (e.g., Cas12a), and even different platforms (e.g., base editors). A distinguishing feature of HT-PAMDA is that the enzymes are expressed in a cell type or organism of interest (e.g., mammalian cells), permitting scalable characterization and comparison of hundreds of enzymes in a relevant setting. HT-PAMDA does not require specialized equipment or expertise and is cost effective for multiplexed characterization of many enzymes. The protocol enables comprehensive PAM characterization of dozens or hundreds of Cas enzymes in parallel in <2 weeks.}, }
@article {pmid33544853, year = {2021}, author = {León, LM and Park, AE and Borges, AL and Zhang, JY and Bondy-Denomy, J}, title = {Mobile element warfare via CRISPR and anti-CRISPR in Pseudomonas aeruginosa.}, journal = {Nucleic acids research}, volume = {}, number = {}, pages = {}, doi = {10.1093/nar/gkab006}, pmid = {33544853}, issn = {1362-4962}, abstract = {Bacteria deploy multiple defenses to prevent mobile genetic element (MGEs) invasion. CRISPR-Cas immune systems use RNA-guided nucleases to target MGEs, which counter with anti-CRISPR (Acr) proteins. Our understanding of the biology and co-evolutionary dynamics of the common Type I-C CRISPR-Cas subtype has lagged because it lacks an in vivo phage-host model system. Here, we show the anti-phage function of a Pseudomonas aeruginosa Type I-C CRISPR-Cas system encoded on a conjugative pKLC102 island, and its Acr-mediated inhibition by distinct MGEs. Seven genes with anti-Type I-C function (acrIC genes) were identified, many with highly acidic amino acid content, including previously described DNA mimic AcrIF2. Four of the acr genes were broad spectrum, also inhibiting I-E or I-F P. aeruginosa CRISPR-Cas subtypes. Dual inhibition comes at a cost, however, as simultaneous expression of Type I-C and I-F systems renders phages expressing the dual inhibitor AcrIF2 more sensitive to targeting. Mutagenesis of numerous acidic residues in AcrIF2 did not impair anti-I-C or anti-I-F function per se but did exacerbate inhibition defects during competition, suggesting that excess negative charge may buffer DNA mimics against competition. Like AcrIF2, five of the Acr proteins block Cascade from binding DNA, while two function downstream, likely preventing Cas3 recruitment or activity. One such inhibitor, AcrIC3, is found in an 'anti-Cas3' cluster within conjugative elements, encoded alongside bona fide Cas3 inhibitors AcrIF3 and AcrIE1. Our findings demonstrate an active battle between an MGE-encoded CRISPR-Cas system and its diverse MGE targets.}, }
@article {pmid33544493, year = {2020}, author = {Ryan, D and Prezza, G and Westermann, AJ}, title = {An RNA-centric view on gut Bacteroidetes.}, journal = {Biological chemistry}, volume = {402}, number = {1}, pages = {55-72}, doi = {10.1515/hsz-2020-0230}, pmid = {33544493}, issn = {1437-4315}, abstract = {Bacteria employ noncoding RNAs to maintain cellular physiology, adapt global gene expression to fluctuating environments, sense nutrients, coordinate their interaction with companion microbes and host cells, and protect themselves against bacteriophages. While bacterial RNA research has made fundamental contributions to biomedicine and biotechnology, the bulk of our knowledge of RNA biology stems from the study of a handful of aerobic model species. In comparison, RNA research is lagging in many medically relevant obligate anaerobic species, in particular the numerous commensal bacteria comprising our gut microbiota. This review presents a guide to RNA-based regulatory mechanisms in the phylum Bacteroidetes, focusing on the most abundant bacterial genus in the human gut, Bacteroides spp. This includes recent case reports on riboswitches, an mRNA leader, cis- and trans-encoded small RNAs (sRNAs) in Bacteroides spp., and a survey of CRISPR-Cas systems across Bacteroidetes. Recent work from our laboratory now suggests the existence of hundreds of noncoding RNA candidates in Bacteroides thetaiotaomicron, the emerging model organism for functional microbiota research. Based on these collective observations, we predict mechanistic and functional commonalities and differences between Bacteroides sRNAs and those of other model bacteria, and outline open questions and tools needed to boost Bacteroidetes RNA research.}, }
@article {pmid33542471, year = {2021}, author = {Liu, S and Cheng, Q and Wei, T and Yu, X and Johnson, LT and Farbiak, L and Siegwart, DJ}, title = {Membrane-destabilizing ionizable phospholipids for organ-selective mRNA delivery and CRISPR-Cas gene editing.}, journal = {Nature materials}, volume = {}, number = {}, pages = {}, pmid = {33542471}, issn = {1476-1122}, support = {RP160157//Cancer Prevention and Research Institute of Texas (Cancer Prevention Research Institute of Texas)/ ; RP160157//Cancer Prevention and Research Institute of Texas (Cancer Prevention Research Institute of Texas)/ ; R01 EB025192-01A1//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; RSG-17-012-01//American Cancer Society (American Cancer Society, Inc.)/ ; I-1855//Welch Foundation/ ; SIEGWA18XX0//Cystic Fibrosis Foundation (CF Foundation)/ ; 5P30CA142543//U.S. Department of Health &amp; Human Services | NIH | National Cancer Institute (NCI)/ ; }, abstract = {Endosomal escape remains a fundamental barrier hindering the advancement of nucleic acid therapeutics. Taking inspiration from natural phospholipids that comprise biological membranes, we report the combinatorial synthesis of multi-tailed ionizable phospholipids (iPhos) capable of delivering messenger RNA or mRNA/single-guide RNA for gene editing in vivo. Optimized iPhos lipids are composed of one pH-switchable zwitterion and three hydrophobic tails, which adopt a cone shape in endosomal acidic environments to facilitate membrane hexagonal transformation and subsequent cargo release from endosomes. Structure-activity relationships reveal that iPhos chemical structure can control in vivo efficacy and organ selectivity. iPhos lipids synergistically function with various helper lipids to formulate multi-component lipid nanoparticles (called iPLNPs) for selective organ targeting. Zwitterionic, ionizable cationic and permanently cationic helper lipids enable tissue-selective mRNA delivery and CRISPR-Cas9 gene editing in spleen, liver and lungs (respectively) following intravenous administration. This rational design of functional phospholipids demonstrates substantial value for gene editing research and therapeutic applications.}, }
@article {pmid33500419, year = {2021}, author = {Shorrocks, AK and Jones, SE and Tsukada, K and Morrow, CA and Belblidia, Z and Shen, J and Vendrell, I and Fischer, R and Kessler, BM and Blackford, AN}, title = {The Bloom syndrome complex senses RPA-coated single-stranded DNA to restart stalled replication forks.}, journal = {Nature communications}, volume = {12}, number = {1}, pages = {585}, pmid = {33500419}, issn = {2041-1723}, support = {C29215/A20772/CRUK_/Cancer Research UK/United Kingdom ; MC_UU_12009/MRC_/Medical Research Council/United Kingdom ; MC_UU_12010/MRC_/Medical Research Council/United Kingdom ; 107457/Z/15Z/WT_/Wellcome Trust/United Kingdom ; IS-BRC-1215-20008/DH_/Department of Health/United Kingdom ; G0902418/MRC_/Medical Research Council/United Kingdom ; MC_UU_12025/MRC_/Medical Research Council/United Kingdom ; MR/N013468/1/MRC_/Medical Research Council/United Kingdom ; }, mesh = {Amino Acid Motifs/genetics ; Bloom Syndrome/*genetics ; CRISPR-Cas Systems/genetics ; DNA Damage ; *DNA Replication ; DNA Topoisomerases, Type I/metabolism ; DNA, Single-Stranded/genetics/*metabolism ; DNA-Binding Proteins/genetics/metabolism ; Gene Knockdown Techniques ; HeLa Cells ; Humans ; Mitosis/genetics ; Mutation ; Protein Binding/genetics ; Protein Domains/genetics ; RecQ Helicases/genetics/*metabolism ; Recombinant Proteins/genetics/metabolism ; Recombinational DNA Repair/genetics ; Replication Protein A/*metabolism ; }, abstract = {The Bloom syndrome helicase BLM interacts with topoisomerase IIIα (TOP3A), RMI1 and RMI2 to form the BTR complex, which dissolves double Holliday junctions to produce non-crossover homologous recombination (HR) products. BLM also promotes DNA-end resection, restart of stalled replication forks, and processing of ultra-fine DNA bridges in mitosis. How these activities of the BTR complex are regulated in cells is still unclear. Here, we identify multiple conserved motifs within the BTR complex that interact cooperatively with the single-stranded DNA (ssDNA)-binding protein RPA. Furthermore, we demonstrate that RPA-binding is required for stable BLM recruitment to sites of DNA replication stress and for fork restart, but not for its roles in HR or mitosis. Our findings suggest a model in which the BTR complex contains the intrinsic ability to sense levels of RPA-ssDNA at replication forks, which controls BLM recruitment and activation in response to replication stress.}, }
@article {pmid32811438, year = {2020}, author = {Shen, L and Zhang, Q and Wang, Z and Wen, H and Hu, G and Ren, D and Hu, J and Zhu, L and Gao, Z and Zhang, G and Guo, L and Zeng, D and Qian, Q}, title = {OsCAF2 contains two CRM domains and is necessary for chloroplast development in rice.}, journal = {BMC plant biology}, volume = {20}, number = {1}, pages = {381}, pmid = {32811438}, issn = {1471-2229}, mesh = {CRISPR-Cas Systems ; Chloroplasts/*physiology ; Gene Expression Profiling ; Introns ; Mutation ; Organelle Biogenesis ; Oryza/genetics/*physiology ; Phenotype ; Plant Proteins/chemistry/genetics/*physiology ; Protein Domains ; RNA Splicing Factors/chemistry/genetics/*physiology ; }, abstract = {BACKGROUND: Chloroplasts play an important role in plant growth and development. The chloroplast genome contains approximately twenty group II introns that are spliced due to proteins encoded by nuclear genes. CAF2 is one of these splicing factors that has been shown to splice group IIB introns in maize and Arabidopsis thaliana. However, the research of the OsCAF2 gene in rice is very little, and the effects of OsCAF2 genes on chloroplasts development are not well characterized.<br><br>RESULTS: In this study, oscaf2 mutants were obtained by editing the OsCAF2 gene in the Nipponbare variety of rice. Phenotypic analysis showed that mutations to OsCAF2 led to albino leaves at the seeding stage that eventually caused plant death, and oscaf2 mutant plants had fewer chloroplasts and damaged chloroplast structure. We speculated that OsCAF2 might participate in the splicing of group IIA and IIB introns, which differs from its orthologs in A. thaliana and maize. Through yeast two-hybrid experiments, we found that the C-terminal region of OsCAF2 interacted with OsCRS2 and formed an OsCAF2-OsCRS2 complex. In addition, the N-terminal region of OsCAF2 interacted with itself to form homodimers.<br><br>CONCLUSION: Taken together, this study improved our understanding of the OsCAF2 protein, and revealed additional information about the molecular mechanism of OsCAF2 in regulating of chloroplast development in rice.}, }
@article {pmid32617133, year = {2020}, author = {Godoy, PRDV and Pour Khavari, A and Rizzo, M and Sakamoto-Hojo, ET and Haghdoost, S}, title = {Targeting NRF2, Regulator of Antioxidant System, to Sensitize Glioblastoma Neurosphere Cells to Radiation-Induced Oxidative Stress.}, journal = {Oxidative medicine and cellular longevity}, volume = {2020}, number = {}, pages = {2534643}, pmid = {32617133}, issn = {1942-0994}, mesh = {Antioxidants/*metabolism ; CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; Cell Survival/radiation effects ; Dose-Response Relationship, Radiation ; *Gamma Rays ; Glioblastoma/*pathology ; Humans ; Models, Biological ; NF-E2-Related Factor 2/*metabolism ; Neoplasm Proteins/metabolism ; Neoplastic Stem Cells/metabolism/pathology ; Nerve Tissue Proteins/metabolism ; Oxidative Stress/*radiation effects ; RNA-Binding Proteins/metabolism ; Radiation Tolerance/radiation effects ; Spheroids, Cellular/*pathology/radiation effects ; }, abstract = {The presence of glioma stem cells (GSCs), which are enriched in neurospheres, may be connected to the radioresistance of glioblastoma (GBM) due to their enhanced antioxidant defense and elevated DNA repair capacity. The aim was to evaluate the responses to different radiation qualities and to reduce radioresistance of U87MG cells, a GBM cell line. U87MG cells were cultured in a 3D model and irradiated with low (24 mGy/h) and high (0.39 Gy/min) dose rates of low LET gamma and high LET carbon ions (1-2 Gy/min). Thereafter, expression of proteins related to oxidative stress response, extracellular 8-oxo-dG, and neurospheres were determined. LD50 for carbon ions was significantly lower compared to LD50 of high and low dose rate gamma radiation. A significantly higher level of 8-oxo-dG was detected in the media of cells exposed to a low dose rate as compared to a high dose rate of gamma or carbon ions. A downregulation of oxidative stress proteins was also observed (NRF2, hMTH1, and SOD1). The NRF2 gene was knocked down by CRISPR/Cas9 in neurosphere cells, resulting in less self-renewal, more differentiated cells, and less proliferation capacity after irradiation with low and high dose rate gamma rays. Overall, U87MG glioma neurospheres presented differential responses to distinct radiation qualities and NRF2 plays an important role in cellular sensitivity to radiation.}, }
@article {pmid32541004, year = {2020}, author = {Al Tanoury, Z and Rao, J and Tassy, O and Gobert, B and Gapon, S and Garnier, JM and Wagner, E and Hick, A and Hall, A and Gussoni, E and Pourquié, O}, title = {Differentiation of the human PAX7-positive myogenic precursors/satellite cell lineage in vitro.}, journal = {Development (Cambridge, England)}, volume = {147}, number = {12}, pages = {}, pmid = {32541004}, issn = {1477-9129}, support = {R01 AR074526/AR/NIAMS NIH HHS/United States ; }, mesh = {CRISPR-Cas Systems/genetics ; *Cell Differentiation ; Cell Lineage ; Cell Self Renewal ; Cells, Cultured ; Genes, Reporter ; Genetic Loci ; Humans ; Induced Pluripotent Stem Cells/cytology/metabolism ; Muscle Fibers, Skeletal/cytology/metabolism ; Myogenin/genetics ; PAX7 Transcription Factor/genetics/*metabolism ; RNA, Guide/metabolism ; Satellite Cells, Skeletal Muscle/cytology/*metabolism ; }, abstract = {Satellite cells (SC) are muscle stem cells that can regenerate adult muscles upon injury. Most SC originate from PAX7+ myogenic precursors set aside during development. Although myogenesis has been studied in mouse and chicken embryos, little is known about human muscle development. Here, we report the generation of human induced pluripotent stem cell (iPSC) reporter lines in which fluorescent proteins have been introduced into the PAX7 and MYOG loci. We use single cell RNA sequencing to analyze the developmental trajectory of the iPSC-derived PAX7+ myogenic precursors. We show that the PAX7+ cells generated in culture can produce myofibers and self-renew in vitro and in vivo Together, we demonstrate that cells exhibiting characteristics of human fetal satellite cells can be produced in vitro from iPSC, opening interesting avenues for muscular dystrophy cell therapy. This work provides significant insights into the development of the human myogenic lineage.}, }
@article {pmid32487737, year = {2020}, author = {Rao, C and Malaguti, M and Mason, JO and Lowell, S}, title = {The transcription factor E2A drives neural differentiation in pluripotent cells.}, journal = {Development (Cambridge, England)}, volume = {147}, number = {12}, pages = {}, pmid = {32487737}, issn = {1477-9129}, support = {/WT_/Wellcome Trust/United Kingdom ; WT103789AIA/WT_/Wellcome Trust/United Kingdom ; MR/K501293/1/MRC_/Medical Research Council/United Kingdom ; 108906/Z/15/Z/WT_/Wellcome Trust/United Kingdom ; }, mesh = {3' Untranslated Regions ; Animals ; Basic Helix-Loop-Helix Transcription Factors/deficiency/genetics/*metabolism ; CRISPR-Cas Systems/genetics ; *Cell Differentiation ; Cell Lineage ; Cell Self Renewal ; Dimerization ; Mice ; Mouse Embryonic Stem Cells/cytology/metabolism ; Neurons/cytology/*metabolism ; Octamer Transcription Factor-3/deficiency/genetics/metabolism ; RNA, Guide/metabolism ; SOXB1 Transcription Factors/deficiency/genetics/metabolism ; Transcriptome ; Up-Regulation ; }, abstract = {The intrinsic mechanisms that link extracellular signalling to the onset of neural differentiation are not well understood. In pluripotent mouse cells, BMP blocks entry into the neural lineage via transcriptional upregulation of inhibitor of differentiation (Id) factors. We have previously identified the major binding partner of Id proteins in pluripotent cells as the basic helix-loop-helix (bHLH) transcription factor (TF) E2A. Id1 can prevent E2A from forming heterodimers with bHLH TFs or from forming homodimers. Here, we show that overexpression of a forced E2A homodimer is sufficient to drive robust neural commitment in pluripotent cells, even under non-permissive conditions. Conversely, we find that E2A null cells display a defect in their neural differentiation capacity. E2A acts as an upstream activator of neural lineage genes, including Sox1 and Foxd4, and as a repressor of Nodal signalling. Our results suggest a crucial role for E2A in establishing neural lineage commitment in pluripotent cells.}, }
@article {pmid32439761, year = {2020}, author = {Brandt, ZJ and Echert, AE and Bostrom, JR and North, PN and Link, BA}, title = {Core Hippo pathway components act as a brake on Yap and Taz in the development and maintenance of the biliary network.}, journal = {Development (Cambridge, England)}, volume = {147}, number = {12}, pages = {}, pmid = {32439761}, issn = {1477-9129}, support = {R01 EY029267/EY/NEI NIH HHS/United States ; }, mesh = {Animals ; Animals, Genetically Modified/growth &amp; development/metabolism ; Biliary Tract/anatomy &amp; histology/growth &amp; development/*metabolism ; CRISPR-Cas Systems/genetics ; Carboxylic Ester Hydrolases/metabolism ; Gallbladder/anatomy &amp; histology/growth &amp; development/metabolism ; Larva/growth &amp; development/metabolism ; Liver/anatomy &amp; histology/metabolism ; Phenotype ; Protein-Serine-Threonine Kinases/deficiency/genetics/*metabolism ; Signal Transduction ; Trans-Activators/genetics/*metabolism ; Transcription Factors/genetics/*metabolism ; Zebrafish/growth &amp; development/metabolism ; Zebrafish Proteins/deficiency/genetics/*metabolism ; }, abstract = {The development of the biliary system is a complex yet poorly understood process, with relevance to multiple diseases, including biliary atresia, choledochal cysts and gallbladder agenesis. We present here a crucial role for Hippo-Yap/Taz signaling in this context. Analysis of sav1 mutant zebrafish revealed dysplastic morphology and expansion of both intrahepatic and extrahepatic biliary cells, and ultimately larval lethality. Biliary dysgenesis, but not larval lethality, is driven primarily by Yap signaling. Re-expression of Sav1 protein in sav1-/- hepatocytes is able to overcome these initial deficits and allows sav1-/- fish to survive, suggesting cell non-autonomous signaling from hepatocytes. Examination of sav1-/- rescued adults reveals loss of gallbladder and formation of dysplastic cell masses expressing biliary markers, suggesting roles for Hippo signaling in extrahepatic biliary carcinomas. Deletion of stk3 revealed that the phenotypes observed in sav1 mutant fish function primarily through canonical Hippo signaling and supports a role for phosphatase PP2A, but also suggests Sav1 has functions in addition to facilitating Stk3 activity. Overall, this study defines a role for Hippo-Yap signaling in the maintenance of both intra- and extrahepatic biliary ducts.}, }
@article {pmid32223888, year = {2020}, author = {Schelling, MA and Sashital, DG}, title = {An adaptable defense.}, journal = {eLife}, volume = {9}, number = {}, pages = {}, pmid = {32223888}, issn = {2050-084X}, mesh = {Bacteria/genetics ; *Bacteriophages/genetics ; CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; }, abstract = {The response of bacteria to the threat posed by phages depends on their local environment.}, }
@article {pmid32200531, year = {2020}, author = {Chen, B and Niu, Y and Wang, H and Wang, K and Yang, H and Li, W}, title = {Recent advances in CRISPR research.}, journal = {Protein &amp; cell}, volume = {11}, number = {11}, pages = {786-791}, pmid = {32200531}, issn = {1674-8018}, mesh = {Animals ; *CRISPR-Cas Systems ; *Gene Editing ; Humans ; }, }
@article {pmid32197113, year = {2020}, author = {Petrus-Reurer, S and Winblad, N and Kumar, P and Gorchs, L and Chrobok, M and Wagner, AK and Bartuma, H and Lardner, E and Aronsson, M and Plaza Reyes, Á and André, H and Alici, E and Kaipe, H and Kvanta, A and Lanner, F}, title = {Generation of Retinal Pigment Epithelial Cells Derived from Human Embryonic Stem Cells Lacking Human Leukocyte Antigen Class I and II.}, journal = {Stem cell reports}, volume = {14}, number = {4}, pages = {648-662}, pmid = {32197113}, issn = {2213-6711}, mesh = {CRISPR-Associated Protein 9/metabolism ; CRISPR-Cas Systems/genetics ; Cytotoxicity, Immunologic ; Epithelial Cells/*metabolism ; Heterografts ; Histocompatibility Antigens Class I/*metabolism ; Histocompatibility Antigens Class II/*metabolism ; Human Embryonic Stem Cells/*cytology/metabolism ; Humans ; Immunomodulation ; Nuclear Proteins/metabolism ; Polymorphism, Single Nucleotide/genetics ; Retinal Pigment Epithelium/*cytology ; T-Lymphocytes/metabolism ; Trans-Activators/metabolism ; beta 2-Microglobulin/metabolism ; }, abstract = {Human embryonic stem cell-derived retinal pigment epithelial (hESC-RPE) cells could serve as a replacement therapy in advanced stages of age-related macular degeneration. However, allogenic hESC-RPE transplants trigger immune rejection, supporting a strategy to evade their immune recognition. We established single-knockout beta-2 microglobulin (SKO-B2M), class II major histocompatibility complex transactivator (SKO-CIITA) and double-knockout (DKO) hESC lines that were further differentiated into corresponding hESC-RPE lines lacking either surface human leukocyte antigen class I (HLA-I) or HLA-II, or both. Activation of CD4+ and CD8+ T-cells was markedly lower by hESC-RPE DKO cells, while natural killer cell cytotoxic response was not increased. After transplantation of SKO-B2M, SKO-CIITA, or DKO hESC-RPEs in a preclinical rabbit model, donor cell rejection was reduced and delayed. In conclusion, we have developed cell lines that lack both HLA-I and -II antigens, which evoke reduced T-cell responses in vitro together with reduced rejection in a large-eyed animal model.}, }
@article {pmid31654413, year = {2020}, author = {Zhang, L and Zhang, H and Liu, Y and Zhou, J and Shen, W and Liu, L and Li, Q and Chen, X}, title = {A CRISPR-Cas9 system for multiple genome editing and pathway assembly in Candida tropicalis.}, journal = {Biotechnology and bioengineering}, volume = {117}, number = {2}, pages = {531-542}, doi = {10.1002/bit.27207}, pmid = {31654413}, issn = {1097-0290}, support = {BK20171138//Natural Science Foundation of Jiangsu Province/International ; 111-2-06//111 Project/International ; none//Top-notch Academic Programs Project of Jiangsu Higher Education Institutions/International ; }, mesh = {CRISPR-Cas Systems/*genetics ; Candida tropicalis/*genetics/metabolism ; DNA, Fungal/genetics ; Gene Editing/*methods ; Genome, Fungal/*genetics ; Promoter Regions, Genetic/genetics ; }, abstract = {Genetic manipulation is among the most important tools for synthetic biology; however, modifying multiple genes is extremely time-consuming and can sometimes be impossible when dealing with gene families. Here, we present a clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated protein 9 (Cas9) system for use in the diploid yeast Candida tropicalis that is vastly superior to traditional techniques. This system enables the rapid and reliable introduction of multiple genetic deletions or mutations, as well as a stable expression using an integrated CRISPR-Cas9 cassette or a transient CRISPR-Cas9 cassette, together with a short donor DNA. We further show that the system can be used to promote the in vivo assembly of multiple DNA fragments and their stable integration into a target locus (or loci) in C. tropicalis. Based on this system, we present a platform for the biosynthesis of β-carotene and its derivatives. These results enable the practical application of C. tropicalis and the application of the system to other organisms.}, }
@article {pmid31631325, year = {2020}, author = {Duroy, PO and Bosshard, S and Schmid-Siegert, E and Neuenschwander, S and Arib, G and Lemercier, P and Masternak, J and Roesch, L and Buron, F and Girod, PA and Xenarios, I and Mermod, N}, title = {Characterization and mutagenesis of Chinese hamster ovary cells endogenous retroviruses to inactivate viral particle release.}, journal = {Biotechnology and bioengineering}, volume = {117}, number = {2}, pages = {466-485}, pmid = {31631325}, issn = {1097-0290}, support = {17196.1 PFLS-LS//Swiss Government Commission for Technology and Innovation/International ; //Université de Lausanne/International ; //Selexis SA/International ; }, mesh = {Animals ; CHO Cells/*virology ; CRISPR-Cas Systems ; Cricetinae ; Cricetulus ; Drug Contamination/prevention &amp; control ; *Endogenous Retroviruses/genetics/metabolism ; Gene Editing ; Genome, Viral/genetics ; Loss of Function Mutation/genetics ; Mutagenesis, Site-Directed/*methods ; *RNA, Viral/genetics/metabolism ; Virion/*genetics ; }, abstract = {The Chinese hamster ovary (CHO) cells used to produce biopharmaceutical proteins are known to contain type-C endogenous retrovirus (ERV) sequences in their genome and to release retroviral-like particles. Although evidence for their infectivity is missing, this has raised safety concerns. As the genomic origin of these particles remained unclear, we characterized type-C ERV elements at the genome, transcriptome, and viral particle RNA levels. We identified 173 type-C ERV sequences clustering into three functionally conserved groups. Transcripts from one type-C ERV group were full-length, with intact open reading frames, and cognate viral genome RNA was loaded into retroviral-like particles, suggesting that this ERV group may produce functional viruses. CRISPR-Cas9 genome editing was used to disrupt the gag gene of the expressed type-C ERV group. Comparison of CRISPR-derived mutations at the DNA and RNA level led to the identification of a single ERV as the main source of the release of RNA-loaded viral particles. Clones bearing a Gag loss-of-function mutation in this ERV showed a reduction of RNA-containing viral particle release down to detection limits, without compromising cell growth or therapeutic protein production. Overall, our study provides a strategy to mitigate potential viral particle contaminations resulting from ERVs during biopharmaceutical manufacturing.}, }
@article {pmid31631317, year = {2020}, author = {Karottki, KJC and Hefzi, H and Xiong, K and Shamie, I and Hansen, AH and Li, S and Pedersen, LE and Li, S and Lee, JS and Lee, GM and Kildegaard, HF and Lewis, NE}, title = {Awakening dormant glycosyltransferases in CHO cells with CRISPRa.}, journal = {Biotechnology and bioengineering}, volume = {117}, number = {2}, pages = {593-598}, pmid = {31631317}, issn = {1097-0290}, support = {R35 GM119850/GM/NIGMS NIH HHS/United States ; NNF16OC0021638//Novo Nordisk Foundation/International ; NNF10CC1016517//Novo Nordisk Foundation/International ; }, mesh = {Animals ; CHO Cells ; CRISPR-Cas Systems/*genetics ; Cricetinae ; Cricetulus ; Gene Editing/*methods ; Glycosylation ; Glycosyltransferases/*genetics ; Phenotype ; Polysaccharides/analysis/chemistry ; }, abstract = {Chinese hamster ovary (CHO) cells are the preferred workhorse for the biopharmaceutical industry, and CRISPR/Cas9 has proven powerful for generating targeted gene perturbations in CHO cells. Here, we expand the CRISPR engineering toolbox with CRISPR activation (CRISPRa) to increase transcription of endogenous genes. We successfully increased transcription of Mgat3 and St6gal1, and verified their activity on a functional level by subsequently detecting that the appropriate glycan structures were produced. This study demonstrates that CRISPRa can make targeted alterations of CHO cells for desired phenotypes.}, }
@article {pmid33540323, year = {2021}, author = {Lee, CY and Degani, I and Cheong, J and Lee, JH and Choi, HJ and Cheon, J and Lee, H}, title = {Fluorescence polarization system for rapid COVID-19 diagnosis.}, journal = {Biosensors &amp; bioelectronics}, volume = {178}, number = {}, pages = {113049}, doi = {10.1016/j.bios.2021.113049}, pmid = {33540323}, issn = {1873-4235}, abstract = {Prompt diagnosis, patient isolation, and contact tracing are key measures to contain the coronavirus disease 2019 (COVID-19). Molecular tests are the current gold standard for COVID-19 detection, but are carried out at central laboratories, delaying treatment and control decisions. Here we describe a portable assay system for rapid, onsite COVID-19 diagnosis. Termed CODA (CRISPR Optical Detection of Anisotropy), the method combined isothermal nucleic acid amplification, activation of CRISPR/Cas12a, and signal generation in a single assay, eliminating extra manual steps. Importantly, signal detection was based on the ratiometric measurement of fluorescent anisotropy, which allowed CODA to achieve a high signal-to-noise ratio. For point-of-care operation, we built a compact, standalone CODA device integrating optoelectronics, an embedded heater, and a microcontroller for data processing. The developed system completed SARS-CoV-2 RNA detection within 20 min of sample loading; the limit of detection reached 3 copy/μL. When applied to clinical samples (10 confirmed COVID-19 patients; 10 controls), the rapid CODA test accurately classified COVID-19 status, in concordance with gold-standard clinical diagnostics.}, }
@article {pmid33538637, year = {2021}, author = {Vicencio, J and Cerón, J}, title = {A Living Organism in your CRISPR Toolbox: Caenorhabditis elegans Is a Rapid and Efficient Model for Developing CRISPR-Cas Technologies.}, journal = {The CRISPR journal}, volume = {}, number = {}, pages = {}, doi = {10.1089/crispr.2020.0103}, pmid = {33538637}, issn = {2573-1602}, abstract = {The Cas9 nuclease from Streptococcus pyogenes (SpCas9) is the most popular enzyme for CRISPR technologies. However, considering the wide diversity of microorganisms (discovered and still unknown), a massive number of CRISPR effectors are being and will be identified and characterized in the search of optimal Cas variants for each of the many applications of CRISPR. In this context, a versatile and efficient multicellular system for CRISPR editing such as Caenorhabditis elegans would be of great help in the development of these effectors. Here, we highlight the benefits of using C. elegans for the rapid evaluation of new CRISPR effectors, and for optimizing CRISPR efficiency in animals in several ways such as by modulating the balance between repair pathways, modifying chromatin accessibility, or controlling the expression and activity of nucleases and guide RNAs.}, }
@article {pmid33537681, year = {2021}, author = {Zhao, R and Yang, Y and Yang, K and Han, W}, title = {Expression, purification, and characterization of a membrane-associated cyclic oligo-adenylate degrader from Sulfolobus islandicus.}, journal = {STAR protocols}, volume = {2}, number = {1}, pages = {100299}, doi = {10.1016/j.xpro.2021.100299}, pmid = {33537681}, issn = {2666-1667}, abstract = {Type III CRISPR-cas systems initiate cyclic oligo-adenylate (cOA) signaling to initiate immune response. Previously, we identified that a membrane-associated DHH-DHHA1 family protein from Sulfolobus islandicus efficiently degrades cOA. Here, we provide detailed protocols for expression and purification of the protein from its native host and a cOA degradation assay with the purified enzyme. The methodology should be of interest for researchers studying Sulfolobus, membrane-associated proteins, or type III CRISPR-cas systems. For complete details on the use and execution of this protocol, please refer to Zhao et al. (2020).}, }
@article {pmid33537251, year = {2021}, author = {Lee, H}, title = {Rapid Way to Generate Mouse Models for In Vivo Studies of the Endothelium.}, journal = {Journal of lipid and atherosclerosis}, volume = {10}, number = {1}, pages = {24-41}, doi = {10.12997/jla.2021.10.1.24}, pmid = {33537251}, issn = {2287-2892}, abstract = {A single layer of squamous endothelial cells (ECs), the endothelium, regulates the flow of substance and fluid into and out of a tissue. The endothelium is also involved in vasculogenesis, the formation of new blood vessels, which is a crucial process for organ development in the embryo and fetus. Because most murine mutations of genes involved in EC development cause early embryo lethality, EC-specific conditional knockout (cKO) mouse models are indispensable for in vivo studies. cKO mice including the floxed allele can be generated through advanced approaches including embryonic stem cell-mediated gene targeting or the CRISPR/Cas system. EC-specific mouse models can be generated through further breeding of floxed mice with a Cre driver line, the latest information of which is available in the Jackson Cre Repository or the EUCOMMTOOLS project. Because it takes a long time (generally 1-2 years) to generate EC-specific mouse models, researchers must thoroughly design and plan a breeding strategy before full-scale mouse experiments, which saves time and money for in vivo study. In summary, revolutionary technical advances in embryo manipulation and assisted reproduction technologies have made it easier to generate EC-specific mouse models, which have been used as essential resources for in vivo studies of the endothelium.}, }
@article {pmid33299166, year = {2020}, author = {Ledford, H}, title = {CRISPR gene therapy shows promise against blood diseases.}, journal = {Nature}, volume = {588}, number = {7838}, pages = {383}, pmid = {33299166}, issn = {1476-4687}, mesh = {*Anemia, Sickle Cell ; CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Editing ; Genetic Therapy ; *Hematologic Diseases ; Humans ; *beta-Thalassemia ; }, }
@article {pmid33283989, year = {2021}, author = {Frangoul, H and Altshuler, D and Cappellini, MD and Chen, YS and Domm, J and Eustace, BK and Foell, J and de la Fuente, J and Grupp, S and Handgretinger, R and Ho, TW and Kattamis, A and Kernytsky, A and Lekstrom-Himes, J and Li, AM and Locatelli, F and Mapara, MY and de Montalembert, M and Rondelli, D and Sharma, A and Sheth, S and Soni, S and Steinberg, MH and Wall, D and Yen, A and Corbacioglu, S}, title = {CRISPR-Cas9 Gene Editing for Sickle Cell Disease and β-Thalassemia.}, journal = {The New England journal of medicine}, volume = {384}, number = {3}, pages = {252-260}, doi = {10.1056/NEJMoa2031054}, pmid = {33283989}, issn = {1533-4406}, mesh = {Adult ; Anemia, Sickle Cell/genetics/*therapy ; *CRISPR-Cas Systems ; Female ; Fetal Hemoglobin/*biosynthesis/genetics ; Gene Editing/*methods ; *Genetic Therapy ; Humans ; Repressor Proteins/*genetics/metabolism ; Young Adult ; beta-Thalassemia/genetics/*therapy ; }, abstract = {Transfusion-dependent β-thalassemia (TDT) and sickle cell disease (SCD) are severe monogenic diseases with severe and potentially life-threatening manifestations. BCL11A is a transcription factor that represses γ-globin expression and fetal hemoglobin in erythroid cells. We performed electroporation of CD34+ hematopoietic stem and progenitor cells obtained from healthy donors, with CRISPR-Cas9 targeting the BCL11A erythroid-specific enhancer. Approximately 80% of the alleles at this locus were modified, with no evidence of off-target editing. After undergoing myeloablation, two patients - one with TDT and the other with SCD - received autologous CD34+ cells edited with CRISPR-Cas9 targeting the same BCL11A enhancer. More than a year later, both patients had high levels of allelic editing in bone marrow and blood, increases in fetal hemoglobin that were distributed pancellularly, transfusion independence, and (in the patient with SCD) elimination of vaso-occlusive episodes. (Funded by CRISPR Therapeutics and Vertex Pharmaceuticals; ClinicalTrials.gov numbers, NCT03655678 for CLIMB THAL-111 and NCT03745287 for CLIMB SCD-121.).}, }
@article {pmid32633292, year = {2020}, author = {Gong, S and Chen, Y and Pan, W and Li, N and Tang, B}, title = {An in vitro site-specific cleavage assay of CRISPR-Cas9 using a personal glucose meter.}, journal = {Chemical communications (Cambridge, England)}, volume = {56}, number = {62}, pages = {8850-8853}, doi = {10.1039/d0cc03505j}, pmid = {32633292}, issn = {1364-548X}, mesh = {Base Sequence ; Blood Glucose Self-Monitoring/economics/*methods ; CRISPR-Cas Systems/*genetics ; Cost-Benefit Analysis ; }, abstract = {A sensitive and cost-effective assay based on a personal glucose meter was developed to detect the in vitro site-specific cleavage efficiency of CRISPR-Cas9.}, }
@article {pmid32160520, year = {2020}, author = {Shinohara, T and Kanatsu-Shinohara, M}, title = {Transgenesis and Genome Editing of Mouse Spermatogonial Stem Cells by Lentivirus Pseudotyped with Sendai Virus F Protein.}, journal = {Stem cell reports}, volume = {14}, number = {3}, pages = {447-461}, pmid = {32160520}, issn = {2213-6711}, mesh = {Animals ; Base Sequence ; CRISPR-Cas Systems/genetics ; *Gene Editing ; *Gene Transfer Techniques ; *Genome ; Kinetics ; Lentivirus/*metabolism ; Male ; Mice, Transgenic ; Phenotype ; Sendai virus/*metabolism ; Sertoli Cells/metabolism ; Spermatogenesis/genetics ; Spermatogonia/*cytology ; Stem Cells/*metabolism ; Viral Fusion Proteins/*metabolism ; Virus Integration ; }, abstract = {Spermatogonial stem cells (SSCs) serve as a resource for producing genetically modified animals. However, genetic manipulation of SSCs has met with limited success. Here, we show efficient gene transfer into SSCs via a lentivirus (FV-LV) using a fusion protein (F), a Sendai virus (SV) envelope protein involved in virion/cell membrane fusion. FV-LVs transduced cultured SSCs more efficiently than conventional LVs. Although SSCs infected with SV failed to produce offspring, those transduced with FV-LVs were fertile. In vivo microinjection showed that FV-LVs could penetrate not only the basement membrane of the seminiferous tubules but also the blood-testis barrier, which resulted in successful transduction of both spermatogenic cells and testicular somatic cells. Cultured SSCs transfected with FV-LVs that express drug-inducible CRISPR/Cas9 against Kit or Sycp3 showed impaired spermatogenesis upon transplantation and drug treatment in vivo. Thus, FV-LVs provide an efficient method for functional analysis of genes involved in SSCs and spermatogenesis.}, }
@article {pmid32057858, year = {2020}, author = {Sun, Y and Liu, M and Yan, C and Yang, H and Wu, Z and Liu, Y and Su, N and Hou, J and Zhang, J and Yang, F and Zhang, J}, title = {CRISPR/Cas9-mediated deletion of β, β-carotene 9', 10'-oxygenase gene (EcBCO2) from Exopalaemon carinicauda.}, journal = {International journal of biological macromolecules}, volume = {151}, number = {}, pages = {168-177}, doi = {10.1016/j.ijbiomac.2020.02.073}, pmid = {32057858}, issn = {1879-0003}, mesh = {Amino Acid Sequence ; Animals ; Base Sequence ; *CRISPR-Cas Systems ; Computational Biology/methods ; *Gene Deletion ; Gene Expression ; Gene Expression Profiling ; Gene Knockdown Techniques ; Gene Targeting ; Oxygenases/*genetics ; Palaemonidae/*genetics ; Phylogeny ; RNA, Guide ; RNA, Messenger/genetics ; }, abstract = {CRISPR/Cas9 technology is an efficient genome editing tool for producing genetically modified animals. Carotenoids color the world around us and their accumulation in animals could be used to culture colorful new verities in animal breeding. β, β-carotene 9', 10'-oxygenase (BCO2) is an important enzyme during β-carotene metabolism. In this research, one full-length cDNA sequence encoding BCO2 (named EcBCO2) were obtained from Exopalaemon carinicauda. The genomic structure analysis showed that EcBCO2 gene was composed of 9 exons and 8 introns. Then, the CRISPR/Cas9-mediated deletion of EcBCO2 gene was generated by co-microinjection of Cas9 mRNA and EcBCO2 sgRNA into one-cell stage embryos of E. carinicauda. Subsequently, the phenotype of EcBCO2-KO prawns was compared with that of wild-type prawns, which showed that EcBCO2-KO resulted in the color change in the hepatopancreas of prawns. In addition, the EcBCO2-KO prawns had a higher survival rate than wild-type prawns when the prawns were challenged with Vibrio parahaemolyticus or Aeromonas hydrophila. These results indicate that BCO2 gene could be used as a candidate gene in molecular marker-assistant breeding of prawns.}, }
@article {pmid31923323, year = {2020}, author = {Du, M and Zhou, K and Liu, Y and Deng, L and Zhang, X and Lin, L and Zhou, M and Zhao, W and Wen, C and Xing, J and Li, CB and Li, C}, title = {A biotechnology-based male-sterility system for hybrid seed production in tomato.}, journal = {The Plant journal : for cell and molecular biology}, volume = {102}, number = {5}, pages = {1090-1100}, pmid = {31923323}, issn = {1365-313X}, mesh = {Biotechnology/*methods ; CRISPR-Cas Systems ; Lycopersicon esculentum/metabolism/*physiology ; Plant Infertility/genetics/physiology ; Seeds/metabolism/*physiology ; }, abstract = {Incorporating male sterility into hybrid seed production reduces its cost and ensures high varietal purity. Despite these advantages, male-sterile lines have not been widely used to produce tomato (Solanum lycopersicum) hybrid seeds. We describe the development of a biotechnology-based breeding platform that utilized genic male sterility to produce hybrid seeds. In this platform, we generated a novel male-sterile tomato line by clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated mutagenesis of a stamen-specific gene SlSTR1 and devised a transgenic maintainer by transforming male-sterile plants with a fertility-restoration gene linked to a seedling-colour gene. Offspring of crosses between a hemizygous maintainer and the homozygous male-sterile plant segregated into 50% non-transgenic male-sterile plants and 50% male-fertile maintainer plants, which could be easily distinguished by seedling colour. This system has great practical potential for hybrid seed breeding and production as it overcomes the problems intrinsic to other male-sterility systems and can be easily adapted for a range of tomato cultivars and diverse vegetable crops.}, }
@article {pmid31748711, year = {2020}, author = {Botkin, JR}, title = {The case for banning heritable genome editing.}, journal = {Genetics in medicine : official journal of the American College of Medical Genetics}, volume = {22}, number = {3}, pages = {487-489}, pmid = {31748711}, issn = {1530-0366}, mesh = {CRISPR-Cas Systems/*genetics ; *Ethicists ; Gene Editing/*ethics/legislation &amp; jurisprudence ; Genome, Human/*genetics ; Humans ; }, }
@article {pmid33534660, year = {2021}, author = {Philippe, C and Moineau, S}, title = {The endless battle between phages and CRISPR-Cas systems in Streptococcus thermophilus.}, journal = {Biochemistry and cell biology = Biochimie et biologie cellulaire}, volume = {}, number = {}, pages = {}, doi = {10.1139/bcb-2020-0593}, pmid = {33534660}, issn = {1208-6002}, abstract = {This review describes the contribution of basic research on phage-bacteria interactions to the understanding of CRISPR-Cas systems as well as its various applications. It focuses on the natural function of CRISPR-Cas system as an adaptive defense mechanism against mobile genetic elements such as bacteriophage genomes and plasmids. Some of the advances in the characterization of the type II-A CRISPR-Cas system of Streptococcus thermophilus and Streptococcus pyogenes later led to the development of the CRISPR-Cas9 genome editing technology. We mostly discuss the three stages of the CRISPR-Cas system in S. thermophilus, namely the adaptation stage, which is unique to this resistance mechanism, the CRISPR RNA biogenesis as well as the DNA cutting activity in the interference stage to protect bacteria against phages. Finally, we looked into applications of CRISPR-Cas in microbiology, including overcoming limitations in genome editing.}, }
@article {pmid33398349, year = {2021}, author = {Wang, Q and Liu, J and Janssen, JM and Le Bouteiller, M and Frock, RL and Gonçalves, MAFV}, title = {Precise and broad scope genome editing based on high-specificity Cas9 nickases.}, journal = {Nucleic acids research}, volume = {49}, number = {2}, pages = {1173-1198}, pmid = {33398349}, issn = {1362-4962}, mesh = {Bacterial Proteins/genetics/*metabolism ; Base Sequence ; CRISPR-Associated Protein 9/genetics/*metabolism ; *CRISPR-Cas Systems ; Clone Cells ; Deoxyribonuclease I/genetics/*metabolism ; Gene Editing/*methods ; Gene Knock-In Techniques ; Gene Knockout Techniques ; Genes, Reporter ; Genotyping Techniques ; HEK293 Cells ; HeLa Cells ; Heterochromatin/genetics ; High-Throughput Nucleotide Sequencing ; Humans ; Induced Pluripotent Stem Cells ; Polymorphism, Genetic ; RNA, Guide/genetics ; Recombinant Proteins/metabolism ; Streptococcus pyogenes/enzymology ; Substrate Specificity ; Transfection ; }, abstract = {RNA-guided nucleases (RGNs) based on CRISPR systems permit installing short and large edits within eukaryotic genomes. However, precise genome editing is often hindered due to nuclease off-target activities and the multiple-copy character of the vast majority of chromosomal sequences. Dual nicking RGNs and high-specificity RGNs both exhibit low off-target activities. Here, we report that high-specificity Cas9 nucleases are convertible into nicking Cas9D10A variants whose precision is superior to that of the commonly used Cas9D10A nickase. Dual nicking RGNs based on a selected group of these Cas9D10A variants can yield gene knockouts and gene knock-ins at frequencies similar to or higher than those achieved by their conventional counterparts. Moreover, high-specificity dual nicking RGNs are capable of distinguishing highly similar sequences by 'tiptoeing' over pre-existing single base-pair polymorphisms. Finally, high-specificity RNA-guided nicking complexes generally preserve genomic integrity, as demonstrated by unbiased genome-wide high-throughput sequencing assays. Thus, in addition to substantially enlarging the Cas9 nickase toolkit, we demonstrate the feasibility in expanding the range and precision of DNA knockout and knock-in procedures. The herein introduced tools and multi-tier high-specificity genome editing strategies might be particularly beneficial whenever predictability and/or safety of genetic manipulations are paramount.}, }
@article {pmid33398345, year = {2021}, author = {Doi, G and Okada, S and Yasukawa, T and Sugiyama, Y and Bala, S and Miyazaki, S and Kang, D and Ito, T}, title = {Catalytically inactive Cas9 impairs DNA replication fork progression to induce focal genomic instability.}, journal = {Nucleic acids research}, volume = {49}, number = {2}, pages = {954-968}, pmid = {33398345}, issn = {1362-4962}, mesh = {CRISPR-Associated Protein 9/chemistry/*metabolism ; *CRISPR-Cas Systems ; DNA Copy Number Variations ; *DNA Replication ; DNA, Fungal/*genetics/metabolism ; DNA, Single-Stranded/genetics ; Gene Editing ; Genes, Fungal ; *Genomic Instability ; Metallothionein/genetics ; Mutagenesis ; Niacinamide/pharmacology ; Rad52 DNA Repair and Recombination Protein/metabolism ; Saccharomyces cerevisiae/enzymology/*genetics ; Saccharomyces cerevisiae Proteins/chemistry/genetics/*metabolism ; Tandem Repeat Sequences ; }, abstract = {Catalytically inactive Cas9 (dCas9) has become an increasingly popular tool for targeted gene activation/inactivation, live-cell imaging, and base editing. While dCas9 was reported to induce base substitutions and indels, it has not been associated with structural variations. Here, we show that dCas9 impedes replication fork progression to destabilize tandem repeats in budding yeast. When targeted to the CUP1 array comprising ∼16 repeat units, dCas9 induced its contraction in most cells, especially in the presence of nicotinamide. Replication intermediate analysis demonstrated replication fork stalling in the vicinity of dCas9-bound sites. Genetic analysis indicated that while destabilization is counteracted by the replisome progression complex components Ctf4 and Mrc1 and the accessory helicase Rrm3, it involves single-strand annealing by the recombination proteins Rad52 and Rad59. Although dCas9-mediated replication fork stalling is a potential risk in conventional applications, it may serve as a novel tool for both mechanistic studies and manipulation of genomic instability.}, }
@article {pmid33398341, year = {2021}, author = {Fu, YW and Dai, XY and Wang, WT and Yang, ZX and Zhao, JJ and Zhang, JP and Wen, W and Zhang, F and Oberg, KC and Zhang, L and Cheng, T and Zhang, XB}, title = {Dynamics and competition of CRISPR-Cas9 ribonucleoproteins and AAV donor-mediated NHEJ, MMEJ and HDR editing.}, journal = {Nucleic acids research}, volume = {49}, number = {2}, pages = {969-985}, pmid = {33398341}, issn = {1362-4962}, mesh = {Adult ; *CRISPR-Cas Systems ; Cell Line, Tumor ; DNA Copy Number Variations ; *DNA End-Joining Repair/drug effects ; *Gene Editing ; Genetic Vectors/*genetics ; HEK293 Cells ; Humans ; Hydroxamic Acids/pharmacology ; INDEL Mutation ; Induced Pluripotent Stem Cells ; Kinetics ; Parvovirinae/*genetics ; RNA, Guide/genetics ; *Recombinational DNA Repair/drug effects ; Ribonucleoproteins/*metabolism ; T-Lymphocytes ; Transduction, Genetic ; }, abstract = {Investigations of CRISPR gene knockout editing profiles have contributed to enhanced precision of editing outcomes. However, for homology-directed repair (HDR) in particular, the editing dynamics and patterns in clinically relevant cells, such as human iPSCs and primary T cells, are poorly understood. Here, we explore the editing dynamics and DNA repair profiles after the delivery of Cas9-guide RNA ribonucleoprotein (RNP) with or without the adeno-associated virus serotype 6 (AAV6) as HDR donors in four cell types. We show that editing profiles have distinct differences among cell lines. We also reveal the kinetics of HDR mediated by the AAV6 donor template. Quantification of T50 (time to reach half of the maximum editing frequency) indicates that short indels (especially +A/T) occur faster than longer (>2 bp) deletions, while the kinetics of HDR falls between NHEJ (non-homologous end-joining) and MMEJ (microhomology-mediated end-joining). As such, AAV6-mediated HDR effectively outcompetes the longer MMEJ-mediated deletions but not NHEJ-mediated indels. Notably, a combination of small molecular compounds M3814 and Trichostatin A (TSA), which potently inhibits predominant NHEJ repairs, leads to a 3-fold increase in HDR efficiency.}, }
@article {pmid33068434, year = {2020}, author = {Lin, J and Liu, Y and Lai, P and Ye, H and Xu, L}, title = {Conditional guide RNA through two intermediate hairpins for programmable CRISPR/Cas9 function: building regulatory connections between endogenous RNA expressions.}, journal = {Nucleic acids research}, volume = {48}, number = {20}, pages = {11773-11784}, pmid = {33068434}, issn = {1362-4962}, mesh = {*CRISPR-Associated Protein 9 ; *CRISPR-Cas Systems ; Escherichia coli/genetics ; HEK293 Cells ; Humans ; MicroRNAs/metabolism ; RNA/chemistry/*metabolism ; RNA, Small Untranslated/metabolism ; Transcriptional Activation ; }, abstract = {A variety of nanodevices developed for nucleic acid computation provide great opportunities to construct versatile synthetic circuits for manipulation of gene expressions. In our study, by employing a two-hairpin mediated nucleic acid strand displacement as a processing joint for conditional guide RNA, we aim to build artificial connections between naturally occurring RNA expressions through programmable CRISPR/Cas9 function. This two-hairpin joint possesses a sequence-switching machinery, in which a random trigger strand can be processed to release an unconstrained sequence-independent strand and consequently activate the self-inhibitory guide RNA for conditional gene regulation. This intermediate processor was characterized by the fluorescence reporter system and applied for regulation of the CRISPR/Cas9 binding activity. Using plasmids to generate this sequence-switching machinery in situ, we achieved the autonomous genetic regulation of endogenous RNA expressions controlled by other unrelated endogenous RNAs in both E. coli and human cells. Unlike previously reported strand-displacement genetic circuits, this advanced nucleic acid nanomachine provides a novel approach that can establish regulatory connections between naturally occurring endogenous RNAs. In addition to CRISPR systems, we anticipate this two-hairpin machine can serve as a general processing joint for wide applications in the development of other RNA-based genetic circuits.}, }
@article {pmid32702356, year = {2020}, author = {Gao, Y and Zhang, X and Zhang, X and Yuan, J and Xiang, J and Li, F}, title = {CRISPR/Cas9-mediated mutation reveals Pax6 is essential for development of the compound eye in Decapoda Exopalaemon carinicauda.}, journal = {Developmental biology}, volume = {465}, number = {2}, pages = {157-167}, doi = {10.1016/j.ydbio.2020.07.001}, pmid = {32702356}, issn = {1095-564X}, mesh = {Animals ; *Arthropod Proteins/genetics/metabolism ; *CRISPR-Cas Systems ; Compound Eye, Arthropod/*embryology ; *Decapoda/embryology/genetics ; Gene Editing ; *Mutation ; *PAX6 Transcription Factor/genetics/metabolism ; }, abstract = {The compound eye in crustaceans is a main eye type in the animal kingdom, knowledge about the mechanism to determine the development of compound eye is very limited. Paired box protein 6 (Pax6) is generally regarded as a master regulator for eye development. In the present study, a genome-based analysis of the Pax6 gene in the ridge tail white prawn Exopalaemon carinicauda was performed and two members of Pax6 homologs, named Ec-Eyeless (EcEy) and Ec-Twin of eyeless (EcToy) were identified. To understand the function of these two homologs of Pax6 gene in the prawn, the CRISPR/Cas9 genome editing technique was applied to generate EcEy and EcToy knock-out (KO) prawns and their phenotypes were analyzed. The surviving EcEy-KO embryos and larvae exhibited severe abnormal eye morphology, suggesting that EcEy is necessary for the compound eye development in prawn, while no mutant phenotype was found in EcToy-KO individuals. These findings highlighted the conservative role of Pax6 gene in the compound eye formation, and the functional differentiation between EcEy and EcToy gene may reveal a novel regulating mechanism of Pax6 on the compound eye development in the decapods. These data will provide important information for understanding the regulation mechanism for crustacean compound eye development.}, }
@article {pmid32478678, year = {2020}, author = {Chemello, F and Bassel-Duby, R and Olson, EN}, title = {Correction of muscular dystrophies by CRISPR gene editing.}, journal = {The Journal of clinical investigation}, volume = {130}, number = {6}, pages = {2766-2776}, pmid = {32478678}, issn = {1558-8238}, support = {U54 HD087351/HD/NICHD NIH HHS/United States ; }, mesh = {Animals ; *CRISPR-Cas Systems ; *Dystrophin/biosynthesis/genetics ; *Gene Editing ; Humans ; *Muscular Dystrophy, Duchenne/genetics/metabolism/therapy ; *Mutation ; }, abstract = {Muscular dystrophies are debilitating disorders that result in progressive weakness and degeneration of skeletal muscle. Although the genetic mutations and clinical abnormalities of a variety of neuromuscular diseases are well known, no curative therapies have been developed to date. The advent of genome editing technology provides new opportunities to correct the underlying mutations responsible for many monogenic neuromuscular diseases. For example, Duchenne muscular dystrophy, which is caused by mutations in the dystrophin gene, has been successfully corrected in mice, dogs, and human cells through CRISPR/Cas9 editing. In this Review, we focus on the potential for, and challenges of, correcting muscular dystrophies by editing disease-causing mutations at the genomic level. Ideally, because muscle tissues are extremely long-lived, CRISPR technology could offer a one-time treatment for muscular dystrophies by correcting the culprit genomic mutations and enabling normal expression of the repaired gene.}, }
@article {pmid32404434, year = {2020}, author = {Kumar, N and Sharma, S and Kumar, R and Tripathi, BN and Barua, S and Ly, H and Rouse, BT}, title = {Host-Directed Antiviral Therapy.}, journal = {Clinical microbiology reviews}, volume = {33}, number = {3}, pages = {}, pmid = {32404434}, issn = {1098-6618}, mesh = {Animals ; *CRISPR-Cas Systems ; *Drug Development ; Gene Targeting ; Host-Derived Cellular Factors/*antagonists &amp; inhibitors/genetics ; Host-Pathogen Interactions/genetics ; Humans ; Mice ; *RNA, Small Interfering ; Virus Replication/*genetics ; Viruses/genetics ; }, abstract = {Antiviral drugs have traditionally been developed by directly targeting essential viral components. However, this strategy often fails due to the rapid generation of drug-resistant viruses. Recent genome-wide approaches, such as those employing small interfering RNA (siRNA) or clustered regularly interspaced short palindromic repeats (CRISPR) or those using small molecule chemical inhibitors targeting the cellular &quot;kinome,&quot; have been used successfully to identify cellular factors that can support virus replication. Since some of these cellular factors are critical for virus replication, but are dispensable for the host, they can serve as novel targets for antiviral drug development. In addition, potentiation of immune responses, regulation of cytokine storms, and modulation of epigenetic changes upon virus infections are also feasible approaches to control infections. Because it is less likely that viruses will mutate to replace missing cellular functions, the chance of generating drug-resistant mutants with host-targeted inhibitor approaches is minimized. However, drug resistance against some host-directed agents can, in fact, occur under certain circumstances, such as long-term selection pressure of a host-directed antiviral agent that can allow the virus the opportunity to adapt to use an alternate host factor or to alter its affinity toward the target that confers resistance. This review describes novel approaches for antiviral drug development with a focus on host-directed therapies and the potential mechanisms that may account for the acquisition of antiviral drug resistance against host-directed agents.}, }
@article {pmid32346757, year = {2020}, author = {Schaffert, L and Schneiker-Bekel, S and Gierhake, J and Droste, J and Persicke, M and Rosen, W and Pühler, A and Kalinowski, J}, title = {Absence of the highly expressed small carbohydrate-binding protein Cgt improves the acarbose formation in Actinoplanes sp. SE50/110.}, journal = {Applied microbiology and biotechnology}, volume = {104}, number = {12}, pages = {5395-5408}, pmid = {32346757}, issn = {1432-0614}, mesh = {ATP-Binding Cassette Transporters/*genetics/metabolism ; Acarbose/*metabolism ; Actinoplanes/*genetics/*metabolism ; Bacterial Proteins/genetics/*metabolism ; CRISPR-Cas Systems ; Carbohydrate Metabolism ; Gene Deletion ; Multigene Family ; Protein Binding ; Proteome/metabolism ; Starch/metabolism ; }, abstract = {Actinoplanes sp. SE50/110 (ATCC 31044) is the wild type of industrial producer strains of acarbose. Acarbose has been used since the early 1990s as an inhibitor of intestinal human α-glucosidases in the medical treatment of type II diabetes mellitus. The small secreted protein Cgt, which consists of a single carbohydrate-binding module (CBM) 20-domain, was found to be highly expressed in Actinoplanes sp. SE50/110 in previous studies, but neither its function nor a possible role in the acarbose formation was explored, yet. Here, we demonstrated the starch-binding function of the Cgt protein in a binding assay. Transcription analysis showed that the cgt gene was strongly repressed in the presence of glucose or lactose. Due to this and its high abundance in the extracellular proteome of Actinoplanes, a functional role within the sugar metabolism or in the environmental stress protection was assumed. However, the gene deletion mutant ∆cgt, constructed by CRISPR/Cas9 technology, displayed no apparent phenotype in screening experiments testing for pH and osmolality stress, limited carbon source starch, and the excess of seven different sugars in liquid culture and further 97 carbon sources in the Omnilog Phenotypic Microarray System of Biolog. Therefore, a protective function as a surface protein or a function within the retainment and the utilization of carbon sources could not be experimentally validated. Remarkably, enhanced production of acarbose was determined yielding into 8-16% higher product titers when grown in maltose-containing medium.}, }
@article {pmid32277804, year = {2020}, author = {Nakayama, T and Grainger, RM and Cha, SW}, title = {Simple embryo injection of long single-stranded donor templates with the CRISPR/Cas9 system leads to homology-directed repair in Xenopus tropicalis and Xenopus laevis.}, journal = {Genesis (New York, N.Y. : 2000)}, volume = {58}, number = {6}, pages = {e23366}, doi = {10.1002/dvg.23366}, pmid = {32277804}, issn = {1526-968X}, support = {EY022954/NH/NIH HHS/United States ; K01 DK101618/DK/NIDDK NIH HHS/United States ; }, mesh = {Animals ; *CRISPR-Cas Systems ; DNA, Single-Stranded/chemistry/*genetics ; Gene Knock-In Techniques/*methods ; Green Fluorescent Proteins/genetics/metabolism ; Larva/metabolism ; Melanocytes/metabolism ; Membrane Transport Proteins/*genetics/metabolism ; Phosphorothioate Oligonucleotides/chemistry/genetics ; *Recombinational DNA Repair ; Skin Pigmentation ; Xenopus laevis ; Zygote/metabolism ; }, abstract = {We report model experiments in which simple microinjection of fertilized eggs has been used to effectively perform homology-directed repair (HDR)-mediated gene editing in the two Xenopus species used most frequently for research: X. tropicalis and X. laevis. We have used long single-stranded DNAs having phosphorothioate modifications as donor templates for HDR at targeted genomic sites using the Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system. First, X. tropicalis tyr mutant (i.e., albino) embryos were successfully rescued: partially pigmented tadpoles were seen in up to 35% of injected embryos, demonstrating the potential for efficient insertion of targeted point mutations. Second, in order to demonstrate the ability to tag genes with fluorescent proteins (FPs), we targeted the melanocyte-specific gene slc45a2.L of X. laevis to label it with the Superfolder green FP (sfGFP), seeing mosaic expression of sfGFP in melanophores in up to 20% of injected tadpoles. Tadpoles generated by these two approaches were raised to sexual maturity, and shown to successfully transmit HDR constructs through the germline with precise targeting and seamless recombination. F1 embryos showed rescue of the tyr mutation (X. tropicalis) and tagging in the appropriate pigment cell-specific manner of slc45a2.L with sfGFP (X. laevis).}, }
@article {pmid32175691, year = {2020}, author = {Choudhury, A and Fenster, JA and Fankhauser, RG and Kaar, JL and Tenaillon, O and Gill, RT}, title = {CRISPR/Cas9 recombineering-mediated deep mutational scanning of essential genes in Escherichia coli.}, journal = {Molecular systems biology}, volume = {16}, number = {3}, pages = {e9265}, pmid = {32175691}, issn = {1744-4292}, mesh = {CRISPR-Cas Systems ; DNA-Directed RNA Polymerases/genetics ; Escherichia coli/*genetics ; Escherichia coli Proteins/genetics ; *Genes, Essential ; Genetic Engineering/*methods ; *Mutation ; RNA, Guide/pharmacology ; Recombination, Genetic ; }, abstract = {Deep mutational scanning can provide significant insights into the function of essential genes in bacteria. Here, we developed a high-throughput method for mutating essential genes of Escherichia coli in their native genetic context. We used Cas9-mediated recombineering to introduce a library of mutations, created by error-prone PCR, within a gene fragment on the genome using a single gRNA pre-validated for high efficiency. Tracking mutation frequency through deep sequencing revealed biases in the position and the number of the introduced mutations. We overcame these biases by increasing the homology arm length and blocking mismatch repair to achieve a mutation efficiency of 85% for non-essential genes and 55% for essential genes. These experiments also improved our understanding of poorly characterized recombineering process using dsDNA donors with single nucleotide changes. Finally, we applied our technology to target rpoB, the beta subunit of RNA polymerase, to study resistance against rifampicin. In a single experiment, we validate multiple biochemical and clinical observations made in the previous decades and provide insights into resistance compensation with the study of double mutants.}, }
@article {pmid32002787, year = {2020}, author = {Loi, M and Trazzi, S and Fuchs, C and Galvani, G and Medici, G and Gennaccaro, L and Tassinari, M and Ciani, E}, title = {Increased DNA Damage and Apoptosis in CDKL5-Deficient Neurons.}, journal = {Molecular neurobiology}, volume = {57}, number = {5}, pages = {2244-2262}, pmid = {32002787}, issn = {1559-1182}, mesh = {Animals ; *Apoptosis/drug effects ; Brain-Derived Neurotrophic Factor/pharmacology ; CRISPR-Cas Systems ; Cell Division ; Cell Line, Tumor ; Cells, Cultured ; *DNA Damage ; Epileptic Syndromes/*genetics/pathology ; Gene Editing ; Hippocampus/cytology ; Histones/analysis ; Humans ; Hydrogen Peroxide/pharmacology ; Kainic Acid/pharmacology ; MAP Kinase Signaling System ; Mice, Inbred C57BL ; Mice, Knockout ; Neurons/drug effects/metabolism/*pathology ; Protein-Serine-Threonine Kinases/*deficiency ; Proto-Oncogene Proteins c-akt ; Signal Transduction ; Spasms, Infantile/*genetics/pathology ; Tretinoin/pharmacology ; }, abstract = {Mutations in the CDKL5 gene, which encodes a serine/threonine kinase, causes a rare encephalopathy, characterized by early-onset epilepsy and severe intellectual disability, named CDKL5 deficiency disorder (CDD). In vitro and in vivo studies in mouse models of Cdkl5 deficiency have highlighted the role of CDKL5 in brain development and, in particular, in the morphogenesis and synaptic connectivity of hippocampal and cortical neurons. Interestingly, Cdkl5 deficiency in mice increases vulnerability to excitotoxic stress in hippocampal neurons. However, the mechanism by which CDKL5 controls neuronal survival is far from being understood. To investigate further the function of CDKL5 and dissect the molecular mechanisms underlying neuronal survival, we generated a human neuronal model of CDKL5 deficiency, using CRISPR/Cas9-mediated genome editing. We demonstrated that CDKL5 deletion in human neuroblastoma SH-SY5Y cells not only impairs neuronal maturation but also reduces cell proliferation and survival, with alterations in the AKT and ERK signaling pathways and an increase in the proapoptotic BAX protein and in DNA damage-associated biomarkers (i.e., γH2AX, RAD50, and PARP1). Furthermore, CDKL5-deficient cells were hypersensitive to DNA damage-associated stress, accumulated more DNA damage foci (γH2AX positive) and were more prone to cell death than the controls. Importantly, increased kainic acid-induced cell death of hippocampal neurons of Cdkl5 KO mice correlated with an increased γH2AX immunostaining. The results suggest a previously unknown role for CDKL5 in DNA damage response that could underlie the pro-survival function of CDKL5.}, }
@article {pmid33529861, year = {2021}, author = {Sheng, Y and Zhang, T and Zhang, S and Johnston, M and Zheng, X and Shan, Y and Liu, T and Huang, Z and Qian, F and Xie, Z and Ai, Y and Zhong, H and Kuang, T and Dincer, C and Urban, GA and Hu, J}, title = {A CRISPR/Cas13a-powered catalytic electrochemical biosensor for successive and highly sensitive RNA diagnostics.}, journal = {Biosensors &amp; bioelectronics}, volume = {178}, number = {}, pages = {113027}, doi = {10.1016/j.bios.2021.113027}, pmid = {33529861}, issn = {1873-4235}, abstract = {Rapid and specific quantitation of a variety of RNAs with low expression levels in early-stage cancer is highly desirable but remains a challenge. Here, we present a dual signal amplification strategy consisting of the CRISPR/Cas13a system and a catalytic hairpin DNA circuit (CHDC), integrated on a reusable electrochemical biosensor for rapid and accurate detection of RNAs. Signal amplification is accomplished through the unique combination of the CRISPR/Cas13a system with CHDC, achieving a limit of detection of 50 aM within a readout time of 6 min and an overall process time of 36 min, using a measuring volume of 10 μL. Enzymatic regeneration of the sensor surface and ratiometric correction of background signal allow up to 37 sequential RNA quantifications by square-wave voltammetry on a single biosensor chip without loss of sensitivity. The reusable biosensor platform could selectively (specificity = 0.952) and sensitively (sensitivity = 0.900) identify low expression RNA targets in human serum, distinguishing early-stage patients (n = 20) suffering from non-small-cell lung carcinoma (NSCLC) from healthy subjects (n = 30) and patients with benign lung disease (n = 12). Measurement of six NSCLC-related RNAs (miR-17, miR-155, TTF-1 mRNA, miR-19b, miR-210 and EGFR mRNA) shows the ability of the electrochemical CRISPR/CHDC system to be a fast, low-cost and highly accurate tool for early cancer diagnostics.}, }
@article {pmid33529466, year = {2021}, author = {Sweat, M and Sweat, Y and Yu, W and Su, D and Leonard, RJ and Eliason, SL and Amendt, BA}, title = {The miR-200 family is required for ectodermal organ development through the regulation of the epithelial stem cell niche.}, journal = {Stem cells (Dayton, Ohio)}, volume = {}, number = {}, pages = {}, doi = {10.1002/stem.3342}, pmid = {33529466}, issn = {1549-4918}, abstract = {The murine lower incisor ectodermal organ contains a single epithelial stem cell (SC) niche that provides epithelial progenitor cells to the continuously growing rodent incisor. The dental stem cell niche gives rise to several cell types and we demonstrate that the miR-200 family regulates these cell fates. The miR-200 family is highly enriched in the differentiated dental epithelium and absent in the stem cell niche. In this study, we inhibited the miR-200 family in developing murine embryos using new technology, resulting in an expanded epithelial stem cell niche and lack of cell differentiation. Inhibition of individual miRs within the miR-200 cluster resulted in differential developmental and cell morphology defects. miR-200 inhibition increased the expression of dental epithelial stem cell markers, expanded the stem cell niche and decreased progenitor cell differentiation. RNA-seq. identified miR-200 regulatory pathways involved in cell differentiation and compartmentalization of the stem cell niche. The miR-200 family regulates signaling pathways required for cell differentiation and cell cycle progression. The inhibition of miR-200 decreased the size of the lower incisor due to increased autophagy and cell death. New miR-200 targets demonstrate gene networks and pathways controlling cell differentiation and maintenance of the stem cell niche. This is the first report demonstrating how the miR-200 family is required for in vivo progenitor cell proliferation and differentiation. © AlphaMed Press 2021 SIGNIFICANCE STATEMENT: Current microRNA (miR) inhibition methods cannot be used to study in vivo developmental stem cell processes. CRISPR-Cas genome editing cannot specifically knockout a miR within a cluster. Furthermore, not all miRs, especially within introns can be targeted by the CRISPR method without affecting gene expression. ES cells have been profiled for miR expression; however, cell-based assays using oligonucleotides targeting miRs are not specific and are toxic. The authors developed a highly specific, effective miR inhibitor that can be used to knockdown miRs during embryonic development to determine their effect on stem cells, cell proliferation, and differentiation. The authors show that the miR-200 family acts to compartmentalize an ectodermal stem cell niche by regulating progenitor cell differentiation during development.}, }
@article {pmid33528277, year = {2021}, author = {Suryaletha, K and Chandrika, SK and Thomas, S}, title = {Comprehensive genomics depict accessory genes encoding pathogenicity and biofilm determinants in Enterococcus faecalis.}, journal = {Future microbiology}, volume = {}, number = {}, pages = {}, doi = {10.2217/fmb-2020-0111}, pmid = {33528277}, issn = {1746-0921}, abstract = {Aim:Enterococcus faecalis is a leading nosocomial pathogen in biofilm-associated polymicrobial infections. The study aims to understand pathogenicity and biofilm determinants of the pathogen by genome analysis. Methodology: Genome sequencing of a strong biofilm forming clinical isolate E. faecalis SK460 devoid of Fsr quorum-signaling system, was performed and comparative genomics was carried out among a set of pathogenic biofilm formers and nonpathogenic weak biofilm formers. Results: Analysis revealed a pool of virulence and adhesion related factors associated with pathogenicity. Absence of CRISPR-Cas system facilitated acquisition of pheromone responsive plasmid, pathogenicity island and phages. Comprehensive analysis identified a subset of accessory genes encoding polysaccharide lyase, sugar phosphotransferase system, phage proteins and transcriptional regulators exclusively in pathogenic biofilm formers. Conclusion: The study identified a set of genes specific to pathogenic biofilm formers and these can act as targets which in turn help to develop future treatment endeavors against enterococcal infections.}, }
@article {pmid33525825, year = {2018}, author = {Zhang, Y and Lin, J and Feng, M and She, Q}, title = {Molecular mechanisms of III-B CRISPR-Cas systems in archaea.}, journal = {Emerging topics in life sciences}, volume = {2}, number = {4}, pages = {483-491}, doi = {10.1042/ETLS20180023}, pmid = {33525825}, issn = {2397-8554}, abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) systems provide the adaptive antiviral immunity against invasive genetic elements in archaea and bacteria. These immune systems are divided into at least six different types, among which Type III CRISPR-Cas systems show several distinct antiviral activities as demonstrated from the investigation of bacterial III-A and archaeal III-B systems in the past decade. First, although initial experiments suggested that III-A systems provided DNA interference activity, whereas III-B system was active only in RNA interference, these immune systems were subsequently found to mediate the transcription-dependent DNA interference and the dual DNA/RNA interference. Second, their ribonucleoprotein (RNP) complexes show target RNA (tgRNA) cleavage by a ruler mechanism and RNA-activated indiscriminate single-stranded DNA cleavage, the latter of which is subjected to spatiotemporal regulation such that the DNase activity occurs only at the right place in the right time. Third, RNPs of Type III systems catalyse the synthesis of cyclic oligoadenylates (cOAs) that function as second messengers to activate Csm6 and Csx1, both of which are potent Cas accessory RNases after activation. To date, Type III CRISPR systems are the only known antiviral immunity that utilizes multiple interference mechanisms for antiviral defence.}, }
@article {pmid33525759, year = {2017}, author = {Van Eck, J}, title = {Gene editing in tomatoes.}, journal = {Emerging topics in life sciences}, volume = {1}, number = {2}, pages = {183-191}, doi = {10.1042/ETLS20170056}, pmid = {33525759}, issn = {2397-8554}, abstract = {Tomato is an effective model plant species because it possesses the qualities necessary for genetic and functional studies, but is also a food crop making what is learned more translatable for crop improvement when compared with other non-food crop models. The availability of genome sequences for many genotypes and amenability to transformation methodologies (Agrobacterium-mediated, direct DNA uptake via protoplasts, biolistics) make tomato the perfect platform to study the application of gene-editing technologies. This review includes information related to tomato transformation methodology, one of the necessary requirements for gene editing, along with the status of site-directed mutagenesis by TALENs (transcription activator-like effector nucleases) and CRISPR/Cas (clustered regularly interspaced short palindromic repeats and CRISPR-associated Proteins). In addition to the reports on proof-of-concept experiments to demonstrate the feasibility of gene editing in tomato, there are many reports that show the power of these technologies for modification of traits, such as fruit characteristics (ripening, size, and parthenocarpy), pathogen susceptibility, architecture (plant and inflorescence), and metabolic engineering. Also highlighted in this review are reports on the application of a recent CRISPR technology called base editing that allows the modification of one base pair in a gene sequence and a strategy that takes advantage of a geminivirus replicon for delivery of DNA repair template.}, }
@article {pmid33378644, year = {2021}, author = {Clarke, R and Terry, AR and Pennington, H and Hasty, C and MacDougall, MS and Regan, M and Merrill, BJ}, title = {Sequential Activation of Guide RNAs to Enable Successive CRISPR-Cas9 Activities.}, journal = {Molecular cell}, volume = {81}, number = {2}, pages = {226-238.e5}, doi = {10.1016/j.molcel.2020.12.003}, pmid = {33378644}, issn = {1097-4164}, support = {R21 OD027080/OD/NIH HHS/United States ; F30 CA225058/CA/NCI NIH HHS/United States ; F30 HD090938/HD/NICHD NIH HHS/United States ; }, mesh = {Animals ; Base Pairing ; Base Sequence ; CRISPR-Associated Protein 9/*genetics/metabolism ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing/*methods ; Genes, Reporter ; Green Fluorescent Proteins/genetics/metabolism ; HEK293 Cells ; Humans ; Mice ; Mouse Embryonic Stem Cells/cytology/metabolism ; Nucleic Acid Conformation ; Plasmids/chemistry/metabolism ; Promoter Regions, Genetic ; RNA, Guide/chemistry/*genetics/metabolism ; Streptococcus pyogenes/chemistry/enzymology ; }, abstract = {Currently, either highly multiplexed genetic manipulations can be delivered to mammalian cells all at once or extensive engineering of gene regulatory sequences can be used to conditionally activate a few manipulations. Here, we provide proof of principle for a new system enabling multiple genetic manipulations to be executed as a preprogrammed cascade of events. The system leverages the programmability of the S. pyogenes Cas9 and is based on flexible arrangements of individual modules of activity. The basic module consists of an inactive single-guide RNA (sgRNA)-like component that is converted to an active state through the effects of another sgRNA. Modules can be arranged to bring about an algorithmic program of sequential genetic manipulations without the need for engineering cell-type-specific promoters or gene regulatory sequences. With the expanding diversity of available tools that use spCas9, this sgRNA-based system provides multiple levels of interfacing with mammalian cell biology.}, }
@article {pmid33303593, year = {2020}, author = {Kaiser, J}, title = {Tweaking genes with CRISPR or viruses fixes blood disorders.}, journal = {Science (New York, N.Y.)}, volume = {370}, number = {6522}, pages = {1254-1255}, doi = {10.1126/science.370.6522.1254}, pmid = {33303593}, issn = {1095-9203}, mesh = {Anemia, Sickle Cell/genetics/*therapy ; *CRISPR-Cas Systems ; Gene Editing/*methods ; Genetic Therapy/*methods ; Humans ; Repressor Proteins/*genetics ; *Viruses/genetics ; beta-Thalassemia/genetics/*therapy ; }, }
@article {pmid33303588, year = {2020}, author = {Muller, R and Meacham, ZA and Ferguson, L and Ingolia, NT}, title = {CiBER-seq dissects genetic networks by quantitative CRISPRi profiling of expression phenotypes.}, journal = {Science (New York, N.Y.)}, volume = {370}, number = {6522}, pages = {}, pmid = {33303588}, issn = {1095-9203}, support = {DP2 CA195768/CA/NCI NIH HHS/United States ; R01 GM130996/GM/NIGMS NIH HHS/United States ; R01 GM135233/GM/NIGMS NIH HHS/United States ; S10 OD018174/OD/NIH HHS/United States ; }, mesh = {Alcohol Oxidoreductases/genetics ; Aminohydrolases/genetics ; *CRISPR-Associated Protein 9 ; *CRISPR-Cas Systems ; Eukaryotic Initiation Factor-2/metabolism ; *Gene Expression ; Gene Expression Profiling/*methods ; *Gene Regulatory Networks ; Phenotype ; Phosphorylation ; Protein-Serine-Threonine Kinases/metabolism ; Pyrophosphatases/genetics ; RNA, Guide/genetics ; RNA, Transfer/genetics/metabolism ; Saccharomyces cerevisiae/*genetics ; Saccharomyces cerevisiae Proteins/genetics/metabolism ; }, abstract = {To realize the promise of CRISPR-Cas9-based genetics, approaches are needed to quantify a specific, molecular phenotype across genome-wide libraries of genetic perturbations. We addressed this challenge by profiling transcriptional, translational, and posttranslational reporters using CRISPR interference (CRISPRi) with barcoded expression reporter sequencing (CiBER-seq). Our barcoding approach allowed us to connect an entire library of guides to their individual phenotypic consequences using pooled sequencing. CiBER-seq profiling fully recapitulated the integrated stress response (ISR) pathway in yeast. Genetic perturbations causing uncharged transfer RNA (tRNA) accumulation activated ISR reporter transcription. Notably, tRNA insufficiency also activated the reporter, independent of the uncharged tRNA sensor. By uncovering alternate triggers for ISR activation, we illustrate how precise, comprehensive CiBER-seq profiling provides a powerful and broadly applicable tool for dissecting genetic networks.}, }
@article {pmid33301730, year = {2021}, author = {Lan, X and Ren, R and Feng, R and Ly, LC and Lan, Y and Zhang, Z and Aboreden, N and Qin, K and Horton, JR and Grevet, JD and Mayuranathan, T and Abdulmalik, O and Keller, CA and Giardine, B and Hardison, RC and Crossley, M and Weiss, MJ and Cheng, X and Shi, J and Blobel, GA}, title = {ZNF410 Uniquely Activates the NuRD Component CHD4 to Silence Fetal Hemoglobin Expression.}, journal = {Molecular cell}, volume = {81}, number = {2}, pages = {239-254.e8}, doi = {10.1016/j.molcel.2020.11.006}, pmid = {33301730}, issn = {1097-4164}, support = {R24 DK106766/DK/NIDDK NIH HHS/United States ; R01 HL119479/HL/NHLBI NIH HHS/United States ; R35 GM134744/GM/NIGMS NIH HHS/United States ; }, mesh = {Animals ; Binding Sites ; COS Cells ; CRISPR-Cas Systems ; Chlorocebus aethiops ; DNA/*genetics/metabolism ; Erythroid Precursor Cells/cytology/*metabolism/transplantation ; Fetal Blood/cytology/metabolism ; Fetal Hemoglobin/*genetics/metabolism ; Fetus ; Gene Editing ; HEK293 Cells ; Heterografts ; Humans ; Mi-2 Nucleosome Remodeling and Deacetylase Complex/chemistry/*genetics/metabolism ; Mice ; Models, Molecular ; Mouse Embryonic Stem Cells/cytology ; Protein Binding ; Protein Conformation, alpha-Helical ; Protein Conformation, beta-Strand ; Protein Interaction Domains and Motifs ; Transcription Factors/chemistry/*genetics/metabolism ; Transcriptional Activation ; }, abstract = {Metazoan transcription factors typically regulate large numbers of genes. Here we identify via a CRISPR-Cas9 genetic screen ZNF410, a pentadactyl DNA-binding protein that in human erythroid cells directly activates only a single gene, the NuRD component CHD4. Specificity is conveyed by two highly evolutionarily conserved clusters of ZNF410 binding sites near the CHD4 gene with no counterparts elsewhere in the genome. Loss of ZNF410 in adult-type human erythroid cell culture systems and xenotransplantation settings diminishes CHD4 levels and derepresses the fetal hemoglobin genes. While previously known to be silenced by CHD4, the fetal globin genes are exposed here as among the most sensitive to reduced CHD4 levels.. In vitro DNA binding assays and crystallographic studies reveal the ZNF410-DNA binding mode. ZNF410 is a remarkably selective transcriptional activator in erythroid cells, and its perturbation might offer new opportunities for treatment of hemoglobinopathies.}, }
@article {pmid33162067, year = {2021}, author = {Edick, AM and Audette, J and Burgos, SA}, title = {CRISPR-Cas9-mediated knockout of GCN2 reveals a critical role in sensing amino acid deprivation in bovine mammary epithelial cells.}, journal = {Journal of dairy science}, volume = {104}, number = {1}, pages = {1123-1135}, doi = {10.3168/jds.2020-18700}, pmid = {33162067}, issn = {1525-3198}, mesh = {Amino Acids/*metabolism ; Animals ; CRISPR-Cas Systems ; Cattle/genetics/*metabolism ; Epithelial Cells/metabolism ; Eukaryotic Initiation Factor-2/genetics/metabolism ; Female ; Gene Knockout Techniques ; Lactation ; Mammary Glands, Animal/*metabolism ; Methionine/metabolism ; Phosphorylation ; Protein-Serine-Threonine Kinases/genetics/*metabolism ; Up-Regulation ; }, abstract = {The objective of this study was to determine the role of GCN2 in the response to AA deprivation of primary bovine mammary epithelial cells (BMEC). Cells were isolated from the mammary tissue of 2 lactating Holstein cows by enzymatic digestion, expanded, and induced to differentiate for 5 to 7 d. Relative mRNA expression was measured by real-time quantitative PCR. Protein abundance and site-specific phosphorylation were measured by immunoblotting. Knockout of GCN2 in BMEC was accomplished by lentiviral delivery of a targeted single guide RNA and endonuclease Cas9. To investigate the role of GCN2, we treated lactogenic differentiated BMEC with either culture medium lacking Arg, Leu, and Lys combined or lacking only one of the 3 AA of interest, in comparison to a control with a full complement of AA. Activation of GCN2 was inferred by the phosphorylation status of its downstream target eIF2α Ser51. We found that GCN2 was activated by both the deprivation of Arg, Leu, and Lys combined and of Arg alone, as shown by a 2.73- and 2.82-fold increase in phosphorylated eIF2α Ser51 after 1 h of deprivation, respectively. In addition, activation of GCN2 as measured by increased phosphorylation of eIF2α Ser51 during the deprivation of Arg, Leu, and Lys combined and of Arg alone was sustained for up to 8 h of deprivation. Phosphorylated eIF2α selectively upregulates translation of transcription factor ATF4, among others, during AA deprivation, which then targets genes necessary for restoring AA homeostasis. Therefore, we investigated the expression of ATF4 transcriptional targets, AA enzyme ASNS and AA transporters SLC7A1 and SLC38A2. We found that ASNS was upregulated in response to combined AA deprivation and by Arg deprivation alone by 3.6- and 4.51-fold, respectively, at 24 h of treatment. We found that SLC7A1 was upregulated in response to combined AA deprivation and deprivation of Arg alone by 2.0- and 2.36-fold, respectively, at 8 h of treatment. To establish the role of GCN2 (encoded by EIF2AK4) in the response to AA deprivation, we ablated GCN2 in BMEC using clustered regularly interspaced short palindromic repeats-Cas9. We showed that BMEC transduced with single guide RNA targeting EIF2AK4 were not as responsive to combined AA deprivation, compared with BMEC transduced with nontargeting single guide RNA. Taken together, our results demonstrate a critical role for GCN2 in sensing AA deprivation in BMEC.}, }
@article {pmid33080227, year = {2020}, author = {Xing, L and Kalebic, N and Namba, T and Vaid, S and Wimberger, P and Huttner, WB}, title = {Serotonin Receptor 2A Activation Promotes Evolutionarily Relevant Basal Progenitor Proliferation in the Developing Neocortex.}, journal = {Neuron}, volume = {108}, number = {6}, pages = {1113-1129.e6}, doi = {10.1016/j.neuron.2020.09.034}, pmid = {33080227}, issn = {1097-4199}, mesh = {Animals ; CRISPR-Cas Systems ; Cell Proliferation/drug effects/*physiology ; Ferrets ; Gene Expression Regulation, Developmental ; Lateral Ventricles/*cytology/drug effects/metabolism ; Mice ; Neocortex/*cytology/drug effects/metabolism ; Neural Stem Cells/*cytology/drug effects/metabolism ; Neurogenesis/drug effects/physiology ; Receptor, Serotonin, 5-HT2A/genetics/*metabolism ; Serotonin/pharmacology ; }, abstract = {Evolutionary expansion of the mammalian neocortex (Ncx) has been linked to increased abundance and proliferative capacity of basal progenitors (BPs) in the subventricular zone during development. BP proliferation is governed by both intrinsic and extrinsic signals, several of which have been identified. However, a role of neurotransmitters, a canonical class of extrinsic signaling molecules, in BP proliferation remains to be established. Here, we show that serotonin (5-HT), via its receptor HTR2A, promotes BP proliferation in an evolutionarily relevant manner. HTR2A is not expressed in embryonic mouse Ncx; accordingly, 5-HT does not increase mouse BP proliferation. However, ectopic HTR2A expression can increase mouse BP proliferation. Conversely, CRISPR/Cas9-mediated knockout of endogenous HTR2A in embryonic ferret Ncx reduces BP proliferation. Pharmacological activation of endogenous HTR2A in fetal human Ncx ex vivo increases BP proliferation via HER2/ERK signaling. Hence, 5-HT emerges as an important extrinsic pro-proliferative signal for BPs, which may have contributed to evolutionary Ncx expansion.}, }
@article {pmid32943441, year = {2020}, author = {Bowen, C and Shibata, M and Zhang, H and Bergren, SK and Shen, MM and Gelmann, EP}, title = {CRISPR/Cas9-Mediated Point Mutation in Nkx3.1 Prolongs Protein Half-Life and Reverses Effects Nkx3.1 Allelic Loss.}, journal = {Cancer research}, volume = {80}, number = {21}, pages = {4805-4814}, pmid = {32943441}, issn = {1538-7445}, support = {K99 CA194287/CA/NCI NIH HHS/United States ; P01 CA154293/CA/NCI NIH HHS/United States ; P30 CA013696/CA/NCI NIH HHS/United States ; R01 CA238005/CA/NCI NIH HHS/United States ; P30 CA023074/CA/NCI NIH HHS/United States ; }, mesh = {Animals ; *CRISPR-Cas Systems ; Gene Editing/*methods ; Homeodomain Proteins/*genetics ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Mutant Strains ; Point Mutation ; Prostatic Neoplasms/*genetics/pathology ; Transcription Factors/*genetics ; }, abstract = {NKX3.1 is the most commonly deleted gene in prostate cancer and is a gatekeeper suppressor. NKX3.1 is haploinsufficient, and pathogenic reduction in protein levels may result from genetic loss, decreased transcription, and increased protein degradation caused by inflammation or PTEN loss. NKX3.1 acts by retarding proliferation, activating antioxidants, and enhancing DNA repair. DYRK1B-mediated phosphorylation at serine 185 of NKX3.1 leads to its polyubiquitination and proteasomal degradation. Because NKX3.1 protein levels are reduced, but never entirely lost, in prostate adenocarcinoma, enhancement of NKX3.1 protein levels represents a potential therapeutic strategy. As a proof of principle, we used CRISPR/Cas9-mediated editing to engineer in vivo a point mutation in murine Nkx3.1 to code for a serine to alanine missense at amino acid 186, the target for Dyrk1b phosphorylation. Nkx3.1S186A/-, Nkx3.1+/- , and Nkx3.1+/+ mice were analyzed over one year to determine the levels of Nkx3.1 expression and effects of the mutant protein on the prostate. Allelic loss of Nkx3.1 caused reduced levels of Nkx3.1 protein, increased proliferation, and prostate hyperplasia and dysplasia, whereas Nkx3.1S186A/- mouse prostates had increased levels of Nkx3.1 protein, reduced prostate size, normal histology, reduced proliferation, and increased DNA end labeling. At 2 months of age, when all mice had normal prostate histology, Nkx3.1+/- mice demonstrated indices of metabolic activation, DNA damage response, and stress response. These data suggest that modulation of Nkx3.1 levels alone can exert long-term control over premalignant changes and susceptibility to DNA damage in the prostate. SIGNIFICANCE: These findings show that prolonging the half-life of Nkx3.1 reduces proliferation, enhances DNA end-labeling, and protects from DNA damage, ultimately blocking the proneoplastic effects of Nkx3.1 allelic loss.}, }
@article {pmid32543746, year = {2020}, author = {McCann, JV and Bischoff, SR and Zhang, Y and Cowley, DO and Sanchez-Gonzalez, V and Daaboul, GD and Dudley, AC}, title = {Reporter mice for isolating and auditing cell type-specific extracellular vesicles in vivo.}, journal = {Genesis (New York, N.Y. : 2000)}, volume = {58}, number = {7}, pages = {e23369}, pmid = {32543746}, issn = {1526-968X}, support = {F31 CA213793/CA/NCI NIH HHS/United States ; RO1-CA177874/CA/NCI NIH HHS/United States ; R01 CA177875/CA/NCI NIH HHS/United States ; P30 CA044579/CA/NCI NIH HHS/United States ; }, mesh = {Animals ; CRISPR-Cas Systems ; Cells, Cultured ; Endothelium, Vascular/drug effects/metabolism ; Extracellular Vesicles/*metabolism ; Gene Knock-In Techniques/*methods ; Green Fluorescent Proteins/genetics/metabolism ; HEK293 Cells ; Humans ; Integrases/genetics/metabolism ; Mice ; Mice, Inbred C57BL ; Tetraspanin 30/*genetics/metabolism ; }, abstract = {Extracellular vesicles (EVs) are abundant, lipid-enclosed vectors that contain nucleic acids and proteins, they can be secreted from donor cells and freely circulate, and they can be engulfed by recipient cells thus enabling systemic communication between heterotypic cell types. However, genetic tools for labeling, isolating, and auditing cell type-specific EVs in vivo, without prior in vitro manipulation, are lacking. We have used CRISPR-Cas9-mediated genome editing to generate mice bearing a CD63-emGFPloxP/stop/loxP knock-in cassette that enables the specific labeling of circulating CD63+ vesicles from any cell type when crossed with lineage-specific Cre recombinase driver mice. As proof-of-principle, we have crossed these mice with Cdh5-CreERT2 mice to generate CD63emGFP+ vasculature. Using these mice, we show that developing vasculature is marked with emerald GFP (emGFP) following tamoxifen administration to pregnant females. In adult mice, quiescent vasculature and angiogenic vasculature (in tumors) is also marked with emGFP. Moreover, whole plasma-purified EVs contain a subpopulation of emGFP+ vesicles that are derived from the endothelium, co-express additional EV (e.g., CD9 and CD81) and endothelial cell (e.g., CD105) markers, and they harbor specific miRNAs (e.g., miR-126, miR-30c, and miR-125b). This new mouse strain should be a useful genetic tool for generating cell type-specific, CD63+ EVs that freely circulate in serum and can subsequently be isolated and characterized using standard methodologies.}, }
@article {pmid32499184, year = {2021}, author = {Oliver, JD and Duscher, D and Hu, MS}, title = {Engineering a Future with VCA: Applying Genetic Circuits to Engineer Tissues for Vascularized Composite Allotransplantation.}, journal = {Journal of plastic, reconstructive &amp; aesthetic surgery : JPRAS}, volume = {74}, number = {1}, pages = {223-243}, doi = {10.1016/j.bjps.2020.05.056}, pmid = {32499184}, issn = {1878-0539}, mesh = {CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; Humans ; Immunity/genetics ; *Immunosuppression ; *Vascularized Composite Allotransplantation ; }, }
@article {pmid32487115, year = {2020}, author = {Wu, X and Ma, W and Mei, C and Chen, X and Yao, Y and Liu, Y and Qin, X and Yuan, Y}, title = {Description of CRISPR/Cas9 development and its prospect in hepatocellular carcinoma treatment.}, journal = {Journal of experimental &amp; clinical cancer research : CR}, volume = {39}, number = {1}, pages = {97}, pmid = {32487115}, issn = {1756-9966}, support = {ChiCTR1900025427//Chinese Clinical Trial Registry/ ; }, mesh = {Animals ; *CRISPR-Cas Systems ; Carcinoma, Hepatocellular/genetics/pathology/*therapy ; *Gene Editing ; *Genetic Therapy ; Humans ; Liver Neoplasms/genetics/pathology/*therapy ; Neoplasm Proteins/antagonists &amp; inhibitors/*genetics ; }, abstract = {Hepatocellular carcinoma (HCC) is one of the most common malignancies today. Patients suffer from HCC since its high malignancy and limited treatment means. With the development of genetic research, new therapeutic strategy comes up in the way of gene editing. Clustered regularly interspaced short palindromic repeat/CRISPR-associated nuclease 9 (CRISPR/Cas9) was discovered as an immune sequence in bacteria and archaea. After artificial transformation and follow-up research, it is widely used as a gene editing tool. In this review, the development of CRISPR/Cas9 is summarized in retrospect. Through the evaluation of novel research in HCC, it is concluded that CRISPR/Cas9 would promote cancer research and provide a new tool for genetic treatment in prospect.}, }
@article {pmid32453716, year = {2020}, author = {Latour, BL and Van De Weghe, JC and Rusterholz, TD and Letteboer, SJ and Gomez, A and Shaheen, R and Gesemann, M and Karamzade, A and Asadollahi, M and Barroso-Gil, M and Chitre, M and Grout, ME and van Reeuwijk, J and van Beersum, SE and Miller, CV and Dempsey, JC and Morsy, H and ,  and Bamshad, MJ and ,  and Nickerson, DA and Neuhauss, SC and Boldt, K and Ueffing, M and Keramatipour, M and Sayer, JA and Alkuraya, FS and Bachmann-Gagescu, R and Roepman, R and Doherty, D}, title = {Dysfunction of the ciliary ARMC9/TOGARAM1 protein module causes Joubert syndrome.}, journal = {The Journal of clinical investigation}, volume = {130}, number = {8}, pages = {4423-4439}, pmid = {32453716}, issn = {1558-8238}, support = {F32 HD095599/HD/NICHD NIH HHS/United States ; U54 HD083091/HD/NICHD NIH HHS/United States ; U54 HG006493/HG/NHGRI NIH HHS/United States ; /DH_/Department of Health/United Kingdom ; }, mesh = {*Abnormalities, Multiple/genetics/metabolism ; Acetylation ; Animals ; *Armadillo Domain Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Cerebellum/*abnormalities/metabolism ; *Cilia/genetics/metabolism ; Disease Models, Animal ; *Eye Abnormalities/genetics/metabolism ; Humans ; *Kidney Diseases, Cystic/genetics/metabolism ; Peptides/genetics/metabolism ; Retina/*abnormalities/metabolism ; *Zebrafish/genetics/metabolism ; *Zebrafish Proteins/genetics/metabolism ; }, abstract = {Joubert syndrome (JBTS) is a recessive neurodevelopmental ciliopathy characterized by a pathognomonic hindbrain malformation. All known JBTS genes encode proteins involved in the structure or function of primary cilia, ubiquitous antenna-like organelles essential for cellular signal transduction. Here, we used the recently identified JBTS-associated protein armadillo repeat motif-containing 9 (ARMC9) in tandem-affinity purification and yeast 2-hybrid screens to identify a ciliary module whose dysfunction underlies JBTS. In addition to the known JBTS-associated proteins CEP104 and CSPP1, we identified coiled-coil domain containing 66 (CCDC66) and TOG array regulator of axonemal microtubules 1 (TOGARAM1) as ARMC9 interaction partners. We found that TOGARAM1 variants cause JBTS and disrupt TOGARAM1 interaction with ARMC9. Using a combination of protein interaction analyses, characterization of patient-derived fibroblasts, and analysis of CRISPR/Cas9-engineered zebrafish and hTERT-RPE1 cells, we demonstrated that dysfunction of ARMC9 or TOGARAM1 resulted in short cilia with decreased axonemal acetylation and polyglutamylation, but relatively intact transition zone function. Aberrant serum-induced ciliary resorption and cold-induced depolymerization in ARMC9 and TOGARAM1 patient cell lines suggest a role for this new JBTS-associated protein module in ciliary stability.}, }
@article {pmid32336701, year = {2020}, author = {Lin, J and Li, Z and Feng, Z and Fang, Z and Chen, J and Chen, W and Liang, W and Chen, Q}, title = {Pseudorabies virus (PRV) strain with defects in gE, gC, and TK genes protects piglets against an emerging PRV variant.}, journal = {The Journal of veterinary medical science}, volume = {82}, number = {6}, pages = {846-855}, pmid = {32336701}, issn = {1347-7439}, mesh = {Animals ; CRISPR-Cas Systems ; Gene Deletion ; Genes, Viral ; HEK293 Cells ; Herpesvirus 1, Suid/*genetics/*immunology ; Humans ; Immunization ; Injections, Intramuscular ; Mice, Inbred BALB C ; Pseudorabies/*prevention &amp; control/virology ; Pseudorabies Vaccines/*administration &amp; dosage/immunology ; Swine ; Swine Diseases/*prevention &amp; control/virology ; Vaccines, Attenuated ; }, abstract = {The prevalence of an emerging variant of the pseudorabies virus (PRV) has been causing serious losses to farmers in China. Moreover, the commercially available PRV vaccine often fails to provide thorough protection. Therefore, in this study, we generated a PRV-∆gC\gE∆TK strain with defects in gC, gE, and TK of PRV. Compared to the parental PRV strain and the single gene deletion strains (PRV-∆gC, PRV-∆gE, and PRV-∆TK), PRV-∆gC\gE∆TK grew slowly, and exhibited fewer and smaller plaques on swine testis (ST) cells. Furthermore, animal experiment results showed that mice that were immunized intramuscularly with PRV-∆gC\gE∆TK, survived throughout the experiment with no observed clinical symptoms, and were completely protected against PRV challenge. Additionally, deletion of the gC, gE, and TK genes significantly alleviated viral damage in the brain. Furthermore, one-day-old weaned piglets immunized intramuscularly with PRV-∆gC\gE∆TK elicited higher levels of gB antibodies against both the emerging PRV variant and the parental PRV, exhibited full protection against challenge with both variants, and showed neutralization capacity against PRV. These data suggest that PRV-∆gC\gE∆TK is a promising vaccine candidate for the control of pseudorabies.}, }
@article {pmid32277572, year = {2020}, author = {Li, C and Liu, H and Hu, YC and Lan, Y and Jiang, R}, title = {Generation and characterization of Six2 conditional mice.}, journal = {Genesis (New York, N.Y. : 2000)}, volume = {58}, number = {7}, pages = {e23365}, pmid = {32277572}, issn = {1526-968X}, support = {R01 DE027046/DE/NIDCR NIH HHS/United States ; }, mesh = {Animals ; CRISPR-Cas Systems ; Craniofacial Abnormalities/*genetics/pathology ; Female ; Gene Editing/methods ; Gene Targeting/methods ; Homeodomain Proteins/*genetics/metabolism ; Integrases/genetics/metabolism ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Transgenic ; Phenotype ; Transcription Factors/*genetics/metabolism ; Wnt1 Protein/genetics/metabolism ; }, abstract = {Heterozygous deletion of Six2, which encodes a member of sine oculis homeobox family transcription factors, has recently been associated with the frontonasal dysplasia syndrome FND4. Previous studies showed that Six2 is expressed in multiple tissues during craniofacial development in mice, including embryonic head mesoderm, postmigratory frontonasal neural crest cells, and epithelial and mesenchymal cells of the developing palate and nasal structures. Whereas Six2 -/- mice exhibited cranial base defects but did not recapitulate frontonasal phenotypes of FND4 patients, Six1 -/- Six2 -/- double mutant mice showed severe craniofacial defects including midline facial clefting. The complex phenotypes of FND4 patients and of Six1 -/- Six2 -/- mutant mice indicate that Six2 plays crucial roles in distinct cell types at multiple stages of craniofacial morphogenesis. Here we report generation of mice carrying insertions of a pair of loxP sites flanking exon-1 of the Six2 gene (Six2 f allele) using CRISPR/Cas9-mediated genome editing. We show that the Six2 f allele functions normally and is effectively inactivated by Cre-mediated recombination in vivo. Furthermore, we show that Six2 f/f ;Wnt1-Cre mice recapitulated cranial base defects but not neonatal lethality of Six2 -/- mice. These results indicate that Six2 f/f mice enable systematic investigation of cell type- and stage-specific Six2 function in development and disease.}, }
@article {pmid32078250, year = {2020}, author = {Beckermann, TM and Welch, RC and Williams, FM and Mortlock, DP and Sha, F and Ikizler, TA and Woodard, LE and Wilson, MH}, title = {CRISPR/Cas9 engineering of albino cystinuria Type A mice.}, journal = {Genesis (New York, N.Y. : 2000)}, volume = {58}, number = {5}, pages = {e23357}, pmid = {32078250}, issn = {1526-968X}, support = {I01 BX004258/BX/BLRD VA/United States ; IK2 BX002797/BX/BLRD VA/United States ; I01 BX002190/BX/BLRD VA/United States ; P30 CA068485/CA/NCI NIH HHS/United States ; P30 DK114809/DK/NIDDK NIH HHS/United States ; P30 DK020593/DK/NIDDK NIH HHS/United States ; R01 DK093660/DK/NIDDK NIH HHS/United States ; }, mesh = {Amino Acid Transport Systems, Basic/*genetics ; Amino Acid Transport Systems, Neutral/*genetics ; Animals ; CRISPR-Cas Systems ; Cysteine/urine ; Cystinuria/*genetics/pathology ; Disease Models, Animal ; Mice ; Mice, Inbred C57BL ; *Mutation ; }, abstract = {Cystinuria Type A is a relatively common genetic kidney disease occurring in 1 in 7,000 people worldwide that results from mutation of the cystine transporter rBAT encoded by Slc3a1. We used CRISPR/Cas9 technology to engineer cystinuria Type A mice via genome editing of the C57BL/6NHsd background. These mice are an improvement on currently available models as they are on a coisogenic genetic background and have a single defined mutation. In order to use albinism to track Cas9 activity, we co-injected gRNAs targeting Slc3a1 and tyrosinase (Tyr) with Cas9 expressing plasmid DNA into mouse embryos. Two different Slc3a1 mutational alleles were derived, with homozygous mice of both demonstrating elevated urinary cystine levels, cystine crystals, and bladder stones. We used whole genome sequencing to evaluate for potential off-target editing. No off-target indels were observed for the top 10 predicted off-targets for Slc3a1 or Tyr. Therefore, we used CRISPR/Cas9 to generate coisogenic albino cystinuria Type A mice that could be used for in vivo imaging, further study, or developing new treatments of cystinuria.}, }
@article {pmid31819203, year = {2020}, author = {Duan, W and Guo, M and Yi, L and Liu, Y and Li, Z and Ma, Y and Zhang, G and Liu, Y and Bu, H and Song, X and Li, C}, title = {The deletion of mutant SOD1 via CRISPR/Cas9/sgRNA prolongs survival in an amyotrophic lateral sclerosis mouse model.}, journal = {Gene therapy}, volume = {27}, number = {3-4}, pages = {157-169}, doi = {10.1038/s41434-019-0116-1}, pmid = {31819203}, issn = {1476-5462}, mesh = {Amyotrophic Lateral Sclerosis/genetics/*therapy ; Animals ; *CRISPR-Cas Systems ; Female ; *Gene Deletion ; Gene Editing/*methods ; Genetic Therapy/*methods ; HEK293 Cells ; Humans ; Male ; Mice ; Muscle, Skeletal/metabolism/pathology ; Mutation, Missense ; RNA, Guide/genetics/metabolism ; Superoxide Dismutase-1/*genetics/metabolism ; }, abstract = {The superoxide dismutase 1 (SOD1) mutation is one of the most notable causes of amyotrophic lateral sclerosis (ALS), and modifying the mutant SOD1 gene is the best approach for the treatment of patients with ALS linked to the mutations in this gene. Clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas9)/sgRNA delivered by the adeno-associated virus (AAV) system is a powerful tool for genome editing in the central nervous system (CNS). Here, we tested the capacity of the AAV-SaCas9-sgRNA system to modify mutant SOD1 in SOD1G93A transgenic mice and found that AAV9-SaCas9-sgRNA5 deleted the SOD1 gene, improved the lifespan of SOD1G93A mice by 54.6%, and notably ameliorated the performance of ALS transgenic mice. An immunochemical analysis showed that the expression of mutant SOD1 was very weak in motor neurons expressing SaCas9-sgRNA5. Consequently, the area showing muscle atrophy was more notably restored in the group treated with SaCas9-sgRNA5 compared with the group treated with SaCas9-sgLacZ. In addition, deep sequencing did not show the indel mutation in the gene highly matched to sgRNA5. Hence, AAV9-SaCas9-sgRNA-based gene editing is a feasible potential treatment for patients with ALS linked to SOD1 mutations.}, }
@article {pmid31654427, year = {2020}, author = {Watanabe, M and Toyomura, T and Wake, H and Liu, K and Teshigawara, K and Takahashi, H and Nishibori, M and Mori, S}, title = {Differential contribution of possible pattern-recognition receptors to advanced glycation end product-induced cellular responses in macrophage-like RAW264.7 cells.}, journal = {Biotechnology and applied biochemistry}, volume = {67}, number = {2}, pages = {265-272}, doi = {10.1002/bab.1843}, pmid = {31654427}, issn = {1470-8744}, support = {18K06807//JSPS/ ; 18K14969//JSPS/ ; //the Wesco Scientific Promotion Foundation/ ; //Ryobi Teien Memory Foundation/ ; //the Okayama Medical Foundation/ ; }, mesh = {Animals ; CRISPR-Associated Protein 9/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Gene Editing ; Glycation End Products, Advanced/genetics/*metabolism ; Macrophages/*metabolism ; Mice ; RAW 264.7 Cells ; Receptor for Advanced Glycation End Products/genetics/*metabolism ; Toll-Like Receptors/genetics/metabolism ; }, abstract = {Advanced glycation end products (AGEs) are considered to be related to the pathogenesis of some inflammatory diseases. AGEs were reported to stimulate the receptor for AGEs (RAGE), which causes inflammatory reactions. However, recently, toll-like receptors (TLRs), in addition to RAGE, have been reported to be related to AGE-mediated cellular responses, and it remains unclear which receptor is responsible for AGE recognition. To reveal the role of pattern-recognition receptors, including TLRs and/or RAGE, in AGE-mediated cellular responses, we generated macrophage-like RAW264.7 knockout (KO) cells lacking these receptors by genome editing using the CRISPR/Cas9 system and assessed AGE-stimulated changes in these cells. Comparison of the established clones suggested that RAGE partially affects the expression of TLRs. In the KO clone lacking TLR4 and TLR2, AGE-stimulated tumor necrosis factor alpha (TNF-α) expression and phosphorylation of IκBα, p38, and extracellular signal-regulated kinase (ERK) were significantly attenuated, suggesting that AGE-mediated responses are largely dependent on TLRs. On the other hand, on comparison of the AGE-stimulated responses between the KO clone lacking TLR4 and TLR2, and the clone lacking TLR4, TLR2, and RAGE, RAGE played little role in AGE-stimulated TNF-α transcription and ERK phosphorylation. Taken together, this study suggested that AGE-stimulated inflammatory responses occur mainly through TLRs rather than RAGE.}, }
@article {pmid33524499, year = {2021}, author = {Shrestha, P and Han, SR and Lee, JH and Park, H and Oh, TJ}, title = {A computational approach to identify CRISPR-Cas loci in the complete genomes of the lichen-associated Burkholderia sp. PAMC28687 and PAMC26561.}, journal = {Genomics}, volume = {}, number = {}, pages = {}, doi = {10.1016/j.ygeno.2021.01.019}, pmid = {33524499}, issn = {1089-8646}, abstract = {The genus Burkholderia and its strains PAMC28687 and PAMC26561 are lichen-associated bacteria isolated from the Antarctic region. Our study is the first to provide the genome sequence of the Burkholderia sp. PAMC26561 strain. The genus Burkholderia includes bacteria that are pathogenic to plants, animals, and humans. Computational analysis of complete genomes of strains from the uncategorized Burkholderia group was performed using the NCBI databank and PATRIC (for genome sequence information) and CRISPRCasFinder (online and offline versions) software in order to predict the CRISPR loci and Cas genes. The RNAfold Webserver online software was used to predict RNA secondary structures. Our study showed that strain MSMB0852 (plasmid) possesses CRISPR-Cas system Class 2, and two lichen-associated strains, PAMC28687 (chromosome I) and PAMC26561 (chromosome I), possess CRISPR-Cas system Class 1. Additionally, only the two lichen-associated strains possess a variety of Cas genes.}, }
@article {pmid33524138, year = {2021}, author = {Xiao, Y and Uh, K and Negrón-Pérez, VM and Haines, H and Lee, K and Hansen, PJ}, title = {Regulation of gene expression in the bovine blastocyst by CSF2 is disrupted by CRISPR/Cas9-mediated deletion of CSF2RA.}, journal = {Biology of reproduction}, volume = {}, number = {}, pages = {}, doi = {10.1093/biolre/ioab015}, pmid = {33524138}, issn = {1529-7268}, abstract = {Colony stimulating factor 2 (CSF2) functions in the reproductive tract to modulate function of the preimplantation embryo. The β subunit of the CSF2 receptor (CSF2RB) is not expressed in the embryo and signal transduction is therefore different than for myeloid cells where the receptor is composed of α (CSF2RA) and β subunits. Here, we produced embryos in which exons 5 and 6 of CSF2RA were disrupted using the CRISPR/Cas 9 system to test whether CSF2RA signaling was essential for actions of CSF2 in the bovine embryo. Wildtype and CSF2RA knockout embryos were treated with 10 ng/mL CSF2 or vehicle at day 5 of development. Blastocysts were harvested at day 8 to determine transcript abundance of 90 genes by real time PCR. Responses in female blastocysts were examined separately from male blastocysts because actions of CSF2 are sex-dependent. For wildtype embryos, CSF2 altered expression of 10 genes in females and 20 in males. Only three genes were affected by CSF2 in a similar manner for both sexes. Disruption of CSF2RA prevented the effect of CSF2 on expression for 9 of 10 CSF2-regulated genes in females and 19 of 20 genes in males. Results confirm the importance of CSF2RA for regulation of gene expression by CSF2 in the blastocyst.}, }
@article {pmid33523494, year = {2021}, author = {Parameshwaran, HP and Babu, K and Tran, C and Guan, K and Allen, A and Kathiresan, V and Qin, PZ and Rajan, R}, title = {The bridge helix of Cas12a imparts selectivity in cis-DNA cleavage and regulates trans-DNA cleavage.}, journal = {FEBS letters}, volume = {}, number = {}, pages = {}, doi = {10.1002/1873-3468.14051}, pmid = {33523494}, issn = {1873-3468}, abstract = {Cas12a is an RNA-guided DNA endonuclease of the type V-A CRISPR-Cas system that has evolved convergently with the type II Cas9 protein. We previously showed that proline substitutions in the bridge helix (BH) impart target DNA cleavage selectivity in Streptococcus pyogenes (Spy) Cas9. Here, we examined a BH variant of Cas12a from Francisella novicida (FnoCas12aKD2P) to test mechanistic conservation. Our results show that for RNA-guided DNA cleavage (cis-activity), FnoCas12aKD2P accumulates nicked products while cleaving supercoiled DNA substrates with mismatches, with certain mismatch positions being more detrimental for linearization. FnoCas12aKD2P also possess reduced trans single-stranded DNA cleavage activity. These results implicate the BH in substrate selectivity in both cis- and trans- cleavages and show its conserved role in target discrimination among Cas nucleases.}, }
@article {pmid33523177, year = {2019}, author = {Green, AA}, title = {Synthetic bionanotechnology: synthetic biology finds a toehold in nanotechnology.}, journal = {Emerging topics in life sciences}, volume = {3}, number = {5}, pages = {507-516}, doi = {10.1042/ETLS20190100}, pmid = {33523177}, issn = {2397-8554}, abstract = {Enabled by its central role in the molecular networks that govern cell function, RNA has been widely used for constructing components used in biological circuits for synthetic biology. Nucleic acid nanotechnology, which exploits predictable nucleic acid interactions to implement programmable molecular systems, has seen remarkable advances in in vitro nanoscale self-assembly and molecular computation, enabling the production of complex nanostructures and DNA-based neural networks. Living cells genetically engineered to execute nucleic acid nanotechnology programs thus have outstanding potential to significantly extend the current limits of synthetic biology. This perspective discusses the recent developments and future challenges in the field of synthetic bionanotechnology. Thus far, researchers in this emerging area have implemented dozens of programmable RNA nanodevices that provide precise control over gene expression at the transcriptional and translational levels and through CRISPR/Cas effectors. Moreover, they have employed synthetic self-assembling RNA networks in engineered bacteria to carry out computations featuring up to a dozen inputs and to substantially enhance the rate of chemical synthesis. Continued advancement of the field will benefit from improved in vivo strategies for streamlining nucleic acid network synthesis and new approaches for enhancing network function. As the field matures and the complexity gap between in vitro and in vivo systems narrows, synthetic bionanotechnology promises to have diverse potential applications ranging from intracellular circuits that detect and treat disease to synthetic enzymatic pathways that efficiently produce novel drug molecules.}, }
@article {pmid33523169, year = {2019}, author = {Berger, I and Tölzer, C and Gupta, K}, title = {The MultiBac system: a perspective.}, journal = {Emerging topics in life sciences}, volume = {3}, number = {5}, pages = {477-482}, doi = {10.1042/ETLS20190084}, pmid = {33523169}, issn = {2397-8554}, abstract = {Baculovirus expression is a time-tested technique to produce proteins in insect cells, in high quality and quantity for a range of applications. MultiBac is a baculovirus expression system we developed originally for producing multiprotein complexes comprising many subunits, for structural and mechanistic studies. First introduced in 2004, MultiBac is now in use in many laboratories worldwide, accelerating research programmes in academia and industry. We have continuously optimized our MultiBac system, providing customized reagents and standard operating protocols to facilitate its use also by non-specialists. More recently, we have generated MultiBac genomes tailored for specific purposes, for example, to produce humanized glycoproteins, high-value pharmaceutical targets including kinases, viral polymerases, and virus-like particles (VLPs) as promising vaccine candidates. By altering the host tropism of the baculovirion, we created MultiBacMam, a heterologous DNA delivery toolkit to target mammalian cells, tissues and organisms. Introducing CRISPR/Cas modalities, we set the stage for large-scale genomic engineering applications utilizing this high-capacity DNA delivery tool. Exploiting synthetic biology approaches and bottom-up design, we engage in optimizing the properties of our baculoviral genome, also to improve manufacturing at scale. Here we provide a perspective of our MultiBac system and its developments, past, present and future.}, }
@article {pmid33523137, year = {2019}, author = {Romito, M and Rai, R and Thrasher, AJ and Cavazza, A}, title = {Genome editing for blood disorders: state of the art and recent advances.}, journal = {Emerging topics in life sciences}, volume = {3}, number = {3}, pages = {289-299}, doi = {10.1042/ETLS20180147}, pmid = {33523137}, issn = {2397-8554}, abstract = {In recent years, tremendous advances have been made in the use of gene editing to precisely engineer the genome. This technology relies on the activity of a wide range of nuclease platforms - such as zinc-finger nucleases, transcription activator-like effector nucleases, and the CRISPR-Cas system - that can cleave and repair specific DNA regions, providing a unique and flexible tool to study gene function and correct disease-causing mutations. Preclinical studies using gene editing to tackle genetic and infectious diseases have highlighted the therapeutic potential of this technology. This review summarizes the progresses made towards the development of gene editing tools for the treatment of haematological disorders and the hurdles that need to be overcome to achieve clinical success.}, }
@article {pmid33519884, year = {2020}, author = {Nadakuduti, SS and Enciso-Rodríguez, F}, title = {Advances in Genome Editing With CRISPR Systems and Transformation Technologies for Plant DNA Manipulation.}, journal = {Frontiers in plant science}, volume = {11}, number = {}, pages = {637159}, doi = {10.3389/fpls.2020.637159}, pmid = {33519884}, issn = {1664-462X}, abstract = {The year 2020 marks a decade since the first gene-edited plants were generated using homing endonucleases and zinc finger nucleases. The advent of CRISPR/Cas9 for gene-editing in 2012 was a major science breakthrough that revolutionized both basic and applied research in various organisms including plants and consequently honored with &quot;The Nobel Prize in Chemistry, 2020.&quot; CRISPR technology is a rapidly evolving field and multiple CRISPR-Cas derived reagents collectively offer a wide range of applications for gene-editing and beyond. While most of these technological advances are successfully adopted in plants to advance functional genomics research and development of innovative crops, others await optimization. One of the biggest bottlenecks in plant gene-editing has been the delivery of gene-editing reagents, since genetic transformation methods are only established in a limited number of species. Recently, alternative methods of delivering CRISPR reagents to plants are being explored. This review mainly focuses on the most recent advances in plant gene-editing including (1) the current Cas effectors and Cas variants with a wide target range, reduced size and increased specificity along with tissue specific genome editing tool kit (2) cytosine, adenine, and glycosylase base editors that can precisely install all possible transition and transversion mutations in target sites (3) prime editing that can directly copy the desired edit into target DNA by search and replace method and (4) CRISPR delivery mechanisms for plant gene-editing that bypass tissue culture and regeneration procedures including de novo meristem induction, delivery using viral vectors and prospects of nanotechnology-based approaches.}, }
@article {pmid33517983, year = {2020}, author = {Bhargava, R and Lopezcolorado, FW and Tsai, LJ and Stark, JM}, title = {The canonical non-homologous end joining factor XLF promotes chromosomal deletion rearrangements in human cells.}, journal = {The Journal of biological chemistry}, volume = {295}, number = {1}, pages = {125-137}, doi = {10.1074/jbc.RA119.010421}, pmid = {33517983}, issn = {1083-351X}, abstract = {Clastogen exposure can result in chromosomal rearrangements, including large deletions and inversions that are associated with cancer development. To examine such rearrangements in human cells, here we developed a reporter assay based on endogenous genes on chromosome 12. Using the RNA-guided nuclease Cas9, we induced two DNA double-strand breaks, one each in the GAPDH and CD4 genes, that caused a deletion rearrangement leading to CD4 expression from the GAPDH promoter. We observed that this GAPDH-CD4 deletion rearrangement activates CD4+ cells that can be readily detected by flow cytometry. Similarly, double-strand breaks in the LPCAT3 and CD4 genes induced an LPCAT3-CD4 inversion rearrangement resulting in CD4 expression. Studying the GAPDH-CD4 deletion rearrangement in multiple cell lines, we found that the canonical non-homologous end joining (C-NHEJ) factor XLF promotes these rearrangements. Junction analysis uncovered that the relative contribution of C-NHEJ appears lower in U2OS than in HEK293 and A549 cells. Furthermore, an ATM kinase inhibitor increased C-NHEJ-mediated rearrangements only in U2OS cells. We also found that an XLF residue that is critical for an interaction with the C-NHEJ factor X-ray repair cross-complementing 4 (XRCC4), and XRCC4 itself are each important for promoting both this deletion rearrangement and end joining without insertion/deletion mutations. In summary, a reporter assay based on endogenous genes on chromosome 12 reveals that XLF-dependent C-NHEJ promotes deletion rearrangements in human cells and that cell type-specific differences in the contribution of C-NHEJ and ATM kinase inhibition influence these rearrangements.}, }
@article {pmid33516347, year = {2020}, author = {Ling, L and Mulaka, M and Munro, J and Dass, S and Mather, MW and Riscoe, MK and Llinás, M and Zhou, J and Ke, H}, title = {Genetic ablation of the mitoribosome in the malaria parasite Plasmodium falciparum sensitizes it to antimalarials that target mitochondrial functions.}, journal = {The Journal of biological chemistry}, volume = {295}, number = {21}, pages = {7235-7248}, doi = {10.1074/jbc.RA120.012646}, pmid = {33516347}, issn = {1083-351X}, abstract = {The mitochondrion of malaria parasites contains several clinically validated drug targets. Within Plasmodium spp., the causative agents of malaria, the mitochondrial DNA (mtDNA) is only 6 kb long, being the smallest mitochondrial genome among all eukaryotes. The mtDNA encodes only three proteins of the mitochondrial electron transport chain and ∼27 small, fragmented rRNA genes having lengths of 22-195 nucleotides. The rRNA fragments are thought to form a mitochondrial ribosome (mitoribosome), together with ribosomal proteins imported from the cytosol. The mitoribosome of Plasmodium falciparum is essential for maintenance of the mitochondrial membrane potential and parasite viability. However, the role of the mitoribosome in sustaining the metabolic status of the parasite mitochondrion remains unclear. The small ribosomal subunit in P. falciparum has 14 annotated mitoribosomal proteins, and employing a CRISPR/Cas9-based conditional knockdown tool, here we verified the location and tested the essentiality of three candidates (PfmtRPS12, PfmtRPS17, and PfmtRPS18). Using immuno-EM, we provide evidence that the P. falciparum mitoribosome is closely associated with the mitochondrial inner membrane. Upon knockdown of the mitoribosome, parasites became hypersensitive to inhibitors targeting mitochondrial Complex III (bc1), dihydroorotate dehydrogenase (DHOD), and the F1F0-ATP synthase complex. Furthermore, the mitoribosome knockdown blocked the pyrimidine biosynthesis pathway and reduced the cellular pool of pyrimidine nucleotides. These results suggest that disruption of the P. falciparum mitoribosome compromises the metabolic capacity of the mitochondrion, rendering the parasite hypersensitive to a panel of inhibitors that target mitochondrial functions.}, }
@article {pmid33516307, year = {2020}, author = {Suzuki, E and Ogawa, N and Takeda, TA and Nishito, Y and Tanaka, YK and Fujiwara, T and Matsunaga, M and Ueda, S and Kubo, N and Tsuji, T and Fukunaka, A and Yamazaki, T and Taylor, KM and Ogra, Y and Kambe, T}, title = {Detailed analyses of the crucial functions of Zn transporter proteins in alkaline phosphatase activation.}, journal = {The Journal of biological chemistry}, volume = {295}, number = {17}, pages = {5669-5684}, doi = {10.1074/jbc.RA120.012610}, pmid = {33516307}, issn = {1083-351X}, abstract = {Numerous zinc ectoenzymes are metalated by zinc and activated in the compartments of the early secretory pathway before reaching their destination. Zn transporter (ZNT) proteins located in these compartments are essential for ectoenzyme activation. We have previously reported that ZNT proteins, specifically ZNT5-ZNT6 heterodimers and ZNT7 homodimers, play critical roles in the activation of zinc ectoenzymes, such as alkaline phosphatases (ALPs), by mobilizing cytosolic zinc into these compartments. However, this process remains incompletely understood. Here, using genetically-engineered chicken DT40 cells, we first determined that Zrt/Irt-like protein (ZIP) transporters that are localized to the compartments of the early secretory pathway play only a minor role in the ALP activation process. These transporters included ZIP7, ZIP9, and ZIP13, performing pivotal functions in maintaining cellular homeostasis by effluxing zinc out of the compartments. Next, using purified ALP proteins, we showed that zinc metalation on ALP produced in DT40 cells lacking ZNT5-ZNT6 heterodimers and ZNT7 homodimers is impaired. Finally, by genetically disrupting both ZNT5 and ZNT7 in human HAP1 cells, we directly demonstrated that the tissue-nonspecific ALP-activating functions of both ZNT complexes are conserved in human cells. Furthermore, using mutant HAP1 cells, we uncovered a previously-unrecognized and unique spatial regulation of ZNT5-ZNT6 heterodimer formation, wherein ZNT5 recruits ZNT6 to the Golgi apparatus to form the heterodimeric complex. These findings fill in major gaps in our understanding of the molecular mechanisms underlying zinc ectoenzyme activation in the compartments of the early secretory pathway.}, }
@article {pmid33516297, year = {2020}, author = {Murugan, K and Seetharam, AS and Severin, AJ and Sashital, DG}, title = {CRISPR-Cas12a has widespread off-target and dsDNA-nicking effects.}, journal = {The Journal of biological chemistry}, volume = {295}, number = {17}, pages = {5538-5553}, doi = {10.1074/jbc.RA120.012933}, pmid = {33516297}, issn = {1083-351X}, abstract = {Cas12a (Cpf1) is an RNA-guided endonuclease in the bacterial type V-A CRISPR-Cas anti-phage immune system that can be repurposed for genome editing. Cas12a can bind and cut dsDNA targets with high specificity in vivo, making it an ideal candidate for expanding the arsenal of enzymes used in precise genome editing. However, this reported high specificity contradicts Cas12a's natural role as an immune effector against rapidly evolving phages. Here, we employed high-throughput in vitro cleavage assays to determine and compare the native cleavage specificities and activities of three different natural Cas12a orthologs (FnCas12a, LbCas12a, and AsCas12a). Surprisingly, we observed pervasive sequence-specific nicking of randomized target libraries, with strong nicking of DNA sequences containing up to four mismatches in the Cas12a-targeted DNA-RNA hybrid sequences. We also found that these nicking and cleavage activities depend on mismatch type and position and vary with Cas12a ortholog and CRISPR RNA sequence. Our analysis further revealed robust nonspecific nicking of dsDNA when Cas12a is activated by binding to a target DNA. Together, our findings reveal that Cas12a has multiple nicking activities against dsDNA substrates and that these activities vary among different Cas12a orthologs.}, }
@article {pmid33509763, year = {2021}, author = {Zhou, L and Ye, Y and Yuan, H and Wu, C and Wu, S}, title = {[Construction of macrophage RAW 264.7 cells with gsdmd gene knockout by CRISPR/Cas9 system].}, journal = {Nan fang yi ke da xue xue bao = Journal of Southern Medical University}, volume = {41}, number = {1}, pages = {116-122}, doi = {10.12122/j.issn.1673-4254.2021.01.17}, pmid = {33509763}, issn = {1673-4254}, mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Gene Knockout Techniques ; Mice ; Plasmids ; RAW 264.7 Cells ; *RNA, Guide ; }, abstract = {OBJECTIVE: To construct a cell model of gsdmd gene knockout in macrophage RAW 264.7 cells using CRISPR/Cas9 system.<br><br>METHODS: Four specific single guide RNAs (sgRNAs) targeting gsdmd were designed to construct pGL3-sgRNA recombinant plasmids, which were identified by PCR amplification and sequencing.Cas9 and the recombinant plasmids were transfected into RAW 264.7 cells in two steps, and the cellular expression of cas9 was detected with real-time quantitative PCR (qPCR).The positive cell clones with gsdmd gene knockout were screened using puromycin and verified by sequencing and Western blotting.Annexin Ⅴ/PI staining and LDH release assay were performed in gsdmd-/-RAW 264.7 cells after being co-cultured with Salmonella Typhimurium.<br><br>RESULTS: qPCR results showed that cas9 gene was stably expressed in RAW 264.7-Cas9 cells (P< 0.01).PCR and sequencing results demonstrated successful construction of the recombinant plasmid pGL3-sgRNA. The results of PCR, sequencing and Western blotting all confirmed that gsdmd-/-RAW 264.7 cells were successfully constructed. Annexin Ⅴ/PI staining and LDH release assay showed that gsdmd gene knockout significantly inhibited macrophage death caused by S.Typhimurium infection (P < 0.01).<br><br>CONCLUSIONS: gsdmd-/-RAW 264.7 cells provide a cell model for studying the mechanisms underlying GSDMD-mediated macrophage death.}, }
@article {pmid33473139, year = {2021}, author = {Sharma, R and Dever, DP and Lee, CM and Azizi, A and Pan, Y and Camarena, J and Köhnke, T and Bao, G and Porteus, MH and Majeti, R}, title = {The TRACE-Seq method tracks recombination alleles and identifies clonal reconstitution dynamics of gene targeted human hematopoietic stem cells.}, journal = {Nature communications}, volume = {12}, number = {1}, pages = {472}, pmid = {33473139}, issn = {2041-1723}, support = {R01 HL135607/HL/NHLBI NIH HHS/United States ; R01 CA188055/CA/NCI NIH HHS/United States ; R01 AI120766/AI/NIAID NIH HHS/United States ; R01 AI097320/AI/NIAID NIH HHS/United States ; R01 HL142637/HL/NHLBI NIH HHS/United States ; }, mesh = {*Alleles ; Animals ; CRISPR-Cas Systems ; *Clone Cells ; Clustered Regularly Interspaced Short Palindromic Repeats ; Female ; Gene Editing/methods ; Gene Targeting/*methods ; Genetic Therapy/methods ; *Hematopoietic Stem Cells ; Humans ; Mice ; Mutation ; *Recombination, Genetic ; Targeted Gene Repair/methods ; }, abstract = {Targeted DNA correction of disease-causing mutations in hematopoietic stem and progenitor cells (HSPCs) may enable the treatment of genetic diseases of the blood and immune system. It is now possible to correct mutations at high frequencies in HSPCs by combining CRISPR/Cas9 with homologous DNA donors. Because of the precision of gene correction, these approaches preclude clonal tracking of gene-targeted HSPCs. Here, we describe Tracking Recombination Alleles in Clonal Engraftment using sequencing (TRACE-Seq), a methodology that utilizes barcoded AAV6 donor template libraries, carrying in-frame silent mutations or semi-randomized nucleotides outside the coding region, to track the in vivo lineage contribution of gene-targeted HSPC clones. By targeting the HBB gene with an AAV6 donor template library consisting of ~20,000 possible unique exon 1 in-frame silent mutations, we track the hematopoietic reconstitution of HBB targeted myeloid-skewed, lymphoid-skewed, and balanced multi-lineage repopulating human HSPC clones in mice. We anticipate this methodology could potentially be used for HSPC clonal tracking of Cas9 RNP and AAV6-mediated gene targeting outcomes in translational and basic research settings.}, }
@article {pmid33473114, year = {2021}, author = {Butterfield, NC and Curry, KF and Steinberg, J and Dewhurst, H and Komla-Ebri, D and Mannan, NS and Adoum, AT and Leitch, VD and Logan, JG and Waung, JA and Ghirardello, E and Southam, L and Youlten, SE and Wilkinson, JM and McAninch, EA and Vancollie, VE and Kussy, F and White, JK and Lelliott, CJ and Adams, DJ and Jacques, R and Bianco, AC and Boyde, A and Zeggini, E and Croucher, PI and Williams, GR and Bassett, JHD}, title = {Accelerating functional gene discovery in osteoarthritis.}, journal = {Nature communications}, volume = {12}, number = {1}, pages = {467}, pmid = {33473114}, issn = {2041-1723}, support = {110140/WT_/Wellcome Trust/United Kingdom ; 110141/WT_/Wellcome Trust/United Kingdom ; 101123/WT_/Wellcome Trust/United Kingdom ; 098051/WT_/Wellcome Trust/United Kingdom ; 206194/WT_/Wellcome Trust/United Kingdom ; }, mesh = {Animals ; Bone and Bones/pathology ; CRISPR-Cas Systems ; Cartilage/pathology ; Clustered Regularly Interspaced Short Palindromic Repeats ; Disease Models, Animal ; Drug Discovery ; Gene Editing ; *Genetic Association Studies ; Genetic Predisposition to Disease/*genetics ; Gonadotropin-Releasing Hormone/genetics ; Iodide Peroxidase ; Mice ; Mice, Knockout ; Osteoarthritis/*genetics/pathology/surgery ; Paired Box Transcription Factors/genetics ; Phenotype ; }, abstract = {Osteoarthritis causes debilitating pain and disability, resulting in a considerable socioeconomic burden, yet no drugs are available that prevent disease onset or progression. Here, we develop, validate and use rapid-throughput imaging techniques to identify abnormal joint phenotypes in randomly selected mutant mice generated by the International Knockout Mouse Consortium. We identify 14 genes with functional involvement in osteoarthritis pathogenesis, including the homeobox gene Pitx1, and functionally characterize 6 candidate human osteoarthritis genes in mouse models. We demonstrate sensitivity of the methods by identifying age-related degenerative joint damage in wild-type mice. Finally, we phenotype previously generated mutant mice with an osteoarthritis-associated polymorphism in the Dio2 gene by CRISPR/Cas9 genome editing and demonstrate a protective role in disease onset with public health implications. We hope this expanding resource of mutant mice will accelerate functional gene discovery in osteoarthritis and offer drug discovery opportunities for this common, incapacitating chronic disease.}, }
@article {pmid33469032, year = {2021}, author = {Hörnblad, A and Bastide, S and Langenfeld, K and Langa, F and Spitz, F}, title = {Dissection of the Fgf8 regulatory landscape by in vivo CRISPR-editing reveals extensive intra- and inter-enhancer redundancy.}, journal = {Nature communications}, volume = {12}, number = {1}, pages = {439}, pmid = {33469032}, issn = {2041-1723}, mesh = {Animals ; CRISPR-Cas Systems/genetics ; Ectoderm/embryology ; Embryo, Mammalian ; Embryonic Development/*genetics ; Enhancer Elements, Genetic/*genetics ; Extremities/embryology ; Feasibility Studies ; Female ; Fibroblast Growth Factor 8/*genetics/metabolism ; *Gene Expression Regulation, Developmental ; Gene Regulatory Networks ; Genetic Engineering/*methods ; Male ; Mesencephalon/embryology ; Mice ; Mice, Transgenic ; Rhombencephalon/embryology ; }, abstract = {Developmental genes are often regulated by multiple elements with overlapping activity. Yet, in most cases, the relative function of those elements and their contribution to endogenous gene expression remain poorly characterized. An example of this phenomenon is that distinct sets of enhancers have been proposed to direct Fgf8 in the limb apical ectodermal ridge and the midbrain-hindbrain boundary. Using in vivo CRISPR/Cas9 genome engineering, we functionally dissect this complex regulatory ensemble and demonstrate two distinct regulatory logics. In the apical ectodermal ridge, the control of Fgf8 expression appears distributed between different enhancers. In contrast, we find that in the midbrain-hindbrain boundary, one of the three active enhancers is essential while the other two are dispensable. We further dissect the essential midbrain-hindbrain boundary enhancer to reveal that it is also composed by a mixture of essential and dispensable modules. Cross-species transgenic analysis of this enhancer suggests that its composition may have changed in the vertebrate lineage.}, }
@article {pmid33469008, year = {2021}, author = {Chen, X and Lloyd, SM and Kweon, J and Gamalong, GM and Bao, X}, title = {Epidermal progenitors suppress GRHL3-mediated differentiation through intronic polyadenylation promoted by CPSF-HNRNPA3 collaboration.}, journal = {Nature communications}, volume = {12}, number = {1}, pages = {448}, pmid = {33469008}, issn = {2041-1723}, support = {P30 AR075049/AR/NIAMS NIH HHS/United States ; R00 AR065480/AR/NIAMS NIH HHS/United States ; R01 AR075015/AR/NIAMS NIH HHS/United States ; T32 GM008061/GM/NIGMS NIH HHS/United States ; }, mesh = {CRISPR-Cas Systems/genetics ; Cell Differentiation/genetics ; Cell Self Renewal/genetics ; Cleavage And Polyadenylation Specificity Factor/genetics/*metabolism ; DNA-Binding Proteins/genetics/*metabolism ; Gene Expression Regulation ; Gene Knockdown Techniques ; Gene Knockout Techniques ; HEK293 Cells ; Heterogeneous-Nuclear Ribonucleoprotein Group A-B/genetics/*metabolism ; Humans ; Introns/genetics ; Keratinocytes/*physiology ; Polyadenylation/genetics ; Primary Cell Culture ; Re-Epithelialization/*genetics ; Stem Cells/*physiology ; Transcription Factors/genetics/*metabolism ; }, abstract = {In self-renewing somatic tissue such as skin epidermis, terminal differentiation genes must be suppressed in progenitors to sustain regenerative capacity. Here we show that hundreds of intronic polyadenylation (IpA) sites are differentially used during keratinocyte differentiation, which is accompanied by downregulation of the Cleavage and Polyadenylation Specificity Factor (CPSF) complex. Sustained CPSF expression in undifferentiated keratinocytes requires the contribution from the transcription factor MYC. In keratinocytes cultured in undifferentiation condition, CSPF knockdown induces premature differentiation and partially affects dynamically used IpA sites. These sites include an IpA site located in the first intron of the differentiation activator GRHL3. CRISPR knockout of GRHL3 IpA increased full-length GRHL3 mRNA expression. Using a targeted genetic screen, we identify that HNRNPA3 interacts with CPSF and enhances GRHL3 IpA. Our data suggest a model where the interaction between CPSF and RNA-binding proteins, such as HNRNPA3, promotes site-specific IpA and suppresses premature differentiation in progenitors.}, }
@article {pmid33441553, year = {2021}, author = {Goldberg, GW and Spencer, JM and Giganti, DO and Camellato, BR and Agmon, N and Ichikawa, DM and Boeke, JD and Noyes, MB}, title = {Engineered dual selection for directed evolution of SpCas9 PAM specificity.}, journal = {Nature communications}, volume = {12}, number = {1}, pages = {349}, pmid = {33441553}, issn = {2041-1723}, support = {F32 GM137482/GM/NIGMS NIH HHS/United States ; R01 GM118851/GM/NIGMS NIH HHS/United States ; R01 GM133936/GM/NIGMS NIH HHS/United States ; RM1 HG009491/HG/NHGRI NIH HHS/United States ; }, mesh = {Amino Acid Sequence ; CRISPR-Associated Protein 9/genetics/*metabolism ; *CRISPR-Cas Systems ; Cell Line, Tumor ; DNA/chemistry/genetics/*metabolism ; Directed Molecular Evolution/methods ; Gene Editing/*methods ; Humans ; Mutation ; Nucleic Acid Conformation ; Nucleotide Motifs/genetics ; Protein Engineering/methods ; RNA, Guide/genetics/*metabolism ; Streptococcus pyogenes/genetics/*metabolism ; Substrate Specificity ; }, abstract = {The widely used Streptococcus pyogenes Cas9 (SpCas9) nuclease derives its DNA targeting specificity from protein-DNA contacts with protospacer adjacent motif (PAM) sequences, in addition to base-pairing interactions between its guide RNA and target DNA. Previous reports have established that the PAM specificity of SpCas9 can be altered via positive selection procedures for directed evolution or other protein engineering strategies. Here we exploit in vivo directed evolution systems that incorporate simultaneous positive and negative selection to evolve SpCas9 variants with commensurate or improved activity on NAG PAMs relative to wild type and reduced activity on NGG PAMs, particularly YGG PAMs. We also show that the PAM preferences of available evolutionary intermediates effectively determine whether similar counterselection PAMs elicit different selection stringencies, and demonstrate that negative selection can be specifically increased in a yeast selection system through the fusion of compensatory zinc fingers to SpCas9.}, }
@article {pmid33257571, year = {2020}, author = {Zhou, M and Kuruvilla, L and Shi, X and Viviano, S and Ahearn, IM and Amendola, CR and Su, W and Badri, S and Mahaffey, J and Fehrenbacher, N and Skok, J and Schlessinger, J and Turk, BE and Calderwood, DA and Philips, MR}, title = {Scaffold association factor B (SAFB) is required for expression of prenyltransferases and RAS membrane association.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {117}, number = {50}, pages = {31914-31922}, pmid = {33257571}, issn = {1091-6490}, support = {R01 CA116034/CA/NCI NIH HHS/United States ; R01 CA163489/CA/NCI NIH HHS/United States ; R35 GM122515/GM/NIGMS NIH HHS/United States ; R35 CA253178/CA/NCI NIH HHS/United States ; P50 CA196530/CA/NCI NIH HHS/United States ; }, mesh = {CRISPR-Cas Systems/genetics ; Cell Membrane/*metabolism ; Computational Biology ; Datasets as Topic ; Dimethylallyltranstransferase/*metabolism ; Gene Knockdown Techniques ; Humans ; Matrix Attachment Region Binding Proteins/genetics/*metabolism ; Neoplasms/genetics/*pathology ; Nuclear Matrix-Associated Proteins/genetics/*metabolism ; Protein Prenylation ; Protein Subunits/metabolism ; Proto-Oncogene Proteins p21(ras)/genetics/*metabolism ; Receptors, Estrogen/genetics/*metabolism ; }, abstract = {Inhibiting membrane association of RAS has long been considered a rational approach to anticancer therapy, which led to the development of farnesyltransferase inhibitors (FTIs). However, FTIs proved ineffective against KRAS-driven tumors. To reveal alternative therapeutic strategies, we carried out a genome-wide CRISPR-Cas9 screen designed to identify genes required for KRAS4B membrane association. We identified five enzymes in the prenylation pathway and SAFB, a nuclear protein with both DNA and RNA binding domains. Silencing SAFB led to marked mislocalization of all RAS isoforms as well as RAP1A but not RAB7A, a pattern that phenocopied silencing FNTA, the prenyltransferase α subunit shared by farnesyltransferase and geranylgeranyltransferase type I. We found that SAFB promoted RAS membrane association by controlling FNTA expression. SAFB knockdown decreased GTP loading of RAS, abrogated alternative prenylation, and sensitized RAS-mutant cells to growth inhibition by FTI. Our work establishes the prenylation pathway as paramount in KRAS membrane association, reveals a regulator of prenyltransferase expression, and suggests that reduction in FNTA expression may enhance the efficacy of FTIs.}, }
@article {pmid33221881, year = {2021}, author = {Mathis, AD and Otto, RM and Reynolds, KA}, title = {A simplified strategy for titrating gene expression reveals new relationships between genotype, environment, and bacterial growth.}, journal = {Nucleic acids research}, volume = {49}, number = {1}, pages = {e6}, pmid = {33221881}, issn = {1362-4962}, support = {R01 GM136842/GM/NIGMS NIH HHS/United States ; }, mesh = {CRISPR-Cas Systems/*genetics ; Cell Division/genetics ; Computational Biology/*methods ; Escherichia coli/*genetics/growth &amp; development ; *Gene Expression Regulation, Bacterial ; Gene-Environment Interaction ; Genetic Techniques ; Genotype ; Mutation ; RNA, Guide/*genetics ; }, abstract = {A lack of high-throughput techniques for making titrated, gene-specific changes in expression limits our understanding of the relationship between gene expression and cell phenotype. Here, we present a generalizable approach for quantifying growth rate as a function of titrated changes in gene expression level. The approach works by performing CRISPRi with a series of mutated single guide RNAs (sgRNAs) that modulate gene expression. To evaluate sgRNA mutation strategies, we constructed a library of 5927 sgRNAs targeting 88 genes in Escherichia coli MG1655 and measured the effects on growth rate. We found that a compounding mutational strategy, through which mutations are incrementally added to the sgRNA, presented a straightforward way to generate a monotonic and gradated relationship between mutation number and growth rate effect. We also implemented molecular barcoding to detect and correct for mutations that 'escape' the CRISPRi targeting machinery; this strategy unmasked deleterious growth rate effects obscured by the standard approach of ignoring escapers. Finally, we performed controlled environmental variations and observed that many gene-by-environment interactions go completely undetected at the limit of maximum knockdown, but instead manifest at intermediate expression perturbation strengths. Overall, our work provides an experimental platform for quantifying the phenotypic response to gene expression variation.}, }
@article {pmid33137185, year = {2021}, author = {Choi, A and Jang, I and Han, H and Kim, MS and Choi, J and Lee, J and Cho, SY and Jun, Y and Lee, C and Kim, J and Lee, B and Lee, S}, title = {iCSDB: an integrated database of CRISPR screens.}, journal = {Nucleic acids research}, volume = {49}, number = {D1}, pages = {D956-D961}, pmid = {33137185}, issn = {1362-4962}, mesh = {CRISPR-Cas Systems/*genetics ; Cell Line, Tumor ; *Databases, Genetic ; Gene Editing/*methods ; Gene Targeting/*methods ; Genome, Human/*genetics ; Genome-Wide Association Study/*methods ; Genomics/*methods ; Humans ; Internet ; Web Browser ; }, abstract = {High-throughput screening based on CRISPR-Cas9 libraries has become an attractive and powerful technique to identify target genes for functional studies. However, accessibility of public data is limited due to the lack of user-friendly utilities and up-to-date resources covering experiments from third parties. Here, we describe iCSDB, an integrated database of CRISPR screening experiments using human cell lines. We compiled two major sources of CRISPR-Cas9 screening: the DepMap portal and BioGRID ORCS. DepMap portal itself is an integrated database that includes three large-scale projects of CRISPR screening. We additionally aggregated CRISPR screens from BioGRID ORCS that is a collection of screening results from PubMed articles. Currently, iCSDB contains 1375 genome-wide screens across 976 human cell lines, covering 28 tissues and 70 cancer types. Importantly, the batch effects from different CRISPR libraries were removed and the screening scores were converted into a single metric to estimate the knockout efficiency. Clinical and molecular information were also integrated to help users to select cell lines of interest readily. Furthermore, we have implemented various interactive tools and viewers to facilitate users to choose, examine and compare the screen results both at the gene and guide RNA levels. iCSDB is available at https://www.kobic.re.kr/icsdb/.}, }
@article {pmid33085911, year = {2021}, author = {Zimmer, AM and Mandic, M and Yew, HM and Kunert, E and Pan, YK and Ha, J and Kwong, RWM and Gilmour, KM and Perry, SF}, title = {Use of a carbonic anhydrase Ca17a knockout to investigate mechanisms of ion uptake in zebrafish (Danio rerio).}, journal = {American journal of physiology. Regulatory, integrative and comparative physiology}, volume = {320}, number = {1}, pages = {R55-R68}, doi = {10.1152/ajpregu.00215.2020}, pmid = {33085911}, issn = {1522-1490}, mesh = {*Acid-Base Equilibrium ; Animals ; Animals, Genetically Modified ; CRISPR-Cas Systems ; Carbonic Anhydrases/*deficiency/genetics ; Chlorides/*metabolism ; *Gene Knockout Techniques ; Hydrogen-Ion Concentration ; Ion Transport ; Mutation ; Sodium/*metabolism ; Zebrafish/embryology/genetics/*metabolism ; Zebrafish Proteins/*deficiency/genetics ; }, abstract = {In fishes, branchial cytosolic carbonic anhydrase (CA) plays an important role in ion and acid-base regulation. The Ca17a isoform in zebrafish (Danio rerio) is expressed abundantly in Na+-absorbing/H+-secreting H+-ATPase-rich (HR) cells. The present study aimed to identify the role of Ca17a in ion and acid-base regulation across life stages using CRISPR/Cas9 gene editing. However, in preliminary experiments, we established that ca17a knockout is lethal with ca17a-/- mutants exhibiting a significant decrease in survival beginning at ∼12 days postfertilization (dpf) and with no individuals surviving past 19 dpf. Based on these findings, we hypothesized that ca17a-/- mutants would display alterations in ion and acid-base balance and that these physiological disturbances might underlie their early demise. Na+ uptake rates were significantly increased by up to 300% in homozygous mutants compared with wild-type individuals at 4 and 9 dpf; however, whole body Na+ content remained constant. While Cl- uptake was significantly reduced in ca17a-/- mutants, Cl- content was unaffected. Reduction of CA activity by Ca17a morpholino knockdown or ethoxzolamide treatments similarly reduced Cl- uptake, implicating Ca17a in the mechanism of Cl- uptake by larval zebrafish. H+ secretion, O2 consumption, CO2 excretion, and ammonia excretion were generally unaltered in ca17a-/- mutants. In conclusion, while the loss of Ca17a caused marked changes in ion uptake rates, providing strong evidence for a Ca17a-dependent Cl- uptake mechanism, the underlying causes of the lethality of this mutation in zebrafish remain unclear.}, }
@article {pmid33084874, year = {2021}, author = {Gurumayum, S and Jiang, P and Hao, X and Campos, TL and Young, ND and Korhonen, PK and Gasser, RB and Bork, P and Zhao, XM and He, LJ and Chen, WH}, title = {OGEE v3: Online GEne Essentiality database with increased coverage of organisms and human cell lines.}, journal = {Nucleic acids research}, volume = {49}, number = {D1}, pages = {D998-D1003}, pmid = {33084874}, issn = {1362-4962}, mesh = {Animals ; CRISPR-Cas Systems ; Cell Line, Tumor ; Computational Biology/*methods ; Data Mining/methods ; *Databases, Genetic ; Genes, Essential/*genetics ; Genetic Predisposition to Disease/genetics ; Genomics/*methods ; Humans ; Internet ; Neoplasms/*genetics/pathology ; Oncogenes/*genetics ; RNA Interference ; }, abstract = {OGEE is an Online GEne Essentiality database. Gene essentiality is not a static and binary property, rather a context-dependent and evolvable property in all forms of life. In OGEE we collect not only experimentally tested essential and non-essential genes, but also associated gene properties that contributes to gene essentiality. We tagged conditionally essential genes that show variable essentiality statuses across datasets to highlight complex interplays between gene functions and environmental/experimental perturbations. OGEE v3 contains gene essentiality datasets for 91 species; almost doubled from 48 species in previous version. To accommodate recent advances on human cancer essential genes (as known as tumor dependency genes) that could serve as targets for cancer treatment and/or drug development, we expanded the collection of human essential genes from 16 cell lines in previous to 581. These human cancer cell lines were tested with high-throughput experiments such as CRISPR-Cas9 and RNAi; in total, 150 of which were tested by both techniques. We also included factors known to contribute to gene essentiality for these cell lines, such as genomic mutation, methylation and gene expression, along with extensive graphical visualizations for ease of understanding of these factors. OGEE v3 can be accessible freely at https://v3.ogee.info.}, }
@article {pmid32642859, year = {2020}, author = {Wolabu, TW and Park, JJ and Chen, M and Cong, L and Ge, Y and Jiang, Q and Debnath, S and Li, G and Wen, J and Wang, Z}, title = {Improving the genome editing efficiency of CRISPR/Cas9 in Arabidopsis and Medicago truncatula.}, journal = {Planta}, volume = {252}, number = {2}, pages = {15}, pmid = {32642859}, issn = {1432-2048}, mesh = {Agrobacterium/genetics ; Alleles ; Arabidopsis/*genetics ; CRISPR-Cas Systems/*genetics ; *Gene Editing ; Homozygote ; Medicago truncatula/*genetics ; Mutation ; Phenotype ; Promoter Regions, Genetic/genetics ; }, abstract = {MAIN CONCLUSION: An improved CRISPR/Cas9 system with the Arabidopsis UBQ10 promoter-driven Cas9 exhibits consistently high mutation efficiency in Arabidopsis and M. truncatula. CRISPR/Cas9 is a powerful genome editing technology that has been applied in several crop species for trait improvement due to its simplicity, versatility, and specificity. However, the mutation efficiency of CRISPR/Cas9 in Arabidopsis and M. truncatula (Mt) is still challenging and inconsistent. To analyze the functionality of the CRISPR/Cas9 system in two model dicot species, four different promoter-driven Cas9 systems to target phytoene desaturase (PDS) genes were designed. Agrobacterium-mediated transformation was used for the delivery of constructed vectors to host plants. Phenotypic and genotypic analyses revealed that the Arabidopsis UBQ10 promoter-driven Cas9 significantly improves the mutation efficiency to 95% in Arabidopsis and 70% in M. truncatula. Moreover, the UBQ10-Cas9 system yielded 11% homozygous mutants in the T1 generation in Arabidopsis. Sequencing analyses of mutation events indicated that single-nucleotide insertions are the most frequent events in Arabidopsis, whereas multi-nucleotide deletions are dominant in bi-allelic and mono-allelic homozygous mutants in M. truncatula. Taken together, the UBQ10 promoter facilitates the best improvement in the CRISPR/Cas9 efficiency in PDS gene editing, followed by the EC1.2 promoter. Consistently, the improved UBQ10-Cas9 vector highly enhanced the mutation efficiency by four-fold over the commonly used 35S promoter in both dicot species.}, }
@article {pmid32583364, year = {2020}, author = {Liu, Z and Liao, Z and Chen, Y and Han, L and Yin, Q and Xiao, H}, title = {Application of Various Delivery Methods for CRISPR/dCas9.}, journal = {Molecular biotechnology}, volume = {62}, number = {8}, pages = {355-363}, doi = {10.1007/s12033-020-00258-8}, pmid = {32583364}, issn = {1559-0305}, support = {Grant nos. 30305030606 and 30305030859//the Young Scholars Foundation of Sichuan Provincial People's Hospital/ ; Grant no.30305030698//the National Key Specialty Construction Project of Clinical Pharmacy/ ; Grant nos. 2009SZ0226, 2014FZ0103, 2015JQO027, and 2015ZR0160//the Science &amp; Technology Program of Sichuan Province/ ; Grant nos. 100491, 120111, and 17ZD038//the Health Department of Sichuan Province/ ; Grant no. YB2019001//Sichuan Cancer Hospital/ ; Grant no.11PPYB010SF-289//Chengdu City Science and Technology Project/ ; Grant No. 2019-801//the Cadre Health Care Research Project of Sichuan Province/ ; Grant No. 201905//ZambonPharmaceutical Scientific Research Foundation of the Chengdu Pharmaceutical Association/ ; }, mesh = {Animals ; *CRISPR-Cas Systems ; Drug Carriers ; *Drug Delivery Systems ; Electroporation ; *Gene Editing ; Genetic Vectors ; Humans ; Mice ; }, abstract = {As gene-editing technology has become more and more popular in the life sciences, CRISPR has brought good news to scientific researchers because of its efficiency, convenience, and wide application. Its wide application has also promoted the development of basic scientific research, agriculture, basic medicine, and clinical treatment. However, how the CRISPR/dCas9 system is effectively delivered to the target organs or cells is still unknown. This paper briefly introduces the CRISPR/dCas9 system and then lists some common delivery methods and their characteristics.}, }
@article {pmid32274563, year = {2020}, author = {Brito, LF and Schultenkämper, K and Passaglia, LMP and Wendisch, VF}, title = {CRISPR interference-based gene repression in the plant growth promoter Paenibacillus sonchi genomovar Riograndensis SBR5.}, journal = {Applied microbiology and biotechnology}, volume = {104}, number = {11}, pages = {5095-5106}, pmid = {32274563}, issn = {1432-0614}, support = {ERA CoBioTech project C1Pro (No. 722361)//ERA CoBioTech/ ; }, mesh = {Biofilms/growth &amp; development ; *CRISPR-Cas Systems ; Chromosomes ; *Gene Expression ; L-Iditol 2-Dehydrogenase/metabolism ; Paenibacillus/enzymology/*genetics ; Promoter Regions, Genetic ; RNA, Guide ; Sorbitol/metabolism ; }, abstract = {Gene repression using the endonucleolytically deactivated dCas9 protein and sgRNAs (CRISPR interference or CRISPRi) is a useful approach to study gene functions. Here, we established CRISPRi in Paenibacillus sonchi genomovar Riograndensis SBR5, a plant growth promoting bacterium. CRISPRi system with sgRNAs targeting SBR5 endogenous genes spo0A, yaaT and ydjJ and plasmid-borne gfpUV was constructed and analyzed. Flow cytometry analysis revealed a significant decrease of reporter protein GFPUV signal in P. sonchi strains expressing gfpUV sgRNA in comparison with non-targeting controls. CRISPRi-based repression of chromosomal genes for regulation of sporulation spo0A and yaaT decreased sporulation and increased biofilm formation in SBR5. Repression of the sorbitol catabolic gene ydjJ revealed decreased specific activity of YdjJ in crude cell extracts and reduced biomass formation from sorbitol in growth experiments. Our work on CRISPRi-based gene repression serves as basis for gene function studies of the plant growth promoter P. sonchi SBR5. To our knowledge, the present study presents the first tool for gene repression established in Paenibacillus species.Key points• CRISPRi toward gene repression was applied for the first time in Paenibacillus.• CRISPRi of spo0A and yaaT depleted spores and increased biofilms in SBR5.• CRISPRi-based ydjJ repression decreased specific activity of sorbitol dehydrogenase.}, }
@article {pmid32221923, year = {2020}, author = {Zhang, WW and Lypaczewski, P and Matlashewski, G}, title = {Application of CRISPR/Cas9-Mediated Genome Editing in Leishmania.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2116}, number = {}, pages = {199-224}, doi = {10.1007/978-1-0716-0294-2_14}, pmid = {32221923}, issn = {1940-6029}, support = {MOP125996//CIHR/Canada ; }, mesh = {CRISPR-Cas Systems/genetics ; Cloning, Molecular/methods ; Gene Editing/*methods ; Genes, Protozoan/*genetics ; Genetic Vectors/genetics ; Humans ; Leishmania/*genetics/isolation &amp; purification ; Mutation ; Parasitology/methods ; RNA, Guide/genetics ; Transfection/methods ; }, abstract = {CRISPR-Cas9 is an RNA guided endonuclease derived from the bacterium Streptococcus pyogenes. Due to its simplicity, versatility, and high efficiency, it has been widely used for genome editing in a variety of organisms including the protozoan parasite Leishmania, the causative agent of human leishmaniasis. Compared to the traditional homologous recombination gene targeting method, CRISPR-Cas9 has been shown to be a more efficient method to delete or disrupt Leishmania genes, generate point mutations, and add tags to endogenous genes. Notably, the stable CRISPR expression systems were shown to delete multicopy family Leishmania genes and genes present in multiploid chromosomes, identify essential Leishmania genes, and create specific chromosome translocations. In this chapter, we describe detailed procedures on using the stable CRISPR expression system for genome editing in Leishmania. These procedures include CRISPR targeting site selection, gRNA design, cloning single and double gRNA coding sequences into the Leishmania CRISPR vector pLdCN, oligonucleotide donor and drug resistance selection donor design, Leishmania cell transfection, screening, and isolation of CRISPR-edited mutants. As the principles of gene editing are generally similar, many of these procedures could also apply to the transient Leishmania CRISPR systems described by other labs.}, }
@article {pmid32221922, year = {2020}, author = {Lander, N and Chiurillo, MA and Docampo, R}, title = {CRISPR/Cas9 Technology Applied to the Study of Proteins Involved in Calcium Signaling in Trypanosoma cruzi.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2116}, number = {}, pages = {177-197}, doi = {10.1007/978-1-0716-0294-2_13}, pmid = {32221922}, issn = {1940-6029}, mesh = {CRISPR-Cas Systems/genetics ; Calcium Channels/genetics/metabolism ; Calcium Signaling/*genetics ; Chagas Disease/parasitology ; Energy Metabolism/genetics ; Gene Editing/*methods ; Gene Knockout Techniques/methods ; Genes, Protozoan/*genetics ; Genetic Vectors/genetics ; Humans ; Inositol 1,4,5-Trisphosphate Receptors/genetics/metabolism ; Life Cycle Stages ; Parasitology/methods ; Phosphoprotein Phosphatases/genetics/metabolism ; Protozoan Proteins/*genetics/metabolism ; RNA, Guide/genetics ; Trypanosoma cruzi/*genetics/metabolism ; }, abstract = {Chagas disease is a vector-borne tropical disease affecting millions of people worldwide, for which there is no vaccine or satisfactory treatment available. It is caused by the protozoan parasite Trypanosoma cruzi and considered endemic from North to South America. This parasite has unique metabolic and structural characteristics that make it an attractive organism for basic research. The genetic manipulation of T. cruzi has been historically challenging, as compared to other pathogenic protozoans. However, the use of the prokaryotic CRISPR/Cas9 system for genome editing has significantly improved the ability to generate genetically modified T. cruzi cell lines, becoming a powerful tool for the functional study of proteins in different stages of this parasite's life cycle, including infective trypomastigotes and intracellular amastigotes. Using the CRISPR/Cas9 method that we adapted to T. cruzi, it has been possible to perform knockout, complementation and in situ tagging of T. cruzi genes. In our system we cotransfect T. cruzi epimastigotes with an expression vector containing the Cas9 sequence and a single guide RNA, together with a donor DNA template to promote DNA break repair by homologous recombination. As a result, we have obtained homogeneous populations of mutant epimastigotes using a single resistance marker to modify both alleles of the gene. Mitochondrial Ca2+ transport in trypanosomes is critical for shaping the dynamics of cytosolic Ca2+ increases, for the bioenergetics of the cells, and for viability and infectivity. In this chapter we describe the most effective methods to achieve genome editing in T. cruzi using as example the generation of mutant cell lines to study proteins involved in calcium homeostasis. Specifically, we describe the methods we have used for the study of three proteins involved in the calcium signaling cascade of T. cruzi: the inositol 1,4,5-trisphosphate receptor (TcIP3R), the mitochondrial calcium uniporter (TcMCU) and the calcium-sensitive pyruvate dehydrogenase phosphatase (TcPDP), using CRISPR/Cas9 technology as an approach to establish their role in the regulation of energy metabolism.}, }
@article {pmid32006418, year = {2020}, author = {Dannenmann, B and Nasri, M and Welte, K and Skokowa, J}, title = {CRISPR/Cas9 Genome Editing of Human-Induced Pluripotent Stem Cells Followed by Granulocytic Differentiation.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2115}, number = {}, pages = {471-483}, doi = {10.1007/978-1-0716-0290-4_27}, pmid = {32006418}, issn = {1940-6029}, mesh = {*CRISPR-Cas Systems ; Cell Line ; Electroporation/methods ; Gene Editing/*methods ; Granulocytes/*cytology/metabolism ; *Hematopoiesis ; Humans ; Induced Pluripotent Stem Cells/*cytology/metabolism ; Transfection/methods ; }, abstract = {Research on patient-derived induced pluripotent stem cells (iPSCs) could immensely benefit from the implementation of CRISPR/Cas9 genome editing of iPSCs, creating unique opportunities such as the establishment of isogenic iPSC lines for disease modeling or personalized patient-specific drug screenings. Here we describe a stepwise protocol of safe, efficient, and selection-free CRISPR/Cas9-mediated gene correction or knockout in human iPSCs followed by 3D spin-embryoid body (EB)-based hematopoietic/neutrophilic iPSC-differentiation.}, }
@article {pmid32006417, year = {2020}, author = {Mir, P and Ritter, M and Welte, K and Skokowa, J and Klimiankou, M}, title = {Gene Knockout in Hematopoietic Stem and Progenitor Cells Followed by Granulocytic Differentiation.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2115}, number = {}, pages = {455-469}, doi = {10.1007/978-1-0716-0290-4_26}, pmid = {32006417}, issn = {1940-6029}, mesh = {*CRISPR-Cas Systems ; Cells, Cultured ; Gene Editing/*methods ; Gene Knockout Techniques/methods ; Granulocytes/*cytology/metabolism ; Hematopoietic Stem Cells/*cytology/metabolism ; Humans ; *Leukopoiesis ; RNA, Guide/genetics ; }, abstract = {In this chapter, we present an optimized CRISPR/Cas9 RNP nucleofection approach for gene knockout (KO) in hematopoietic stem and progenitor cells (HSPCs). With experimentally proved active locus-specific sgRNAs, we routinely reach over 80% gene KO in HSPCs, thus avoiding the need for cell sorting or enrichment of targeted cell population. Additionally, we provide a protocol for in vitro granulocytic differentiation of HSPCs after gene KO and detailed description of granulocyte function tests which can be applied to study the effects of a particular gene KO.}, }
@article {pmid32006416, year = {2020}, author = {Bai, B and Myklebust, JH and Wälchli, S}, title = {Gene Editing in B-Lymphoma Cell Lines Using CRISPR/Cas9 Technology.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2115}, number = {}, pages = {445-454}, doi = {10.1007/978-1-0716-0290-4_25}, pmid = {32006416}, issn = {1940-6029}, mesh = {B-Lymphocytes/metabolism ; CRISPR-Associated Protein 9/genetics ; *CRISPR-Cas Systems ; Cell Line, Tumor ; Clustered Regularly Interspaced Short Palindromic Repeats ; Electroporation/methods ; Gene Editing/*methods ; Gene Transfer Techniques ; Humans ; Lymphoma, B-Cell/*genetics/therapy ; RNA, Guide/genetics ; Transduction, Genetic/methods ; }, abstract = {Genome editing in eukaryotes has greatly improved through the application of targeted editing tools. The development of the CRISPR/Cas9 technology has facilitated genome editing in mammalian cells. However, efficient delivery of CRISPR components into cells growing in suspension remains a challenge. Here, we present a strategy for sequential delivery of the two essential components, Cas9 and sgRNA, into B-lymphoid cell lines. Stable Cas9 expression is obtained by retroviral transduction, before sgRNA is transiently delivered into the Cas9+ cells. This method improves the on-target efficiency of genome editing and, through the transient presence of sgRNA, reduces the potential off-target sites. The current method can be easily applied to other cell types that are difficult to edit with CRISPR/Cas9.}, }
@article {pmid32006415, year = {2020}, author = {Laoharawee, K and Johnson, MJ and Moriarity, BS}, title = {CRISPR/Cas9-Mediated Genome Engineering of Primary Human B Cells.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2115}, number = {}, pages = {435-444}, doi = {10.1007/978-1-0716-0290-4_24}, pmid = {32006415}, issn = {1940-6029}, mesh = {B-Lymphocytes/cytology/*metabolism ; *CRISPR-Cas Systems ; Cells, Cultured ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing/*methods ; Humans ; Leukocytes, Mononuclear/cytology/metabolism ; }, abstract = {The CRISPR/Cas9 system allows for site-specific gene editing and genome engineering of primary human cells. Here we describe methods for gene editing and genome engineering of B cells isolated from human peripheral blood mononuclear cells using CRISPR/Cas9. Editing frequencies of up to 90% and integration rates greater than 60% can be achieved with this method.}, }
@article {pmid32006413, year = {2020}, author = {Wälchli, S and Sioud, M}, title = {Next Generation of Adoptive T Cell Therapy Using CRISPR/Cas9 Technology: Universal or Boosted?.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2115}, number = {}, pages = {407-417}, doi = {10.1007/978-1-0716-0290-4_22}, pmid = {32006413}, issn = {1940-6029}, mesh = {Animals ; *CRISPR-Cas Systems ; Gene Editing/*methods ; Humans ; Immunotherapy, Adoptive/adverse effects/*methods ; T-Lymphocytes/metabolism/transplantation ; }, abstract = {Adoptive T cell therapy (ACT) using either chimeric antigen receptor (CAR)- or T cell receptor (TCR)-engineered lymphocytes has emerged as a promising strategy to treat cancer. However, this therapy is still facing enormous challenges such as poor quality of autologous T cells, T cell exhaustion, and the immune suppressive tumor microenvironments. Additionally, graft-versus-host disease is an issue that must be addressed to allow the use of allogeneic T cells. Strategies to overcome these therapeutic challenges using gene editing technology are now being developed. One strategy is to disrupt TCR and/or MHC expression in healthy donor T cells to generate T cells for universal use. Another strategy is to improve the quality of patient's T cells by eliminating either the expression of selected immune checkpoint receptors or negative regulators of TCR signaling and/or T-cell homeostasis. Here, we review the use of CRISPR-Cas9 platform in T cell engineering with a focus on the development of universal T cells and boosted autologous cells for next-generation ACT.}, }
@article {pmid32006412, year = {2020}, author = {Prykhozhij, SV and Cordeiro-Santanach, A and Caceres, L and Berman, JN}, title = {Genome Editing in Zebrafish Using High-Fidelity Cas9 Nucleases: Choosing the Right Nuclease for the Task.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2115}, number = {}, pages = {385-405}, doi = {10.1007/978-1-0716-0290-4_21}, pmid = {32006412}, issn = {1940-6029}, mesh = {Animals ; CRISPR-Associated Protein 9/*genetics ; *CRISPR-Cas Systems ; Gene Editing/*methods ; Microinjections ; Mutagenesis, Site-Directed ; RNA, Guide/genetics ; Zebrafish/*genetics ; }, abstract = {Shortly after the development of the CRISPR/Cas9 system, it was recognized that it is prone to induce off-target mutations at significant frequencies. Therefore, there is a strong motivation to develop Cas9 enzymes with reduced off-target activity. Multiple rational design or selection approaches have been applied to develop several Cas9 versions with reduced off-target activities (high fidelity). To make these high-fidelity Cas9s available for model systems other than human cells and bacterial strains, as, for example, in zebrafish, new specialized expression vectors need to be developed. In this chapter, we focused on the HypaCas9 and HiFi Cas9 high-fidelity enzymes and incorporated the mutations of these Cas9 versions into a codon-optimized zebrafish Cas9 vector. This optimized vector was further improved by introducing an artificial polyadenine insert (A71) since polyadenylation is known to enhance mRNA translational efficiency. The Hypa-nCas9n and HiFi-nCas9n vectors were produced by single-site mutagenesis from pT3TS-nCas9n-A71 vector. We then tested the polyadenylated mRNAs for nCas9n, Hypa-nCas9n, HiFi-nCas9n, and HiFi-Cas9 protein for editing efficiency in five genome editing strategies and found that these high-fidelity Cas9 versions had different performances ranging from activity at 2-4 sites, where the wild-type nCas9n is active, indicating that these Cas9 versions have different sgRNA preferences. In summary, the developed new high-fidelity Cas9 vectors will enable researchers to perform much more accurate genome editing.}, }
@article {pmid32006411, year = {2020}, author = {Sahoo, N and Cuello, V and Udawant, S and Litif, C and Mustard, JA and Keniry, M}, title = {CRISPR-Cas9 Genome Editing in Human Cell Lines with Donor Vector Made by Gibson Assembly.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2115}, number = {}, pages = {365-383}, pmid = {32006411}, issn = {1940-6029}, support = {SC3 GM132053/GM/NIGMS NIH HHS/United States ; }, mesh = {CRISPR-Associated Protein 9/genetics ; *CRISPR-Cas Systems ; Cell Line ; Clustered Regularly Interspaced Short Palindromic Repeats ; Forkhead Box Protein O3/genetics ; Gene Editing/*methods ; Genetic Vectors/genetics ; Humans ; Mutation ; RNA, Guide/genetics ; }, abstract = {CRISPR Cas9 genome editing allows researchers to modify genes in a multitude of ways including to obtain deletions, epitope-tagged loci, and knock-in mutations. Within 6 years of its initial application, CRISPR-Cas9 genome editing has been widely employed, but disadvantages to this method, such as low modification efficiencies and off-target effects, need careful consideration. Obtaining custom donor vectors can also be expensive and time-consuming. This chapter details strategies to overcome barriers to CRISPR-Cas9 genome editing as well as recent developments in employing this technique.}, }
@article {pmid32006410, year = {2020}, author = {Hiranniramol, K and Chen, Y and Wang, X}, title = {CRISPR/Cas9 Guide RNA Design Rules for Predicting Activity.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2115}, number = {}, pages = {351-364}, doi = {10.1007/978-1-0716-0290-4_19}, pmid = {32006410}, issn = {1940-6029}, support = {R01 DE026471/DE/NIDCR NIH HHS/United States ; R01 GM089784/GM/NIGMS NIH HHS/United States ; }, mesh = {Animals ; CRISPR-Associated Protein 9/genetics ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing/methods ; Genomics/methods ; Humans ; Models, Genetic ; RNA, Guide/*genetics ; }, abstract = {A critical stage in performing gene editing experiments using the CRISPR/Cas9 system is the design of guide RNA (gRNA). In this chapter, we conduct a review of the current gRNA design rules for maximizing on-target Cas9 activity while minimizing off-target activity. In addition, we present some of the currently available computational tools for gRNA activity prediction and assay design.}, }
@article {pmid32006392, year = {2020}, author = {Sioud, M}, title = {RNA and CRISPR Interferences: Past, Present, and Future Perspectives.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2115}, number = {}, pages = {1-22}, doi = {10.1007/978-1-0716-0290-4_1}, pmid = {32006392}, issn = {1940-6029}, mesh = {Animals ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing/*methods ; Gene Transfer Techniques ; Genetic Therapy/methods ; Humans ; RNA/genetics ; *RNA Interference ; RNA, Small Interfering/genetics ; }, abstract = {RNA interference (RNAi), a natural gene silencing process, is a widely used technique in basic research, preclinical studies, and drug development strategies. Although the technique has great potential to generate new human therapies and treat undruggable diseases, the clinical application of RNAi is still challenging primarily because of the delivery problem and potential off-target effects. Over the past two decades, great efforts have been undertaken to develop delivery agents and chemical modifications to overcome these challenges. Such advances in RNA delivery and chemical modifications have benefited researchers who are developing gene-editing therapies based on CRISPR-Cas9, an RNA-guided endonuclease, which is already having a major impact on biology and medicine. Here, I review the discovery of these two interference tools, identify the technical challenges yet to be overcome and provide some perspectives on how these two RNA-based technologies can be harnessed to treat human diseases.}, }
@article {pmid31988004, year = {2020}, author = {Zaynab, M and Sharif, Y and Fatima, M and Afzal, MZ and Aslam, MM and Raza, MF and Anwar, M and Raza, MA and Sajjad, N and Yang, X and Li, S}, title = {CRISPR/Cas9 to generate plant immunity against pathogen.}, journal = {Microbial pathogenesis}, volume = {141}, number = {}, pages = {103996}, doi = {10.1016/j.micpath.2020.103996}, pmid = {31988004}, issn = {1096-1208}, mesh = {Bacterial Infections ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Crops, Agricultural/genetics ; Disease Resistance/*genetics ; Gene Editing/*methods ; Mycoses ; Plant Immunity/genetics ; Plants, Genetically Modified ; Virus Diseases ; }, abstract = {Different types of molecular approaches have been used for improving resistance against pathogens to secure food. Efficient and advanced genome editing tool as paralleled to earlier techniques like Zinc Finger Nuclease (ZFN), transcription activator-like effector nucleases (TALENs), and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR). The approach of CRISPR/Cas9 has updated our abilities of genetic manipulation in many crops. The assembly of purposes that can be achieved through CRISPR/Cas9 and its related products make it a powerful system that will expose novel prospects in the complex domain of plant-pathogen interactions and will help to develop crop resistance against pathogens. CRISPR/Cas9 engineering permits DNA endonuclease guided by an RNA for a range of genome engineering applications across various eukaryotic species and provides an effective platform to create resistance against bacteria, viruses, insects, and fungi. In this review, we discuss CRISPR-Cas9 engineered crop plants resistant to specific pathogens.}, }
@article {pmid31476099, year = {2020}, author = {Xing, WQ and Ma, SY and Liu, YY and Xia, QY}, title = {CRISPR/dCas9-mediated imaging of endogenous genomic loci in living Bombyx mori cells.}, journal = {Insect science}, volume = {27}, number = {6}, pages = {1360-1364}, doi = {10.1111/1744-7917.12722}, pmid = {31476099}, issn = {1744-7917}, support = {31530071//National Natural Science Foundation of China/ ; 31802011//National Natural Science Foundation of China/ ; }, mesh = {Animals ; Bombyx/*genetics ; *CRISPR-Associated Protein 9 ; *CRISPR-Cas Systems ; Cell Line ; Fibroins/*genetics ; Genes, Insect ; Genome ; Molecular Imaging/methods ; }, }
@article {pmid33514840, year = {2021}, author = {Wang, Z and Maluenda, J and Giraut, L and Vieille, T and Lefevre, A and Salthouse, D and Radou, G and Moulinas, R and Astete, S and D'Avezac, P and Smith, G and André, C and Allemand, JF and Bensimon, D and Croquette, V and Ouellet, J and Hamilton, G}, title = {Detection of genetic variation and base modifications at base-pair resolution on both DNA and RNA.}, journal = {Communications biology}, volume = {4}, number = {1}, pages = {128}, pmid = {33514840}, issn = {2399-3642}, support = {9830//EC | Eurostars/ ; ANR-15-CE12-0015//Agence Nationale de la Recherche (French National Research Agency)/ ; 829965//EC | Horizon 2020 Framework Programme (EU Framework Programme for Research and Innovation H2020)/ ; }, abstract = {Accurate decoding of nucleic acid variation is critical to understand the complexity and regulation of genome function. Here we use a single-molecule magnetic tweezer (MT) platform to identify sequence variation and map a range of important epigenetic base modifications with high sensitivity, specificity, and precision in the same single molecules of DNA or RNA. We have also developed a highly specific amplification-free CRISPR-Cas enrichment strategy to isolate genomic regions from native DNA. We demonstrate enrichment of DNA from both E. coli and the FMR1 5'UTR coming from cells derived from a Fragile X carrier. From these kilobase-length enriched molecules we could characterize the differential levels of adenine and cytosine base modifications on E. coli, and the repeat expansion length and methylation status of FMR1. Together these results demonstrate that our platform can detect a variety of genetic, epigenetic, and base modification changes concomitantly within the same single molecules.}, }
@article {pmid33511745, year = {2021}, author = {Huang, TK and Armstrong, B and Schindele, P and Puchta, H}, title = {Efficient gene targeting in Nicotiana tabacum using CRISPR/SaCas9 and temperature tolerant LbCas12a.}, journal = {Plant biotechnology journal}, volume = {}, number = {}, pages = {}, doi = {10.1111/pbi.13546}, pmid = {33511745}, issn = {1467-7652}, support = {MOST 106-2917-I-564-007-A1//Ministry of Science and Technology, Taiwan/ ; 760331-2//Horizon 2020 Framework Programme/ ; }, abstract = {Nicotiana tabacum is a non-food herb that has the potential to be utilized as bio-factory for generating medicines, vaccines or valuable small metabolites. To achieve these goals, the improvement of genetic tools for pre-designed genome modifications is indispensable. The development of CRISPR/Cas nucleases allows the induction of site-specific double-strand breaks to enhance homologous recombination-mediated gene targeting (GT). However, the efficiency of GT is still a challenging obstacle for many crops including tobacco. Recently, studies in several plant species indicated that by replacing SpCas9 with other CRISPR/Cas-based nucleases, GT efficiencies might be enhanced considerably. Therefore, we tested SaCas9 as well as a temperature-insensitive version of LbCas12a (ttLbCas12a) for targeting the tobacco SuRB gene. At the same time, we also optimized the protocol for Agrobacterium-mediated tobacco transformation and tissue culture. In this way, we could improve GT efficiencies to up to a third of the inoculated cotyledons when using ttLbCas12a, which outperformed SaCas9 considerably. In addition, we could show that the conversion tract length of the GT reaction can be up to 606 bp long and in the majority of cases, it is longer than 250 bp. We obtained multiple heritable GT events, mostly heterozygous, but also biallelic GT events and some without T-DNA integration. Thus, we were not only able to obtain CRISPR/Cas-based heritable GT events in allotetraploid Nicotiana tabacum for the first time, but our results also indicate that ttLbCas12a might be a superior alternative for gene editing and GT in tobacco as well as in other crops.}, }
@article {pmid33511075, year = {2020}, author = {Laugi, H}, title = {Discovery of Hepatitis C Virus: 2020 Nobel Prize in Medicine.}, journal = {Euroasian journal of hepato-gastroenterology}, volume = {10}, number = {2}, pages = {105-108}, doi = {10.5005/jp-journals-10018-1326}, pmid = {33511075}, issn = {2231-5047}, abstract = {Hepatitis C virus (HCV) accounts for hepatitis, liver cirrhosis, hepatocellular carcinoma, and liver transplantation. This virus is a single-stranded RNA virus that belongs to the Flaviviridae family. According to the WHO, about 71 million people have chronic HCV infections around the globe in 2020, and hence, it is a plague of humankind. The credit of discovery of HCV goes to Michael Houghton, Harvey Alter, and Charles Rice for which they are awarded 2020 Nobel Prize in Medicine. Their contribution has given better hope to mankind to cure HCV for the first time in the history. With the use of pegylated interferon and ribavirin jointly, higher SVR has been found comparatively, even in patients with chronic liver diseases. However, due to excessive pain tolerated by patients, interferon (IFN)-based therapy is rapidly being replaced with IFN-free DAA regimens. With the onset of resistance to DAA drugs, CRISPR-Cas system can be used to modify the viral genome to impair their ability to develop resistance. How to cite this article: Laugi H. Discovery of Hepatitis C Virus: 2020 Nobel Prize in Medicine. Euroasian J Hepato-Gastroenterol 2020;10(2): 105-108.}, }
@article {pmid33306959, year = {2021}, author = {Fozouni, P and Son, S and Díaz de León Derby, M and Knott, GJ and Gray, CN and D'Ambrosio, MV and Zhao, C and Switz, NA and Kumar, GR and Stephens, SI and Boehm, D and Tsou, CL and Shu, J and Bhuiya, A and Armstrong, M and Harris, AR and Chen, PY and Osterloh, JM and Meyer-Franke, A and Joehnk, B and Walcott, K and Sil, A and Langelier, C and Pollard, KS and Crawford, ED and Puschnik, AS and Phelps, M and Kistler, A and DeRisi, JL and Doudna, JA and Fletcher, DA and Ott, M}, title = {Amplification-free detection of SARS-CoV-2 with CRISPR-Cas13a and mobile phone microscopy.}, journal = {Cell}, volume = {184}, number = {2}, pages = {323-333.e9}, pmid = {33306959}, issn = {1097-4172}, support = {U54 HL143541/HL/NHLBI NIH HHS/United States ; R61 AI140465/AI/NIAID NIH HHS/United States ; R61 DA048444/DA/NIDA NIH HHS/United States ; T32 GM007618/GM/NIGMS NIH HHS/United States ; F30 AI143401/AI/NIAID NIH HHS/United States ; }, mesh = {Animals ; COVID-19 Nucleic Acid Testing/economics/instrumentation/*methods ; CRISPR-Cas Systems ; Cell Line ; Cell Phone/*instrumentation ; Coronavirus Nucleocapsid Proteins/genetics ; Humans ; Nasopharynx/virology ; Optical Imaging/instrumentation/*methods ; Phosphoproteins/genetics ; Point-of-Care Testing ; RNA Interference ; RNA, Viral/*analysis/genetics ; Sensitivity and Specificity ; Viral Load/economics/instrumentation/*methods ; }, abstract = {The December 2019 outbreak of a novel respiratory virus, SARS-CoV-2, has become an ongoing global pandemic due in part to the challenge of identifying symptomatic, asymptomatic, and pre-symptomatic carriers of the virus. CRISPR diagnostics can augment gold-standard PCR-based testing if they can be made rapid, portable, and accurate. Here, we report the development of an amplification-free CRISPR-Cas13a assay for direct detection of SARS-CoV-2 from nasal swab RNA that can be read with a mobile phone microscope. The assay achieved ∼100 copies/μL sensitivity in under 30 min of measurement time and accurately detected pre-extracted RNA from a set of positive clinical samples in under 5 min. We combined crRNAs targeting SARS-CoV-2 RNA to improve sensitivity and specificity and directly quantified viral load using enzyme kinetics. Integrated with a reader device based on a mobile phone, this assay has the potential to enable rapid, low-cost, point-of-care screening for SARS-CoV-2.}, }
@article {pmid33257546, year = {2020}, author = {Bhave, M and Mino, RE and Wang, X and Lee, J and Grossman, HM and Lakoduk, AM and Danuser, G and Schmid, SL and Mettlen, M}, title = {Functional characterization of 67 endocytic accessory proteins using multiparametric quantitative analysis of CCP dynamics.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {117}, number = {50}, pages = {31591-31602}, pmid = {33257546}, issn = {1091-6490}, support = {R01 GM073165/GM/NIGMS NIH HHS/United States ; R01 MH061345/MH/NIMH NIH HHS/United States ; R37 MH061345/MH/NIMH NIH HHS/United States ; }, mesh = {Adaptor Proteins, Vesicular Transport/genetics/*metabolism ; CRISPR-Cas Systems/genetics ; Cell Line ; Cell Membrane/*metabolism ; Clathrin/*metabolism ; Cluster Analysis ; Coated Pits, Cell-Membrane/*metabolism ; Endocytosis/*physiology ; Gene Knockdown Techniques ; Green Fluorescent Proteins/chemistry/genetics ; Humans ; Intravital Microscopy/methods ; Luminescent Agents/chemistry ; Microscopy, Fluorescence/methods ; Molecular Imaging/methods ; RNA, Small Interfering/metabolism ; }, abstract = {Clathrin-mediated endocytosis (CME) begins with the nucleation of clathrin assembly on the plasma membrane, followed by stabilization and growth/maturation of clathrin-coated pits (CCPs) that eventually pinch off and internalize as clathrin-coated vesicles. This highly regulated process involves a myriad of endocytic accessory proteins (EAPs), many of which are multidomain proteins that encode a wide range of biochemical activities. Although domain-specific activities of EAPs have been extensively studied, their precise stage-specific functions have been identified in only a few cases. Using single-guide RNA (sgRNA)/dCas9 and small interfering RNA (siRNA)-mediated protein knockdown, combined with an image-based analysis pipeline, we have determined the phenotypic signature of 67 EAPs throughout the maturation process of CCPs. Based on these data, we show that EAPs can be partitioned into phenotypic clusters, which differentially affect CCP maturation and dynamics. Importantly, these clusters do not correlate with functional modules based on biochemical activities. Furthermore, we discover a critical role for SNARE proteins and their adaptors during early stages of CCP nucleation and stabilization and highlight the importance of GAK throughout CCP maturation that is consistent with GAK's multifunctional domain architecture. Together, these findings provide systematic, mechanistic insights into the plasticity and robustness of CME.}, }
@article {pmid33202035, year = {2020}, author = {Potts, MA and McDonald, JA and Sutherland, KD and Herold, MJ}, title = {Critical cancer vulnerabilities identified by unbiased CRISPR/Cas9 screens inform on efficient cancer Immunotherapy.}, journal = {European journal of immunology}, volume = {50}, number = {12}, pages = {1871-1884}, doi = {10.1002/eji.202048712}, pmid = {33202035}, issn = {1521-4141}, mesh = {Animals ; CRISPR-Cas Systems/*genetics/immunology ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics/immunology ; Gene Editing/methods ; Humans ; Immunotherapy/methods ; Neoplasms/*genetics/*immunology/therapy ; }, abstract = {The mutational landscape of human cancers is highly complex. While next generation sequencing aims to comprehensively catalogue somatic alterations in tumor cells, it fails to delineate driver from passenger mutations. Functional genomic approaches, particularly CRISPR/Cas9, enable both gene discovery, and annotation of gene function. Indeed, recent CRISPR/Cas9 technologies have flourished with the development of more sophisticated and versatile platforms capable of gene knockouts to high throughput genome wide editing of a single nucleotide base. With new platforms constantly emerging, it can be challenging to navigate what CRISPR tools are available and how they can be effectively applied to understand cancer biology. This review provides an overview of current and emerging CRISPR technologies and their power to model cancer and identify novel treatments. Specifically, how CRISPR screening approaches have been exploited to enhance immunotherapies through the identification of tumor intrinsic and extrinsic mechanisms to escape immune recognition will be discussed.}, }
@article {pmid33060800, year = {2020}, author = {}, title = {Henceforth CRISPR.}, journal = {Nature biomedical engineering}, volume = {4}, number = {11}, pages = {1023}, pmid = {33060800}, issn = {2157-846X}, mesh = {CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Editing/history/*methods ; History, 21st Century ; Humans ; Nobel Prize ; }, }
@article {pmid32168764, year = {2020}, author = {Xu, Y and Liu, H and Pan, H and Wang, X and Zhang, Y and Yao, B and Li, N and Lai, L and Li, Z}, title = {CRISPR/Cas9-mediated Disruption of Fibroblast Growth Factor 5 in Rabbits Results in a Systemic Long Hair Phenotype by Prolonging Anagen.}, journal = {Genes}, volume = {11}, number = {3}, pages = {}, pmid = {32168764}, issn = {2073-4425}, mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Exons/genetics ; Fibroblast Growth Factor 5/*genetics ; Hair/*growth &amp; development ; Hair Follicle/*growth &amp; development/metabolism ; Phenotype ; RNA, Messenger/genetics ; Rabbits ; }, abstract = {Hair growth and morphology are generally regulated by the hair cycle in mammals. Fibroblast Growth Factor 5 (FGF5), which is a hair cycle regulator, has a role in regulating the hair cycle during the transition from the anagen phase to the catagen phase, and a hereditary long hair phenotype has been widely reported when FGF5 is mutated in humans and other species. However, there has been no such report in rabbits. Thus, the first exon of rabbit FGF5 was disrupted by the CRISPR/Cas9 system, and the phenotype of FGF5-/- rabbits was characterized while using hematoxylin and eosin (H&amp;E) staining, immunohistochemistry, quantitative PCR, scanning electron microscopy, and western blotting. The results showed a significant and systemic long hair phenotype in the FGF5-/- rabbits, which indicated that FGF5 is a negative regulator of hair growth. In addition, a decreased diameter of the fiber and a higher area proportion of hair follicle clusters were determined in FGF5-/- rabbits as compared with the WT rabbits. Further investigation verified that prolonging the anagen phase in rabbits, with decreased BMP2/4 pathway signaling and increased VERSICAN pathway signaling, caused the systemic long hair phenotype. Taken together, these results indicate a systemic long hair phenotype by prolonging anagen in FGF5-/- rabbits, which could be widely used for Fur production and an ideal model for studying the mechanism of long hair in the future.}, }
@article {pmid32138192, year = {2020}, author = {Bouzroud, S and Gasparini, K and Hu, G and Barbosa, MAM and Rosa, BL and Fahr, M and Bendaou, N and Bouzayen, M and Zsögön, A and Smouni, A and Zouine, M}, title = {Down Regulation and Loss of Auxin Response Factor 4 Function Using CRISPR/Cas9 Alters Plant Growth, Stomatal Function and Improves Tomato Tolerance to Salinity and Osmotic Stress.}, journal = {Genes}, volume = {11}, number = {3}, pages = {}, pmid = {32138192}, issn = {2073-4425}, mesh = {Abscisic Acid/metabolism ; CRISPR-Cas Systems/genetics ; Gene Expression Regulation, Plant/genetics ; Lycopersicon esculentum/*genetics/growth &amp; development ; Osmotic Pressure/physiology ; Plant Development/genetics ; Plant Leaves/genetics/growth &amp; development ; Plant Proteins/*genetics ; Plants, Genetically Modified/genetics/growth &amp; development ; Salinity ; Salt Stress/*genetics ; Salt Tolerance/*genetics ; }, abstract = {Auxin controls multiple aspects of plant growth and development. However, its role in stress responses remains poorly understood. Auxin acts on the transcriptional regulation of target genes, mainly through Auxin Response Factors (ARF). This study focuses on the involvement of SlARF4 in tomato tolerance to salinity and osmotic stress. Using a reverse genetic approach, we found that the antisense down-regulation of SlARF4 promotes root development and density, increases soluble sugars content and maintains chlorophyll content at high levels under stress conditions. Furthermore, ARF4-as displayed higher tolerance to salt and osmotic stress through reduced stomatal conductance coupled with increased leaf relative water content and Abscisic acid (ABA) content under normal and stressful conditions. This increase in ABA content was correlated with the activation of ABA biosynthesis genes and the repression of ABA catabolism genes. Cu/ZnSOD and mdhar genes were up-regulated in ARF4-as plants which can result in a better tolerance to salt and osmotic stress. A CRISPR/Cas9 induced SlARF4 mutant showed similar growth and stomatal responses as ARF4-as plants, which suggest that arf4-cr can tolerate salt and osmotic stresses. Our data support the involvement of ARF4 as a key factor in tomato tolerance to salt and osmotic stresses and confirm the use of CRISPR technology as an efficient tool for functional reverse genetics studies.}, }
@article {pmid31996360, year = {2020}, author = {Lund, C and Yellapragada, V and Vuoristo, S and Balboa, D and Trova, S and Allet, C and Eskici, N and Pulli, K and Giacobini, P and Tuuri, T and Raivio, T}, title = {Characterization of the human GnRH neuron developmental transcriptome using a GNRH1-TdTomato reporter line in human pluripotent stem cells.}, journal = {Disease models &amp; mechanisms}, volume = {13}, number = {3}, pages = {}, pmid = {31996360}, issn = {1754-8411}, mesh = {CRISPR-Associated Protein 9/metabolism ; CRISPR-Cas Systems/genetics ; Cell Line ; Fetus/cytology ; Fibroblast Growth Factor 8/pharmacology ; *Genes, Reporter ; Gonadotropin-Releasing Hormone/*metabolism ; Humans ; Hypogonadism/genetics ; LIM-Homeodomain Proteins/metabolism ; Neurons/drug effects/*metabolism ; Pluripotent Stem Cells/drug effects/*metabolism ; RNA, Messenger/genetics/metabolism ; Transcription Factors/metabolism ; Transcriptome/*genetics ; Up-Regulation/drug effects/genetics ; }, abstract = {Gonadotropin-releasing hormone (GnRH) neurons provide a fundamental signal for the onset of puberty and subsequent reproductive functions by secretion of gonadotropin-releasing hormone. Their disrupted development or function leads to congenital hypogonadotropic hypogonadism (CHH). To model the development of human GnRH neurons, we generated a stable GNRH1-TdTomato reporter cell line in human pluripotent stem cells (hPSCs) using CRISPR-Cas9 genome editing. RNA-sequencing of the reporter clone, differentiated into GnRH neurons by dual SMAD inhibition and FGF8 treatment, revealed 6461 differentially expressed genes between progenitors and GnRH neurons. Expression of the transcription factor ISL1, one of the top 50 most upregulated genes in the TdTomato-expressing GnRH neurons, was confirmed in 10.5 gestational week-old human fetal GnRH neurons. Among the differentially expressed genes, we detected 15 genes that are implicated in CHH and several genes that are implicated in human puberty timing. Finally, FGF8 treatment in the neuronal progenitor pool led to upregulation of 37 genes expressed both in progenitors and in TdTomato-expressing GnRH neurons, which suggests upstream regulation of these genes by FGF8 signaling during GnRH neuron differentiation. These results illustrate how hPSC-derived human GnRH neuron transcriptomic analysis can be utilized to dissect signaling pathways and gene regulatory networks involved in human GnRH neuron development.This article has an associated First Person interview with the first author of the paper.}, }
@article {pmid31950445, year = {2020}, author = {Dong, J and Kan, B and Liu, H and Zhan, M and Wang, S and Xu, G and Han, R and Ni, Y}, title = {CRISPR-Cpf1-Assisted Engineering of Corynebacterium glutamicum SNK118 for Enhanced L-Ornithine Production by NADP-Dependent Glyceraldehyde-3-Phosphate Dehydrogenase and NADH-Dependent Glutamate Dehydrogenase.}, journal = {Applied biochemistry and biotechnology}, volume = {191}, number = {3}, pages = {955-967}, doi = {10.1007/s12010-020-03231-y}, pmid = {31950445}, issn = {1559-0291}, support = {31601463//National Natural Science Foundation of China/ ; LITE2018-07//National First-class Discipline Program of Light Industry Technology and Engineering/ ; 2018YFA0901700//National Key R&amp;D Program/ ; }, mesh = {Arginine/metabolism ; Bioreactors ; *CRISPR-Cas Systems ; Citrulline/metabolism ; Corynebacterium glutamicum/enzymology/*genetics ; Escherichia coli/genetics/metabolism ; Fermentation ; Genome, Bacterial ; Glucose/metabolism ; Glutamate Synthase (NADH)/*metabolism ; Glyceraldehyde-3-Phosphate Dehydrogenases/*metabolism ; Glycolysis ; Industrial Microbiology ; Metabolic Engineering ; NADP/metabolism ; Ornithine/*biosynthesis ; Plasmids/genetics ; Recombinant Proteins/metabolism ; Transcription, Genetic ; }, abstract = {Here, Corynebacterium glutamicum SNK118 was metabolically engineered for L-ornithine production through CRISPR-Cpf1-based genome manipulation and plasmid-based heterologous overexpression. Genes argF, argR, and ncgl2228 were deleted to block the degradation of L-ornithine, eliminate the global transcriptional repression, and alleviate the competitive branch pathway, respectively. Overexpression of CsgapC (NADP-dependent glyceraldehyde 3-phosphate dehydrogenases gene from Clostridium saccharobutylicum DSM 13864) and BsrocG (NADH-dependent glutamate dehydrogenase gene from Bacillus subtilis HB-1) resulted markedly increased ornithine biosynthesis. Eventually, the engineered strain KBJ11 (SNK118ΔargRΔargFΔncgl2228/pXMJ19-CsgapC-BsrocG) was constructed for L-ornithine overproduction. In fed-batch fermentation, L-ornithine of 88.26 g/L with productivity of 1.23 g/L/h (over 72 h) and yield of 0.414 g/g glucose was achieved by strain KBJ11 in a 10-L bioreactor. Our result represents the highest titer and yield of L-ornithine production by microbial fermentation. This study suggests that heterologous expression of CsgapC and BsrocG could promote L-ornithine production by C. glutamicum strains.}, }
@article {pmid31745578, year = {2020}, author = {Sun, S and Wang, X and Wang, K and Cui, X}, title = {Dissection of complex traits of tomato in the post-genome era.}, journal = {TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik}, volume = {133}, number = {5}, pages = {1763-1776}, doi = {10.1007/s00122-019-03478-y}, pmid = {31745578}, issn = {1432-2242}, mesh = {CRISPR-Cas Systems ; Chromosome Mapping ; *Gene Editing ; *Genome, Plant ; Lycopersicon esculentum/*genetics/*growth &amp; development ; *Multifactorial Inheritance ; Plant Breeding/*methods ; *Quantitative Trait Loci ; }, abstract = {KEY MESSAGE: We present the main advances of dissection of complex traits in tomato by omics, the genes identified to control complex traits and the application of CRISPR/Cas9 in tomato breeding. Complex traits are believed to be under the control of multiple genes, each with different effects and interaction with environmental factors. Advance development of sequencing and molecular technologies has enabled the recognition of the genomic structure of most organisms and the identification of a nearly limitless number of markers that have made it to accelerate the speed of QTL identification and gene cloning. Meanwhile, multiomics have been used to identify the genetic variations among different tomato species, determine the expression profiles of genes in different tissues and at distinct developmental stages, and detect metabolites in different pathways and processes. The combination of these data facilitates to reveal mechanism underlying complex traits. Moreover, mutants generated by mutagens and genome editing provide relatively rich genetic variation for deciphering the complex traits and exploiting them in tomato breeding. In this article, we present the main advances of complex trait dissection in tomato by omics since the release of the tomato genome sequence in 2012. We provide further insight into some tomato complex traits because of the causal genetic variations discovered so far and explore the utilization of CRISPR/Cas9 for the modification of tomato complex traits.}, }
@article {pmid31744829, year = {2020}, author = {Li, F and Huang, Q and Luster, TA and Hu, H and Zhang, H and Ng, WL and Khodadadi-Jamayran, A and Wang, W and Chen, T and Deng, J and Ranieri, M and Fang, Z and Pyon, V and Dowling, CM and Bagdatlioglu, E and Almonte, C and Labbe, K and Silver, H and Rabin, AR and Jani, K and Tsirigos, A and Papagiannakopoulos, T and Hammerman, PS and Velcheti, V and Freeman, GJ and Qi, J and Miller, G and Wong, KK}, title = {In Vivo Epigenetic CRISPR Screen Identifies Asf1a as an Immunotherapeutic Target in Kras-Mutant Lung Adenocarcinoma.}, journal = {Cancer discovery}, volume = {10}, number = {2}, pages = {270-287}, pmid = {31744829}, issn = {2159-8290}, support = {R01 CA216188/CA/NCI NIH HHS/United States ; R01 CA219670/CA/NCI NIH HHS/United States ; U01 CA233084/CA/NCI NIH HHS/United States ; R01 CA166480/CA/NCI NIH HHS/United States ; R01 CA222218/CA/NCI NIH HHS/United States ; }, mesh = {Adenocarcinoma of Lung/*drug therapy/genetics/immunology/pathology ; Animals ; CRISPR-Cas Systems/genetics ; Cell Cycle Proteins/genetics/*metabolism ; Cell Differentiation/drug effects/genetics ; Cell Line, Tumor ; Disease Models, Animal ; Drug Resistance, Neoplasm/*genetics ; Epigenesis, Genetic/immunology ; Gene Expression Regulation, Neoplastic/immunology ; Gene Knockout Techniques ; HEK293 Cells ; Humans ; Immune Checkpoint Inhibitors/*pharmacology/therapeutic use ; Lung/pathology ; Lung Neoplasms/*drug therapy/genetics/immunology/pathology ; Macrophages/drug effects/immunology/metabolism ; Male ; Mice ; Molecular Chaperones/genetics/*metabolism ; Programmed Cell Death 1 Receptor/antagonists &amp; inhibitors/immunology ; Proto-Oncogene Proteins p21(ras)/genetics ; RNA, Guide/genetics ; RNA, Small Interfering/metabolism ; RNA-Seq ; Tumor Microenvironment/drug effects/immunology ; Tumor Suppressor Protein p53/genetics ; }, abstract = {Despite substantial progress in lung cancer immunotherapy, the overall response rate in patients with KRAS-mutant lung adenocarcinoma (LUAD) remains low. Combining standard immunotherapy with adjuvant approaches that enhance adaptive immune responses-such as epigenetic modulation of antitumor immunity-is therefore an attractive strategy. To identify epigenetic regulators of tumor immunity, we constructed an epigenetic-focused single guide RNA library and performed an in vivo CRISPR screen in a KrasG12D/Trp53-/- LUAD model. Our data showed that loss of the histone chaperone Asf1a in tumor cells sensitizes tumors to anti-PD-1 treatment. Mechanistic studies revealed that tumor cell-intrinsic Asf1a deficiency induced immunogenic macrophage differentiation in the tumor microenvironment by upregulating GM-CSF expression and potentiated T-cell activation in combination with anti-PD-1. Our results provide a rationale for a novel combination therapy consisting of ASF1A inhibition and anti-PD-1 immunotherapy. SIGNIFICANCE: Using an in vivo epigenetic CRISPR screen, we identified Asf1a as a critical regulator of LUAD sensitivity to anti-PD-1 therapy. Asf1a deficiency synergized with anti-PD-1 immunotherapy by promoting M1-like macrophage polarization and T-cell activation. Thus, we provide a new immunotherapeutic strategy for this subtype of patients with LUAD.See related commentary by Menzel and Black, p. 179.This article is highlighted in the In This Issue feature, p. 161.}, }
@article {pmid31654081, year = {2020}, author = {Wang, K and Gong, Q and Ye, X}, title = {Recent developments and applications of genetic transformation and genome editing technologies in wheat.}, journal = {TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik}, volume = {133}, number = {5}, pages = {1603-1622}, doi = {10.1007/s00122-019-03464-4}, pmid = {31654081}, issn = {1432-2242}, support = {2016ZX08010004//Ministry of Agriculture of China/ ; 2016ZX08009001//Ministry of Agriculture of China/ ; 2019BBF02020//Science and Technology Department of Ningxia in China/ ; 2060302-2-19//Chinese Academy of Agricultural Sciences in China/ ; }, mesh = {CRISPR-Cas Systems ; *Gene Editing ; *Gene Expression Regulation, Plant ; *Genetic Engineering ; *Genome, Plant ; Plant Proteins/genetics/*metabolism ; *Transformation, Genetic ; Triticum/genetics/*growth &amp; development/metabolism ; }, abstract = {Wheat (Triticum aestivum) is a staple crop across the world and plays a remarkable role in food supplying security. Over the past few decades, basic and applied research on wheat has lagged behind other cereal crops due to the complex and polyploid genome and difficulties in genetic transformation. A breakthrough called as PureWheat was made in the genetic transformation of wheat in 2014 in Asia, leading to a noticeable progress of wheat genome editing. Due to this great achievement, it is predicated that wheat biotechnology revolution is arriving. Genome editing technologies using zinc finger nucleases, transcription activator-like effector nuclease, and clustered regularly interspaced short palindromic repeats-associated endonucleases (CRISR/Cas) are becoming powerful tools for crop modification which can help biologists and biotechnologists better understand the processes of mutagenesis and genomic alteration. Among the three genome editing systems, CRISR/Cas has high specificity and activity, and therefore it is widely used in genetic engineering. Generally, the genome editing technologies depend on an efficient genetic transformation system. In this paper, we summarize recent progresses and applications on genetic transformation and genome editing in wheat. We also examine the future aspects of genetic transformation and genome editing. We believe that the technologies for wheat efficient genetic engineering and functional studies will become routine with the emergence of high-quality genomic sequences.}, }
@article {pmid33505962, year = {2020}, author = {Nguyen, T and Urrutia-Cabrera, D and Liou, RH and Luu, CD and Guymer, R and Wong, RC}, title = {New Technologies to Study Functional Genomics of Age-Related Macular Degeneration.}, journal = {Frontiers in cell and developmental biology}, volume = {8}, number = {}, pages = {604220}, doi = {10.3389/fcell.2020.604220}, pmid = {33505962}, issn = {2296-634X}, abstract = {Age-related macular degeneration (AMD) is the most common cause of irreversible vision loss in people over 50 years old in developed countries. Currently, we still lack a comprehensive understanding of the genetic factors contributing to AMD, which is critical to identify effective therapeutic targets to improve treatment outcomes for AMD patients. Here we discuss the latest technologies that can facilitate the identification and functional study of putative genes in AMD pathology. We review improved genomic methods to identify novel AMD genes, advances in single cell transcriptomics to profile gene expression in specific retinal cell types, and summarize recent development of in vitro models for studying AMD using induced pluripotent stem cells, organoids and biomaterials, as well as new molecular technologies using CRISPR/Cas that could facilitate functional studies of AMD-associated genes.}, }
@article {pmid33433564, year = {2021}, author = {Abbasi, J}, title = {Promising Strategies for Sickle Cell Disease and β-Thalassemia.}, journal = {JAMA}, volume = {325}, number = {2}, pages = {121}, doi = {10.1001/jama.2020.26232}, pmid = {33433564}, issn = {1538-3598}, mesh = {Anemia, Sickle Cell/genetics/*therapy ; *CRISPR-Cas Systems ; *Gene Editing ; Humans ; Repressor Proteins/genetics ; beta-Thalassemia/genetics/*therapy ; }, }
@article {pmid32448328, year = {2020}, author = {Su, B and Song, D and Zhu, H}, title = {Homology-dependent recombination of large synthetic pathways into E. coli genome via λ-Red and CRISPR/Cas9 dependent selection methodology.}, journal = {Microbial cell factories}, volume = {19}, number = {1}, pages = {108}, pmid = {32448328}, issn = {1475-2859}, support = {2018B020206001//Guangdong Province Science and Technology Innovation Strategy Special Fund/ ; 2020GDASYL-20200302002//GDAS' Special Project of Science and Technology Development/ ; 2016A010105013//Science and Technology Plan Project of Guangdong Province/ ; 2019B030316017//Science and Technology Plan Project of Guangdong Province/ ; }, mesh = {CRISPR-Cas Systems ; *Chromosomes, Bacterial ; Escherichia coli/*genetics ; *Gene Editing ; *Homologous Recombination ; *Metabolic Engineering ; }, abstract = {BACKGROUND: Metabolic engineering frequently needs genomic integration of many heterologous genes for biosynthetic pathway assembly. Despite great progresses in genome editing for the model microorganism Escherichia coli, the integration of large pathway into genome for stabilized chemical production is still challenging compared with small DNA integration.<br><br>RESULTS: We have developed a λ-Red assisted homology-dependent recombination for large synthetic pathway integration in E. coli. With this approach, we can integrate as large as 12 kb DNA module into the chromosome of E. coli W3110 in a single step. The efficiency of this method can reach 100%, thus markedly improve the integration efficiency and overcome the limitation of the integration size adopted the common method. Furthermore, the limiting step in the methylerythritol 4-phosphate (MEP) pathway and lycopene synthetic pathway were integrated into the W3110 genome using our system. Subsequently, the yields of the final strain were increased 106 and 4.4-fold compared to the initial strain and the reference strain, respectively.<br><br>CONCLUSIONS: In addition to pre-existing method, our system presents an optional strategy for avoiding using plasmids and a valuable tool for large synthetic pathway assembly in E. coli.}, }
@article {pmid32297291, year = {2020}, author = {Ghorbani Faal, P and Farsi, M and Seifi, A and Mirshamsi Kakhki, A}, title = {Virus-induced CRISPR-Cas9 system improved resistance against tomato yellow leaf curl virus.}, journal = {Molecular biology reports}, volume = {47}, number = {5}, pages = {3369-3376}, doi = {10.1007/s11033-020-05409-3}, pmid = {32297291}, issn = {1573-4978}, support = {3/41501//Ferdowsi University of Mashhad/ ; 950709//Biotechnology Development Council of the Islamic Republic of Iran/ ; }, mesh = {Begomovirus/*genetics/pathogenicity ; CRISPR-Cas Systems ; Crops, Agricultural/genetics ; Lycopersicon esculentum/*genetics ; Plant Diseases/genetics ; Plant Viruses/genetics ; Plants, Genetically Modified/genetics ; Promoter Regions, Genetic/genetics ; }, abstract = {Plant viruses are the most significant factors associated with massive economical losses in agricultural industries worldwide. Accordingly, many studies are dedicated to making virus-resistant crop varieties each year due to the ever-changing nature of viruses. Recently genome engineering methods have been used to confer interference against eukaryotic viruses. Research results on genome editing technics, in particular, CRISPR-Cas9, promises a feasible solution to make virus-resistant crops. In this research, we explored the possibility of utilizing CRISPR-Cas9 to obtain TYLCV resistant tomato varieties. Moreover, to overcome any potential off-target effects of Cas9, we used an inducible promoter to initiate Cas9 activity in case of the virus attack. Cas9 vector was transformed by the rgsCaM promoter, known as an endogenous silencer of RNAi and overexpressed after a virus attack. The golden gate cloning method was applied to construct sgRNAs. Intergenic region and coat protein-coding sequences of TYLCV were used to design sgRNAs. Infiltrated sensitive Money Maker varieties analyzed by real-time PCR, showed a significant reduction or delayed accumulation of viral DNA compared to the control plants. This result demonstrates the efficiency of using an inducible promoter in CRISPR-Cas9 constructs.}, }
@article {pmid32264803, year = {2020}, author = {Akram, F and Ikram Ul Haq,  and Ahmed, Z and Khan, H and Ali, MS}, title = {CRISPR-Cas9, A Promising Therapeutic Tool for Cancer Therapy: A Review.}, journal = {Protein and peptide letters}, volume = {27}, number = {10}, pages = {931-944}, doi = {10.2174/0929866527666200407112432}, pmid = {32264803}, issn = {1875-5305}, mesh = {*CRISPR-Cas Systems ; *Gene Editing ; *Genetic Therapy ; Humans ; *Neoplasms/genetics/therapy ; }, abstract = {Cancer is one of the most leading causes of mortality all over the world and remains a foremost social and economic burden. Mutations in the genome of individuals are taking place more frequently due to the excessive progress of xenobiotics and industrialization in the present world. With the progress in the field of molecular biology, it is possible to alter the genome and to observe the functional changes derived from genetic modulation using gene-editing technologies. Several therapies have been applied for the treatment of malignancy which affect the normal body cells; however, more effort is required to develop vsome latest therapeutic approaches for cancer biology and oncology exploiting these molecular biology advances. Recently, the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) associated protein 9 (Cas9) system has emerged as a powerful technology for cancer therapy because of its great accuracy and efficiency. Genome editing technologies have demonstrated a plethora of benefits to the biological sciences. CRISPR- Cas9, a versatile gene editing tool, has become a robust strategy for making alterations to the genome of organisms and a potent weapon in the arsenal of tumor treatment. It has revealed an excellent clinical potential for cancer therapy by discovering novel targets and has provided the researchers with the perception about how tumors respond to drug therapy. Stern efforts are in progress to enhance its efficiency of sequence specific targeting and consequently repressing offtarget effects. CRISPR-Cas9 uses specific proteins to convalesce mutations at genetic level. In CRISPR-Cas9 system, RNA-guided Cas9 endonuclease harnesses gene mutation, DNA deletion or insertion, transcriptional activation or repression, multiplex targeting only by manipulating 20-nucleotide components of RNA. Originally, CRISPR-Cas9 system was used by bacteria for their defense against different bacteriophages, and recently this system is receiving noteworthy appreciation due to its emerging role in the treatment of genetic disorders and carcinogenesis. CRISPR-Cas9 can be employed to promptly engineer oncolytic viruses and immune cells for cancer therapeutic applications. More notably, it has the ability to precisely edit genes not only in model organisms but also in human being that permits its use in therapeutic analysis. It also plays a significant role in the development of complete genomic libraries for cancer patients. In this review, we have highlighted the involvement of CRISPR-Cas9 system in cancer therapy accompanied by its prospective applications in various types of malignancy and cancer biology. In addition, some other conspicuous functions of this unique system have also been discussed beyond genome editing.}, }
@article {pmid32102844, year = {2020}, author = {Liu, HJ and Jian, L and Xu, J and Zhang, Q and Zhang, M and Jin, M and Peng, Y and Yan, J and Han, B and Liu, J and Gao, F and Liu, X and Huang, L and Wei, W and Ding, Y and Yang, X and Li, Z and Zhang, M and Sun, J and Bai, M and Song, W and Chen, H and Sun, X and Li, W and Lu, Y and Liu, Y and Zhao, J and Qian, Y and Jackson, D and Fernie, AR and Yan, J}, title = {High-Throughput CRISPR/Cas9 Mutagenesis Streamlines Trait Gene Identification in Maize.}, journal = {The Plant cell}, volume = {32}, number = {5}, pages = {1397-1413}, pmid = {32102844}, issn = {1532-298X}, mesh = {Base Sequence ; CRISPR-Associated Protein 9/*metabolism ; CRISPR-Cas Systems/*genetics ; DNA Repair/genetics ; Gene Editing ; *Genes, Plant ; Mutagenesis/*genetics ; Mutation/genetics ; Plants, Genetically Modified ; Plasmids/genetics ; *Quantitative Trait, Heritable ; RNA, Guide/genetics ; Reproducibility of Results ; Templates, Genetic ; Transformation, Genetic ; Zea mays/*genetics ; }, abstract = {Maize (Zea mays) is one of the most important crops in the world. However, few agronomically important maize genes have been cloned and used for trait improvement, due to its complex genome and genetic architecture. Here, we integrated multiplexed CRISPR/Cas9-based high-throughput targeted mutagenesis with genetic mapping and genomic approaches to successfully target 743 candidate genes corresponding to traits relevant for agronomy and nutrition. After low-cost barcode-based deep sequencing, 412 edited sequences covering 118 genes were precisely identified from individuals showing clear phenotypic changes. The profiles of the associated gene-editing events were similar to those identified in human cell lines and consequently are predictable using an existing algorithm originally designed for human studies. We observed unexpected but frequent homology-directed repair through endogenous templates that was likely caused by spatial contact between distinct chromosomes. Based on the characterization and interpretation of gene function from several examples, we demonstrate that the integration of forward and reverse genetics via a targeted mutagenesis library promises rapid validation of important agronomic genes for crops with complex genomes. Beyond specific findings, this study also guides further optimization of high-throughput CRISPR experiments in plants.}, }
@article {pmid32017998, year = {2020}, author = {Li, C and Li, YH and Li, Y and Lu, H and Hong, H and Tian, Y and Li, H and Zhao, T and Zhou, X and Liu, J and Zhou, X and Jackson, SA and Liu, B and Qiu, LJ}, title = {A Domestication-Associated Gene GmPRR3b Regulates the Circadian Clock and Flowering Time in Soybean.}, journal = {Molecular plant}, volume = {13}, number = {5}, pages = {745-759}, doi = {10.1016/j.molp.2020.01.014}, pmid = {32017998}, issn = {1752-9867}, mesh = {CRISPR-Cas Systems/genetics ; Circadian Clocks/*genetics ; *Domestication ; Evolution, Molecular ; Flowers/*genetics ; *Gene Expression Regulation, Plant ; *Genes, Plant ; Genome-Wide Association Study ; Geography ; Haplotypes/genetics ; Mutation/genetics ; Plant Breeding ; Plant Proteins/*genetics/metabolism ; Quantitative Trait Loci/genetics ; RNA, Messenger/genetics/metabolism ; Soybeans/*genetics ; Subcellular Fractions/metabolism ; Time Factors ; Transcription, Genetic ; }, abstract = {Improved soybean cultivars have been adapted to grow at a wide range of latitudes, enabling expansion of cultivation worldwide. However, the genetic basis of this broad adaptation is still not clear. Here, we report the identification of GmPRR3b as a major flowering time regulatory gene that has been selected during domestication and genetic improvement for geographic expansion. Through a genome-wide association study of a diverse soybean landrace panel consisting of 279 accessions, we identified 16 candidate quantitative loci associated with flowering time and maturity time. The strongest signal resides in the known flowering gene E2, verifying the effectiveness of our approach. We detected strong signals associated with both flowering and maturity time in a genomic region containing GmPRR3b. Haplotype analysis revealed that GmPRR3bH6 is the major form of GmPRR3b that has been utilized during recent breeding of modern cultivars. mRNA profiling analysis showed that GmPRR3bH6 displays rhythmic and photoperiod-dependent expression and is preferentially induced under long-day conditions. Overexpression of GmPRR3bH6 increased main stem node number and yield, while knockout of GmPRR3bH6 using CRISPR/Cas9 technology delayed growth and the floral transition. GmPRR3bH6 appears to act as a transcriptional repressor of multiple predicted circadian clock genes, including GmCCA1a, which directly upregulates J/GmELF3a to modulate flowering time. The causal SNP (Chr12:5520945) likely endows GmPRR3bH6 a moderate but appropriate level of activity, leading to early flowering and vigorous growth traits preferentially selected during broad adaptation of landraces and improvement of cultivars.}, }
@article {pmid31260171, year = {2020}, author = {Zou, B and Desmidt, AA and Mittal, R and Yan, D and Richmond, M and Tekin, M and Liu, XZ and Lu, Z}, title = {The Generation of Zebrafish Mariner Model Using the CRISPR/Cas9 System.}, journal = {Anatomical record (Hoboken, N.J. : 2007)}, volume = {303}, number = {3}, pages = {556-562}, doi = {10.1002/ar.24221}, pmid = {31260171}, issn = {1932-8494}, support = {R01DC009645/DC/NIDCD NIH HHS/United States ; R01DC012836/DC/NIDCD NIH HHS/United States ; R21DC009879/DC/NIDCD NIH HHS/United States ; R01DC012115/DC/NIDCD NIH HHS/United States ; R01DC012546/DC/NIDCD NIH HHS/United States ; R01DC005575/DC/NIDCD NIH HHS/United States ; }, mesh = {Animals ; Behavior, Animal/physiology ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Deafness/*genetics ; Disease Models, Animal ; Gene Editing/*methods ; Myosins/genetics ; *Phenotype ; Zebrafish/genetics ; Zebrafish Proteins/*genetics ; }, abstract = {Targeted genome editing mediated by clustered, regularly interspaced, short palindromic repeat (CRISPR)/CRISPR-associated nuclease 9 (Cas9) technology has emerged as a powerful tool for gene function studies and has great potential for gene therapy. Although CRISPR/Cas9 has been widely used in many research fields, only a few successful zebrafish models have been established using this technology in hearing research. In this study, we successfully created zebrafish mariner mutants by targeting the motor head domain of Myo7aa using CRISPR/Cas9. The CRISPR/Cas9-generated mutants showed unbalanced swimming behavior and disorganized sterocilia of inner ear hair cells, which resemble the phenotype of the zebrafish mariner mutants. In addition, we found that CRISPR/Cas9-generated mutants have reduced number of stereociliary bundles of inner ear hair cells and have significant hearing loss. Furthermore, phenotypic analysis was performed on F0 larvae within the first week post fertilization, which dramatically shortens data collection period. Therefore, results of this study showed that CRISPR/Cas9 is a quick and effective method to generate zebrafish mutants as a model for studying human genetic deafness. Anat Rec, 303:556-562, 2020. © 2019 American Association for Anatomy.}, }
@article {pmid32620100, year = {2020}, author = {Zeng, X and Luo, Y and Vu, NTQ and Shen, S and Xia, K and Zhang, M}, title = {CRISPR/Cas9-mediated mutation of OsSWEET14 in rice cv. Zhonghua11 confers resistance to Xanthomonas oryzae pv. oryzae without yield penalty.}, journal = {BMC plant biology}, volume = {20}, number = {1}, pages = {313}, pmid = {32620100}, issn = {1471-2229}, support = {31701403//National Natural Science Foundation of China/ ; 31772384//National Natural Science Foundation of China/ ; 2017YFD0100101//National Key Research and Development Program of China/ ; XDA24030201//the Chinese Academy of Sciences/ ; 2019A1515011975//Natural Science Foundation of Guangdong Province (CN)/ ; }, mesh = {CRISPR-Cas Systems ; Disease Resistance/genetics ; Monosaccharide Transport Proteins/*genetics/immunology/metabolism ; Mutagenesis ; Oryza/*genetics/growth &amp; development ; Plant Diseases/genetics/*microbiology ; Transcriptome ; *Xanthomonas ; }, abstract = {BACKGROUND: Bacterial blight of rice, caused by Xanthomonas oryzae pv. oryzae (Xoo), is a devastating rice disease in Southeast Asia and West Africa. OsSWEET14, encoding a sugar transporter, is known to be a major susceptible gene of bacterial blight targeted by four different transcription activator-like (TAL) effectors from either Asian or African Xoo strains. However, the OsSWEET14 single knockout or promoter mutants in the Kitaake background are moderately resistant or even susceptible to African Xoo strains. Therefore, in this study, we knocked out OsSWEET14 in rice cv. Zhonghua 11 background for disease assessment.<br><br>RESULTS: In this study, CRISPR/Cas9 was utilized to disrupt the function of OsSWEET14 by modifying its corresponding coding region in the genome of rice cv. Zhonghua 11 (CR-S14). In total, we obtained nine different OsSWEET14-mutant alleles. Besides conferring broad-spectrum resistance to Asian Xoo strains, tested mutant alleles also showed strong resistance to African Xoo strain AXO1947. Moreover, the expression of OsSWEET14 was detected in vascular tissues, including the stem, leaf sheath, leaf blade and root. The disruption of OsSWEET14 led to increased plant height without a reduction in yield.<br><br>CONCLUSIONS: Disruption of OsSWEET14 in the Zhonghua 11 background is able to confer strong resistance to African Xoo strain AXO1947 and Asian Xoo strain PXO86. CR-S14 has normal reproductive growth and enhanced plant height under normal growth conditions. These results imply that CR-S14 may serve as a better tester line than sweet14 single-knockout mutant in the Kitaake background for the diagnostic kit for rice blight resistance. The genetic background and increased plant height need to be taken into consideration when utilizing OsSWEET14 for resistant rice breeding.}, }
@article {pmid32563468, year = {2020}, author = {Sun, M and Li, H and Li, Y and Xiang, H and Liu, Y and He, Y and Qi, M and Li, T}, title = {Tomato YABBY2b controls plant height through regulating indole-3-acetic acid-amido synthetase (GH3.8) expression.}, journal = {Plant science : an international journal of experimental plant biology}, volume = {297}, number = {}, pages = {110530}, doi = {10.1016/j.plantsci.2020.110530}, pmid = {32563468}, issn = {1873-2259}, mesh = {CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Flowers/growth &amp; development ; Fruit/growth &amp; development ; Gene Editing ; Gene Expression Profiling ; Indoleacetic Acids/metabolism ; Ligases/metabolism/*physiology ; Lycopersicon esculentum/enzymology/genetics/*growth &amp; development ; Plant Growth Regulators/metabolism ; Plant Proteins/metabolism/*physiology ; Polymerase Chain Reaction ; Transcription Factors/metabolism/*physiology ; Two-Hybrid System Techniques ; }, abstract = {Dwarfing is a desirable agronomic trait in cultivation management. Dwarf plants are lodging-resistant, compact, and perform well under high-density planting. The use of dwarf genetic resources is one approach to improve crop yield. YABBY2b in tomato (Solanum lycopersicum) encodes a transcription factor that regulates plant height. In this study, we created YABBY2b knockout mutant lines, and the resulting yabby2b plants exhibited reduced height and smaller flowers and fruits. The RNA-seq analysis showed that 17 genes responding to gibberellin and auxin were differentially expressed. We hypothesized that indole-3-acetic acid-amido synthetase GH3.8 (GH3.8) played a crucial role in the resulting yabby2b dwarf phenotype. Further analysis showed that YABBY2b suppresses GH3.8 gene expression by directly binding to its promoter, and that this contributes to auxin-mediated repression of GH3.8. Moreover, the silencing of GH3.8 led to increased plant height. Combined, our data suggest that YABBY2b may positively regulate plant height in tomato by inhibiting the expression of growth suppressor GH3.8.}, }
@article {pmid32563463, year = {2020}, author = {Zhang, C and Wang, J and Wang, X and Li, C and Ye, Z and Zhang, J}, title = {UF, a WOX gene, regulates a novel phenotype of un-fused flower in tomato.}, journal = {Plant science : an international journal of experimental plant biology}, volume = {297}, number = {}, pages = {110523}, doi = {10.1016/j.plantsci.2020.110523}, pmid = {32563463}, issn = {1873-2259}, mesh = {CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Chromosome Mapping ; Chromosomes, Plant/genetics ; Flowers/anatomy &amp; histology/genetics/*growth &amp; development ; Gene Editing ; Genes, Plant/genetics/*physiology ; Lycopersicon esculentum/anatomy &amp; histology/*genetics/growth &amp; development ; Microscopy, Electron, Scanning ; Phenotype ; Phylogeny ; Plant Leaves/anatomy &amp; histology/genetics/growth &amp; development ; Real-Time Polymerase Chain Reaction ; Transcription Factors/genetics/physiology ; }, abstract = {Flower formation is a basic condition for fruit set in all flowering plants. The normal stamen of tomato flower fused together to form a yellow cylinder surrounding the carpels. In this study, we identified an un-fused flower (uf) tomato mutant that is defective in petal, carpal and stamen fusion and lateral outgrowth. After RNA-seq-based BSA (BSR), the candidate region location was identified in the long arm of chromosome 3. Using map-based cloning with InDel and CAPS markers, the UF candidate gene was mapped in a 104 kb region. In this region, a WOX (WUSCHEL-related homeobox) transcription factor SlWOX1 was considered as a candidate of UF as there is a 72bp deletion in its second exon in uf mutant. The mutations of SlWOX1 generated by CRISPR/CAS9 approach under wild-type background reproduced the phenotypes of uf mutant, indicating that the SlWOX1 gene is indeed UF. Interestingly, expression analysis of organ lateral polarity determinant genes showed that abaxial genes (SlYABBY5 and SlARF4) and adaxial genes (AS and HD-ZIPIII) were significantly down-regulated in the uf mutant, which is different to that in Arabidopsis and petunia. In conclusion, this work revealed a novel function of SlWOX1 in the regulation of flower development in tomato.}, }
@article {pmid32563459, year = {2020}, author = {Wang, S and Li, Q and Zhao, L and Fu, S and Qin, L and Wei, Y and Fu, YB and Wang, H}, title = {Arabidopsis UBC22, an E2 able to catalyze lysine-11 specific ubiquitin linkage formation, has multiple functions in plant growth and immunity.}, journal = {Plant science : an international journal of experimental plant biology}, volume = {297}, number = {}, pages = {110520}, doi = {10.1016/j.plantsci.2020.110520}, pmid = {32563459}, issn = {1873-2259}, mesh = {Arabidopsis/*enzymology/genetics/growth &amp; development/immunology ; Arabidopsis Proteins/genetics/*physiology ; Botrytis ; CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; DNA, Plant/genetics ; Gene Editing ; Gene Expression Profiling ; Gene Knockout Techniques ; Plant Diseases/immunology/microbiology ; RNA, Plant/genetics ; Real-Time Polymerase Chain Reaction ; Ubiquitin-Conjugating Enzymes/genetics/*physiology ; Ubiquitination/genetics ; }, abstract = {Protein ubiquitination is critical for various biological processes in eukaryotes. A ubiquitin (Ub) chain can be linked through one of the seven lysine (K) residues or the N-terminus methionine of the Ub, and the Ub-conjugating enzymes called E2s play a critical role in determining the linkage specificity of Ub chains. Further, while K48-linked polyubiquitin chain is important for protein degradation, much less is known about the functions of other types of polyubiquitin chains in plants. We showed previously that UBC22 is unique in its ability to catalyze K11-dependent Ub dimer formation in vitro and ubc22 knockout mutants had defects in megasporogenesis. In this study, further analyses of the Arabidopsis ubc22 mutants revealed four subtypes of plants based on the phenotypic changes in vegetative growth. These four subtypes appeared consistently in the plants of three independent ubc22 mutants. Transcriptomic analysis showed that transcript levels of genes related to several pathways were altered differently in different subtypes of mutant plants. In one subtype, the mutant plants had increased expression of genes related to plant defenses and showed enhanced resistance to a necrotrophic plant pathogen. These results suggest multiple functions of UBC22 during plant development and stress response.}, }
@article {pmid32007615, year = {2020}, author = {Cao, M and Fatma, Z and Song, X and Hsieh, PH and Tran, VG and Lyon, WL and Sayadi, M and Shao, Z and Yoshikuni, Y and Zhao, H}, title = {A genetic toolbox for metabolic engineering of Issatchenkia orientalis.}, journal = {Metabolic engineering}, volume = {59}, number = {}, pages = {87-97}, doi = {10.1016/j.ymben.2020.01.005}, pmid = {32007615}, issn = {1096-7184}, mesh = {*CRISPR-Cas Systems ; *DNA, Fungal/genetics/metabolism ; *Metabolic Engineering ; *Pichia/genetics/metabolism ; Saccharomyces cerevisiae/genetics ; }, abstract = {The nonconventional yeast Issatchenkia orientalis can grow under highly acidic conditions and has been explored for production of various organic acids. However, its broader application is hampered by the lack of efficient genetic tools to enable sophisticated metabolic manipulations. We recently constructed an episomal plasmid based on the autonomously replicating sequence (ARS) from Saccharomyces cerevisiae (ScARS) in I. orientalis and developed a CRISPR/Cas9 system for multiplexed gene deletions. Here we report three additional genetic tools including: (1) identification of a 0.8 kb centromere-like (CEN-L) sequence from the I. orientalis genome by using bioinformatics and functional screening; (2) discovery and characterization of a set of constitutive promoters and terminators under different culture conditions by using RNA-Seq analysis and a fluorescent reporter; and (3) development of a rapid and efficient in vivo DNA assembly method in I. orientalis, which exhibited ~100% fidelity when assembling a 7 kb-plasmid from seven DNA fragments ranging from 0.7 kb to 1.7 kb. As proof of concept, we used these genetic tools to rapidly construct a functional xylose utilization pathway in I. orientalis.}, }
@article {pmid32002907, year = {2020}, author = {Güell, M}, title = {Genome-Wide PERV Inactivation in Pigs Using CRISPR/Cas9.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2110}, number = {}, pages = {139-149}, doi = {10.1007/978-1-0716-0255-3_10}, pmid = {32002907}, issn = {1940-6029}, mesh = {Algorithms ; Animals ; *CRISPR-Cas Systems ; Cell Line ; Computational Biology/methods ; Endogenous Retroviruses/*genetics ; Gene Dosage ; *Gene Editing ; Gene Expression ; Genes, Reporter ; Genetic Vectors ; Genome-Wide Association Study/*methods ; Heterografts ; Humans ; RNA, Guide ; Swine ; Transplantation, Heterologous/adverse effects ; Virus Activation/genetics ; }, abstract = {The shortage of organs for transplantation is probably the biggest unmet medical need. A potential problem with the clinical use of porcine xenografts is the risk that porcine endogenous retroviruses (PERVs) could infect human cells. In the past, we determined the PERV copy number in the porcine kidney epithelial cell line PK15 and in primary fibroblasts. Using CRISPR-Cas9, we disrupted the catalytic center of pol, which is essential for virus replication. Next, we isolated cells in which 100% of the PERV elements had been inactivated. This method enables the possibility of eradicating PERVs in vitro for application to pig-to-human xenotransplantation. Here we describe the methodological bases of this work.}, }
@article {pmid32002906, year = {2020}, author = {Muñoz-Santos, D and Montoliu, L and Fernández, A}, title = {Generation of Genetically Modified Mice Using CRISPR/Cas9.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2110}, number = {}, pages = {129-138}, doi = {10.1007/978-1-0716-0255-3_9}, pmid = {32002906}, issn = {1940-6029}, mesh = {Animals ; *CRISPR-Cas Systems ; *Gene Editing ; Gene Knock-In Techniques ; Gene Knockout Techniques ; Gene Targeting ; Genetic Engineering/methods ; Genotyping Techniques ; Mice ; *Mice, Transgenic ; Models, Animal ; Oocytes ; RNA, Guide ; }, abstract = {Using CRISPR-based genome-editing techniques, we are able to generate a variety of new mouse models of several types of diseases. These animal models will be instrumental not only for enabling the comprehension of a particular disease and its underlying molecular mechanism but also as unique recipients for testing novel and innovative therapeutic approaches that are being currently explored. This chapter describes detailed step-by-step protocols, reagents, and equipment required for successful generation of genome-edited mice using CRISPR tools.}, }
@article {pmid31939194, year = {2020}, author = {Huo, Z and Tu, J and Lee, DF and Zhao, R}, title = {Engineering Mutation Clones in Mammalian Cells with CRISPR/Cas9.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2108}, number = {}, pages = {355-369}, doi = {10.1007/978-1-0716-0247-8_29}, pmid = {31939194}, issn = {1940-6029}, mesh = {Animals ; *CRISPR-Cas Systems ; Cell Line ; Cloning, Molecular ; *Gene Editing ; Gene Order ; *Gene Targeting ; *Genetic Engineering ; Genetic Vectors ; Humans ; *Mutation ; Plasmids ; RNA, Guide ; }, abstract = {CRISPR, Clustered Regularly Interspaced Short Palindromic Repeat, as a powerful genome engineering system has been widely accepted and employed in gene editing of a vast range of cell types. Comparing to zinc finger nucleases (ZFNs) or transcription-activator-like effector nucleases (TALENs), CRISPR shows less complicated process and higher efficiency. With the development of different CRISPR systems, it can be used not only to knock out a gene, but also to make precise modifications, activate or repress target genes with epigenetic modifications, and even for genome-wide screening. Here we will describe the procedure of generating stable cell lines with a knock-in mutation created by CRISPR. Specifically, this protocol demonstrated how to apply CRISPR to create the point mutation of R249 to S249 on TP53 exon 7 in human embryonic stem cells (hESC) H9 line, which includes three major steps: (1) design CRISPR system targeting TP53 genomic region, (2) deliver the system to H9 hESC and clone selection, and (3) examination and selection of positive clones.}, }
@article {pmid31939193, year = {2020}, author = {Hu, O and Provvido, A and Zhu, Y}, title = {Generation of IL17RB Knockout Cell Lines Using CRISPR/Cas9-Based Genome Editing.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2108}, number = {}, pages = {345-353}, doi = {10.1007/978-1-0716-0247-8_28}, pmid = {31939193}, issn = {1940-6029}, support = {R15 CA213426/CA/NCI NIH HHS/United States ; }, mesh = {Animals ; *CRISPR-Cas Systems ; Cell Line ; *Gene Editing ; Gene Expression ; *Gene Knockout Techniques ; Genetic Vectors/genetics ; Humans ; Receptors, Interleukin-17/*genetics ; Single-Cell Analysis ; Transfection ; }, abstract = {CRISPR/Cas9-based genome editing is an inexpensive and efficient tool for genetic modification. Here we present a methodological approach of establishing interleukin-17 receptor B (IL17RB) knockout cell lines using CRISPR/Cas9-mediated genomic deletion. IL17RB gene encodes for a cytokine receptor that specifically binds to IL17B and IL17E and overexpressed in various cancers. The method involves CRISPR design, CRISPR cloning, delivery of CRISPR clone into cells, and verification of IL17RB gene deletion by deletion screening primer design, genomic DNA extraction, and polymerase chain reaction (PCR). Similar approaches can be used for generating mammalian cell lines with gene knockout for other genes of interest.}, }
@article {pmid31933198, year = {2020}, author = {Shang, W and Wang, F and Zhu, Q and Wang, L and Wang, H}, title = {CRISPR/Cas9-Based Genetic Screening to Study T-Cell Function.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2111}, number = {}, pages = {59-70}, doi = {10.1007/978-1-0716-0266-9_5}, pmid = {31933198}, issn = {1940-6029}, mesh = {CRISPR-Cas Systems ; Gene Editing ; *Gene Regulatory Networks ; Genetic Testing ; High-Throughput Nucleotide Sequencing ; Humans ; Jurkat Cells ; Lentivirus/genetics ; Lymphocyte Activation ; RNA, Guide/*pharmacology ; Sequence Analysis, DNA/*methods ; Signal Transduction ; T-Lymphocytes/*immunology ; Transduction, Genetic ; }, abstract = {T-cell-based cancer immunotherapies have emerged as a promising approach for cancer treatment, highlighting the importance of understanding the regulation of T-cell function. However, the molecular mechanisms underlying T-cell activation are not fully understood. The CRISPR/Cas9 system can serve as a robust method to systematically study signaling pathways. In this chapter, we describe details of using the CRISPR screen to identify regulators in TCR signaling, from the sgRNA library construction to genomic DNA sequencing. We also add some notes to further help readers performing the CRISPR screen. This approach can be readily adapted to study the activation of other immune cells, including B cells and dendritic cells.}, }
@article {pmid31610242, year = {2020}, author = {Liang, Y and Jiao, S and Wang, M and Yu, H and Shen, Z}, title = {A CRISPR/Cas9-based genome editing system for Rhodococcus ruber TH.}, journal = {Metabolic engineering}, volume = {57}, number = {}, pages = {13-22}, doi = {10.1016/j.ymben.2019.10.003}, pmid = {31610242}, issn = {1096-7184}, mesh = {Acrylamide/*metabolism ; *Biocatalysis ; *CRISPR-Cas Systems ; *Gene Editing ; *Metabolic Engineering ; *Rhodococcus/genetics/metabolism ; }, abstract = {Rhodococcus spp. are organic solvent-tolerant strains with strong adaptive abilities and diverse metabolic activities, and are therefore widely utilized in bioconversion, biosynthesis and bioremediation. However, due to the high GC-content of the genome (~70%), together with low transformation and recombination efficiency, the efficient genome editing of Rhodococcus remains challenging. In this study, we report for the first time the successful establishment of a CRISPR/Cas9-based genome editing system for R. ruber. With a bypass of the restriction-modification system, the transformation efficiency of R. ruber was enhanced by 89-fold, making it feasible to obtain enough colonies for screening of mutants. By introducing a pair of bacteriophage recombinases, Che9c60 and Che9c61, the editing efficiency was improved from 1% to 75%. A CRISPR/Cas9-mediated triple-plasmid recombineering system was developed with high efficiency of gene deletion, insertion and mutation. Finally, this new genome editing method was successfully applied to engineer R. ruber for the bio-production of acrylamide. By deletion of a byproduct-related gene and in-situ subsititution of the natural nitrile hydratase gene with a stable mutant, an engineered strain R. ruber THY was obtained with reduced byproduct formation and enhanced catalytic stability. Compared with the use of wild-type R. ruber TH, utilization of R. ruber THY as biocatalyst increased the acrylamide concentration from 405 g/L to 500 g/L, reduced the byproduct concentration from 2.54 g/L to 0.5 g/L, and enhanced the number of times that cells could be recycled from 1 batch to 4 batches.}, }
@article {pmid33497205, year = {2021}, author = {Li, H and Li, M and Yang, Y and Wang, F and Wang, F and Li, C}, title = {Aptamer-Linked CRISPR/Cas12a-Based Immunoassay.}, journal = {Analytical chemistry}, volume = {}, number = {}, pages = {}, doi = {10.1021/acs.analchem.0c04687}, pmid = {33497205}, issn = {1520-6882}, abstract = {The clustered regularly interspaced short palindromic repeat (CRISPR)/Cas system has shown great promising applications in the area of nucleic acid biosensing. However, because of the dearth of versatile signal transduction strategies, this system is usually compromised to low versatility, moderate sensitivity, and complex operation for non-nucleic acid targets, limiting its clinical transition. Herein, we describe a direct method to establish the correlation between non-nucleic acid analytes and the CRISPR/Cas12a system using a series of rationally designed, aptamer-flanked activator DNA strands, which enable ultrasensitive detection of biomarkers from different species, greatly broadening the possibility of the CRISPR/Cas system in bioanalysis. Meanwhile, the signal output is highly optional and the sensing principle is akin to the traditional enzyme-linked immunosorbent assay (ELISA), so it can be directly imposed on the currently available ELISA platform, further facilitating its application in medical diagnostics.}, }
@article {pmid33496064, year = {2021}, author = {Manjunath, M and Choudhary, B and Raghavan, SC}, title = {SCR7, a potent cancer therapeutic agent and a biochemical inhibitor of nonhomologous DNA end-joining.}, journal = {Cancer reports (Hoboken, N.J.)}, volume = {}, number = {}, pages = {e1341}, doi = {10.1002/cnr2.1341}, pmid = {33496064}, issn = {2573-8348}, abstract = {BACKGROUND: DNA double-strand breaks (DSBs) are harmful to the cell as it could lead to genomic instability and cell death when left unrepaired. Homologous recombination and nonhomologous end-joining (NHEJ) are two major DSB repair pathways, responsible for ensuring genome integrity in mammals. There have been multiple efforts using small molecule inhibitors to target these DNA repair pathways in cancers. SCR7 is a very well-studied anticancer molecule that blocks NHEJ by targeting one of the critical enzymes, Ligase IV.<br><br>RECENT FINDINGS: In this review, we have highlighted the anticancer effects of SCR7 as a single agent and in combination with other chemotherapeutic agents and radiation. SCR7 blocked NHEJ effectively both in vitro and ex vivo. SCR7 has been used for biochemical studies like chromosomal territory resetting and in understanding the role of repair proteins in cell cycle phases. Various forms of SCR7 and its derivatives are discussed. SCR7 is also used as a potent biochemical inhibitor of NHEJ, which has found its application in improving genome editing using a CRISPR-Cas system.<br><br>CONCLUSION: SCR7 is a potent NHEJ inhibitor with unique properties and wide applications as an anticancer agent. Most importantly, SCR7 has become a handy aid for improving genome editing across different model systems.}, }
@article {pmid33495647, year = {2021}, author = {Li, Z and Zhang, H and Xiao, R and Han, R and Chang, L}, title = {Cryo-EM structure of the RNA-guided ribonuclease Cas12g.}, journal = {Nature chemical biology}, volume = {}, number = {}, pages = {}, pmid = {33495647}, issn = {1552-4469}, support = {R01GM138675//U.S. Department of Health &amp; Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; }, abstract = {Cas12g, the type V-G CRISPR-Cas effector, is an RNA-guided ribonuclease that targets single-stranded RNA substrate. The CRISPR-Cas12g system offers a potential platform for transcriptome engineering and diagnostic applications. We determined the structures of Cas12g-guide RNA complexes in the absence and presence of target RNA by cryo-EM to a resolution of 3.1 Å and 4.8 Å, respectively. Cas12g adopts a bilobed structure with miniature REC2 and Nuc domains, whereas the guide RNAs fold into a flipped 'F' shape, which is primarily recognized by the REC lobe. Target RNA and the CRISPR RNA (crRNA) guide form a duplex that inserts into the central cavity between the REC and NUC lobes, inducing conformational changes in both lobes to activate Cas12g. The structural insights would facilitate the development of Cas12g-based applications.}, }
@article {pmid33068406, year = {2021}, author = {Dwane, L and Behan, FM and Gonçalves, E and Lightfoot, H and Yang, W and van der Meer, D and Shepherd, R and Pignatelli, M and Iorio, F and Garnett, MJ}, title = {Project Score database: a resource for investigating cancer cell dependencies and prioritizing therapeutic targets.}, journal = {Nucleic acids research}, volume = {49}, number = {D1}, pages = {D1365-D1372}, pmid = {33068406}, issn = {1362-4962}, support = {206194/WT_/Wellcome Trust/United Kingdom ; }, mesh = {Antineoplastic Agents/therapeutic use ; Biomarkers, Tumor/*genetics ; CRISPR-Cas Systems ; Carcinogenesis/drug effects/genetics/metabolism/pathology ; Cell Line, Tumor ; *Databases, Factual ; *Gene Expression Regulation, Neoplastic ; Genetic Fitness ; *Genome, Human ; Humans ; Internet ; Molecular Targeted Therapy ; Neoplasms/drug therapy/*genetics/metabolism/pathology ; Oncogenes ; *Software ; }, abstract = {CRISPR genetic screens in cancer cell models are a powerful tool to elucidate oncogenic mechanisms and to identify promising therapeutic targets. The Project Score database (https://score.depmap.sanger.ac.uk/) uses genome-wide CRISPR-Cas9 dropout screening data in hundreds of highly annotated cancer cell models to identify genes required for cell fitness and prioritize novel oncology targets. The Project Score database currently allows users to investigate the fitness effect of 18 009 genes tested across 323 cancer cell models. Through interactive interfaces, users can investigate data by selecting a specific gene, cancer cell model or tissue type, as well as browsing all gene fitness scores. Additionally, users can identify and rank candidate drug targets based on an established oncology target prioritization pipeline, incorporating genetic biomarkers and clinical datasets for each target, and including suitability for drug development based on pharmaceutical tractability. Data are freely available and downloadable. To enhance analyses, links to other key resources including Open Targets, COSMIC, the Cell Model Passports, UniProt and the Genomics of Drug Sensitivity in Cancer are provided. The Project Score database is a valuable new tool for investigating genetic dependencies in cancer cells and the identification of candidate oncology targets.}, }
@article {pmid33010154, year = {2021}, author = {Cui, Y and Cheng, X and Chen, Q and Song, B and Chiu, A and Gao, Y and Dawson, T and Chao, L and Zhang, W and Li, D and Zeng, Z and Yu, J and Li, Z and Fei, T and Peng, S and Li, W}, title = {CRISP-view: a database of functional genetic screens spanning multiple phenotypes.}, journal = {Nucleic acids research}, volume = {49}, number = {D1}, pages = {D848-D854}, pmid = {33010154}, issn = {1362-4962}, mesh = {CRISPR-Cas Systems/*genetics ; *Databases, Genetic ; *Genetic Testing ; Metadata ; Molecular Sequence Annotation ; Phenotype ; User-Computer Interface ; }, abstract = {High-throughput genetic screening based on CRISPR/Cas9 or RNA-interference (RNAi) enables the exploration of genes associated with the phenotype of interest on a large scale. The rapid accumulation of public available genetic screening data provides a wealth of knowledge about genotype-to-phenotype relationships and a valuable resource for the systematic analysis of gene functions. Here we present CRISP-view, a comprehensive database of CRISPR/Cas9 and RNAi screening datasets that span multiple phenotypes, including in vitro and in vivo cell proliferation and viability, response to cancer immunotherapy, virus response, protein expression, etc. By 22 September 2020, CRISP-view has collected 10 321 human samples and 825 mouse samples from 167 papers. All the datasets have been curated, annotated, and processed by a standard MAGeCK-VISPR analysis pipeline with quality control (QC) metrics. We also developed a user-friendly webserver to visualize, explore, and search these datasets. The webserver is freely available at http://crispview.weililab.org.}, }
@article {pmid32342312, year = {2020}, author = {Silaeva, YY and Kalmykov, VA and Varlamova, EA and Korshunov, EN and Korshunova, DS and Kubekina, MV and Shtil, AA and Roninson, IB and Deykin, AV}, title = {Genome Editing As an Approach to the Study of in Vivo Transcription Reprogramming.}, journal = {Doklady. Biochemistry and biophysics}, volume = {490}, number = {1}, pages = {43-46}, pmid = {32342312}, issn = {1608-3091}, mesh = {Animals ; CRISPR-Cas Systems ; Catalytic Domain ; Cyclin-Dependent Kinase 8/*metabolism ; Exons ; Gene Editing/*methods ; *Genome ; Heterozygote ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Inbred CBA ; Mutation ; Oligonucleotides/genetics ; *RNA, Guide ; *Transcription, Genetic ; }, abstract = {CDK8-mediated transcriptional reprogramming is essential for an extensive gene expression. Constitutive knockouts of the cdk8 gene are lethal at the morula stage. For modeling transcriptional reprogramming in an adult organism, we investigated the possibility to attenuate the CDK8 kinase activity with a F97G mutation in exon 3 of the cdk8 gene. According to preliminary experimental data, this mutation should lead to a decrease in CDK8 kinase activity. To edit the genome of laboratory mice, the CRISPR/Cas9 technology was used, in which the introduction of a double-stranded gap occurred at a distance of 128 nucleotide pairs from the planned site of the introduced mutation. To introduce the mutation, a matrix for homologous repair was used as part of plasmid DNA, with homologous arms 903 and 484 bp in the 5'-3' region from the point of double-stranded rupture, respectively. As a result, mice with site-specific target mutations in exon 3 of the cdk8 gene were obtained. We for the first time demonstrated a high efficacy of the mutation 128 bp apart from the site of double-strand break. Viable animals with the F97G mutation in the catalytic domain of CDK8 kinase were obtained for the first time. The resulting cdk8 mutant mice will be used in subsequent studies to simulate the processes involving transcription reprogramming.}, }
@article {pmid32250339, year = {2020}, author = {Hatcher, A and Yu, K and Meyer, J and Aiba, I and Deneen, B and Noebels, JL}, title = {Pathogenesis of peritumoral hyperexcitability in an immunocompetent CRISPR-based glioblastoma model.}, journal = {The Journal of clinical investigation}, volume = {130}, number = {5}, pages = {2286-2300}, pmid = {32250339}, issn = {1558-8238}, support = {R01 CA223388/CA/NCI NIH HHS/United States ; R01 NS029709/NS/NINDS NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; }, mesh = {Animals ; *Brain/metabolism/pathology/physiopathology ; *Brain Neoplasms/genetics/metabolism/pathology/physiopathology ; *CRISPR-Cas Systems ; Gene Deletion ; *Glioblastoma/genetics/metabolism/pathology/physiopathology ; Mice ; Mice, Knockout ; *Neoplasms, Experimental/genetics/metabolism/pathology/physiopathology ; *Seizures/genetics/metabolism/pathology/physiopathology ; *Synaptic Transmission ; }, abstract = {Seizures often herald the clinical appearance of gliomas or appear at later stages. Dissecting their precise evolution and cellular pathogenesis in brain malignancies could inform the development of staged therapies for these highly pharmaco-resistant epilepsies. Studies in immunodeficient xenograft models have identified local interneuron loss and excess glial glutamate release as chief contributors to network disinhibition, but how hyperexcitability in the peritumoral microenvironment evolves in an immunocompetent brain is unclear. We generated gliomas in WT mice via in utero deletion of key tumor suppressor genes and serially monitored cortical epileptogenesis during tumor infiltration with in vivo electrophysiology and GCAMP7 calcium imaging, revealing a reproducible progression from hyperexcitability to convulsive seizures. Long before seizures, coincident with loss of inhibitory cells and their protective scaffolding, gain of glial glutamate antiporter xCT expression, and reactive astrocytosis, we detected local Iba1+ microglial inflammation that intensified and later extended far beyond tumor boundaries. Hitherto unrecognized episodes of cortical spreading depolarization that arose frequently from the peritumoral region may provide a mechanism for transient neurological deficits. Early blockade of glial xCT activity inhibited later seizures, and genomic reduction of host brain excitability by deleting MapT suppressed molecular markers of epileptogenesis and seizures. Our studies confirmed xenograft tumor-driven pathobiology and revealed early and late components of tumor-related epileptogenesis in a genetically tractable, immunocompetent mouse model of glioma, allowing the complex dissection of tumor versus host pathogenic seizure mechanisms.}, }
@article {pmid32215707, year = {2020}, author = {Mo, XH and Zhang, H and Wang, TM and Zhang, C and Zhang, C and Xing, XH and Yang, S}, title = {Establishment of CRISPR interference in Methylorubrum extorquens and application of rapidly mining a new phytoene desaturase involved in carotenoid biosynthesis.}, journal = {Applied microbiology and biotechnology}, volume = {104}, number = {10}, pages = {4515-4532}, doi = {10.1007/s00253-020-10543-w}, pmid = {32215707}, issn = {1432-0614}, support = {2018YFA0901500//the National Key R&amp;D Program of China/ ; 21776149//the National Natural Science Foundation of China/ ; 2016GSF117026//Shandong Provincial Key Research and Development Project of China/ ; ZR2015CM017//Shandong Provincial Natural Science Foundation of China/ ; }, mesh = {Biosynthetic Pathways/*genetics ; CRISPR-Associated Protein 9/genetics ; *CRISPR-Cas Systems ; Carotenoids/*metabolism ; Gene Knockdown Techniques ; Metabolic Networks and Pathways ; Methylobacterium extorquens/*enzymology/*genetics ; Oxidoreductases/*genetics/metabolism ; Phylogeny ; Promoter Regions, Genetic ; RNA, Guide/genetics ; Streptococcus pyogenes/enzymology/genetics ; }, abstract = {The methylotrophic bacterium Methylorubrum extorquens AM1 holds a great potential of a microbial cell factory in producing high value chemicals with methanol as the sole carbon and energy source. However, many gene functions remain unknown, hampering further rewiring of metabolic networks. Clustered regularly interspaced short palindromic repeat interference (CRISPRi) has been demonstrated to be a robust tool for gene knockdown in diverse organisms. In this study, we developed an efficient CRISPRi system through optimizing the promoter strength of Streptococcus pyogenes-derived deactivated cas9 (dcas9). When the dcas9 and sgRNA were respectively controlled by medium PR/tetO and strong PmxaF-g promoters, dynamic repression efficacy of cell growth through disturbing a central metabolism gene glyA was achieved from 41.9 to 96.6% dependent on the sgRNA targeting sites. Furthermore, the optimized CRISPRi system was shown to effectively decrease the abundance of exogenous fluorescent protein gene mCherry over 50% and to reduce the expression of phytoene desaturase gene crtI by 97.7%. We then used CRISPRi technology combined with 26 sgRNAs pool to rapidly discover a new phytoene desaturase gene META1_3670 from 2470 recombinant mutants. The gene function was further verified through gene deletion and complementation as well as phylogenetic tree analysis. In addition, we applied CRISPRi to repress the transcriptional level of squalene-hopene cyclase gene shc involved in hopanoid biosynthesis by 64.9%, which resulted in enhancing 1.9-fold higher of carotenoid production without defective cell growth. Thus, the CRISPRi system developed here provides a useful tool in mining functional gene of M. extorquens as well as in biotechnology for producing high-valued chemicals from methanol. KEY POINTS: Developing an efficient CRISPRi to knockdown gene expression in C1-utilizing bacteria CRISPRi combined with sgRNAs pool to rapidly discover a new phytoene desaturase gene Improvement of carotenoid production by repressing a competitive pathway.}, }
@article {pmid32215703, year = {2020}, author = {Wu, D and Xie, W and Li, X and Cai, G and Lu, J and Xie, G}, title = {Metabolic engineering of Saccharomyces cerevisiae using the CRISPR/Cas9 system to minimize ethyl carbamate accumulation during Chinese rice wine fermentation.}, journal = {Applied microbiology and biotechnology}, volume = {104}, number = {10}, pages = {4435-4444}, doi = {10.1007/s00253-020-10549-4}, pmid = {32215703}, issn = {1432-0614}, support = {BK20170178//the Natural Science Foundation of Jiangsu Province, China/ ; 31701588//the National Natural Science Foundation of China/ ; 31701730//the National Natural Science Foundation of China/ ; JUSRP11965//the Fundamental Research Funds for the Central Universities/ ; }, mesh = {*CRISPR-Cas Systems ; *Fermentation ; Genome, Fungal ; Membrane Transport Proteins/genetics/metabolism ; Metabolic Engineering ; Oryza/*microbiology ; Recombination, Genetic ; Saccharomyces cerevisiae/*genetics/metabolism ; Saccharomyces cerevisiae Proteins/genetics/metabolism ; Urethane/*metabolism ; Wine/*analysis ; }, abstract = {Ethyl carbamate (EC) is a potential carcinogen to humans that is mainly produced through the spontaneous reaction between urea and ethanol during Chinese rice wine brewing. We metabolically engineered a strain by over-expressing the DUR3 gene in a previously modified strain using an improved CRISPR/Cas9 system to further decrease the EC level. Homologous recombination of the DUR3 over-expression cassette was performed at the HO locus by individual transformation of the constructed plasmid CRISPR-DUR3-gBlock-HO, generating the engineered strain N85DUR1,2/DUR3-c. Consequently, the DUR3 expression level was significantly enhanced in the modified strain, resulting in increased utilization of urea. The brewing test showed that N85DUR1,2/DUR3-c reduced urea and EC concentrations by 92.0% and 58.5%, respectively, compared with those of the original N85 strain. Moreover, the engineered strain showed good genetic stability in reducing urea content during the repeated brewing experiments. Importantly, the genetic manipulation had a negligible effect on the growth and fermentation characteristics of the yeast strain. Therefore, the constructed strain is potentially suitable for application to reduce urea and EC contents during production of Chinese rice wine. KEY POINTS: • An efficient CRISPR vector was constructed and applied for DUR3 over-expression. • Multi-modification of urea cycle had synergistic effect on reducing EC level. • Fermentation performance of engineered strain was similar with the parental strain. • No residual heterologous genes were left in the genome after genetic manipulation. • An efficient CRISPR vector was constructed and applied for DUR3 over-expression. • Multi-modification of urea cycle had synergistic effect on reducing EC level. • Fermentation performance of engineered strain was similar with the parental strain. • No residual heterologous genes were left in the genome after genetic manipulation.}, }
@article {pmid32086048, year = {2020}, author = {Kalds, P and Gao, Y and Zhou, S and Cai, B and Huang, X and Wang, X and Chen, Y}, title = {Redesigning small ruminant genomes with CRISPR toolkit: Overview and perspectives.}, journal = {Theriogenology}, volume = {147}, number = {}, pages = {25-33}, doi = {10.1016/j.theriogenology.2020.02.015}, pmid = {32086048}, issn = {1879-3231}, mesh = {Animals ; *CRISPR-Cas Systems ; *Genome ; Goats/*genetics ; Humans ; Sheep/*genetics ; }, abstract = {Genetic modification is a rapidly developing field in which numerous significant breakthroughs have been achieved. Over the last few decades, genetic modification has evolved from insertional transgenesis to gene targeting and editing and, more recently, to base and prime editing using CRISPR-derived systems. Currently, CRISPR-based genome editing systems are showing great potential for generating gene-edited offspring with defined genetic characteristics. Domestic small ruminants (sheep and goats) have shown great potential as large animal models for genome engineering. Ovine and caprine genomes have been engineered using CRISPR-based systems for numerous purposes. These include generating superior agricultural breeds, expression of therapeutic agents in mammary glands, and developing animal models to be used in the study of human genetic disorders and regenerative medicine. The creation of these models has been facilitated by the continuous emergence and development of genetic modification tools. In this review, we provide an overview on how CRISPR-based systems have been used in the generation of gene-edited small ruminants through the two main pathways (embryonic microinjection and somatic cell nuclear transfer) and highlight the ovine and caprine genes that have been targeted via knockout, knockin, HDR-mediated point mutation, and base editing approaches, as well as the aims of these specific manipulations.}, }
@article {pmid33493854, year = {2021}, author = {Bruch, R and Johnston, M and Kling, A and Mattmüller, T and Baaske, J and Partel, S and Madlener, S and Weber, W and Urban, GA and Dincer, C}, title = {CRISPR-powered electrochemical microfluidic multiplexed biosensor for target amplification-free miRNA diagnostics.}, journal = {Biosensors &amp; bioelectronics}, volume = {177}, number = {}, pages = {112887}, doi = {10.1016/j.bios.2020.112887}, pmid = {33493854}, issn = {1873-4235}, abstract = {Recently the use of microRNAs (miRNAs) as biomarkers for a multitude of diseases has gained substantial significance for clinical as well as point-of-care diagnostics. Amongst other challenges, however, it holds the central requirement that the concentration of a given miRNA must be evaluated within the context of other factors in order to unambiguously diagnose one specific disease. In terms of the development of diagnostic methods and devices, this implies an inevitable demand for multiplexing in order to be able to gauge the abundance of several components of interest in a patient's sample in parallel. In this study, we design and implement different multiplexed versions of our electrochemical microfluidic biosensor by dividing its channel into subsections, creating four novel chip designs for the amplification-free and simultaneous quantification of up to eight miRNAs on the CRISPR-Biosensor X ('X' highlighting the multiplexing aspect of the device). We then use a one-step model assay followed by amperometric readout in combination with a 2-min-stop-flow-protocol to explore the fluidic and mechanical characteristics and limitations of the different versions of the device. The sensor showing the best performance, is subsequently used for the Cas13a-powered proof-of-concept measurement of two miRNAs (miRNA-19b and miRNA-20a) from the miRNA-17-92 cluster, which is dysregulated in the blood of pediatric medulloblastoma patients. Quantification of the latter, alongside simultaneous negative control measurements are accomplished on the same device. We thereby confirm the applicability of our platform to the challenge of amplification-free, parallel detection of multiple nucleic acids.}, }
@article {pmid33490975, year = {2021}, author = {Zhou, P and Wan, YK and Chan, BKC and Choi, GCG and Wong, ASL}, title = {Extensible combinatorial CRISPR screening in mammalian cells.}, journal = {STAR protocols}, volume = {2}, number = {1}, pages = {100255}, doi = {10.1016/j.xpro.2020.100255}, pmid = {33490975}, issn = {2666-1667}, abstract = {The CRISPR-Cas system coupled with Combinatorial Genetics En Masse (CombiGEM) enables systematic analysis of high-order genetic perturbations that are important for understanding biological processes and discovering therapeutic target combinations. Here, we present detailed steps and technical considerations for building multiplexed guide RNA libraries and carrying out a combinatorial CRISPR screen in mammalian cells. We also present an analytical pipeline, CombiPIPE, for mapping two- and three-way genetic interactions. For complete details on the use and execution of this protocol, please refer to Zhou et al. (2020).}, }
@article {pmid33488814, year = {2020}, author = {Padayachee, J and Singh, M}, title = {Therapeutic applications of CRISPR/Cas9 in breast cancer and delivery potential of gold nanomaterials.}, journal = {Nanobiomedicine}, volume = {7}, number = {}, pages = {1849543520983196}, doi = {10.1177/1849543520983196}, pmid = {33488814}, issn = {1849-5435}, abstract = {Globally, approximately 1 in 4 cancers in women are diagnosed as breast cancer (BC). Despite significant advances in the diagnosis and therapy BCs, many patients develop metastases or relapses. Hence, novel therapeutic strategies are required, that can selectively and efficiently kill malignant cells. Direct targeting of the genetic and epigenetic aberrations that occur in BC development is a promising strategy to overcome the limitations of current therapies, which target the tumour phenotype. The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system, composed of only an easily modifiable single guide RNA (sgRNA) sequence bound to a Cas9 nuclease, has revolutionised genome editing due to its simplicity and efficiency compared to earlier systems. CRISPR/Cas9 and its associated catalytically inactivated dCas9 variants facilitate the knockout of overexpressed genes, correction of mutations in inactivated genes, and reprogramming of the epigenetic landscape to impair BC growth. To achieve efficient genome editing in vivo, a vector is required to deliver the components to target cells. Gold nanomaterials, including gold nanoparticles and nanoclusters, display many advantageous characteristics that have facilitated their widespread use in theranostics, as delivery vehicles, and imaging and photothermal agents. This review highlights the therapeutic applications of CRISPR/Cas9 in treating BCs, and briefly describes gold nanomaterials and their potential in CRISPR/Cas9 delivery.}, }
@article {pmid33488667, year = {2020}, author = {Shin, U and Brondani, V}, title = {Analysis of Wild Type LbCpf1 Protein, and PAM Recognition Variants, in a Cellular Context.}, journal = {Frontiers in genetics}, volume = {11}, number = {}, pages = {571591}, doi = {10.3389/fgene.2020.571591}, pmid = {33488667}, issn = {1664-8021}, abstract = {Nucleases used in genome engineering induce hydrolysis of DNA phosphate backbone in a sequence-specific manner. So far CRISPR-Cas, the RNA-guided nucleases, is the most advanced genome engineering system. The CRISPR nucleases allows recognition of a particular genomic sequence with two distinct molecular interactions: first, by direct interaction between the nuclease and the protospacer-adjacent motif, wherein discrete amino acids interact with DNA base pairs; and second, by hybridization of the guide RNA with the target DNA sequence. Here we report the application of the single strand annealing cellular assay to analyze and quantify nuclease activity of wild type and mutant CRISPR-Cpf1. Using this heterologous marker system based on GFP activity, we observed a comparable PAM recognition selectivity with the NGS analysis. The heterologous marker system has revealed that LbCpf1 is a more specific nuclease than AsCpf1 in a cellular context. We controlled the in vitro activity of the Cpf1 nuclease complexes expressed in mammalian cells and demonstrated that they are responsible of the DNA cleavage at the target site. In addition, we generated and tested LbCpf1 variants with several combinations of mutations at the PAM-recognition positions G532, K538 and Y542. Finally, we showed that the results of the in vitro DNA cleavage assay with the wild type and mutants LbCpf1 corroborate with the selection of 6TG resistant cells associated to the genomic disruption of hprt gene.}, }
@article {pmid33488567, year = {2020}, author = {Zhao, Y and Wang, F and Wang, C and Zhang, X and Jiang, C and Ding, F and Shen, L and Zhang, Q}, title = {Optimization of CRISPR/Cas System for Improving Genome Editing Efficiency in Plasmodium falciparum.}, journal = {Frontiers in microbiology}, volume = {11}, number = {}, pages = {625862}, doi = {10.3389/fmicb.2020.625862}, pmid = {33488567}, issn = {1664-302X}, abstract = {Studies of molecular mechanisms and related gene functions have long been restricted by limited genome editing technologies in malaria parasites. Recently, a simple and effective genome editing technology, the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated) system, has greatly facilitated these studies in many organisms, including malaria parasites. However, due to the special genome feature of malaria parasites, the manipulation and gene editing efficacy of the CRISPR/Cas system in this pathogen need to be improved, particularly in the human malaria parasite, Plasmodium falciparum. Herein, based on the CRISPR/Cas9 system, we developed an integrating strategy to generate a Cas9i system, which significantly shortened the time for generation of transgenic strains in P. falciparum. Moreover, with this Cas9i system, we have successfully achieved multiplexed genome editing (mutating or tagging) by a single-round transfection in P. falciparum. In addition, we for the first time adapted AsCpf1 (Acidaminococcus sp. Cpf1), an alternative to Cas9, into P. falciparum parasites and examined it for gene editing. These optimizations of the CRISPR/Cas system will further facilitate the mechanistic research of malaria parasites and contribute to eliminating malaria in the future.}, }
@article {pmid33420081, year = {2021}, author = {Hou, G and Harley, ITW and Lu, X and Zhou, T and Xu, N and Yao, C and Qin, Y and Ouyang, Y and Ma, J and Zhu, X and Yu, X and Xu, H and Dai, D and Ding, H and Yin, Z and Ye, Z and Deng, J and Zhou, M and Tang, Y and Namjou, B and Guo, Y and Weirauch, MT and Kottyan, LC and Harley, JB and Shen, N}, title = {SLE non-coding genetic risk variant determines the epigenetic dysfunction of an immune cell specific enhancer that controls disease-critical microRNA expression.}, journal = {Nature communications}, volume = {12}, number = {1}, pages = {135}, pmid = {33420081}, issn = {2041-1723}, support = {T32 AR007534/AR/NIAMS NIH HHS/United States ; }, mesh = {Animals ; CRISPR-Cas Systems ; Case-Control Studies ; Cell Line, Tumor ; Epigenesis, Genetic/*immunology ; Female ; Genetic Predisposition to Disease ; Genotyping Techniques ; HEK293 Cells ; Healthy Volunteers ; Humans ; Interferon Type I/metabolism ; Leukocytes, Mononuclear/transplantation ; Lupus Erythematosus, Systemic/blood/*genetics/immunology ; Mice ; MicroRNAs/*genetics/metabolism ; NF-kappa B/metabolism ; Polymorphism, Single Nucleotide ; Primary Cell Culture ; Promoter Regions, Genetic ; RNA-Seq ; Regulatory Sequences, Nucleic Acid/*genetics ; Signal Transduction/genetics/immunology ; }, abstract = {Since most variants that impact polygenic disease phenotypes localize to non-coding genomic regions, understanding the consequences of regulatory element variants will advance understanding of human disease mechanisms. Here, we report that the systemic lupus erythematosus (SLE) risk variant rs2431697 as likely causal for SLE through disruption of a regulatory element, modulating miR-146a expression. Using epigenomic analysis, genome-editing and 3D chromatin structure analysis, we show that rs2431697 tags a cell-type dependent distal enhancer specific for miR-146a that physically interacts with the miR-146a promoter. NF-kB binds the disease protective allele in a sequence-specific manner, increasing expression of this immunoregulatory microRNA. Finally, CRISPR activation-based modulation of this enhancer in the PBMCs of SLE patients attenuates type I interferon pathway activation by increasing miR-146a expression. Our work provides a strategy to define non-coding RNA functional regulatory elements using disease-associated variants and provides mechanistic links between autoimmune disease risk genetic variation and disease etiology.}, }
@article {pmid33270810, year = {2020}, author = {Ogata, T and Ishizaki, T and Fujita, M and Fujita, Y}, title = {CRISPR/Cas9-targeted mutagenesis of OsERA1 confers enhanced responses to abscisic acid and drought stress and increased primary root growth under nonstressed conditions in rice.}, journal = {PloS one}, volume = {15}, number = {12}, pages = {e0243376}, pmid = {33270810}, issn = {1932-6203}, mesh = {Abscisic Acid/*metabolism ; CRISPR-Cas Systems/genetics ; *Droughts ; Frameshift Mutation ; Mutagenesis ; Oryza/*physiology ; Plant Proteins/genetics/*metabolism ; Plant Roots/growth &amp; development ; Plant Stomata/physiology ; Plants, Genetically Modified/physiology ; *Stress, Physiological ; }, abstract = {Abscisic acid (ABA) signaling components play an important role in the drought stress response in plants. Arabidopsis thaliana ENHANCED RESPONSE TO ABA1 (ERA1) encodes the β-subunit of farnesyltransferase and regulates ABA signaling and the dehydration response. Therefore, ERA1 is an important candidate gene for enhancing drought tolerance in numerous crops. However, a rice (Oryza sativa) ERA1 homolog has not been characterized previously. Here, we show that rice osera1 mutant lines, harboring CRISPR/Cas9-induced frameshift mutations, exhibit similar leaf growth as control plants but increased primary root growth. The osera1 mutant lines also display increased sensitivity to ABA and an enhanced response to drought stress through stomatal regulation. These results illustrate that OsERA1 is a negative regulator of primary root growth under nonstressed conditions and also of responses to ABA and drought stress in rice. These findings improve our understanding of the role of ABA signaling in the drought stress response in rice and suggest a strategy to genetically improve rice.}, }
@article {pmid33270431, year = {2020}, author = {Moon, J and Kwon, HJ and Yong, D and Lee, IC and Kim, H and Kang, H and Lim, EK and Lee, KS and Jung, J and Park, HG and Kang, T}, title = {Colorimetric Detection of SARS-CoV-2 and Drug-Resistant pH1N1 Using CRISPR/dCas9.}, journal = {ACS sensors}, volume = {5}, number = {12}, pages = {4017-4026}, pmid = {33270431}, issn = {2379-3694}, mesh = {CRISPR-Associated Protein 9/*genetics ; CRISPR-Cas Systems/*genetics ; *Colorimetry ; Drug Resistance, Viral/*genetics ; Humans ; Influenza A Virus, H1N1 Subtype/*genetics/*isolation &amp; purification ; SARS-CoV-2/drug effects/*genetics/*isolation &amp; purification ; }, abstract = {Viruses have been a continuous threat to human beings. The coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has led to a pandemic that is still ongoing worldwide. Previous pandemic influenza A virus (pH1N1) might be re-emerging through a drug-resistant mutation. We report a colorimetric viral detection method based on the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 endonuclease dead (dCas9) system. In this method, RNA in the viral lysate was directly recognized by the CRISPR/dCas9 system with biotin-protospacer adjacent motif (PAM)-presenting oligonucleotide (PAMmer). Streptavidin-horseradish peroxidase then bound to biotin-PAMmer, inducing a color change through the oxidation of 3,3',5,5'-tetramethylbenzidine. Using the developed method, we successfully identified SARS-CoV-2, pH1N1, and pH1N1/H275Y viruses by the naked eye. Moreover, the detection of viruses in human nasopharyngeal aspirates and sputum was demonstrated. Finally, clinical samples from COVID-19 patients led to a successful diagnosis. We anticipate that the current method can be employed for simple and accurate diagnosis of viruses.}, }
@article {pmid33122083, year = {2021}, author = {Wang, X and Hayes, JE and Xu, X and Gao, X and Mehta, D and Lilja, HG and Klein, RJ}, title = {Validation of prostate cancer risk variants rs10993994 and rs7098889 by CRISPR/Cas9 mediated genome editing.}, journal = {Gene}, volume = {768}, number = {}, pages = {145265}, pmid = {33122083}, issn = {1879-0038}, support = {P30 CA008748/CA/NCI NIH HHS/United States ; P50 CA092629/CA/NCI NIH HHS/United States ; R01 CA175491/CA/NCI NIH HHS/United States ; U01 HG007033/HG/NHGRI NIH HHS/United States ; }, mesh = {CRISPR-Cas Systems/genetics ; Gene Deletion ; Gene Editing/methods ; Genetic Predisposition to Disease/*genetics ; *Genome-Wide Association Study ; Histone Code/genetics ; Humans ; Linkage Disequilibrium/genetics ; Male ; Nuclear Receptor Coactivators/*biosynthesis/genetics ; Polymorphism, Single Nucleotide/genetics ; Promoter Regions, Genetic/genetics ; Prostatic Neoplasms/*genetics ; Prostatic Secretory Proteins/*genetics ; RNA, Messenger/biosynthesis ; }, abstract = {GWAS have identified numerous SNPs associated with prostate cancer risk. One such SNP is rs10993994. It is located in the β-microseminoprotein (MSMB) promoter region, mediates MSMB prostate secretion levels, and is linked to mRNA expression changes in both MSMB and the adjacent gene NCOA4. In addition, our previous work showed a second SNP, rs7098889, is in positive linkage disequilibrium with rs10993994 and associated with MSMB expression independent of rs10993994. Here, we generate a series of clones with single alleles removed by double guide RNA (gRNA) mediated CRISPR/Cas9 deletions, through which we demonstrate that each of these SNPs independently and greatly alters MSMB expression in an allele-specific manner. We further show that these SNPs have no substantial effect on the expression of NCOA4. These data demonstrate that a single SNP can have a large effect on gene expression and illustrate the importance of functional validation studies to deconvolute observed correlations. The method we have developed is generally applicable to test any SNP for which a relevant heterozygous cell line is available. AUTHOR SUMMARY: In pursuing the underlying biological mechanism of prostate cancer pathogenesis, scientists utilized the existence of common single nucleotide polymorphisms (SNPs) in the human genome as genetic markers to perform large scale genome wide association studies (GWAS) and have so far identified more than a hundred prostate cancer risk variants. Such variants provide an unbiased and systematic new venue to study the disease mechanism, and the next big challenge is to translate these genetic associations to the causal role of altered gene function in oncogenesis. The majority of these variants are waiting to be studied and lots of them may act in oncogenesis through gene expression regulation. To prove the concept, we took rs10993994 and its linked rs7098889 as an example and engineered single cell clones by allelic-specific CRISPR/Cas9 deletion to separate the effect of each allele. We observed that a single nucleotide difference would lead to surprisingly high level of MSMB gene expression change in a gene specific and cell-type specific manner. Our study strongly supports the notion that differential level of gene expression caused by risk variants and their associated genetic locus play a major role in oncogenesis and also highlights the importance of studying the function of MSMB encoded β-MSP in prostate cancer pathogenesis.}, }
@article {pmid33107304, year = {2020}, author = {Saha, A and Arantes, PR and Hsu, RV and Narkhede, YB and Jinek, M and Palermo, G}, title = {Molecular Dynamics Reveals a DNA-Induced Dynamic Switch Triggering Activation of CRISPR-Cas12a.}, journal = {Journal of chemical information and modeling}, volume = {60}, number = {12}, pages = {6427-6437}, pmid = {33107304}, issn = {1549-960X}, support = {R01 EY027440/EY/NEI NIH HHS/United States ; }, mesh = {COVID-19/*diagnosis ; CRISPR-Cas Systems ; Catalytic Domain ; DNA Cleavage ; DNA, Viral/*analysis/*genetics ; Gene Editing ; Humans ; Molecular Dynamics Simulation ; Nucleic Acid Conformation ; Phase Transition ; SARS-CoV-2/*genetics ; Substrate Specificity ; }, abstract = {CRISPR-Cas12a is a genome-editing system, recently also harnessed for nucleic acid detection, which is promising for the diagnosis of the SARS-CoV-2 coronavirus through the DETECTR technology. Here, a collective ensemble of multimicrosecond molecular dynamics characterizes the key dynamic determinants allowing nucleic acid processing in CRISPR-Cas12a. We show that DNA binding induces a switch in the conformational dynamics of Cas12a, which results in the activation of the peripheral REC2 and Nuc domains to enable cleavage of nucleic acids. The simulations reveal that large-amplitude motions of the Nuc domain could favor the conformational activation of the system toward DNA cleavages. In this process, the REC lobe plays a critical role. Accordingly, the joint dynamics of REC and Nuc shows the tendency to prime the conformational transition of the DNA target strand toward the catalytic site. Most notably, the highly coupled dynamics of the REC2 region and Nuc domain suggests that REC2 could act as a regulator of the Nuc function, similar to what was observed previously for the HNH domain in the CRISPR-associated nuclease Cas9. These mutual domain dynamics could be critical for the nonspecific binding of DNA and thereby for the underlying mechanistic functioning of the DETECTR technology. Considering that REC is a key determinant in the system's specificity, our findings provide a rational basis for future biophysical studies aimed at characterizing its function in CRISPR-Cas12a. Overall, our outcomes advance our mechanistic understanding of CRISPR-Cas12a and provide grounds for novel engineering efforts to improve genome editing and viral detection.}, }
@article {pmid33082438, year = {2020}, author = {Lee, J and Sheen, JH and Lim, O and Lee, Y and Ryu, J and Shin, D and Kim, YY and Kim, M}, title = {Abrogation of HLA surface expression using CRISPR/Cas9 genome editing: a step toward universal T cell therapy.}, journal = {Scientific reports}, volume = {10}, number = {1}, pages = {17753}, pmid = {33082438}, issn = {2045-2322}, mesh = {CRISPR-Cas Systems ; Cell- and Tissue-Based Therapy/*methods ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; HLA Antigens/*genetics ; Humans ; T-Lymphocytes/*metabolism ; }, abstract = {As recent advancements in the chimeric antigen receptor-T cells have revolutionized the way blood cancers are handled, potential benefits from producing off-the-shelf, standardized immune cells entail the need for development of allogeneic immune cell therapy. However, host rejection driven by HLA disparity in adoptively transferred allogeneic T cells remains a key obstacle to the universal donor T cell therapy. To evade donor HLA-mediated immune rejection, we attempted to eliminate T cell's HLA through the CRISPR/Cas9 gene editing system. First, we screened 60 gRNAs targeting B2M and multiple sets of gRNA each targeting α chains of HLA-II (DPA, DQA and DRA, respectively) using web-based design tools, and identified specific gRNA sequences highly efficient for target deletion without carrying off-target effects. Multiplex genome editing of primary human T cells achieved by the newly discovered gRNAs yielded HLA-I- or HLA-I/II-deficient T cells that were phenotypically unaltered and functionally intact. The overnight mixed lymphocyte reactions demonstrated the HLA-I-negative cells induced decreased production of IFN-γ and TNF-α in alloreactive T cells, and deficiency of HLA-I/II in T cells further dampened the inflammatory responses. Taken together, our approach will provide an efficacious pathway toward the universal donor cell generation by manipulating HLA expression in therapeutic T cells.}, }
@article {pmid33020655, year = {2020}, author = {Alerasool, N and Segal, D and Lee, H and Taipale, M}, title = {An efficient KRAB domain for CRISPRi applications in human cells.}, journal = {Nature methods}, volume = {17}, number = {11}, pages = {1093-1096}, doi = {10.1038/s41592-020-0966-x}, pmid = {33020655}, issn = {1548-7105}, support = {//CIHR/Canada ; }, mesh = {CRISPR-Associated Protein 9/*genetics ; CRISPR-Cas Systems/*genetics ; Catalytic Domain ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Genes, Reporter ; HEK293 Cells ; Humans ; K562 Cells ; Kruppel-Like Transcription Factors/*genetics ; Promoter Regions, Genetic ; RNA, Guide/genetics ; Repressor Proteins/genetics ; Transcription, Genetic ; }, abstract = {Clustered regularly interspaced short palindromic repeat interference (CRISPRi), based on the fusion of inactive Cas9 (dCas9) to the Krüppel-associated box (KRAB) repressor, is a powerful platform for silencing gene expression. However, it suffers from incomplete silencing of target genes. We assayed 57 KRAB domains for their repressive potency and identified the ZIM3 KRAB domain as an exceptionally potent repressor. We establish that ZIM3 KRAB-dCas9 fusion silences gene expression more efficiently than existing platforms.}, }
@article {pmid32994506, year = {2020}, author = {Owen, JR and Hennig, SL and McNabb, BR and Lin, JC and Young, AE and Murray, JD and Ross, PJ and Van Eenennaam, AL}, title = {Harnessing endogenous repair mechanisms for targeted gene knock-in of bovine embryos.}, journal = {Scientific reports}, volume = {10}, number = {1}, pages = {16031}, pmid = {32994506}, issn = {2045-2322}, mesh = {Animals ; CRISPR-Associated Protein 9/genetics ; CRISPR-Cas Systems/genetics ; Cattle ; DNA End-Joining Repair/physiology ; DNA Repair/genetics ; Gene Editing/*methods ; Gene Knock-In Techniques/*methods ; Genetic Engineering/*methods ; Genome/genetics ; Homologous Recombination/genetics ; Microinjections/methods ; RNA, Guide/genetics ; Recombinational DNA Repair/genetics ; Zygote/physiology ; }, abstract = {Introducing useful traits into livestock breeding programs through gene knock-ins has proven challenging. Typically, targeted insertions have been performed in cell lines, followed by somatic cell nuclear transfer cloning, which can be inefficient. An alternative is to introduce genome editing reagents and a homologous recombination (HR) donor template into embryos to trigger homology directed repair (HDR). However, the HR pathway is primarily restricted to actively dividing cells (S/G2-phase) and its efficiency for the introduction of large DNA sequences in zygotes is low. The homology-mediated end joining (HMEJ) approach has been shown to improve knock-in efficiency in non-dividing cells and to harness HDR after direct injection of embryos. The knock-in efficiency for a 1.8 kb gene was contrasted when combining microinjection of a gRNA/Cas9 ribonucleoprotein complex with a traditional HR donor template or an HMEJ template in bovine zygotes. The HMEJ template resulted in a significantly higher rate of gene knock-in as compared to the HR template (37.0% and 13.8%; P < 0.05). Additionally, more than a third of the knock-in embryos (36.9%) were non-mosaic. This approach will facilitate the one-step introduction of gene constructs at a specific location of the bovine genome and contribute to the next generation of elite cattle.}, }
@article {pmid32989329, year = {2020}, author = {Schumann, K and Raju, SS and Lauber, M and Kolb, S and Shifrut, E and Cortez, JT and Skartsis, N and Nguyen, VQ and Woo, JM and Roth, TL and Yu, R and Nguyen, MLT and Simeonov, DR and Nguyen, DN and Targ, S and Gate, RE and Tang, Q and Bluestone, JA and Spitzer, MH and Ye, CJ and Marson, A}, title = {Functional CRISPR dissection of gene networks controlling human regulatory T cell identity.}, journal = {Nature immunology}, volume = {21}, number = {11}, pages = {1456-1466}, pmid = {32989329}, issn = {1529-2916}, support = {P50 GM082250/GM/NIGMS NIH HHS/United States ; P30 DK063720/DK/NIDDK NIH HHS/United States ; DP3 DK111914/DK/NIDDK NIH HHS/United States ; DP5 OD023056/OD/NIH HHS/United States ; }, mesh = {Biomarkers ; CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Disease Susceptibility ; *Gene Expression Profiling ; *Gene Expression Regulation ; Gene Knockout Techniques ; *Gene Regulatory Networks ; Gene Targeting ; Graft vs Host Disease/etiology ; High-Throughput Nucleotide Sequencing ; Humans ; T-Lymphocytes, Regulatory/*immunology/*metabolism ; *Transcriptome ; }, abstract = {Human regulatory T (Treg) cells are essential for immune homeostasis. The transcription factor FOXP3 maintains Treg cell identity, yet the complete set of key transcription factors that control Treg cell gene expression remains unknown. Here, we used pooled and arrayed Cas9 ribonucleoprotein screens to identify transcription factors that regulate critical proteins in primary human Treg cells under basal and proinflammatory conditions. We then generated 54,424 single-cell transcriptomes from Treg cells subjected to genetic perturbations and cytokine stimulation, which revealed distinct gene networks individually regulated by FOXP3 and PRDM1, in addition to a network coregulated by FOXO1 and IRF4. We also discovered that HIVEP2, to our knowledge not previously implicated in Treg cell function, coregulates another gene network with SATB1 and is important for Treg cell-mediated immunosuppression. By integrating CRISPR screens and single-cell RNA-sequencing profiling, we have uncovered transcriptional regulators and downstream gene networks in human Treg cells that could be targeted for immunotherapies.}, }
@article {pmid32786832, year = {2020}, author = {Guo, D and Ling, X and Zhou, X and Li, X and Wang, J and Qiu, S and Yang, Y and Zhang, B}, title = {Evaluation of the Quality of a High-Resistant Starch and Low-Glutelin Rice (Oryza sativa L.) Generated through CRISPR/Cas9-Mediated Targeted Mutagenesis.}, journal = {Journal of agricultural and food chemistry}, volume = {68}, number = {36}, pages = {9733-9742}, doi = {10.1021/acs.jafc.0c02995}, pmid = {32786832}, issn = {1520-5118}, mesh = {Alleles ; CRISPR-Cas Systems ; Food, Genetically Modified ; Glutens/*analysis/metabolism ; Mutagenesis ; Oryza/chemistry/*genetics/metabolism ; Plant Proteins/genetics/metabolism ; Plants, Genetically Modified/*chemistry/genetics/metabolism ; }, abstract = {A high-resistant starch (RS) and low-glutelin diet is beneficial for the health of patients with diabetes and kidney diseases. Rice is an important food crop worldwide. Previous studies have demonstrated that downregulating the expression of rice starch branching enzyme IIb (SBEIIb) affected the composition and the structure of starch. However, there has been no report about generating the loss-of-function mutants of SBEIIb using low-glutelin rice cultivars as recipients. In this study, we adopted a CRISPR/Cas9 system to induce site-specific mutations at the SBEIIb locus in an elite low-glutelin japonica rice cultivar derived from Low Glutelin Content-1 (LGC-1) and successfully obtained two independent transgene-free sbeIIb/Lgc1 mutant lines. In the mutant lines, the apparent amylose content (AAC) was increased by approximately 1.8-fold and the RS content reached approximately 6%. The glutelin content was approximately 2%, maintaining the low-glutelin trait of the recipient cultivar. The formation mechanism of RS was explored by analyzing the fine structures and the properties of starch. According to the X-ray diffraction pattern and the increased lipid content, the high RS content of the sbeIIb/Lgc1 lines was attributed to the increased content of amylose-lipid complex. Further analyses of the nutritional quality revealed that the soluble sugar and lipid contents, especially sucrose and unsaturated fatty acids, increased in the sbeIIb/Lgc1 lines significantly. This research is expected to facilitate the cultivation and the application of functional rice suitable for patients with diabetes and kidney diseases.}, }
@article {pmid32715554, year = {2020}, author = {Chaverra-Rodriguez, D and Dalla Benetta, E and Heu, CC and Rasgon, JL and Ferree, PM and Akbari, OS}, title = {Germline mutagenesis of Nasonia vitripennis through ovarian delivery of CRISPR-Cas9 ribonucleoprotein.}, journal = {Insect molecular biology}, volume = {29}, number = {6}, pages = {569-577}, doi = {10.1111/imb.12663}, pmid = {32715554}, issn = {1365-2583}, support = {R21AI111175//NIH/NIAID/International ; }, mesh = {Animals ; CRISPR-Associated Protein 9/*genetics ; CRISPR-Cas Systems ; Female ; *Gene Editing ; Germ Cells/*metabolism ; Insect Proteins/*genetics ; Male ; *Mutagenesis ; Ovary ; Ribonucleoproteins/*genetics ; Wasps/*genetics/growth &amp; development ; }, abstract = {CRISPR/Cas9 gene editing is a powerful technology to study the genetics of rising model organisms, such as the jewel wasp Nasonia vitripennis. However, current methods involving embryonic microinjection of CRISPR reagents are challenging. Delivery of Cas9 ribonucleoprotein into female ovaries is an alternative that has only been explored in a small handful of insects, such as mosquitoes, whiteflies and beetles. Here, we developed a simple protocol for germline gene editing by injecting Cas9 ribonucleoprotein in adult N. vitripennis females using either ReMOT control (Receptor-Mediated Ovary Transduction of Cargo) or BAPC (Branched Amphiphilic Peptide Capsules) as ovary delivery methods. For ReMOT Control we used the Drosophila melanogaster-derived peptide 'P2C' fused to EGFP to visualize the ovary delivery, and fused to Cas9 protein for gene editing of the cinnabar gene using saponin as an endosomal escape reagent. For BAPC we optimized the concentrations of protein, sgRNA and the transfection reagent. We demonstrate delivery of protein cargo such as EGFP and Cas9 into developing oocytes via P2C peptide and BAPC. Additionally, somatic and germline gene editing were demonstrated. This approach will greatly facilitate CRISPR-applied genetic manipulation in this and other rising model organisms.}, }
@article {pmid32534063, year = {2020}, author = {Fourier, N and Zolty, M and Azriel, A and Tedesco, D and Levi, BZ}, title = {MafK Mediates Chromatin Remodeling to Silence IRF8 Expression in Non-immune Cells in a Cell Type-SpecificManner.}, journal = {Journal of molecular biology}, volume = {432}, number = {16}, pages = {4544-4560}, doi = {10.1016/j.jmb.2020.06.005}, pmid = {32534063}, issn = {1089-8638}, mesh = {Animals ; Binding Sites ; CRISPR-Cas Systems ; Chromatin/*metabolism ; Chromatin Assembly and Disassembly ; Gene Expression Regulation ; HEK293 Cells ; Humans ; Interferon Regulatory Factors/chemistry/*genetics/metabolism ; MafK Transcription Factor/*genetics/*metabolism ; Mice ; NIH 3T3 Cells ; Organ Specificity ; RAW 264.7 Cells ; RNA, Small Interfering/pharmacology ; }, abstract = {The regulation of gene expression is a result of a complex interplay between chromatin remodeling, transcription factors, and signaling molecules. Cell differentiation is accompanied by chromatin remodeling of specific loci to permanently silence genes that are not essential for the differentiated cell activity. The molecular cues that recruit the chromatin remodeling machinery are not well characterized. IRF8 is an immune-cell specific transcription factor and its expression is augmented by interferon-γ. Therefore, it serves as a model gene to elucidate the molecular mechanisms governing its silencing in non-immune cells. Ahigh-throughput shRNA library screen in IRF8 expression-restrictive cells enabled the identification of MafK as modulator of IRF8 silencing, affecting chromatin architecture. ChIP-Seq analysis revealed three MafK binding regions (-25 kb, -20 kb, and IRF8 6th intron) within the IRF8 locus. These MafK binding sites are sufficient to repress a reporter gene when cloned in genome-integrated lentiviral reporter constructs in only expression-restrictive cells. Conversely, plasmid-based constructs do not demonstrate such repressive effect. These results highlight the role of these MafK binding sites in mediating repressed chromatin assembly. Finally, a more thorough genomic analysis was performed, using CRISPR-Cas9 to delete MafK-int6 binding region in IRF8 expression-restrictive cells. Deleted clones exhibited an accessible chromatin conformation within the IRF8 locus that was accompanied by a significant increase in basal expression of IRF8 that was further induced by interferon-γ. Taken together, we identified and characterized several MafK binding elements within the IRF8 locus that mediate repressive chromatin conformation resulting in the silencing of IRF8 expression in a celltype-specific manner.}, }
@article {pmid32500359, year = {2020}, author = {Benayas, B and Sastre, I and López-Martín, S and Oo, A and Kim, B and Bullido, MJ and Aldudo, J and Yáñez-Mó, M}, title = {Tetraspanin CD81 regulates HSV-1 infection.}, journal = {Medical microbiology and immunology}, volume = {209}, number = {4}, pages = {489-498}, pmid = {32500359}, issn = {1432-1831}, support = {AI136581/NH/NIH HHS/United States ; R01 AI136581/AI/NIAID NIH HHS/United States ; BIO2017-86500-R//Ministerio de Economía y Competitividad/ ; AI150451/NH/NIH HHS/United States ; Contrato Predoctoral Garantía Juvenil//Comunidad de Madrid/ ; SAF2017-85747-R//Ministerio de Economía y Competitividad/ ; }, mesh = {CRISPR-Cas Systems ; Cell Line, Tumor ; Gene Knockout Techniques ; Herpesviridae Infections/*virology ; Herpesvirus 1, Human/pathogenicity/*physiology ; Humans ; SAM Domain and HD Domain-Containing Protein 1/metabolism ; Tetraspanin 28/*genetics/*metabolism ; Viral Envelope Proteins/metabolism ; Virion/metabolism ; Virus Internalization ; Virus Replication ; }, abstract = {Different members of the tetraspanin superfamily have been described to regulate different virus infectious cycles at several stages: viral entry, viral replication or virion exit or infectivity. In addition, tetraspanin CD81 regulates HIV reverse transcription through its association with the dNTP hydrolase SAMHD1. Here we aimed at analysing the role of CD81 in Herpes simplex virus 1 infectivity using a neuroblastoma cell model. For this purpose, we generated a CD81 KO cell line using the CRISPR/Cas9 technology. Despite being CD81 a plasma membrane protein, CD81 KO cells showed no defects in viral entry nor in the expression of early protein markers. In contrast, glycoprotein B and C, which require viral DNA replication for their expression, were significantly reduced in CD81 KO infected cells. Indeed, HSV-1 DNA replication and the formation of new infectious particles were severely compromised in CD81 KO cells. We could not detect significant changes in SAMHD1 total expression levels, but a relocalization into endosomal structures was observed in CD81 KO cells. In summary, CD81 KO cells showed impaired viral DNA replication and produced greatly diminished viral titers.}, }
@article {pmid32276588, year = {2020}, author = {Faulkner, J and Jiang, P and Farris, D and Walker, R and Dai, Z}, title = {CRISPR/CAS9-mediated knockout of Abi1 inhibits p185Bcr-Abl-induced leukemogenesis and signal transduction to ERK and PI3K/Akt pathways.}, journal = {Journal of hematology &amp; oncology}, volume = {13}, number = {1}, pages = {34}, pmid = {32276588}, issn = {1756-8722}, support = {R15 CA191476/CA/NCI NIH HHS/United States ; R21 CA187303/CA/NCI NIH HHS/United States ; }, mesh = {Adaptor Proteins, Signal Transducing/*genetics ; Animals ; CRISPR-Cas Systems ; Carcinogenesis/*genetics/metabolism ; Cell Line, Tumor ; Cytoskeletal Proteins/*genetics ; Female ; Fusion Proteins, bcr-abl/*genetics ; Leukemia/*genetics/metabolism ; MAP Kinase Signaling System ; Mice, Inbred BALB C ; Phosphatidylinositol 3-Kinases/metabolism ; Proto-Oncogene Proteins c-akt/metabolism ; Proto-Oncogene Proteins c-bcr/*genetics ; *Signal Transduction ; }, abstract = {BACKGROUND: Abl interactor 1 (Abi1) is a downstream target of Abl tyrosine kinases and a component of the WAVE regulatory complex (WRC) that plays an important role in regulating actin cytoskeleton remodeling and membrane receptor signaling. While studies using short hairpin RNA (shRNA) have suggested that Abi1 plays a critical role in Bcr-Abl-induced leukemogenesis, the mechanism involved is not clear.<br><br>METHODS: In this study, we knocked out Abi1 expression in p185Bcr-Abl-transformed hematopoietic cells using CRISPR/Cas9-mediated gene editing technology. The effects of Abi1 deficiency on actin cytoskeleton remodeling, the Bcr-Abl signaling, IL-3 independent growth, and SDF-induced chemotaxis in these cells were examined by various in vitro assays. The leukemogenic activity of these cells was evaluated by a syngeneic mouse transplantation model.<br><br>RESULTS: We show here that Abi1 deficiency reduced the IL3-independent growth and SDF-1α-mediated chemotaxis in p185Bcr-Abl-transformed hematopoietic cells and inhibited Bcr-Abl-induced abnormal actin remodeling. Depletion of Abi1 also impaired the Bcr-Abl signaling to the ERK and PI3 kinase/Akt pathways. Remarkably, the p185Bcr-Abl-transformed cells with Abi1 deficiency lost their ability to develop leukemia in syngeneic mice. Even though these cells developed drug tolerance in vitro after prolonged selection with imatinib as their parental cells, the imatinib-tolerant cells remain incapable of leukemogenesis in vivo.<br><br>CONCLUSIONS: Together, this study highlights an essential role of Abi1 in Bcr-Abl-induced leukemogenesis and provides a model system for dissecting the Abi1 signaling in Bcr-Abl-positive leukemia.}, }
@article {pmid32191315, year = {2020}, author = {Hayashi, S and Watanabe, M and Kobayashi, M and Tohge, T and Hashimoto, T and Shoji, T}, title = {Genetic Manipulation of Transcriptional Regulators Alters Nicotine Biosynthesis in Tobacco.}, journal = {Plant &amp; cell physiology}, volume = {61}, number = {6}, pages = {1041-1053}, doi = {10.1093/pcp/pcaa036}, pmid = {32191315}, issn = {1471-9053}, mesh = {CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Gene Editing ; Gene Expression Regulation, Plant/*genetics ; Gene Knockout Techniques ; Metabolic Networks and Pathways/genetics ; Nicotine/*biosynthesis ; Plant Leaves/metabolism ; Plant Proteins/genetics/*metabolism ; Plant Roots/metabolism ; Plants, Genetically Modified ; Tobacco/*genetics ; Transcription Factors/genetics/*metabolism ; }, abstract = {The toxic alkaloid nicotine is produced in the roots of Nicotiana species and primarily accumulates in leaves as a specialized metabolite. A series of metabolic and transport genes involved in the nicotine pathway are coordinately upregulated by a pair of jasmonate-responsive AP2/ERF-family transcription factors, NtERF189 and NtERF199, in the roots of Nicotiana tabacum (tobacco). In this study, we explored the potential of manipulating the expression of these transcriptional regulators to alter nicotine biosynthesis in tobacco. The transient overexpression of NtERF189 led to alkaloid production in the leaves of Nicotiana benthamiana and Nicotiana alata. This ectopic production was further enhanced by co-overexpressing a gene encoding a basic helix-loop-helix-family MYC2 transcription factor. Constitutive and leaf-specific overexpression of NtERF189 increased the accumulation of foliar alkaloids in transgenic tobacco plants but negatively affected plant growth. By contrast, in a knockout mutant of NtERF189 and NtERF199 obtained through CRISPR/Cas9-based genome editing, alkaloid levels were drastically reduced without causing major growth defects. Metabolite profiling revealed the impact of manipulating the nicotine pathway on a wide range of nitrogen- and carbon-containing metabolites. Our findings provide insights into the biotechnological applications of engineering metabolic pathways by targeting transcription factors.}, }
@article {pmid32079488, year = {2020}, author = {Luo, F and Long, K and Li, X and Mai, M and Zhong, Z and Li, S and Li, P and Zhou, S and Zhang, T and Long, X and Jin, L and Li, M}, title = {Deficient of LRRC8A attenuates hypoxia-induced necrosis in 3T3-L1 cells.}, journal = {Bioscience, biotechnology, and biochemistry}, volume = {84}, number = {6}, pages = {1139-1145}, doi = {10.1080/09168451.2020.1730689}, pmid = {32079488}, issn = {1347-6947}, mesh = {3T3-L1 Cells ; Animals ; CRISPR-Cas Systems ; Cell Hypoxia/*genetics ; Cell Survival/genetics ; Female ; *Gene Expression ; Gene Knockout Techniques ; Hypoxia/*genetics ; Membrane Proteins/*genetics/*metabolism ; Mice ; Myoblasts, Cardiac/metabolism ; Necrosis/genetics ; Rats ; Respiratory Mucosa/metabolism ; Swine ; Transfection ; }, abstract = {Under acute hypoxia, multiple ion channels on the cell membrane are activated, causing cell swelling and eventually necrosis. LRRC8A is an indispensable protein of the volume-regulated anion channel (VRAC), which participates in swelling and the acceleration of cell necrosis. In this study, we revealed a dynamic change in the expression level of the LRRC8 family during hypoxia in 3T3-L1 cells. The disruption of LRRC8A in 3T3-L1 cells was also associated with a significant anti-necrotic phenotype upon hypoxia accompanied by the reduced expression of necrosis-related genes. In vivo, differential expression of LRRC8 family members was also identified between high-altitude pigs and their low-altitude relatives. Taken these findings together, this study demonstrates the involvement of LRRC8A in hypoxia-induced cell necrosis.}, }
@article {pmid32069391, year = {2020}, author = {Polino, AJ and Nasamu, AS and Niles, JC and Goldberg, DE}, title = {Assessment of Biological Role and Insight into Druggability of the Plasmodium falciparum Protease Plasmepsin V.}, journal = {ACS infectious diseases}, volume = {6}, number = {4}, pages = {738-746}, pmid = {32069391}, issn = {2373-8227}, support = {R01 AI112508/AI/NIAID NIH HHS/United States ; R01 AI138447/AI/NIAID NIH HHS/United States ; P50 GM098792/GM/NIGMS NIH HHS/United States ; }, mesh = {Aspartic Acid Endopeptidases/*antagonists &amp; inhibitors ; CRISPR-Cas Systems ; Erythrocytes/parasitology ; Humans ; Inhibitory Concentration 50 ; Life Cycle Stages/drug effects ; Plasmodium falciparum/*drug effects/*enzymology/growth &amp; development ; Protein Processing, Post-Translational/genetics ; Protozoan Proteins/*antagonists &amp; inhibitors ; }, abstract = {Upon infecting a red blood cell (RBC), the malaria parasite Plasmodium falciparum drastically remodels its host by exporting hundreds of proteins into the RBC cytosol. This protein export program is essential for parasite survival. Hence export-related proteins could be potential drug targets. One essential enzyme in this pathway is plasmepsin V (PMV), an aspartic protease that processes export-destined proteins in the parasite endoplasmic reticulum (ER) at the Plasmodium export element (PEXEL) motif. Despite long-standing interest in this enzyme, functional studies have been hindered by the inability of previous technologies to produce a regulatable lethal depletion of PMV. To overcome this technical barrier, we designed a system for stringent post-transcriptional regulation allowing a tightly controlled, tunable knockdown of PMV. Using this system, we found that PMV must be dramatically depleted to affect parasite growth, suggesting the parasite maintains this enzyme in substantial excess. Surprisingly, depletion of PMV arrested parasite growth immediately after RBC invasion, significantly before the death from exported protein deficit that has previously been described. The data suggest that PMV inhibitors can halt parasite growth at two distinct points in the parasite life cycle. However, overcoming the functional excess of PMV in the parasite may require inhibitor concentrations far beyond the enzyme's IC50.}, }
@article {pmid31899623, year = {2020}, author = {Kopniczky, MB and Canavan, C and McClymont, DW and Crone, MA and Suckling, L and Goetzmann, B and Siciliano, V and MacDonald, JT and Jensen, K and Freemont, PS}, title = {Cell-Free Protein Synthesis as a Prototyping Platform for Mammalian Synthetic Biology.}, journal = {ACS synthetic biology}, volume = {9}, number = {1}, pages = {144-156}, doi = {10.1021/acssynbio.9b00437}, pmid = {31899623}, issn = {2161-5063}, support = {BB/M025632/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; }, mesh = {Binding Sites ; CRISPR-Associated Protein 9/chemistry/genetics ; CRISPR-Cas Systems ; Cell Engineering/*methods ; Cell-Free System ; Escherichia coli/genetics ; Gene Regulatory Networks ; Genetic Engineering/*methods ; HeLa Cells ; Humans ; Internal Ribosome Entry Sites/genetics ; Plasmids/genetics ; Promoter Regions, Genetic ; Protein Biosynthesis/*genetics ; Reproducibility of Results ; Synthetic Biology/methods ; Transcription, Genetic/genetics ; }, abstract = {The field of mammalian synthetic biology is expanding quickly, and technologies for engineering large synthetic gene circuits are increasingly accessible. However, for mammalian cell engineering, traditional tissue culture methods are slow and cumbersome, and are not suited for high-throughput characterization measurements. Here we have utilized mammalian cell-free protein synthesis (CFPS) assays using HeLa cell extracts and liquid handling automation as an alternative to tissue culture and flow cytometry-based measurements. Our CFPS assays take a few hours, and we have established optimized protocols for small-volume reactions using automated acoustic liquid handling technology. As a proof-of-concept, we characterized diverse types of genetic regulation in CFPS, including T7 constitutive promoter variants, internal ribosomal entry sites (IRES) constitutive translation-initiation sequence variants, CRISPR/dCas9-mediated transcription repression, and L7Ae-mediated translation repression. Our data shows simple regulatory elements for use in mammalian cells can be quickly prototyped in a CFPS model system.}, }
@article {pmid31885264, year = {2020}, author = {East, KW and Newton, JC and Morzan, UN and Narkhede, YB and Acharya, A and Skeens, E and Jogl, G and Batista, VS and Palermo, G and Lisi, GP}, title = {Allosteric Motions of the CRISPR-Cas9 HNH Nuclease Probed by NMR and Molecular Dynamics.}, journal = {Journal of the American Chemical Society}, volume = {142}, number = {3}, pages = {1348-1358}, pmid = {31885264}, issn = {1520-5126}, support = {P20 GM109035/GM/NIGMS NIH HHS/United States ; R01 EY027440/EY/NEI NIH HHS/United States ; }, mesh = {Allosteric Regulation ; *CRISPR-Cas Systems ; Deoxyribonucleases/metabolism ; *Molecular Dynamics Simulation ; Nuclear Magnetic Resonance, Biomolecular/*methods ; }, abstract = {CRISPR-Cas9 is a widely employed genome-editing tool with functionality reliant on the ability of the Cas9 endonuclease to introduce site-specific breaks in double-stranded DNA. In this system, an intriguing allosteric communication has been suggested to control its DNA cleavage activity through flexibility of the catalytic HNH domain. Here, solution NMR experiments and a novel Gaussian-accelerated molecular dynamics (GaMD) simulation method are used to capture the structural and dynamic determinants of allosteric signaling within the HNH domain. We reveal the existence of a millisecond time scale dynamic pathway that spans HNH from the region interfacing the adjacent RuvC nuclease and propagates up to the DNA recognition lobe in full-length CRISPR-Cas9. These findings reveal a potential route of signal transduction within the CRISPR-Cas9 HNH nuclease, advancing our understanding of the allosteric pathway of activation. Further, considering the role of allosteric signaling in the specificity of CRISPR-Cas9, this work poses the mechanistic basis for novel engineering efforts aimed at improving its genome-editing capability.}, }
@article {pmid31829621, year = {2020}, author = {Knoot, CJ and Biswas, S and Pakrasi, HB}, title = {Tunable Repression of Key Photosynthetic Processes Using Cas12a CRISPR Interference in the Fast-Growing Cyanobacterium Synechococcus sp. UTEX 2973.}, journal = {ACS synthetic biology}, volume = {9}, number = {1}, pages = {132-143}, doi = {10.1021/acssynbio.9b00417}, pmid = {31829621}, issn = {2161-5063}, mesh = {Bacterial Proteins/genetics ; Base Sequence ; CRISPR-Associated Protein 9/genetics ; *CRISPR-Cas Systems ; Gene Expression Regulation, Bacterial ; Gene Knockdown Techniques ; Isopropyl Thiogalactoside/pharmacology ; Lac Operon ; Lac Repressors/genetics ; Luminescent Proteins/genetics ; Microorganisms, Genetically-Modified ; Photosynthesis/*genetics ; Photosystem I Protein Complex/genetics ; Plasmids/genetics ; RNA, Guide/genetics ; Synechococcus/*genetics/*growth &amp; development ; }, abstract = {Cyanobacteria are photoautotrophic prokaryotes that serve as key model organisms to study basic photosynthetic processes and are potential carbon-negative production chassis for commodity and high-value chemicals. The development of new synthetic biology tools and improvement of current ones is a requisite for furthering these organisms as models and production vehicles. CRISPR interference (CRISPRi) allows for targeted gene repression using a DNase-dead Cas nuclease (&quot;dCas&quot;). Here, we describe a titratable dCas12a (dCpf1) CRISPRi system and apply it to repress key photosynthetic processes in the fast-growing cyanobacterium Synechococcus sp. UTEX 2973 (S2973). The system relies on a lac repressor system that retains tight regulation in the absence of inducer (0-10% repression) while maintaining the capability for >90% repression of high-abundance gene targets. We determined that dCas12a is less toxic than dCas9. We tested the efficacy of the system toward eYFP and three native targets in S2973: the phycobilisome antenna, glycogen synthesis, and photosystem I (PSI), an essential part of the photosynthetic electron transport chain in oxygenic photoautotrophs. PSI was knocked down indirectly by repressing the protein factor BtpA involved in stabilizing core PSI proteins. We could reduce cellular PSI titer by 87% under photoautotrophic conditions, and we characterized these cells to gain insights into the response of the strain to the low PSI content. The ability to tightly regulate and time the (de)repression of essential genes in trans will allow for the study of photosynthetic processes that are not accessible using knockout mutants.}, }
@article {pmid31825599, year = {2020}, author = {Dalvie, NC and Leal, J and Whittaker, CA and Yang, Y and Brady, JR and Love, KR and Love, JC}, title = {Host-Informed Expression of CRISPR Guide RNA for Genomic Engineering in Komagataella phaffii.}, journal = {ACS synthetic biology}, volume = {9}, number = {1}, pages = {26-35}, pmid = {31825599}, issn = {2161-5063}, support = {P30 CA014051/CA/NCI NIH HHS/United States ; T32 GM008334/GM/NIGMS NIH HHS/United States ; }, mesh = {3' Untranslated Regions ; Base Sequence ; Biological Products ; *CRISPR-Cas Systems ; Fungal Proteins/genetics ; Gene Editing/*methods ; Gene Knockout Techniques ; *Genome, Fungal ; Glycerol Kinase/genetics ; Glycosylation ; Phenotype ; Promoter Regions, Genetic ; Protein Processing, Post-Translational ; RNA Polymerase III/genetics ; RNA, Guide/*genetics ; Saccharomycetales/*genetics ; Transcriptome ; }, abstract = {There is growing interest in the use of nonmodel microorganisms as hosts for biopharmaceutical manufacturing. These hosts require genomic engineering to meet clinically relevant product qualities and titers, but the adaptation of tools for editing genomes, such as CRISPR-Cas9, has been slow for poorly characterized hosts. Specifically, a lack of biochemical characterization of RNA polymerase III transcription has hindered reliable expression of guide RNAs in new hosts. Here, we present a sequencing-based strategy for the design of host-specific cassettes for modular, reliable, expression of guide RNAs. Using this strategy, we achieved up to 95% gene editing efficiency in the methylotrophic yeast Komagataella phaffii. We applied this approach for the rapid, multiplexed engineering of a complex phenotype, achieving humanized product glycosylation in two sequential steps of engineering. Reliable extension of simple gene editing tools to nonmodel manufacturing hosts will enable rapid engineering of manufacturing strains tuned for specific product profiles and potentially decrease the costs and timelines for process development.}, }
@article {pmid31800222, year = {2020}, author = {Kim, H and Ji, CH and Je, HW and Kim, JP and Kang, HS}, title = {mpCRISTAR: Multiple Plasmid Approach for CRISPR/Cas9 and TAR-Mediated Multiplexed Refactoring of Natural Product Biosynthetic Gene Clusters.}, journal = {ACS synthetic biology}, volume = {9}, number = {1}, pages = {175-180}, doi = {10.1021/acssynbio.9b00382}, pmid = {31800222}, issn = {2161-5063}, mesh = {Base Sequence ; Biological Products/*metabolism ; CRISPR-Associated Protein 9/genetics ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Genetic Engineering/*methods ; Genotype ; *Multigene Family ; Plasmids/*genetics ; Promoter Regions, Genetic ; RNA, Guide/metabolism ; Recombination, Genetic ; Saccharomyces cerevisiae/*genetics ; Transcription, Genetic/*genetics ; }, abstract = {Multiplexed refactoring provides a tool for rapid transcriptional optimization of biosynthetic gene clusters (BGCs) through simultaneous replacement of multiple native promoters with synthetic counterparts. Here, we present the mpCRISTAR, a multiple plasmid-based CRISPR/Cas9 and TAR (transformation-associated recombination), that enables a rapid and highly efficient, multiplexed refactoring of natural product BGCs in yeast. A series of CRISPR plasmids with different auxotrophic markers that could be stably maintained in yeast cells were constructed to express multiple gRNAs simultaneously. We demonstrated the multiplexing capacity of mpCRISTAR using the actinorhodin biosynthetic gene cluster as a model cluster. mpCRISTAR1, in which each CRISPR plasmid expresses one gRNA, allows for simultaneous replacement of up to four promoter sites with nearly 100% efficiency. By expressing two gRNAs from one CRISPR plasmid, termed mpCRISTAR2, we simultaneously replaced a total of six and eight promoter sites with 68% and 32% efficiency, respectively. The mpCRISTAR could be performed iteratively using two different auxotrophic markers, allowing for refactoring of any type of BGC regardless of their operon complexities. The mpCRISTAR platform we report here would become a useful tool for the discovery of new natural products from transcriptionally silent biosynthetic gene clusters present in microbial genomes.}, }
@article {pmid31502220, year = {2020}, author = {Grossi, S and Fenini, G and Hennig, P and Di Filippo, M and Beer, HD}, title = {Generation of Knockout Human Primary Keratinocytes by CRISPR/Cas9.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2109}, number = {}, pages = {125-145}, doi = {10.1007/7651_2019_262}, pmid = {31502220}, issn = {1940-6029}, mesh = {3T3-L1 Cells ; Animals ; CRISPR-Cas Systems ; Cells, Cultured ; Feeder Cells/*cytology ; Gene Editing/*methods ; Gene Expression ; Gene Knockout Techniques/*methods ; Humans ; Keratinocytes/*cytology/metabolism ; Mice ; Primary Cell Culture ; }, abstract = {The culture of epidermal human primary keratinocytes (HPKs) represents a well-established model in biological and dermatological research. In addition, HPKs are used in three-dimensional organotypic cultures (OTCs), and gene therapeutic approaches have been reported for the treatment of patients suffering from epidermolysis bullosa, a severe blistering disease that can result in postnatal lethality. Therefore, there is a strong need for the development of techniques for the stable and specific genetic manipulation of HPKs, for example, by genome editing via the CRISPR/Cas9 approach. However, the main disadvantage of working with HPKs is the fact that these cells are prone to terminal differentiation and proliferate only for few passages in monoculture. As it is well known that the co-culture of HPKs with fibroblasts strongly increases the lifetime of the epidermal cells, we developed a protocol for the stable modification of HPKs by CRISPR/Cas9 via lentiviral transduction in the presence of 3T3-J2 fibroblasts as feeder cells. Selection of transduced HPKs is achieved with antibiotics in co-culture with antibiotic-resistant feeder cells. Modified HPKs generated by our protocol have the potential to generate epidermis-like structures in OTCs.}, }
@article {pmid31309518, year = {2020}, author = {Granata, M and Skarmoutsou, E and Mazzarino, MC and Libra, M and D'Amico, F}, title = {Contribution of Immunohistochemistry in Revealing S100A7/JAB1 Colocalization in Psoriatic Epidermal Keratinocyte.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2109}, number = {}, pages = {67-74}, doi = {10.1007/7651_2019_251}, pmid = {31309518}, issn = {1940-6029}, mesh = {Biopsy ; COP9 Signalosome Complex/*metabolism ; CRISPR-Cas Systems ; Case-Control Studies ; Cell Line ; Humans ; Immunohistochemistry ; Intracellular Signaling Peptides and Proteins/*metabolism ; Keratinocytes/metabolism ; Peptide Hydrolases/*metabolism ; Psoriasis/metabolism/*pathology ; S100 Calcium Binding Protein A7/genetics/*metabolism ; Tissue Embedding ; Tissue Fixation ; }, abstract = {The application of immunohistological methods provides an invaluable contribution in revealing the protein colocalization, which may reflect the occurrence of molecular interaction processes.This chapter describes comprehensive protocols for detection of S100A7/JAB1 colocalization by immunohistochemistry in archival formalin-fixed and paraffin-embedded skin biopsies from healthy and psoriatic subjects. In addition, we provide a protocol for immunocytochemical detection of S100A7/JAB1 colocalization in S100A7 CRISPR-activated human keratinocyte cell line.}, }
@article {pmid33484441, year = {2021}, author = {Lopes, AMM and Martins, M and Goldbeck, R}, title = {Heterologous Expression of Lignocellulose-Modifying Enzymes in Microorganisms: Current Status.}, journal = {Molecular biotechnology}, volume = {}, number = {}, pages = {}, pmid = {33484441}, issn = {1559-0305}, support = {2019/08542-3//FAPESP/ ; 001//CAPES/ ; }, abstract = {Heterologous expression of the carbohydrate-active enzymes in microorganisms is a promising approach to produce bio-based compounds, such as fuels, nutraceuticals and other value-added products from sustainable lignocellulosic sources. Several microorganisms, including Saccharomyces cerevisiae, Escherichia coli, and the filamentous fungi Aspergillus nidulans, have unique characteristics desirable for a biorefinery production approach like well-known genetic tools, thermotolerance, high fermentative capacity and product tolerance, and high amount of recombinant enzyme secretion. These microbial factories are already stablished in the heterologous production of the carbohydrate-active enzymes to produce, among others, ethanol, xylooligosaccharides and the valuable coniferol. A complete biocatalyst able to heterologous express the CAZymes of glycoside hydrolases, carbohydrate esterases and auxiliary activities families could release these compounds faster, with higher yield and specificity. Recent advances in the synthetic biology tools could expand the number and diversity of enzymes integrated in these microorganisms, and also modify those already integrated. This review outlines the heterologous expression of carbohydrate-active enzymes in microorganisms, as well as recent updates in synthetic biology.}, }
@article {pmid33484202, year = {2021}, author = {Uranga, M and Aragonés, V and Selma, S and Vázquez-Vilar, M and Orzáez, D and Daròs, JA}, title = {Efficient Cas9 multiplex editing using unspaced sgRNA arrays engineering in a Potato virus X vector.}, journal = {The Plant journal : for cell and molecular biology}, volume = {}, number = {}, pages = {}, doi = {10.1111/tpj.15164}, pmid = {33484202}, issn = {1365-313X}, abstract = {Systems based on the clustered, regularly interspaced, short palindromic repeat (CRISPR) and CRISPR associated proteins (Cas) have revolutionized genome editing in many organisms, including plants. Most CRISPR-Cas strategies in plants rely on genetic transformation using Agrobacterium tumefaciens to supply the gene editing reagents, such as Cas nucleases or the synthetic guide RNA (sgRNA). While Cas nucleases are constant elements in editing approaches, sgRNAs are target-specific and a screening process is usually required to identify those most effective. Plant virus-derived vectors are an alternative for the fast and efficient delivery of sgRNAs into adult plants, due to the virus capacity for genome amplification and systemic movement, a strategy known as virus-induced genome editing (VIGE). We engineered Potato virus X (PVX) to build a vector that easily expresses multiple sgRNAs in adult solanaceous plants. Using the PVX-based vector, Nicotiana benthamiana genes were efficiently targeted, producing nearly 80% indels in a transformed line that constitutively expresses Streptococcus pyogenes Cas9. Interestingly, results showed that the PVX vector allows expression of arrays of unspaced sgRNAs, achieving highly efficient multiplex editing in a few days in adult plant tissues. Moreover, virus-free edited progeny can be obtained from plants regenerated from infected tissues or infected plant seeds, which exhibit a high rate of heritable bi-allelic mutations. In sum, this new PVX vector allows easy, fast and efficient expression of sgRNAs arrays for multiplex CRISPR-Cas genome editing and will be a useful tool for functional gene analysis and precision breeding across diverse plant species, particularly in Solanaceae crops.}, }
@article {pmid33483498, year = {2021}, author = {Collias, D and Beisel, CL}, title = {CRISPR technologies and the search for the PAM-free nuclease.}, journal = {Nature communications}, volume = {12}, number = {1}, pages = {555}, pmid = {33483498}, issn = {2041-1723}, abstract = {The ever-expanding set of CRISPR technologies and their programmable RNA-guided nucleases exhibit remarkable flexibility in DNA targeting. However, this flexibility comes with an ever-present constraint: the requirement for a protospacer adjacent motif (PAM) flanking each target. While PAMs play an essential role in self/nonself discrimination by CRISPR-Cas immune systems, this constraint has launched a far-reaching expedition for nucleases with relaxed PAM requirements. Here, we review ongoing efforts toward realizing PAM-free nucleases through natural ortholog mining and protein engineering. We also address potential consequences of fully eliminating PAM recognition and instead propose an alternative nuclease repertoire covering all possible PAM sequences.}, }
@article {pmid33482972, year = {2020}, author = {Boland, J and Nedelcu, E}, title = {CRISPR/Cas9 for the Clinician: Current uses of gene editing and applications for new therapeutics in oncology.}, journal = {The Permanente journal}, volume = {24}, number = {}, pages = {1-3}, doi = {10.7812/TPP/20.040}, pmid = {33482972}, issn = {1552-5775}, abstract = {Precise genomic editing has given rise to treatments in previously untreatable genetic diseases and has led to revolutions in treatment for cancer. In the past decade, the discovery and development of clustered regularly interspaced short palindromic repeats (CRISPR) technologies has led to advances across medicine and biotechnology. Specifically, the CRISPR/Cas9 system has improved translational discovery and therapeutics for oncology across tumor types. In this review, we briefly summarize the history and development of CRISPR, explain CRISPR-Cas systems and CRISPR gene editing tools, highlight the development and application of CRISPR technologies for translational and therapeutic purposes in different oncologic tumors, and review novel treatment paradigms using CRISPR in immuno-oncology, including checkpoint inhibitors and chimeric antigen receptor T cell therapy.}, }
@article {pmid33479216, year = {2021}, author = {Li, S and Akrap, N and Cerboni, S and Porritt, MJ and Wimberger, S and Lundin, A and Möller, C and Firth, M and Gordon, E and Lazovic, B and Sieńska, A and Pane, LS and Coelho, MA and Ciotta, G and Pellegrini, G and Sini, M and Xu, X and Mitra, S and Bohlooly-Y, M and Taylor, BJM and Sienski, G and Maresca, M}, title = {Universal toxin-based selection for precise genome engineering in human cells.}, journal = {Nature communications}, volume = {12}, number = {1}, pages = {497}, pmid = {33479216}, issn = {2041-1723}, abstract = {Prokaryotic restriction enzymes, recombinases and Cas proteins are powerful DNA engineering and genome editing tools. However, in many primary cell types, the efficiency of genome editing remains low, impeding the development of gene- and cell-based therapeutic applications. A safe strategy for robust and efficient enrichment of precisely genetically engineered cells is urgently required. Here, we screen for mutations in the receptor for Diphtheria Toxin (DT) which protect human cells from DT. Selection for cells with an edited DT receptor variant enriches for simultaneously introduced, precisely targeted gene modifications at a second independent locus, such as nucleotide substitutions and DNA insertions. Our method enables the rapid generation of a homogenous cell population with bi-allelic integration of a DNA cassette at the selection locus, without clonal isolation. Toxin-based selection works in both cancer-transformed and non-transformed cells, including human induced pluripotent stem cells and human primary T-lymphocytes, as well as it is applicable also in vivo, in mice with humanized liver. This work represents a flexible, precise, and efficient selection strategy to engineer cells using CRISPR-Cas and base editing systems.}, }
@article {pmid33478937, year = {2020}, author = {Nakato, M and Shiranaga, N and Tomioka, M and Watanabe, H and Kurisu, J and Kengaku, M and Komura, N and Ando, H and Kimura, Y and Kioka, N and Ueda, K}, title = {ABCA13 dysfunction associated with psychiatric disorders causes impaired cholesterol trafficking.}, journal = {The Journal of biological chemistry}, volume = {296}, number = {}, pages = {100166}, doi = {10.1074/jbc.RA120.015997}, pmid = {33478937}, issn = {1083-351X}, abstract = {ATP-binding cassette subfamily A member 13 (ABCA13) is predicted to be the largest ABC protein, consisting of 5058 amino acids and a long N-terminal region. Mutations in the ABCA13 gene were reported to increase the susceptibility to schizophrenia, bipolar disorder, and major depression. However, little is known about the molecular functions of ABCA13 or how they associate with psychiatric disorders. Here, we examined the biochemical activity of ABCA13 using HEK293 cells transfected with mouse ABCA13. The expression of ABCA13 induced the internalization of cholesterol and gangliosides from the plasma membrane to intracellular vesicles. Cholesterol internalization by ABCA13 required the long N-terminal region and ATP hydrolysis. To examine the physiological roles of ABCA13, we generated Abca13 KO mice using CRISPR/Cas and found that these mice exhibited deficits of prepulse inhibition. Vesicular cholesterol accumulation and synaptic vesicle endocytosis were impaired in primary cultures of Abca13 KO cortical neurons. Furthermore, mutations in ABCA13 gene associated with psychiatric disorders disrupted the protein's subcellular localization and impaired cholesterol trafficking. These findings suggest that ABCA13 accelerates cholesterol internalization by endocytic retrograde transport in neurons and that loss of this function is associated with the pathophysiology of psychiatric disorders.}, }
@article {pmid33478513, year = {2021}, author = {Asemoloye, MD and Marchisio, MA and Gupta, VK and Pecoraro, L}, title = {Genome-based engineering of ligninolytic enzymes in fungi.}, journal = {Microbial cell factories}, volume = {20}, number = {1}, pages = {20}, pmid = {33478513}, issn = {1475-2859}, abstract = {BACKGROUND: Many fungi grow as saprobic organisms and obtain nutrients from a wide range of dead organic materials. Among saprobes, fungal species that grow on wood or in polluted environments have evolved prolific mechanisms for the production of degrading compounds, such as ligninolytic enzymes. These enzymes include arrays of intense redox-potential oxidoreductase, such as laccase, catalase, and peroxidases. The ability to produce ligninolytic enzymes makes a variety of fungal species suitable for application in many industries, including the production of biofuels and antibiotics, bioremediation, and biomedical application as biosensors. However, fungal ligninolytic enzymes are produced naturally in small quantities that may not meet the industrial or market demands. Over the last decade, combined synthetic biology and computational designs have yielded significant results in enhancing the synthesis of natural compounds in fungi. In this review, we gave insights into different protein engineering methods, including rational, semi-rational, and directed evolution approaches that have been employed to enhance the production of some important ligninolytic enzymes in fungi. We described the role of metabolic pathway engineering to optimize the synthesis of chemical compounds of interest in various fields. We highlighted synthetic biology novel techniques for biosynthetic gene cluster (BGC) activation in fungo and heterologous reconstruction of BGC in microbial cells. We also discussed in detail some recombinant ligninolytic enzymes that have been successfully enhanced and expressed in different heterologous hosts. Finally, we described recent advance in CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas (CRISPR associated) protein systems as the most promising biotechnology for large-scale production of ligninolytic enzymes.<br><br>SHORT CONCLUSION: Aggregation, expression, and regulation of ligninolytic enzymes in fungi require very complex procedures with many interfering factors. Synthetic and computational biology strategies, as explained in this review, are powerful tools that can be combined to solve these puzzles. These integrated strategies can lead to the production of enzymes with special abilities, such as wide substrate specifications, thermo-stability, tolerance to long time storage, and stability in different substrate conditions, such as pH and nutrients.}, }
@article {pmid33478128, year = {2021}, author = {Varanda, CM and Félix, MDR and Campos, MD and Patanita, M and Materatski, P}, title = {Plant Viruses: From Targets to Tools for CRISPR.}, journal = {Viruses}, volume = {13}, number = {1}, pages = {}, doi = {10.3390/v13010141}, pmid = {33478128}, issn = {1999-4915}, support = {PTDC/ASP-PLA/28266/2017//Fundação para a Ciência e a Tecnologia/ ; PTDC/ASP-PLA/28263/2017//Fundação para a Ciência e a Tecnologia/ ; UIDB/05183/2020//Fundação para a Ciência e a Tecnologia/ ; ALT20-03-0145-FEDER-028266//European Regional Development Fund/ ; ALT20-03-0145-FEDER-028263//European Regional Development Fund/ ; SFRH/BD/145321/2019//Fundação para a Ciência e a Tecnologia/ ; }, abstract = {Plant viruses cause devastating diseases in many agriculture systems, being a serious threat for the provision of adequate nourishment to a continuous growing population. At the present, there are no chemical products that directly target the viruses, and their control rely mainly on preventive sanitary measures to reduce viral infections that, although important, have proved to be far from enough. The current most effective and sustainable solution is the use of virus-resistant varieties, but which require too much work and time to obtain. In the recent years, the versatile gene editing technology known as CRISPR/Cas has simplified the engineering of crops and has successfully been used for the development of viral resistant plants. CRISPR stands for 'clustered regularly interspaced short palindromic repeats' and CRISPR-associated (Cas) proteins, and is based on a natural adaptive immune system that most archaeal and some bacterial species present to defend themselves against invading bacteriophages. Plant viral resistance using CRISPR/Cas technology can been achieved either through manipulation of plant genome (plant-mediated resistance), by mutating host factors required for viral infection; or through manipulation of virus genome (virus-mediated resistance), for which CRISPR/Cas systems must specifically target and cleave viral DNA or RNA. Viruses present an efficient machinery and comprehensive genome structure and, in a different, beneficial perspective, they have been used as biotechnological tools in several areas such as medicine, materials industry, and agriculture with several purposes. Due to all this potential, it is not surprising that viruses have also been used as vectors for CRISPR technology; namely, to deliver CRISPR components into plants, a crucial step for the success of CRISPR technology. Here we discuss the basic principles of CRISPR/Cas technology, with a special focus on the advances of CRISPR/Cas to engineer plant resistance against DNA and RNA viruses. We also describe several strategies for the delivery of these systems into plant cells, focusing on the advantages and disadvantages of the use of plant viruses as vectors. We conclude by discussing some of the constrains faced by the application of CRISPR/Cas technology in agriculture and future prospects.}, }
@article {pmid33471982, year = {2021}, author = {Malech, HL}, title = {Treatment by CRISPR-Cas9 Gene Editing - A Proof of Principle.}, journal = {The New England journal of medicine}, volume = {384}, number = {3}, pages = {286-287}, doi = {10.1056/NEJMe2034624}, pmid = {33471982}, issn = {1533-4406}, mesh = {*Anemia, Sickle Cell ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; Humans ; *beta-Thalassemia ; }, }
@article {pmid33471981, year = {2021}, author = {Walters, MC}, title = {Induction of Fetal Hemoglobin by Gene Therapy.}, journal = {The New England journal of medicine}, volume = {384}, number = {3}, pages = {284-285}, doi = {10.1056/NEJMe2034338}, pmid = {33471981}, issn = {1533-4406}, mesh = {*Anemia, Sickle Cell/genetics/therapy ; CRISPR-Cas Systems ; Fetal Hemoglobin/genetics ; Gene Editing ; Genetic Therapy ; Humans ; *beta-Thalassemia/genetics ; }, }
@article {pmid33346188, year = {2020}, author = {Li, H and Qin, H and Zhang, N and Zhao, J and Xin, J and Perez-Campo, FM and Liu, H}, title = {Construction of CRISPR Plasmids and Detection of Knockout Efficiency in Mammalian Cells through a Dual Luciferase Reporter System.}, journal = {Journal of visualized experiments : JoVE}, volume = {}, number = {166}, pages = {}, doi = {10.3791/59639}, pmid = {33346188}, issn = {1940-087X}, mesh = {Animals ; Base Sequence ; CRISPR-Cas Systems/*genetics ; Cell Line ; DNA/metabolism ; DNA Repair ; *Gene Knockout Techniques ; *Genes, Reporter ; Genetic Vectors/metabolism ; Luciferases/genetics/*metabolism ; Mammals/*metabolism ; Oligonucleotides/metabolism ; Plasmids/*genetics ; RNA, Guide/genetics ; Reproducibility of Results ; Sheep ; Transformation, Genetic ; }, abstract = {Although highly efficient, modification of a genomic site by the CRISPR enzyme requires the generation of a sgRNA unique to the target site(s) beforehand. This work describes the key steps leading to the construction of efficient sgRNA vectors using a strategy that allows the efficient detection of the positive colonies by PCR prior to DNA sequencing. Since efficient genome editing using the CRISPR system requires a highly efficient sgRNA, a preselection of candidate sgRNA targets is necessary to save time and effort. A dual luciferase reporter system has been developed to evaluate knockout efficiency by examining double-strand break repair via single strand annealing. Here, we use this reporter system to pick up the preferred xCas9/sgRNA target from candidate sgRNA vectors for specific gene editing. The protocol outlined will provide a preferred sgRNA/CRISPR enzyme vector in 10 days (starting with appropriately designed oligonucleotides).}, }
@article {pmid33305314, year = {2021}, author = {Levi, O and Arava, YS}, title = {Pseudouridine-mediated translation control of mRNA by methionine aminoacyl tRNA synthetase.}, journal = {Nucleic acids research}, volume = {49}, number = {1}, pages = {432-443}, pmid = {33305314}, issn = {1362-4962}, mesh = {CRISPR-Cas Systems ; *Gene Expression Regulation, Fungal ; Methionine/metabolism ; Methionine-tRNA Ligase/*metabolism ; Peptide Elongation Factors/*biosynthesis/genetics ; Polyribosomes/metabolism ; Protein Binding ; *Protein Biosynthesis ; Pseudouridine/*physiology ; RNA Processing, Post-Transcriptional ; RNA, Fungal/*genetics ; RNA, Messenger/*genetics ; Saccharomyces cerevisiae/genetics/*metabolism ; Saccharomyces cerevisiae Proteins/biosynthesis/genetics/*metabolism ; }, abstract = {Modification of nucleotides within an mRNA emerges as a key path for gene expression regulation. Pseudouridine is one of the most common RNA modifications; however, only a few mRNA modifiers have been identified to date, and no one mRNA pseudouridine reader is known. Here, we applied a novel genome-wide approach to identify mRNA regions that are bound by yeast methionine aminoacyl tRNAMet synthetase (MetRS). We found a clear enrichment to regions that were previously described to contain pseudouridine (Ψ). Follow-up in vitro and in vivo analyses on a prime target (position 1074 within YEF3 mRNA) demonstrated the importance of pseudouridine for MetRS binding. Furthermore, polysomal and protein analyses revealed that Ψ1074 mediates translation. Modification of this site occurs presumably by Pus6, a pseudouridine synthetase known to modify MetRS cognate tRNA. Consistently, the deletion of Pus6 leads to a decrease in MetRS association with both tRNAMet and YEF3 mRNA. Furthermore, while global protein synthesis decreases in pus6Δ, translation of YEF3 increases. Together, our data imply that Pus6 'writes' modifications on tRNA and mRNA, and both types of RNAs are 'read' by MetRS for translation regulation purposes. This represents a novel integrated path for writing and reading modifications on both tRNA and mRNA, which may lead to coordination between global and gene-specific translational responses.}, }
@article {pmid33283228, year = {2021}, author = {Cipullo, M and Pearce, SF and Lopez Sanchez, IG and Gopalakrishna, S and Krüger, A and Schober, F and Busch, JD and Li, X and Wredenberg, A and Atanassov, I and Rorbach, J}, title = {Human GTPBP5 is involved in the late stage of mitoribosome large subunit assembly.}, journal = {Nucleic acids research}, volume = {49}, number = {1}, pages = {354-370}, pmid = {33283228}, issn = {1362-4962}, mesh = {Bone Neoplasms/pathology ; CRISPR-Cas Systems ; Cell Line, Tumor ; Gene Expression Regulation ; Gene Knockout Techniques ; Guanosine Triphosphate/metabolism ; HEK293 Cells ; Humans ; Mitochondrial Proteins/*metabolism ; Mitochondrial Ribosomes/*metabolism ; Monomeric GTP-Binding Proteins/*physiology ; Osteosarcoma/pathology ; Oxidative Phosphorylation ; Protein Interaction Mapping ; Ribosomal Proteins/*metabolism ; Ribosome Subunits, Large, Eukaryotic/*metabolism ; }, abstract = {Human mitoribosomes are macromolecular complexes essential for translation of 11 mitochondrial mRNAs. The large and the small mitoribosomal subunits undergo a multistep maturation process that requires the involvement of several factors. Among these factors, GTP-binding proteins (GTPBPs) play an important role as GTP hydrolysis can provide energy throughout the assembly stages. In bacteria, many GTPBPs are needed for the maturation of ribosome subunits and, of particular interest for this study, ObgE has been shown to assist in the 50S subunit assembly. Here, we characterize the role of a related human Obg-family member, GTPBP5. We show that GTPBP5 interacts specifically with the large mitoribosomal subunit (mt-LSU) proteins and several late-stage mitoribosome assembly factors, including MTERF4:NSUN4 complex, MRM2 methyltransferase, MALSU1 and MTG1. Interestingly, we find that interaction of GTPBP5 with the mt-LSU is compromised in the presence of a non-hydrolysable analogue of GTP, implying a different mechanism of action of this protein in contrast to that of other Obg-family GTPBPs. GTPBP5 ablation leads to severe impairment in the oxidative phosphorylation system, concurrent with a decrease in mitochondrial translation and reduced monosome formation. Overall, our data indicate an important role of GTPBP5 in mitochondrial function and suggest its involvement in the late-stage of mt-LSU maturation.}, }
@article {pmid32563448, year = {2020}, author = {Navarro-Serna, S and Vilarino, M and Park, I and Gadea, J and Ross, PJ}, title = {Livestock Gene Editing by One-step Embryo Manipulation.}, journal = {Journal of equine veterinary science}, volume = {89}, number = {}, pages = {103025}, doi = {10.1016/j.jevs.2020.103025}, pmid = {32563448}, issn = {0737-0806}, mesh = {Animals ; CRISPR-Associated Protein 9 ; CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing/veterinary ; *Livestock ; }, abstract = {The breakthrough and rapid advance of clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) technology has enabled the efficient generation of gene-edited animals by one-step embryo manipulation. Clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 9 delivery to the livestock embryos has been typically achieved by intracytoplasmic microinjection; however, recent studies show that electroporation may be a reliable, efficient, and practical method for CRISPR/Cas9 delivery. The source of embryos used to generate gene-edited animals varies from in vivo to in vitro produced, depending mostly on the species of interest. In addition, different Cas9 and gRNA reagents can be used for embryo editing, ranging from Cas9-coding plasmid or messenger RNA to Cas9 recombinant protein, which can be combined with in vitro transcribed or synthetic guide RNAs. Mosaicism is reported as one of the main problems with generation of animals by embryo editing. On the other hand, off-target mutations are rarely found in livestock derived from one-step editing. In this review, we discussed these and other aspects of generating gene-edited animals by single-step embryo manipulation.}, }
@article {pmid32458346, year = {2020}, author = {Chen, B and Deng, S and Ge, T and Ye, M and Yu, J and Lin, S and Ma, W and Songyang, Z}, title = {Live cell imaging and proteomic profiling of endogenous NEAT1 lncRNA by CRISPR/Cas9-mediated knock-in.}, journal = {Protein &amp; cell}, volume = {11}, number = {9}, pages = {641-660}, pmid = {32458346}, issn = {1674-8018}, mesh = {*CRISPR-Cas Systems ; *Cell Tracking ; *Gene Expression Profiling ; *Gene Knock-In Techniques ; HEK293 Cells ; Humans ; *Proteomics ; RNA, Long Noncoding/genetics/*metabolism ; }, abstract = {In mammalian cells, long noncoding RNAs (lncRNAs) form complexes with proteins to execute various biological functions such as gene transcription, RNA processing and other signaling activities. However, methods to track endogenous lncRNA dynamics in live cells and screen for lncRNA interacting proteins are limited. Here, we report the development of CERTIS (CRISPR-mediated Endogenous lncRNA Tracking and Immunoprecipitation System) to visualize and isolate endogenous lncRNA, by precisely inserting a 24-repeat MS2 tag into the distal end of lncRNA locus through the CRISPR/Cas9 technology. In this study, we show that CERTIS effectively labeled the paraspeckle lncRNA NEAT1 without disturbing its physiological properties and could monitor the endogenous expression variation of NEAT1. In addition, CERTIS displayed superior performance on both short- and long-term tracking of NEAT1 dynamics in live cells. We found that NEAT1 and paraspeckles were sensitive to topoisomerase I specific inhibitors. Moreover, RNA Immunoprecipitation (RIP) of the MS2-tagged NEAT1 lncRNA successfully revealed several new protein components of paraspeckle. Our results support CERTIS as a tool suitable to track both spatial and temporal lncRNA regulation in live cells as well as study the lncRNA-protein interactomes.}, }
@article {pmid32246439, year = {2020}, author = {Jia, F and Li, X and Zhang, C and Tang, X}, title = {The expanded development and application of CRISPR system for sensitive nucleotide detection.}, journal = {Protein &amp; cell}, volume = {11}, number = {9}, pages = {624-629}, pmid = {32246439}, issn = {1674-8018}, mesh = {Animals ; *CRISPR-Cas Systems ; Humans ; RNA, Viral/*genetics ; Viruses/*genetics ; }, }
@article {pmid32004507, year = {2020}, author = {Papanicolaou, KN and Ashok, D and Liu, T and Bauer, TM and Sun, J and Li, Z and da Costa, E and D'Orleans, CC and Nathan, S and Lefer, DJ and Murphy, E and Paolocci, N and Foster, DB and O'Rourke, B}, title = {Global knockout of ROMK potassium channel worsens cardiac ischemia-reperfusion injury but cardiomyocyte-specific knockout does not: Implications for the identity of mitoKATP.}, journal = {Journal of molecular and cellular cardiology}, volume = {139}, number = {}, pages = {176-189}, doi = {10.1016/j.yjmcc.2020.01.010}, pmid = {32004507}, issn = {1095-8584}, support = {ZIA HL002066/ImNIH/Intramural NIH HHS/United States ; R01 HL136918/HL/NHLBI NIH HHS/United States ; K12 HL141952/HL/NHLBI NIH HHS/United States ; F31 HL134198/HL/NHLBI NIH HHS/United States ; R01 HL137259/HL/NHLBI NIH HHS/United States ; R01 HL092141/HL/NHLBI NIH HHS/United States ; }, mesh = {Animals ; Animals, Newborn ; CRISPR-Cas Systems/genetics ; Calcium/metabolism ; Electrophysiological Phenomena ; Gene Editing ; Gene Knockout Techniques ; Hemodynamics ; Ischemic Preconditioning, Myocardial ; Mice, Knockout ; Mitochondria, Heart/metabolism ; Myocardial Reperfusion Injury/*metabolism/pathology/physiopathology ; Myocardium/metabolism/pathology ; Myocytes, Cardiac/*metabolism/pathology ; Organ Specificity ; Perfusion ; Phenotype ; Potassium Channels/*metabolism ; Potassium Channels, Inwardly Rectifying/*deficiency/metabolism ; }, abstract = {The renal-outer-medullary‑potassium (ROMK) channel, mutated in Bartter's syndrome, regulates ion exchange in kidney, but its extra-renal functions remain unknown. Additionally, ROMK was postulated to be the pore-forming subunit of the mitochondrial ATP-sensitive K+ channel (mitoKATP), a mediator of cardioprotection. Using global and cardiomyocyte-specific knockout mice (ROMK-GKO and ROMK-CKO respectively), we characterize the effects of ROMK knockout on mitochondrial ion handling, the response to pharmacological KATP channel modulators, and ischemia/reperfusion (I/R) injury. Mitochondria from ROMK-GKO hearts exhibited a lower threshold for Ca2+-triggered permeability transition pore (mPTP) opening but normal matrix volume changes during oxidative phosphorylation. Isolated perfused ROMK-GKO hearts exhibited impaired functional recovery and increased infarct size when I/R was preceded by an ischemic preconditioning (IPC) protocol. Because ROMK-GKO mice exhibited severe renal defects and cardiac remodeling, we further characterized ROMK-CKO hearts to avoid confounding systemic effects. Mitochondria from ROMK-CKO hearts had unchanged matrix volume responses during oxidative phosphorylation and still swelled upon addition of a mitoKATP opener, but exhibited a lower threshold for mPTP opening, similar to GKO mitochondria. Nevertheless, I/R induced damage was not exacerbated in ROMK-CKO hearts, either ex vivo or in vivo. Lastly, we examined the response of ROMK-CKO hearts to ex vivo I/R injury with or without IPC and found that IPC still protected these hearts, suggesting that cardiomyocyte ROMK does not participate significantly in the cardioprotective pathway elicited by IPC. Collectively, our findings from these novel strains of mice suggest that cardiomyocyte ROMK is not a central mediator of mitoKATP function, although it can affect mPTP activation threshold.}, }
@article {pmid31895004, year = {2020}, author = {Bennett, HW and Gustavsson, AK and Bayas, CA and Petrov, PN and Mooney, N and Moerner, WE and Jackson, PK}, title = {Novel fibrillar structure in the inversin compartment of primary cilia revealed by 3D single-molecule superresolution microscopy.}, journal = {Molecular biology of the cell}, volume = {31}, number = {7}, pages = {619-639}, pmid = {31895004}, issn = {1939-4586}, support = {R35 GM118067/GM/NIGMS NIH HHS/United States ; R01 GM121565/GM/NIGMS NIH HHS/United States ; R01 GM114276/GM/NIGMS NIH HHS/United States ; K99 GM134187/GM/NIGMS NIH HHS/United States ; T32 AG047126/AG/NIA NIH HHS/United States ; }, mesh = {Biomarkers/metabolism ; CRISPR-Cas Systems/genetics ; Cell Line ; Cilia/*metabolism ; Green Fluorescent Proteins/metabolism ; Humans ; *Imaging, Three-Dimensional ; Kinesin/metabolism ; *Microscopy ; Models, Biological ; Mutation/genetics ; NIMA-Related Kinases/metabolism ; Nuclear Proteins/metabolism ; Protein Transport ; *Single Molecule Imaging ; Transcription Factors/*metabolism ; }, abstract = {Primary cilia in many cell types contain a periaxonemal subcompartment called the inversin compartment. Four proteins have been found to assemble within the inversin compartment: INVS, ANKS6, NEK8, and NPHP3. The function of the inversin compartment is unknown, but it appears to be critical for normal development, including left-right asymmetry and renal tissue homeostasis. Here we combine superresolution imaging of human RPE1 cells, a classic model for studying primary cilia in vitro, with a genetic dissection of the protein-protein binding relationships that organize compartment assembly to develop a new structural model. We observe that INVS is the core structural determinant of a compartment composed of novel fibril-like substructures, which we identify here by three-dimensional single-molecule superresolution imaging. We find that NEK8 and ANKS6 depend on INVS for localization to these fibrillar assemblies and that ANKS6-NEK8 density within the compartment is regulated by NEK8. Together, NEK8 and ANKS6 are required downstream of INVS to localize and concentrate NPHP3 within the compartment. In the absence of these upstream components, NPHP3 is redistributed within cilia. These results provide a more detailed structure for the inversin compartment and introduce a new example of a membraneless compartment organized by protein-protein interactions.}, }
@article {pmid33475257, year = {2021}, author = {Vavassori, V and Mercuri, E and Marcovecchio, GE and Castiello, MC and Schiroli, G and Albano, L and Margulies, C and Buquicchio, F and Fontana, E and Beretta, S and Merelli, I and Cappelleri, A and Rancoita, PM and Lougaris, V and Plebani, A and Kanariou, M and Lankester, A and Ferrua, F and Scanziani, E and Cotta-Ramusino, C and Villa, A and Naldini, L and Genovese, P}, title = {Modeling, optimization, and comparable efficacy of T cell and hematopoietic stem cell gene editing for treating hyper-IgM syndrome.}, journal = {EMBO molecular medicine}, volume = {}, number = {}, pages = {e13545}, doi = {10.15252/emmm.202013545}, pmid = {33475257}, issn = {1757-4684}, support = {TIGET-E3//Fondazione Telethon (Telethon Foundation)/ ; GR-2013-02358956//Ministero della Salute (Ministry of Health, Italy)/ ; GR-2016-02364847//Ministero della Salute (Ministry of Health, Italy)/ ; PE-2016-02363691//Ministero della Salute (Ministry of Health, Italy)/ ; E-Rare-3 JTC 2017//Ministero della Salute (Ministry of Health, Italy)/ ; //Banca d'Italia (Bank of Italy)/ ; PRIN 2017 Prot. 20175XHBPN//Ministero dell'Istruzione, dell'Università e della Ricerca (MIUR)/ ; //Editas Medicine/ ; //EU Horizon 2020 Program/ ; //Liberal contribution/ ; //Louis-Jeantet Foundation/ ; //Jeantet-Collen Prize for Translational Medicine/ ; }, abstract = {Precise correction of the CD40LG gene in T cells and hematopoietic stem/progenitor cells (HSPC) holds promise for treating X-linked hyper-IgM Syndrome (HIGM1), but its actual therapeutic potential remains elusive. Here, we developed a one-size-fits-all editing strategy for effective T-cell correction, selection, and depletion and investigated the therapeutic potential of T-cell and HSPC therapies in the HIGM1 mouse model. Edited patients' derived CD4 T cells restored physiologically regulated CD40L expression and contact-dependent B-cell helper function. Adoptive transfer of wild-type T cells into conditioned HIGM1 mice rescued antigen-specific IgG responses and protected mice from a disease-relevant pathogen. We then obtained ~ 25% CD40LG editing in long-term repopulating human HSPC. Transplanting such proportion of wild-type HSPC in HIGM1 mice rescued immune functions similarly to T-cell therapy. Overall, our findings suggest that autologous edited T cells can provide immediate and substantial benefits to HIGM1 patients and position T-cell ahead of HSPC gene therapy because of easier translation, lower safety concerns and potentially comparable clinical benefits.}, }
@article {pmid33474893, year = {2021}, author = {He, XY and Zhang, AQ and Gong, T and Li, YQ}, title = {[Transcriptomic Analysis of csn2 Gene Mutant Strains of Streptococcus mutans CRISPR-Cas9 System].}, journal = {Sichuan da xue xue bao. Yi xue ban = Journal of Sichuan University. Medical science edition}, volume = {52}, number = {1}, pages = {76-81}, doi = {10.12182/20210160505}, pmid = {33474893}, issn = {1672-173X}, abstract = {Objective: To explore the differences in transcriptional levels between mutant strains of csn2 gene of CRISPR-Cas9 system of Streptococcus mutans(S. mutans) and wild-type strains.<br><br>Methods: The S. mutans UA159, csn2-gene-deleted strains (Δ csn2) and csn2-gene-covering strains (Δ csn2/pDL278- csn2) of S. mutans were cultivated. Total RNA was extracted, and high-throughput sequencing technology was used for transcriptome sequencing. Based on the GO analysis and the KEGG analysis of the differentially expressed genes, the biological processes involved were thoroughly examined. The qRT-PCR method was used to verify the transcriptome sequencing results.<br><br>Results: The transcriptome results showed that, compared with UA159, there were 176 genes in Δ csn2 whose gene expression changed more than one fold (P<0.05), of which 72 were up-regulated and 104 were down-regulated. The GO enrichment analysis and the KEGG enrichment analysis revealed that both the up-regulated and down-regulated differentially expressed genes (DEG) were involved in amino acid transport and metabolism. In addition, the biological processes that up-regulated DEGs participated in were mainly related to carbohydrate metabolism, energy production and conversion, and transcription; down-regulated DEGs were mainly related to lipid metabolism, DNA replication, recombination and repair, signal transduction mechanisms, nucleotide transport and metabolism. The functions of some DEGs were still unclear. Results of qRT-PCR verified that the expressions of leuA, leuC and leuD(genes related to the formation of branched-chain amino acids) were significantly down-regulated in Δ csn2 when compared with UA159 and Δ csn2/pDL278- csn2.<br><br>Conclusion: Through transcriptome sequencing and qRT-PCR verification, it was found that the expression of genes related to branched-chain amino acid synthesis and cell membrane permeability in Δ csn2 changed significantly.}, }
@article {pmid33472516, year = {2021}, author = {Hanson, B and Wood, MJA and Roberts, TC}, title = {Molecular correction of Duchenne muscular dystrophy by splice modulation and gene editing.}, journal = {RNA biology}, volume = {}, number = {}, pages = {1-15}, doi = {10.1080/15476286.2021.1874161}, pmid = {33472516}, issn = {1555-8584}, abstract = {Duchenne muscular dystrophy (DMD) is a currently incurable X-linked neuromuscular disorder, characterized by progressive muscle wasting and premature death, typically as a consequence of cardiac failure. DMD-causing mutations in the dystrophin gene are highly diverse, meaning that the development of a universally-applicable therapy to treat all patients is very challenging. The leading therapeutic strategy for DMD is antisense oligonucleotide-mediated splice modulation, whereby one or more specific exons are excluded from the mature dystrophin mRNA in order to correct the translation reading frame. Indeed, three exon skipping oligonucleotides have received FDA approval for use in DMD patients. Second-generation exon skipping drugs (i.e. peptide-antisense oligonucleotide conjugates) exhibit enhanced potency, and also induce dystrophin restoration in the heart. Similarly, multiple additional antisense oligonucleotide drugs targeting various exons are in clinical development in order to treat a greater proportion of DMD patient mutations. Relatively recent advances in the field of genome engineering (specifically, the development of the CRISPR/Cas system) have provided multiple promising therapeutic approaches for the RNA-directed genetic correction of DMD, including exon excision, exon reframing via the introduction of insertion/deletion mutations, disruption of splice signals to promote exon skipping, and the templated correction of point mutations by seamless homology directed repair or base editing technology. Potential limitations to the clinical translation of the splice modulation and gene editing approaches are discussed, including drug delivery, the importance of uniform dystrophin expression in corrected myofibres, safety issues (e.g. renal toxicity, viral vector immunogenicity, and off-target gene editing), and the high cost of therapy.}, }
@article {pmid33367730, year = {2020}, author = {Li, HL and Wang, XY and Zheng, XL and Lu, W}, title = {Research Progress on Oviposition-Related Genes in Insects.}, journal = {Journal of insect science (Online)}, volume = {20}, number = {6}, pages = {}, pmid = {33367730}, issn = {1536-2442}, mesh = {Animals ; CRISPR-Cas Systems ; Egg Proteins/genetics ; Female ; Gene Expression ; Genes, Insect ; Insect Control/methods ; Insecta/*genetics ; Oogenesis/genetics ; Oviposition/*genetics ; RNA Interference ; Receptors, Cell Surface/genetics ; Vitellogenins/genetics ; }, abstract = {Oviposition-related genes have remained a consistent focus of insect molecular biology. Previous research has gradually clarified our mechanistic understanding of oviposition-related genes, including those related to oviposition-gland-related genes, oogenesis-related genes, oviposition-site-selection-related genes, and genes related to ovulation and hatching. Moreover, some of this research has revealed how the expression of single oviposition-related genes affects the expression of related genes, and more importantly, how individual node genes function to link the expression of upstream and downstream genes. However, the research to date is not sufficient to completely explain the overall interactions among the genes of the insect oviposition system. Through a literature review of a large number of studies, this review provides references for future research on oviposition-related genes in insects and the use of RNAi or CRISPR/Cas9 technology to verify the functions of oviposition-related genes and to prevent and control harmful insects.}, }
@article {pmid33315895, year = {2020}, author = {Ghoshal, B and Vong, B and Picard, CL and Feng, S and Tam, JM and Jacobsen, SE}, title = {A viral guide RNA delivery system for CRISPR-based transcriptional activation and heritable targeted DNA demethylation in Arabidopsis thaliana.}, journal = {PLoS genetics}, volume = {16}, number = {12}, pages = {e1008983}, pmid = {33315895}, issn = {1553-7404}, support = {R35 GM130272/GM/NIGMS NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; }, mesh = {Arabidopsis ; Arabidopsis Proteins/*genetics/metabolism ; *CRISPR-Cas Systems ; *DNA Methylation ; Epigenome ; Gene Editing/*methods ; Gene Targeting/*methods ; Plant Viruses/*genetics ; RNA, Guide/*genetics ; RNA, Transfer/genetics ; Transcriptional Activation ; }, abstract = {Plant RNA viruses are used as delivery vectors for their high level of accumulation and efficient spread during virus multiplication and movement. Utilizing this concept, several viral-based guide RNA delivery platforms for CRISPR-Cas9 genome editing have been developed. The CRISPR-Cas9 system has also been adapted for epigenome editing. While systems have been developed for CRISPR-Cas9 based gene activation or site-specific DNA demethylation, viral delivery of guide RNAs remains to be developed for these purposes. To address this gap we have developed a tobacco rattle virus (TRV)-based single guide RNA delivery system for epigenome editing in Arabidopsis thaliana. Because tRNA-like sequences have been shown to facilitate the cell-to-cell movement of RNAs in plants, we used the tRNA-guide RNA expression system to express guide RNAs from the viral genome to promote heritable epigenome editing. We demonstrate that the tRNA-gRNA system with TRV can be used for both transcriptional activation and targeted DNA demethylation of the FLOWERING WAGENINGEN gene in Arabidopsis. We achieved up to ~8% heritability of the induced demethylation phenotype in the progeny of virus inoculated plants. We did not detect the virus in the next generation, indicating effective clearance of the virus from plant tissues. Thus, TRV delivery, combined with a specific tRNA-gRNA architecture, provides for fast and effective epigenome editing.}, }
@article {pmid33214258, year = {2020}, author = {Pennisi, E}, title = {Like CRISPR, mystery gene editor began as a virus fighter.}, journal = {Science (New York, N.Y.)}, volume = {370}, number = {6519}, pages = {898-899}, doi = {10.1126/science.370.6519.898}, pmid = {33214258}, issn = {1095-9203}, mesh = {Bacteria/*virology ; Bacteriophages/*physiology ; *CRISPR-Cas Systems ; DNA, Bacterial/genetics ; *Gene Editing ; RNA, Bacterial/genetics ; RNA-Directed DNA Polymerase/physiology ; }, }
@article {pmid33208534, year = {2020}, author = {Alphey, LS and Crisanti, A and Randazzo, FF and Akbari, OS}, title = {Opinion: Standardizing the definition of gene drive.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {117}, number = {49}, pages = {30864-30867}, pmid = {33208534}, issn = {1091-6490}, support = {DP2 AI152071/AI/NIAID NIH HHS/United States ; BBS/E/I/00007033/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BBS/E/I/00007034/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; }, mesh = {CRISPR-Cas Systems/genetics ; Gene Drive Technology/*standards ; Gene Frequency/genetics ; Humans ; Reference Standards ; }, }
@article {pmid33122427, year = {2020}, author = {Esk, C and Lindenhofer, D and Haendeler, S and Wester, RA and Pflug, F and Schroeder, B and Bagley, JA and Elling, U and Zuber, J and von Haeseler, A and Knoblich, JA}, title = {A human tissue screen identifies a regulator of ER secretion as a brain-size determinant.}, journal = {Science (New York, N.Y.)}, volume = {370}, number = {6519}, pages = {935-941}, doi = {10.1126/science.abb5390}, pmid = {33122427}, issn = {1095-9203}, mesh = {Brain/*growth &amp; development/metabolism ; CRISPR-Cas Systems ; Carrier Proteins/genetics/*physiology ; Cell Line ; Cell Lineage ; Endoplasmic Reticulum/*metabolism ; Extracellular Matrix Proteins/*metabolism ; Gene Knockout Techniques ; Genetic Testing/*methods ; Humans ; Membrane Proteins/genetics/*physiology ; Microcephaly/*genetics ; Organ Size ; Organoids/growth &amp; development/metabolism ; }, abstract = {Loss-of-function (LOF) screens provide a powerful approach to identify regulators in biological processes. Pioneered in laboratory animals, LOF screens of human genes are currently restricted to two-dimensional cell cultures, which hinders the testing of gene functions requiring tissue context. Here, we present CRISPR-lineage tracing at cellular resolution in heterogeneous tissue (CRISPR-LICHT), which enables parallel LOF studies in human cerebral organoid tissue. We used CRISPR-LICHT to test 173 microcephaly candidate genes, revealing 25 to be involved in known and uncharacterized microcephaly-associated pathways. We characterized IER3IP1, which regulates the endoplasmic reticulum (ER) function and extracellular matrix protein secretion crucial for tissue integrity, the dysregulation of which results in microcephaly. Our human tissue screening technology identifies microcephaly genes and mechanisms involved in brain-size control.}, }
@article {pmid32778842, year = {2020}, author = {Mathiasen, S and Palmisano, T and Perry, NA and Stoveken, HM and Vizurraga, A and McEwen, DP and Okashah, N and Langenhan, T and Inoue, A and Lambert, NA and Tall, GG and Javitch, JA}, title = {G12/13 is activated by acute tethered agonist exposure in the adhesion GPCR ADGRL3.}, journal = {Nature chemical biology}, volume = {16}, number = {12}, pages = {1343-1350}, pmid = {32778842}, issn = {1552-4469}, support = {R01 GM120110/GM/NIGMS NIH HHS/United States ; R01 NS103946/NS/NINDS NIH HHS/United States ; F30 GM131672/GM/NIGMS NIH HHS/United States ; R01 GM130142/GM/NIGMS NIH HHS/United States ; T32 GM007315/GM/NIGMS NIH HHS/United States ; }, mesh = {Activating Transcription Factor 6/agonists/chemistry/genetics/*metabolism ; Animals ; Arrestin/chemistry/genetics/metabolism ; CRISPR-Cas Systems ; Cell Engineering ; GTP-Binding Protein alpha Subunits, G12-G13/chemistry/genetics/*metabolism ; GTP-Binding Protein alpha Subunits, Gq-G11/chemistry/genetics/metabolism ; Gene Expression ; HEK293 Cells ; Humans ; Kinetics ; Mice ; Mitogen-Activated Protein Kinase 1/chemistry/genetics/metabolism ; Mitogen-Activated Protein Kinase 3/chemistry/genetics/metabolism ; Peptides/chemistry/*metabolism/pharmacology ; Protein Binding ; Receptors, G-Protein-Coupled/chemistry/genetics/*metabolism ; Receptors, Peptide/chemistry/genetics/*metabolism ; Recombinant Proteins/chemistry/genetics/metabolism ; Signal Transduction ; }, abstract = {The adhesion G-protein-coupled receptor (GPCR) latrophilin 3 (ADGRL3) has been associated with increased risk of attention deficit hyperactivity disorder (ADHD) and substance use in human genetic studies. Knockdown in multiple species leads to hyperlocomotion and altered dopamine signaling. Thus, ADGRL3 is a potential target for treatment of neuropsychiatric disorders that involve dopamine dysfunction, but its basic signaling properties are poorly understood. Identification of adhesion GPCR signaling partners has been limited by a lack of tools to acutely activate these receptors in living cells. Here, we design a novel acute activation strategy to characterize ADGRL3 signaling by engineering a receptor construct in which we could trigger acute activation enzymatically. Using this assay, we found that ADGRL3 signals through G12/G13 and Gq, with G12/13 the most robustly activated. Gα12/13 is a new player in ADGRL3 biology, opening up unexplored roles for ADGRL3 in the brain. Our methodological advancements should be broadly useful in adhesion GPCR research.}, }
@article {pmid32544385, year = {2020}, author = {Lowey, B and Whiteley, AT and Keszei, AFA and Morehouse, BR and Mathews, IT and Antine, SP and Cabrera, VJ and Kashin, D and Niemann, P and Jain, M and Schwede, F and Mekalanos, JJ and Shao, S and Lee, ASY and Kranzusch, PJ}, title = {CBASS Immunity Uses CARF-Related Effectors to Sense 3'-5'- and 2'-5'-Linked Cyclic Oligonucleotide Signals and Protect Bacteria from Phage Infection.}, journal = {Cell}, volume = {182}, number = {1}, pages = {38-49.e17}, doi = {10.1016/j.cell.2020.05.019}, pmid = {32544385}, issn = {1097-4172}, support = {R01 AI026289/AI/NIAID NIH HHS/United States ; R01 ES027595/ES/NIEHS NIH HHS/United States ; P42 ES010337/ES/NIEHS NIH HHS/United States ; F31 CA236405/CA/NCI NIH HHS/United States ; T32 CA207021/CA/NCI NIH HHS/United States ; R01 AI018045/AI/NIAID NIH HHS/United States ; F32 GM133063/GM/NIGMS NIH HHS/United States ; S10 OD020025/OD/NIH HHS/United States ; }, mesh = {Amino Acid Sequence ; Bacteria/*virology ; Bacterial Proteins/chemistry/metabolism ; Bacteriophages/*metabolism ; *CRISPR-Cas Systems ; Deoxyribonuclease I/metabolism ; *Immunity ; Ligands ; Mutagenesis/genetics ; Nucleotidyltransferases/metabolism ; Oligonucleotides/*metabolism ; Protein Binding ; Second Messenger Systems ; *Signal Transduction ; }, abstract = {cGAS/DncV-like nucleotidyltransferase (CD-NTase) enzymes are immune sensors that synthesize nucleotide second messengers and initiate antiviral responses in bacterial and animal cells. Here, we discover Enterobacter cloacae CD-NTase-associated protein 4 (Cap4) as a founding member of a diverse family of >2,000 bacterial receptors that respond to CD-NTase signals. Structures of Cap4 reveal a promiscuous DNA endonuclease domain activated through ligand-induced oligomerization. Oligonucleotide recognition occurs through an appended SAVED domain that is an unexpected fusion of two CRISPR-associated Rossman fold (CARF) subunits co-opted from type III CRISPR immunity. Like a lock and key, SAVED effectors exquisitely discriminate 2'-5'- and 3'-5'-linked bacterial cyclic oligonucleotide signals and enable specific recognition of at least 180 potential nucleotide second messenger species. Our results reveal SAVED CARF family proteins as major nucleotide second messenger receptors in CBASS and CRISPR immune defense and extend the importance of linkage specificity beyond mammalian cGAS-STING signaling.}, }
@article {pmid32539998, year = {2020}, author = {Miyamoto, T and Takada, R and Tobimatsu, Y and Suzuki, S and Yamamura, M and Osakabe, K and Osakabe, Y and Sakamoto, M and Umezawa, T}, title = {Double knockout of OsWRKY36 and OsWRKY102 boosts lignification with altering culm morphology of rice.}, journal = {Plant science : an international journal of experimental plant biology}, volume = {296}, number = {}, pages = {110466}, doi = {10.1016/j.plantsci.2020.110466}, pmid = {32539998}, issn = {1873-2259}, mesh = {CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Cell Wall/metabolism ; Gene Editing ; Gene Knockout Techniques ; Lignin/*metabolism ; Magnetic Resonance Spectroscopy ; Oryza/*anatomy &amp; histology/genetics/metabolism ; Phylogeny ; Plant Proteins/metabolism/*physiology ; Plant Stems/*anatomy &amp; histology/genetics/metabolism ; Plants, Genetically Modified ; Transcription Factors/metabolism/*physiology ; }, abstract = {Breeding to enrich lignin, a major component of lignocelluloses, in plants contributes to enhanced applications of lignocellulosic biomass into solid biofuels and valuable aromatic chemicals. To collect information on enhancing lignin deposition in grass species, important lignocellulose feedstocks, we generated rice (Oryza sativa) transgenic lines deficient in OsWRKY36 and OsWRKY102, which encode putative transcriptional repressors for secondary cell wall formation. We used CRISPR/Cas9-mediated targeted mutagenesis and closely characterized their altered cell walls using chemical and nuclear magnetic resonance (NMR) methods. Both OsWRKY36 and OsWRKY102 mutations significantly increased lignin content by up to 28 % and 32 %, respectively. Additionally, OsWRKY36/OsWRKY102-double-mutant lines displayed lignin enrichment of cell walls (by up to 41 %) with substantially altered culm morphology over the single-mutant lines as well as the wild-type controls. Our chemical and NMR analyses showed that relative abundances of guaiacyl and p-coumarate units were slightly higher and lower, respectively, in the WRKY mutant lignins compared with those in the wild-type lignins. Our results provide evidence that both OsWRKY36 and OsWRKY102 are associated with repression of rice lignification.}, }
@article {pmid32488144, year = {2020}, author = {Eskandari-Shahraki, M and Prud'homme, B and Bergeron, F and Manjunath, P}, title = {Epididymal proteins Binder of SPerm Homologs 1 and 2 (BSPH1/2) are dispensable for male fertility and sperm motility in mice.}, journal = {Scientific reports}, volume = {10}, number = {1}, pages = {8982}, pmid = {32488144}, issn = {2045-2322}, support = {MOP-130274//Gouvernement du Canada | Canadian Institutes of Health Research (Instituts de Recherche en Santé du Canada)/International ; }, mesh = {Animals ; Animals, Newborn ; Body Weight/genetics ; *CRISPR-Cas Systems ; Female ; Fertility/*genetics ; Male ; Mice, Knockout/*genetics ; Seminal Vesicle Secretory Proteins/*genetics/*physiology ; Sperm Motility/*genetics ; }, abstract = {The binder of sperm family of proteins has been reported to be indispensable for sperm maturation and capacitation. However, their physiological functions in fertility have only been studied in vitro. CRISPR/Cas9 genome editing was utilized to generate double knockout (DKO) mice by simultaneously targeting the two murine binder of sperm genes, Bsph1 and Bsph2. To confirm that the homologous genes and proteins were completely eliminated in the DKO mice, different methods such as reverse transcription polymerase chain reaction, digital droplet-polymerase chain reaction and liquid chromatography tandem mass spectrometry were applied. Bsph1/2 DKO male mice were bred by intercrossing. Compared to wild type counterparts, male Bsph1/2 null mice, lacking BSPH1/2 proteins, were fertile with no differences in sperm motility and sperm count. However, the weights of male pups were significantly increased in Bsph1/2 double knockout mice in a time dependent manner spanning days 6 and 21, as well as 6 weeks of age. No change was detected in the weights of female pups during the same period. Taken together, these data indicate that BSPH1/2 proteins are dispensable for male fertility in mice but may influence growth.}, }
@article {pmid32408049, year = {2020}, author = {Ramongolalaina, C}, title = {Dual-luciferase assay and siRNA silencing for nodD1 to study the competitiveness of Bradyrhizobium diazoefficiens USDA110 in soybean nodulation.}, journal = {Microbiological research}, volume = {237}, number = {}, pages = {126488}, doi = {10.1016/j.micres.2020.126488}, pmid = {32408049}, issn = {1618-0623}, mesh = {Bacterial Proteins/*genetics ; Bradyrhizobium/*genetics ; CRISPR-Cas Systems ; Fluorescent Dyes/analysis ; Genes, Bacterial ; Luciferases, Renilla ; Nitrogen Fixation/genetics ; Plant Development ; *Plant Root Nodulation ; Plant Roots/microbiology ; RNA, Small Interfering ; Soil Microbiology ; Soybeans/*microbiology ; Symbiosis ; Transformation, Bacterial ; }, abstract = {The symbiosis of soybean with Bradyrhizobium diazoefficiens USDA110, which always competes with other rhizobia in the field, is of great agronomic and environmental importance. Herein, a dual-luciferase reporter assay was utilized to monitor the dynamics of two dominant bradyrhizobia infecting roots of soybean. More explicitly, luciferase-tagged B. diazoefficiens USDA110 (USDA110-FLuc) and Bradyrhizobium elkanii USDA 94 (USDA94-RLuc) were designed, co-inoculated into soybean seeds, and observed for their colonization in root nodules by bioluminescence imaging. The results showed that USDA110-FLuc initiated infection earlier than USDA94-RLuc, but its occupancy in the nodules decreased as the plant grew. A nodulation test showed that nodD1 mutant USDA110 strains, including CRISPR engineered mutants, were less competitive than wild type. I constructed siRNAs to knockdown nodD1 at different target sites and transformed them into the bacteria. Surprisingly, although siRNAs - with 3' end target sites - were able to repress up to 65% of nodD1 expression, the profiling of total RNAs with a bioanalyzer revealed that 23S/16S-rRNA ratios of siRNA-transformed and wild type USDA110 strains were similar, but lower than that of nodD1 mutant. In short, the current work - while reporting the competitiveness of B. diazoefficiens USDA110 in early occupancy of soybean nodules and the gene nodD1 as a key determinant of this infection - gives an insight on siRNA silencing in microbes, and demonstrates a highly efficient imaging approach that could entail many new avenues for many biological research fields.}, }
@article {pmid32326099, year = {2020}, author = {Becskei, A}, title = {Tuning up Transcription Factors for Therapy.}, journal = {Molecules (Basel, Switzerland)}, volume = {25}, number = {8}, pages = {}, pmid = {32326099}, issn = {1420-3049}, support = {310030_185001//Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung/ ; }, mesh = {Animals ; Biotechnology/methods ; CRISPR-Cas Systems ; Clinical Trials as Topic ; DNA-Binding Proteins/genetics/metabolism ; Endonucleases/genetics/metabolism ; *Gene Expression Regulation ; Gene Transfer Techniques ; *Genetic Therapy/methods ; Humans ; Protein Binding ; Repressor Proteins/genetics/metabolism ; Transcription Factors/chemistry/*genetics/*metabolism ; *Transcription, Genetic ; }, abstract = {The recent developments in the delivery and design of transcription factors put their therapeutic applications within reach, exemplified by cell replacement, cancer differentiation and T-cell based cancer therapies. The success of such applications depends on the efficacy and precision in the action of transcription factors. The biophysical and genetic characterization of the paradigmatic prokaryotic repressors, LacI and TetR and the designer transcription factors, transcription activator-like effector (TALE) and CRISPR-dCas9 revealed common principles behind their efficacy, which can aid the optimization of transcriptional activators and repressors. Further studies will be required to analyze the linkage between dissociation constants and enzymatic activity, the role of phase separation and squelching in activation and repression and the long-range interaction of transcription factors with epigenetic regulators in the context of the chromosomes. Understanding these mechanisms will help to tailor natural and synthetic transcription factors to the needs of specific applications.}, }
@article {pmid31978714, year = {2020}, author = {Xu, C and Zhou, Z and Liu, C and Kang, X and Zhong, X and Zhang, Q and Xu, Y}, title = {Generation of a DAPK1 knockout first (conditional ready) human embryonic stem cell line (ZSSYe001-A) by CRISPR-Cas9 technology.}, journal = {Stem cell research}, volume = {43}, number = {}, pages = {101693}, doi = {10.1016/j.scr.2019.101693}, pmid = {31978714}, issn = {1876-7753}, mesh = {CRISPR-Cas Systems/*genetics ; Cell Line ; Death-Associated Protein Kinases/*genetics ; Human Embryonic Stem Cells/*metabolism ; Humans ; Male ; }, abstract = {Death-associated protein kinase 1 (DAPK1) is a Ca2+/calmodulin regulated Ser/Thr kinase involved in various cellular processes including cell death, autophagy and inflammation. Its dysregulation has been linked to tumour metastasis, anti-viral responses, Alzheimer's disease and other neurological disorders. To further investigate the role of DAPK1 in these processes, we generated a DAPK1 knockout first (conditional ready) human embryonic stem (hES) cell line in which the endogenous DAPK1 can be easily restored with expression of FLPe. This cell line provides an ideal model to study the role of DAPK1 in human development and various pathologies related to DAPK1 dysregulation in vitro.}, }
@article {pmid33472057, year = {2021}, author = {Wang, B and Zhang, T and Yin, J and Yu, Y and Xu, W and Ding, J and Patel, DJ and Yang, H}, title = {Structural basis for self-cleavage prevention by tag:anti-tag pairing complementarity in type VI Cas13 CRISPR systems.}, journal = {Molecular cell}, volume = {}, number = {}, pages = {}, doi = {10.1016/j.molcel.2020.12.033}, pmid = {33472057}, issn = {1097-4164}, abstract = {Bacteria and archaea apply CRISPR-Cas surveillance complexes to defend against foreign invaders. These invading genetic elements are captured and integrated into the CRISPR array as spacer elements, guiding sequence-specific DNA/RNA targeting and cleavage. Recently, in vivo studies have shown that target RNAs with extended complementarity with repeat sequences flanking the target element (tag:anti-tag pairing) can dramatically reduce RNA cleavage by the type VI-A Cas13a system. Here, we report the cryo-EM structure of Leptotrichia shahii LshCas13acrRNA in complex with target RNA harboring tag:anti-tag pairing complementarity, with the observed conformational changes providing a molecular explanation for inactivation of the composite HEPN domain cleavage activity. These structural insights, together with in vitro biochemical and in vivo cell-based assays on key mutants, define the molecular principles underlying Cas13a's capacity to target and discriminate between self and non-self RNA targets. Our studies illuminate approaches to regulate Cas13a's cleavage activity, thereby influencing Cas13a-mediated biotechnological applications.}, }
@article {pmid33471332, year = {2021}, author = {Tang, X and Qi, Y and Zhang, Y}, title = {Single Transcript Unit CRISPR 2.0 Systems for Genome Editing in Rice.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2238}, number = {}, pages = {193-204}, pmid = {33471332}, issn = {1940-6029}, abstract = {CRISPR-Cas9 and Cas12a (formerly Cpf1), RNA-guided DNA endonucleases found from adaptive immune system in prokaryotes, have been engineered and widely adopted as two of the most powerful genome editing systems in plants. Recently, we developed a single transcript unit (STU) CRISPR 2.0 toolbox for applications in plants, which contains two STU-Cas9 systems and one STU-Cas12a system. Here, we describe a detailed protocol about using the STU CRISPR 2.0 systems to achieve single and multiplex genome editing in rice.}, }
@article {pmid33471330, year = {2021}, author = {Liu, G and Qi, Y and Zhang, T}, title = {Analysis of Off-Target Mutations in CRISPR-Edited Rice Plants Using Whole-Genome Sequencing.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2238}, number = {}, pages = {145-172}, pmid = {33471330}, issn = {1940-6029}, abstract = {The CRISPR/Cas systems have become the most widely used tool for genome editing in plants and beyond. However, CRISPR/Cas systems may cause unexpected off-target mutations due to sgRNA recognizing highly homologous DNA sequence elsewhere in the genome. Whole-genome sequencing (WGS) can be used to identify on- and off-target mutation. Here, we describe a pipeline of analyzing WGS data using a series of open source software for analysis of off-target mutations in CRISPR-edited rice plants. In this pipeline, the adapter is trimmed using SKEWER. Then, the cleaned reads are mapped to reference genome by applying BWA. To avoid mapping bias, the GATK is used to realign reads near indels (insertions and deletions) and recalibrate base quality controls. Whole-genome single nucleotide variations (SNVs) and indels are detected by LoFreq*, Mutect2, VarScan2, and Pindel. Last, SNVs and indels are compared with in silico off-target sites using Cas-OFFinder.}, }
@article {pmid33471328, year = {2021}, author = {Das, A and Ghana, P and Rudrappa, B and Gandhi, R and Tavva, VS and Mohanty, A}, title = {Genome Editing of Rice by CRISPR-Cas: End-to-End Pipeline for Crop Improvement.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2238}, number = {}, pages = {115-134}, pmid = {33471328}, issn = {1940-6029}, abstract = {CRISPR-Cas resonates a revolutionary genome editing technology applicable through a horizon spreading across microbial organism to higher plant and animal. This technology can be harnessed with ease to understand the basic genetics of a living system by altering sequence of individual genes and characterizing their functions. The precision of this technology is unparallel. It allows very precise and targeted base pair level edits in the genome. Here, in the current chapter, we have provided end-to-end process outline on how to generate genome edited plants in crops like rice to evaluate for agronomic traits associated with yield, disease resistance and abiotic stress tolerance, etc. Genome editing process includes designing of gene editing strategy, vector construction, plant transformation, molecular screening, and phenotyping under control environment conditions. Furthermore, its application for development of commercial crop product may require additional processes, including field trials in the target geography for evaluation of product efficacy. Evaluation of genome edited lines in controlled greenhouse/net house or open field condition requires few generations for outcrossing with wild-type parent to eliminate and/or reduce any potential pleiotropic effect in the edited genome which may arise during the process. The genome edited plant selected for advancement shall harbor the genome with only the intended changes, which can be analyzed by various molecular techniques, advanced sequencing methods, and genomic data analysis tools. CRISPR-Cas-based genome editing has opened a plethora of opportunities in agriculture as well as human health.}, }
@article {pmid33469138, year = {2021}, author = {Fernandes, LGV and Hornsby, RL and Nascimento, ALTO and Nally, JE}, title = {Genetic manipulation of pathogenic Leptospira: CRISPR interference (CRISPRi)-mediated gene silencing and rapid mutant recovery at 37 °C.}, journal = {Scientific reports}, volume = {11}, number = {1}, pages = {1768}, pmid = {33469138}, issn = {2045-2322}, support = {2019/20302-8//Fapesp/ ; 2014/50981-0//Fapesp/ ; }, abstract = {Leptospirosis is a neglected, widespread zoonosis caused by pathogenic species of the genus Leptospira, and is responsible for 60,000 deaths per year. Pathogenic mechanisms of leptospirosis remain poorly understood mainly because targeted mutations or gene silencing in pathogenic Leptospira continues to be inherently inefficient, laborious, costly and difficult to implement. In addition, pathogenic leptospires are highly fastidious and the selection of mutants on solid agar media can take up to 6 weeks. The catalytically inactive Cas9 (dCas9) is an RNA-guided DNA-binding protein from the Streptococcus pyogenes CRISPR/Cas system and can be used for gene silencing, in a strategy termed CRISPR interference (CRISPRi). Here, this technique was employed to silence genes encoding major outer membrane proteins of pathogenic L. interrogans. Conjugation protocols were optimized using the newly described HAN media modified for rapid mutant recovery at 37 °C in 3% CO2 within 8 days. Complete silencing of LipL32 and concomitant and complete silencing of both LigA and LigB outer membrane proteins were achieved, revealing for the first time that Lig proteins are involved in pathogenic Leptospira serum resistance. Gene silencing in pathogenic leptospires and rapid mutant recovery will facilitate novel studies to further evaluate and understand pathogenic mechanisms of leptospirosis.}, }
@article {pmid33468705, year = {2021}, author = {Singh, A and Gaur, M and Sharma, V and Khanna, P and Bothra, A and Bhaduri, A and Mondal, AK and Dash, D and Singh, Y and Misra, R}, title = {Comparative Genomic Analysis of Mycobacteriaceae Reveals Horizontal Gene Transfer-Mediated Evolution of the CRISPR-Cas System in the Mycobacterium tuberculosis Complex.}, journal = {mSystems}, volume = {6}, number = {1}, pages = {}, pmid = {33468705}, issn = {2379-5077}, abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) genes are conserved genetic elements in many prokaryotes, including Mycobacterium tuberculosis, the causative agent of tuberculosis. Although knowledge of CRISPR locus variability has been utilized in M. tuberculosis strain genotyping, its evolutionary path in Mycobacteriaceae is not well understood. In this study, we have performed a comparative analysis of 141 mycobacterial genomes and identified the exclusive presence of the CRISPR-Cas type III-A system in M. tuberculosis complex (MTBC). Our global phylogenetic analysis of CRISPR repeats and Cas10 proteins offers evidence of horizontal gene transfer (HGT) of the CRISPR-Cas module in the last common ancestor of MTBC and Mycobacterium canettii from a Streptococcus-like environmental bacterium. Additionally, our results show that the variation of CRISPR-Cas organization in M. tuberculosis lineages, especially in the Beijing sublineage of lineage 2, is due to the transposition of insertion sequence IS6110 The direct repeat (DR) region of the CRISPR-Cas locus acts as a hot spot for IS6110 insertion. We show in M. tuberculosis H37Rv that the repeat at the 5' end of CRISPR1 of the forward strand is an atypical repeat made up partly of IS-terminal inverted repeat and partly CRISPR DR. By tracing an undetectable spacer sequence in the DR region, the two CRISPR loci could theoretically be joined to reconstruct the ancestral single CRISPR-Cas locus organization, as seen in M. canettii This study retracing the evolutionary events of HGT and IS6110-driven genomic deletions helps us to better understand the strain-specific variations in M. tuberculosis lineages.IMPORTANCE Comparative genomic analysis of prokaryotes has led to a better understanding of the biology of several pathogenic microorganisms. One such clinically important pathogen is M. tuberculosis, the leading cause of bacterial infection worldwide. Recent evidence on the functionality of the CRISPR-Cas system in M. tuberculosis has brought back focus on these conserved genetic elements, present in many prokaryotes. Our study advances understanding of mycobacterial CRISPR-Cas origin and its diversity among the different species. We provide phylogenetic evidence of acquisition of CRISPR-Cas type III-A in the last common ancestor shared between MTBC and M. canettii, by HGT-mediated events. The most likely source of HGT was an environmental Firmicutes bacterium. Genomic mapping of the CRISPR loci showed the IS6110 transposition-driven variations in M. tuberculosis strains. Thus, this study offers insights into events related to the evolution of CRISPR-Cas in M. tuberculosis lineages.}, }
@article {pmid33468685, year = {2021}, author = {Xiao, H and Wyler, E and Milek, M and Grewe, B and Kirchner, P and Ekici, A and Silva, ABOV and Jungnickl, D and Full, F and Thomas, M and Landthaler, M and Ensser, A and Überla, K}, title = {CRNKL1 Is a Highly Selective Regulator of Intron-Retaining HIV-1 and Cellular mRNAs.}, journal = {mBio}, volume = {12}, number = {1}, pages = {}, pmid = {33468685}, issn = {2150-7511}, abstract = {The HIV-1 Rev protein is a nuclear export factor for unspliced and incompletely spliced HIV-1 RNAs. Without Rev, these intron-retaining RNAs are trapped in the nucleus. A genome-wide screen identified nine proteins of the spliceosome, which all enhanced expression from the HIV-1 unspliced RNA after CRISPR/Cas knockdown. Depletion of DHX38, WDR70, and four proteins of the Prp19-associated complex (ISY1, BUD31, XAB2, and CRNKL1) resulted in a more than 20-fold enhancement of unspliced HIV-1 RNA levels in the cytoplasm. Targeting of CRNKL1, DHX38, and BUD31 affected nuclear export efficiencies of the HIV-1 unspliced RNA to a much larger extent than splicing. Transcriptomic analyses further revealed that CRNKL1 also suppresses cytoplasmic levels of a subset of cellular mRNAs, including some with selectively retained introns. Thus, CRNKL1-dependent nuclear retention is a novel cellular mechanism for the regulation of cytoplasmic levels of intron-retaining HIV-1 mRNAs, which HIV-1 may have harnessed to direct its complex splicing pattern.IMPORTANCE To regulate its complex splicing pattern, HIV-1 uses the adaptor protein Rev to shuttle unspliced or partially spliced mRNA from the nucleus to the cytoplasm. In the absence of Rev, these RNAs are retained in the nucleus, but it is unclear why. Here we identify cellular proteins whose depletion enhances cytoplasmic levels of the HIV-1 unspliced RNA. Depletion of one of them, CRNKL1, also increases cytoplasmic levels of a subset of intron-retaining cellular mRNA, suggesting that CRNKL1-dependent nuclear retention may be a basic cellular mechanism exploited by HIV-1.}, }
@article {pmid33349126, year = {2021}, author = {Cornu, TI and Mussolino, C and Müller, MC and Wehr, C and Kern, WV and Cathomen, T}, title = {HIV Gene Therapy: An Update.}, journal = {Human gene therapy}, volume = {32}, number = {1-2}, pages = {52-65}, doi = {10.1089/hum.2020.159}, pmid = {33349126}, issn = {1557-7422}, abstract = {Progress in antiretroviral therapy has considerably reduced mortality and notably improved the quality of life of individuals infected with HIV since the pandemic began some 40 years ago. However, drug resistance, treatment-associated toxicity, adherence to medication, and the need for lifelong therapy have remained major challenges. While the development of an HIV vaccine has remained elusive, considerable progress in developing innovative cell and gene therapies to treat HIV infection has been made. This includes immune cell therapies, such as chimeric antigen receptor T cells to target HIV infected cells, as well as gene therapies and genome editing strategies to render the patient's immune system resistant to HIV. Nonetheless, all of these attempts to achieve a functional cure in HIV patients have failed thus far. This review introduces the clinical as well as the technical challenges of treating HIV infection, and summarizes the most promising cell and gene therapy concepts that have aspired to bring about functional cure for people living with HIV. It further discusses socioeconomic aspects as well as future directions for developing cell and gene therapies with a potential to be an effective one-time treatment with minimal toxicity.}, }
@article {pmid33454599, year = {2021}, author = {Ashraf, MU and Salman, HM and Khalid, MF and Khan, MHF and Anwar, S and Afzal, S and Idrees, M and Chaudhary, SU}, title = {CRISPR-Cas13a mediated targeting of hepatitis C virus internal-ribosomal entry site (IRES) as an effective antiviral strategy.}, journal = {Biomedicine &amp; pharmacotherapy = Biomedecine &amp; pharmacotherapie}, volume = {136}, number = {}, pages = {111239}, doi = {10.1016/j.biopha.2021.111239}, pmid = {33454599}, issn = {1950-6007}, abstract = {Hepatitis C is an inflammatory liver disease caused by the single-stranded RNA (ssRNA) hepatitis C virus (HCV). The genetic diversity of the virus and quasispecies produced during replication have resulted in viral resistance to direct-acting antivirals (DAAs) as well as impediments in vaccine development. The recent adaptation of CRISPR-Cas as an alternative antiviral approach has demonstrated degradation of viral nucleic acids in eukaryotes. In particular, the CRISPR-effector Cas13 enzyme has been shown to target ssRNA viruses effectively. In this work, we have employed Cas13a to knockdown HCV in mammalian cells. Using a computational screen, we identified several potential Cas13a target sites within highly conserved regions of the HCV internal ribosomal entry site (IRES). Our results demonstrate significant inhibition of HCV replication as well as translation in huh-7.5 cells with minimal effects on cell viability. These findings were validated using a multi-modality approach involving qRT-PCR, luciferase assay, and MTT cell viability assay. In conclusion, the CRISPR-Cas13a system efficiently targets HCV in vitro, suggesting its potential as a programmable therapeutic antiviral strategy.}, }
@article {pmid33453817, year = {2020}, author = {Pinci, F and Gaidt, MM and Jung, C and Kuut, G and Jackson, MA and Bauernfried, S and Hornung, V}, title = {C-tag TNF: a reporter system to study TNF shedding.}, journal = {The Journal of biological chemistry}, volume = {295}, number = {52}, pages = {18065-18075}, doi = {10.1074/jbc.RA120.015248}, pmid = {33453817}, issn = {1083-351X}, abstract = {TNF is a highly pro-inflammatory cytokine that contributes not only to the regulation of immune responses but also to the development of severe inflammatory diseases. TNF is synthesized as a transmembrane protein, which is further matured via proteolytic cleavage by metalloproteases such as ADAM17, a process known as shedding. At present, TNF is mainly detected by measuring the precursor or the mature cytokine of bulk cell populations by techniques such as ELISA or immunoblotting. However, these methods do not provide information on the exact timing and extent of TNF cleavage at single-cell resolution and they do not allow the live visualization of shedding events. Here, we generated C-tag TNF as a genetically encoded reporter to study TNF shedding at the single-cell level. The functionality of the C-tag TNF reporter is based on the exposure of a cryptic epitope on the C terminus of the transmembrane portion of pro-TNF on cleavage. In both denatured and nondenatured samples, this epitope can be detected by a nanobody in a highly sensitive and specific manner only upon TNF shedding. As such, C-tag TNF can successfully be used for the detection of TNF cleavage in flow cytometry and live-cell imaging applications. We furthermore demonstrate its applicability in a forward genetic screen geared toward the identification of genetic regulators of TNF maturation. In summary, the C-tag TNF reporter can be employed to gain novel insights into the complex regulation of ADAM-dependent TNF shedding.}, }
@article {pmid33461211, year = {2021}, author = {Rostøl, JT and Xie, W and Kuryavyi, V and Maguin, P and Kao, K and Froom, R and Patel, DJ and Marraffini, LA}, title = {The Card1 nuclease provides defence during type-III CRISPR immunity.}, journal = {Nature}, volume = {}, number = {}, pages = {}, doi = {10.1038/s41586-021-03206-x}, pmid = {33461211}, issn = {1476-4687}, abstract = {During the prokaryotic type III CRISPR-Cas immune response, infection triggers the production of cyclic oligoadenylates, which bind and activate CARF domain-containing proteins1,2. Many type III loci are associated with proteins in which the CARF domain is fused to an endonuclease-like domain3,4; however, with the exception of the well-characterized Csm6/Csx1 RNases5,6, whether and how these inducible effectors provide defense is not known. Here we investigated one of such type III CRISPR accessory proteins, Card1. Card1 forms a symmetrical dimer with a large central cavity between its CARF and restriction endonuclease (REase) domains that binds cA4. Ligand binding results in a conformational change where individual monomers rotate relative to each other to form a more compact dimeric scaffold wherein a Mn cation coordinates to the catalytic residues and activates the cleavage of single, but not double, stranded nucleic acids (DNA and RNA). In vivo, Card1 activation induces dormancy of the infected hosts to provide immunity against phage infection and plasmids. Our results highlight the diversity of strategies used by CRISPR systems to provide immunity.}, }
@article {pmid33243861, year = {2020}, author = {Jin, X and Simmons, SK and Guo, A and Shetty, AS and Ko, M and Nguyen, L and Jokhi, V and Robinson, E and Oyler, P and Curry, N and Deangeli, G and Lodato, S and Levin, JZ and Regev, A and Zhang, F and Arlotta, P}, title = {In vivo Perturb-Seq reveals neuronal and glial abnormalities associated with autism risk genes.}, journal = {Science (New York, N.Y.)}, volume = {370}, number = {6520}, pages = {}, doi = {10.1126/science.aaz6063}, pmid = {33243861}, issn = {1095-9203}, support = {U01 MH115727/MH/NIMH NIH HHS/United States ; R01 MH096066/MH/NIMH NIH HHS/United States ; P50 MH094271/MH/NIMH NIH HHS/United States ; R01 HG009761/HG/NHGRI NIH HHS/United States ; R01 MH110049/MH/NIMH NIH HHS/United States ; DP1 HL141201/HL/NHLBI NIH HHS/United States ; //Howard Hughes Medical Insititue/International ; }, mesh = {Animals ; Ankyrins/genetics/metabolism ; Autistic Disorder/*genetics/*pathology ; Brain/*abnormalities ; CRISPR-Cas Systems ; DNA-Binding Proteins/genetics ; Frameshift Mutation ; Gene Expression Profiling ; Genetic Loci ; Humans ; Mice ; Neuroglia/metabolism/*pathology ; Neurons/metabolism/*pathology ; Repressor Proteins/genetics ; Risk ; Transcription Factors/genetics ; }, abstract = {The number of disease risk genes and loci identified through human genetic studies far outstrips the capacity to systematically study their functions. We applied a scalable genetic screening approach, in vivo Perturb-Seq, to functionally evaluate 35 autism spectrum disorder/neurodevelopmental delay (ASD/ND) de novo loss-of-function risk genes. Using CRISPR-Cas9, we introduced frameshift mutations in these risk genes in pools, within the developing mouse brain in utero, followed by single-cell RNA-sequencing of perturbed cells in the postnatal brain. We identified cell type-specific and evolutionarily conserved gene modules from both neuronal and glial cell classes. Recurrent gene modules and cell types are affected across this cohort of perturbations, representing key cellular effects across sets of ASD/ND risk genes. In vivo Perturb-Seq allows us to investigate how diverse mutations affect cell types and states in the developing organism.}, }
@article {pmid33220342, year = {2021}, author = {Katsuma, S and Shoji, K and Suzuki, Y and Kiuchi, T}, title = {CRISPR/Cas9-mediated mutagenesis of Ago2 and Siwi in silkworm cultured cells.}, journal = {Gene}, volume = {768}, number = {}, pages = {145314}, doi = {10.1016/j.gene.2020.145314}, pmid = {33220342}, issn = {1879-0038}, mesh = {Animals ; Argonaute Proteins/*genetics ; Bombyx/*cytology/genetics ; CRISPR-Cas Systems ; Cell Proliferation ; Cells, Cultured ; Gene Editing ; Insect Proteins/genetics ; Loss of Function Mutation ; Mutagenesis, Site-Directed/*methods ; RNA, Small Interfering/genetics ; RNA, Viral/genetics ; Signal Transduction ; Tymoviridae/*genetics ; }, abstract = {The BmN-4 cell line, originated from the silkworm Bombyx mori ovary, possesses endogenous small interfering RNA (siRNA) and PIWI-interacting RNA (piRNA) pathways. We performed CRISPR/Cas9-mediated genome editing of Ago2 and Siwi, which are the core factors for siRNA and piRNA pathways, respectively, to understand the importance of the two distinct small RNA pathways in this cell line. We found that approximately half of the alleles contained loss-of-function mutations in both Ago2- and Siwi-mutated cells. The mutated cells grew at a slower rate compared to the control cells, strongly suggesting that the siRNA and piRNA pathways are both crucial for the normal growth of BmN-4 cells. The amounts of piRNAs decreased markedly in the Siwi-mutated cells, but global de-repression of transposable elements was not observed. Although the RNA amount of latently infected RNA virus, Bombyx mori macula-like virus (BmLV), increased in both Ago2- and Siwi-mutated cells, the siRNA and piRNA pathways showed a bias toward targeting BmLV genomic and subgenomic RNA, respectively. These results indicate the common, specific, and crucial roles of the two small RNA pathways in B. mori cultured cells.}, }
@article {pmid33172989, year = {2020}, author = {Zhang, H and Zoued, A and Liu, X and Sit, B and Waldor, MK}, title = {Type I interferon remodels lysosome function and modifies intestinal epithelial defense.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {117}, number = {47}, pages = {29862-29871}, pmid = {33172989}, issn = {1091-6490}, support = {/HHMI/Howard Hughes Medical Institute/United States ; R01 AI042347/AI/NIAID NIH HHS/United States ; }, mesh = {Animals ; Bacterial Proteins/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Disease Models, Animal ; Epithelial Cells/chemistry/cytology/*immunology/metabolism ; Gene Expression Regulation, Bacterial/immunology ; HT29 Cells ; Host-Pathogen Interactions/genetics/immunology ; Humans ; Hydrogen-Ion Concentration ; Immunity, Innate ; Interferon Type I/*metabolism ; Intestinal Mucosa/cytology/*immunology/microbiology ; Lysosomes/chemistry/immunology/*metabolism ; Mice ; Mice, Knockout ; Necroptosis/immunology ; Peptide Hydrolases/metabolism ; Proteomics ; Receptor, Interferon alpha-beta/genetics/metabolism ; Salmonella Infections/*immunology/microbiology ; Salmonella typhimurium/immunology/pathogenicity ; Signal Transduction/immunology ; Virulence/immunology ; Virulence Factors/genetics/metabolism ; }, abstract = {Organelle remodeling is critical for cellular homeostasis, but host factors that control organelle function during microbial infection remain largely uncharacterized. Here, a genome-scale CRISPR/Cas9 screen in intestinal epithelial cells with the prototypical intracellular bacterial pathogen Salmonella led us to discover that type I IFN (IFN-I) remodels lysosomes. Even in the absence of infection, IFN-I signaling modified the localization, acidification, protease activity, and proteomic profile of lysosomes. Proteomic and genetic analyses revealed that multiple IFN-I-stimulated genes including IFITM3, SLC15A3, and CNP contribute to lysosome acidification. IFN-I-dependent lysosome acidification was associated with elevated intracellular Salmonella virulence gene expression, rupture of the Salmonella-containing vacuole, and host cell death. Moreover, IFN-I signaling promoted in vivo Salmonella pathogenesis in the intestinal epithelium where Salmonella initiates infection, indicating that IFN-I signaling can modify innate defense in the epithelial compartment. We propose that IFN-I control of lysosome function broadly impacts host defense against diverse viral and microbial pathogens.}, }
@article {pmid33168709, year = {2020}, author = {Yu, X and Zhao, Q and Li, X and Chen, Y and Tian, Y and Liu, S and Xiong, W and Huang, P}, title = {Deafness mutation D572N of TMC1 destabilizes TMC1 expression by disrupting LHFPL5 binding.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {117}, number = {47}, pages = {29894-29903}, pmid = {33168709}, issn = {1091-6490}, mesh = {Animals ; COS Cells ; CRISPR-Cas Systems/genetics ; Chlorocebus aethiops ; Deafness/*genetics/pathology ; Disease Models, Animal ; Gene Knock-In Techniques ; HEK293 Cells ; Hair Cells, Auditory, Inner/metabolism/*pathology ; Humans ; Mechanotransduction, Cellular/*genetics ; Membrane Proteins/*genetics/isolation &amp; purification/*metabolism ; Mice ; Mice, Transgenic ; Point Mutation ; Protein Binding/genetics ; Two-Hybrid System Techniques ; }, abstract = {Transmembrane channel-like protein 1 (TMC1) and lipoma HMGIC fusion partner-like 5 (LHFPL5) are recognized as two critical components of the mechanotransduction complex in inner-ear hair cells. However, the physical and functional interactions of TMC1 and LHFPL5 remain largely unexplored. We examined the interaction between TMC1 and LHFPL5 by using multiple approaches, including our recently developed ultrasensitive microbead-based single-molecule pulldown (SiMPull) assay. We demonstrate that LHFPL5 physically interacts with and stabilizes TMC1 in both heterologous expression systems and in the soma and hair bundle of hair cells. Moreover, the semidominant deafness mutation D572N in human TMC1 (D569N in mouse TMC1) severely disrupted LHFPL5 binding and destabilized TMC1 expression. Thus, our findings reveal previously unrecognized physical and functional interactions of TMC1 and LHFPL5 and provide insights into the molecular mechanism by which the D572N mutation causes deafness. Notably, these findings identify a missing link in the currently known physical organization of the mechanotransduction macromolecular complex. Furthermore, this study has demonstrated the power of the microbead-based SiMPull assay for biochemical investigation of rare cells such as hair cells.}, }
@article {pmid32887745, year = {2020}, author = {Llamosas, N and Arora, V and Vij, R and Kilinc, M and Bijoch, L and Rojas, C and Reich, A and Sridharan, B and Willems, E and Piper, DR and Scampavia, L and Spicer, TP and Miller, CA and Holder, JL and Rumbaugh, G}, title = {SYNGAP1 Controls the Maturation of Dendrites, Synaptic Function, and Network Activity in Developing Human Neurons.}, journal = {The Journal of neuroscience : the official journal of the Society for Neuroscience}, volume = {40}, number = {41}, pages = {7980-7994}, pmid = {32887745}, issn = {1529-2401}, support = {R01 MH096847/MH/NIMH NIH HHS/United States ; R01 MH113648/MH/NIMH NIH HHS/United States ; }, mesh = {CRISPR-Cas Systems ; Cell Differentiation/genetics ; Cell Size ; Cells, Cultured ; Dendrites/*physiology ; Excitatory Postsynaptic Potentials/genetics ; Female ; Gene Deletion ; Humans ; Nerve Net/*physiology ; Nervous System/*growth &amp; development ; Neurodevelopmental Disorders/genetics ; Pluripotent Stem Cells ; Synapses/*physiology ; ras GTPase-Activating Proteins/*genetics/*physiology ; }, abstract = {SYNGAP1 is a major genetic risk factor for global developmental delay, autism spectrum disorder, and epileptic encephalopathy. De novo loss-of-function variants in this gene cause a neurodevelopmental disorder defined by cognitive impairment, social-communication disorder, and early-onset seizures. Cell biological studies in mouse and rat neurons have shown that Syngap1 regulates developing excitatory synapse structure and function, with loss-of-function variants driving formation of larger dendritic spines and stronger glutamatergic transmission. However, studies to date have been limited to mouse and rat neurons. Therefore, it remains unknown how SYNGAP1 loss of function impacts the development and function of human neurons. To address this, we used CRISPR/Cas9 technology to ablate SYNGAP1 protein expression in neurons derived from a commercially available induced pluripotent stem cell line (hiPSC) obtained from a human female donor. Reducing SynGAP protein expression in developing hiPSC-derived neurons enhanced dendritic morphogenesis, leading to larger neurons compared with those derived from isogenic controls. Consistent with larger dendritic fields, we also observed a greater number of morphologically defined excitatory synapses in cultures containing these neurons. Moreover, neurons with reduced SynGAP protein had stronger excitatory synapses and expressed synaptic activity earlier in development. Finally, distributed network spiking activity appeared earlier, was substantially elevated, and exhibited greater bursting behavior in SYNGAP1 null neurons. We conclude that SYNGAP1 regulates the postmitotic maturation of human neurons made from hiPSCs, which influences how activity develops within nascent neural networks. Alterations to this fundamental neurodevelopmental process may contribute to the etiology of SYNGAP1-related disorders.SIGNIFICANCE STATEMENTSYNGAP1 is a major genetic risk factor for global developmental delay, autism spectrum disorder, and epileptic encephalopathy. While this gene is well studied in rodent neurons, its function in human neurons remains unknown. We used CRISPR/Cas9 technology to disrupt SYNGAP1 protein expression in neurons derived from an induced pluripotent stem cell line. We found that induced neurons lacking SynGAP expression exhibited accelerated dendritic morphogenesis, increased accumulation of postsynaptic markers, early expression of synapse activity, enhanced excitatory synaptic strength, and early onset of neural network activity. We conclude that SYNGAP1 regulates the postmitotic differentiation rate of developing human neurons and disrupting this process impacts the function of nascent neural networks. These altered developmental processes may contribute to the etiology of SYNGAP1 disorders.}, }
@article {pmid32745561, year = {2020}, author = {Yan, Y and Ziemek, J and Schetelig, MF}, title = {CRISPR/Cas9 mediated disruption of the white gene leads to pigmentation deficiency and copulation failure in Drosophila suzukii.}, journal = {Journal of insect physiology}, volume = {126}, number = {}, pages = {104091}, doi = {10.1016/j.jinsphys.2020.104091}, pmid = {32745561}, issn = {1879-1611}, mesh = {ATP-Binding Cassette Transporters/*genetics ; Animals ; CRISPR-Associated Protein 9 ; *CRISPR-Cas Systems ; Copulation ; Drosophila/*genetics/physiology ; Drosophila Proteins/*genetics ; Eye Proteins/*genetics ; Female ; Genes, Insect ; Insect Control/methods ; Male ; Mutation ; Pigmentation/*genetics ; *Sexual Behavior, Animal ; }, abstract = {The Spotted-wing Drosophila (Drosophila suzukii) is a devastating invasive pest of fruit crops. In D. melanogaster, the white (w) gene was associated with pigmentation and mating behavior, which are also important aspects to understand the invasion biology as well as to develop control strategies for D. suzukii. Here, we show that the generation of D. suzukii white-eyed mutants by CRISPR/Cas9 mutagenesis of the w gene resulted in the complete failure of copulation when w- males were individually paired with w- females in small circular arenas (diameter 0.7 cm) for 24 h. Further analysis showed that the mating defect was associated with w- males and could not be rectified by two years of inbreeding by crossing sibling w- females with w+ males, dim red illumination, male-female sexual training, changing to large arenas (diameter 3.5 cm), or different sex ratios. Profound pigmentation deficiency was detected in the compound eyes, ocelli, Malpighian tubules and testis sheaths in the w- flies. Specifically, testis imaging showed that w- males failed to deposit any pigments into pigment cells of the testis sheath, and produced smaller sperms and less seminal fluid compared to those from wildtype males. Together these observations suggest that the w gene plays an essential role in the regulation of sexual behavior and reproduction in D. suzukii. The similarities and differences in w gene function between D. suzukii and D. melanogaster in the context of pigmentation and mating behavior are discussed.}, }
@article {pmid32518161, year = {2020}, author = {Hu, L and Li, H and Chi, Z and He, J}, title = {Loss of the RNA-binding protein Rbm15 disrupts liver maturation in zebrafish.}, journal = {The Journal of biological chemistry}, volume = {295}, number = {33}, pages = {11466-11472}, pmid = {32518161}, issn = {1083-351X}, mesh = {Animals ; Apoptosis ; CRISPR-Cas Systems ; Cell Differentiation ; Cell Proliferation ; *Gene Deletion ; *Gene Expression Regulation, Developmental ; Hepatocytes/cytology/metabolism ; Liver/cytology/*embryology ; Zebrafish/*embryology/genetics ; }, abstract = {Liver organogenesis begins with hepatic precursors in the foregut endoderm, followed by hepatoblast specification, differentiation, outgrowth, and maturation for the formation of functional hepatocytes. Although several signaling pathways and critical factors that regulate liver specification, differentiation, and proliferation have been identified, little is known about how liver maturation is regulated. Here, we used a screen for mutations affecting liver development in zebrafish and identified a cq96 mutant that exhibits a specific defect in liver maturation. Results from positional cloning revealed that cq96 encodes an RNA-binding protein, Rbm15, which is an evolutionarily conserved Spen family protein and known to play a crucial role in RNA m6A modification, nuclear export, and alternative splicing. However, a function of Rbm15 in embryonic liver development has not been reported. We found that Rbm15 is specifically expressed in the liver after its differentiation. CRISPR/Cas9-mediated loss of rbm15 repressed hepatic maturation, but did not affect hepatoblast specification, differentiation, and hepatocyte proliferation and apoptosis. Additional experiments disclosed that the mTOR complex 1 (mTORC1) pathway is highly activated in rbm15-deficient hepatocytes. Moreover, rapamycin treatment partially restored normal hepatic gene expression as well as the nuclear location of the transcription factor Hnf4a. Taken together, these results reveal an unexpected role of Rbm15 in liver maturation.}, }
@article {pmid32349563, year = {2020}, author = {Tromp, TR and Stroes, ESG and Hovingh, GK}, title = {Gene-based therapy in lipid management: the winding road from promise to practice.}, journal = {Expert opinion on investigational drugs}, volume = {29}, number = {5}, pages = {483-493}, doi = {10.1080/13543784.2020.1757070}, pmid = {32349563}, issn = {1744-7658}, mesh = {Animals ; CRISPR-Cas Systems/genetics ; Cardiovascular Diseases/etiology/*prevention &amp; control ; Genetic Therapy/*methods ; Heart Disease Risk Factors ; Humans ; Hypercholesterolemia/complications/genetics/*therapy ; Lipid Metabolism/genetics ; Oligonucleotides, Antisense/administration &amp; dosage ; }, abstract = {INTRODUCTION: Cardiovascular disease (CVD) is a leading cause of morbidity and mortality. High plasma low-density lipoprotein cholesterol (LDL-C) levels are a key CVD-risk factor. Triglyceride-rich remnant particles and lipoprotein(a) (Lp[a]) are also causally related to CVD. Consequently, therapeutic strategies for lowering LDL-C and triglyceride levels are widely used in routine clinical practice; however, specific Lp(a) lowering agents are not available. Many patients do not achieve guideline-recommended lipid levels with currently available therapies; hence, novel targets and treatment modalities are eagerly sought.<br><br>AREAS COVERED: We discuss the milestones on the trajectory toward the full application of gene-based therapies in daily clinical practice. We describe the different methods, ranging from antisense oligonucleotides to liver-directed gene therapy and Crispr-cas9 modification to target the pivotal players in lipid metabolism: PCSK9, APOB, ANGPTL3, Lp(a), LDLR, and apoC-III.<br><br>EXPERT OPINION: While acknowledging their different stages of development, gene-based therapies are likely to invoke a paradigm shift in lipid management because they allow us to target previously undruggable targets. Moreover, their low dosing frequency, high target selectivity, and relatively predictable adverse event profile are considered major advantages over current lipid-lowering therapies.}, }
@article {pmid32307772, year = {2020}, author = {Chiu, YW and Hori, Y and Ebinuma, I and Sato, H and Hara, N and Ikeuchi, T and Tomita, T}, title = {Identification of calcium and integrin-binding protein 1 as a novel regulator of production of amyloid β peptide using CRISPR/Cas9-based screening system.}, journal = {FASEB journal : official publication of the Federation of American Societies for Experimental Biology}, volume = {34}, number = {6}, pages = {7661-7674}, doi = {10.1096/fj.201902966RR}, pmid = {32307772}, issn = {1530-6860}, mesh = {Alzheimer Disease/metabolism ; Amyloid Precursor Protein Secretases/metabolism ; Amyloid beta-Peptides/*metabolism ; Amyloid beta-Protein Precursor/metabolism ; Animals ; Brain/metabolism ; CRISPR-Cas Systems/*physiology ; Calcium-Binding Proteins/*metabolism ; Carrier Proteins/metabolism ; Cell Line ; Cell Line, Tumor ; Cell Membrane/metabolism ; HEK293 Cells ; Humans ; Mice ; Neurons/metabolism ; Protein Binding/physiology ; Protein Transport/physiology ; Synapsins/metabolism ; Up-Regulation/physiology ; }, abstract = {The aberrant metabolism of amyloid β peptide (Aβ) has been implicated in the etiology of Alzheimer disease (AD). Aβ is produced via the sequential cleavage of amyloid precursor protein (APP) by β- and γ-secretases. However, the precise regulatory mechanism of Aβ generation still remains unclear. To gain a better understanding of the molecular mechanism of Aβ production, we established a genetic screening method based on the CRISPR/Cas9 system to identify novel regulators of Aβ production. We successfully identified calcium and integrin-binding protein 1 (CIB1) as a potential negative regulator of Aβ production. The disruption of Cib1 significantly upregulated Aβ levels. In addition, immunoprecipitation experiments demonstrated that CIB1 interacts with the γ-secretase complex. Moreover, the disruption of Cib1 specifically reduced the cell-surface localization of mature Nicastrin (Nct), which is a component of the γ-secretase complex, without changing the intrinsic activity of γ-secretase. Finally, we confirmed using the single-cell RNA-seq data in human that CIB1 mRNA level in neuron was decreased in the early stage of AD. Taken together, our results indicate that CIB1 regulates Aβ production via controlling the subcellular localization of γ-secretase, suggesting CIB1 is involved in the development of AD.}, }
@article {pmid32302524, year = {2020}, author = {Tian, S and Liu, Y and Wu, H and Liu, H and Zeng, J and Choi, MY and Chen, H and Gerhard, R and Dong, M}, title = {Genome-Wide CRISPR Screen Identifies Semaphorin 6A and 6B as Receptors for Paeniclostridium sordellii Toxin TcsL.}, journal = {Cell host &amp; microbe}, volume = {27}, number = {5}, pages = {782-792.e7}, pmid = {32302524}, issn = {1934-6069}, support = {R21 NS106159/NS/NINDS NIH HHS/United States ; R01 NS080833/NS/NINDS NIH HHS/United States ; R01 HL146134/HL/NHLBI NIH HHS/United States ; R01 HL093242/HL/NHLBI NIH HHS/United States ; R01 AI132387/AI/NIAID NIH HHS/United States ; R01 AI139087/AI/NIAID NIH HHS/United States ; R01 HL130845/HL/NHLBI NIH HHS/United States ; P30 HD018655/HD/NICHD NIH HHS/United States ; P30 DK034854/DK/NIDDK NIH HHS/United States ; }, mesh = {A549 Cells ; Animals ; Bacterial Proteins ; Bacterial Toxins/*metabolism ; Binding Sites ; CRISPR-Cas Systems ; Cell Line, Tumor ; Clostridium sordellii/*genetics/*metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats ; Endothelial Cells/metabolism ; Female ; Gene Knockout Techniques ; HeLa Cells ; Humans ; Lung Neoplasms ; Male ; Mice ; Protein Binding ; Semaphorins/*genetics/*isolation &amp; purification/metabolism ; Virulence Factors/metabolism ; }, abstract = {The exotoxin TcsL is a major virulence factor in Paeniclostridium (Clostridium) sordellii and responsible for the high lethality rate associated with P. sordellii infection. Here, we present a genome-wide CRISPR-Cas9-mediated screen using a human lung carcinoma cell line and identify semaphorin (SEMA) 6A and 6B as receptors for TcsL. Disrupting SEMA6A/6B expression in several distinct human cell lines and primary human endothelial cells results in reduced TcsL sensitivity, while SEMA6A/6B over-expression increases their sensitivity. TcsL recognizes the extracellular domain (ECD) of SEMA6A/6B via a region homologous to the receptor-binding site in Clostridioides difficile toxin B (TcdB), which binds the human receptor Frizzled. Exchanging the receptor-binding interfaces between TcsL and TcdB switches their receptor-binding specificity. Finally, administration of SEMA6A-ECD proteins protects human cells from TcsL toxicity and reduces TcsL-induced damage to lung tissues and the lethality rate in mice. These findings establish SEMA6A and 6B as pathophysiologically relevant receptors for TcsL.}, }
@article {pmid32277852, year = {2020}, author = {Przanowska, RK and Sobierajska, E and Su, Z and Jensen, K and Przanowski, P and Nagdas, S and Kashatus, JA and Kashatus, DF and Bhatnagar, S and Lukens, JR and Dutta, A}, title = {miR-206 family is important for mitochondrial and muscle function, but not essential for myogenesis in vitro.}, journal = {FASEB journal : official publication of the Federation of American Societies for Experimental Biology}, volume = {34}, number = {6}, pages = {7687-7702}, pmid = {32277852}, issn = {1530-6860}, support = {18PRE33990261/AHA/American Heart Association-American Stroke Association/United States ; R01 AR067712/AR/NIAMS NIH HHS/United States ; T32 CA009109/CA/NCI NIH HHS/United States ; T32 GM007267/GM/NIGMS NIH HHS/United States ; }, mesh = {Animals ; CRISPR-Cas Systems/genetics ; Cell Differentiation/genetics ; Cell Line ; Cell Proliferation/genetics ; HEK293 Cells ; Humans ; Mice ; Mice, Knockout ; MicroRNAs/*genetics ; Mitochondria/*genetics ; Muscle Development/*genetics ; Muscle, Skeletal/*physiology ; Muscular Diseases/genetics ; Myoblasts, Skeletal/*physiology ; }, abstract = {miR-206, miR-1a-1, and miR-1a-2 are induced during differentiation of skeletal myoblasts and promote myogenesis in vitro. miR-206 is required for skeletal muscle regeneration in vivo. Although this miRNA family is hypothesized to play an essential role in differentiation, a triple knock-out (tKO) of the three genes has not been done to test this hypothesis. We report that tKO C2C12 myoblasts generated using CRISPR/Cas9 method differentiate despite the expected derepression of the miRNA targets. Surprisingly, their mitochondrial function is diminished. tKO mice demonstrate partial embryonic lethality, most likely due to the role of miR-1a in cardiac muscle differentiation. Two tKO mice survive and grow normally to adulthood with smaller myofiber diameter, diminished physical performance, and an increase in PAX7 positive satellite cells. Thus, unlike other miRNAs important in other differentiation pathways, the miR-206 family is not absolutely essential for myogenesis and is instead a modulator of optimal differentiation of skeletal myoblasts.}, }
@article {pmid32213923, year = {2020}, author = {Lim, KRQ and Nguyen, Q and Dzierlega, K and Huang, Y and Yokota, T}, title = {CRISPR-Generated Animal Models of Duchenne Muscular Dystrophy.}, journal = {Genes}, volume = {11}, number = {3}, pages = {}, pmid = {32213923}, issn = {2073-4425}, support = {FDN 143251//CIHR/Canada ; }, mesh = {Animals ; *CRISPR-Cas Systems ; *Disease Models, Animal ; Gene Editing/methods ; Haplorhini ; Murinae ; Muscular Dystrophy, Duchenne/*genetics/pathology ; Rabbits ; Swine ; }, abstract = {Duchenne muscular dystrophy (DMD) is a fatal X-linked recessive neuromuscular disorder most commonly caused by mutations disrupting the reading frame of the dystrophin (DMD) gene. DMD codes for dystrophin, which is critical for maintaining the integrity of muscle cell membranes. Without dystrophin, muscle cells receive heightened mechanical stress, becoming more susceptible to damage. An active body of research continues to explore therapeutic treatments for DMD as well as to further our understanding of the disease. These efforts rely on having reliable animal models that accurately recapitulate disease presentation in humans. While current animal models of DMD have served this purpose well to some extent, each has its own limitations. To help overcome this, clustered regularly interspaced short palindromic repeat (CRISPR)-based technology has been extremely useful in creating novel animal models for DMD. This review focuses on animal models developed for DMD that have been created using CRISPR, their advantages and disadvantages as well as their applications in the DMD field.}, }
@article {pmid32164255, year = {2020}, author = {Lanigan, TM and Kopera, HC and Saunders, TL}, title = {Principles of Genetic Engineering.}, journal = {Genes}, volume = {11}, number = {3}, pages = {}, pmid = {32164255}, issn = {2073-4425}, mesh = {Animals ; CRISPR-Cas Systems ; Gene Targeting/methods ; Gene Transfer Techniques ; Genetic Engineering/*methods/standards/trends ; Humans ; }, abstract = {Genetic engineering is the use of molecular biology technology to modify DNA sequence(s) in genomes, using a variety of approaches. For example, homologous recombination can be used to target specific sequences in mouse embryonic stem (ES) cell genomes or other cultured cells, but it is cumbersome, poorly efficient, and relies on drug positive/negative selection in cell culture for success. Other routinely applied methods include random integration of DNA after direct transfection (microinjection), transposon-mediated DNA insertion, or DNA insertion mediated by viral vectors for the production of transgenic mice and rats. Random integration of DNA occurs more frequently than homologous recombination, but has numerous drawbacks, despite its efficiency. The most elegant and effective method is technology based on guided endonucleases, because these can target specific DNA sequences. Since the advent of clustered regularly interspaced short palindromic repeats or CRISPR/Cas9 technology, endonuclease-mediated gene targeting has become the most widely applied method to engineer genomes, supplanting the use of zinc finger nucleases, transcription activator-like effector nucleases, and meganucleases. Future improvements in CRISPR/Cas9 gene editing may be achieved by increasing the efficiency of homology-directed repair. Here, we describe principles of genetic engineering and detail: (1) how common elements of current technologies include the need for a chromosome break to occur, (2) the use of specific and sensitive genotyping assays to detect altered genomes, and (3) delivery modalities that impact characterization of gene modifications. In summary, while some principles of genetic engineering remain steadfast, others change as technologies are ever-evolving and continue to revolutionize research in many fields.}, }
@article {pmid32100378, year = {2020}, author = {Kim, GD and Lee, JH and Song, S and Kim, SW and Han, JS and Shin, SP and Park, BC and Park, TS}, title = {Generation of myostatin-knockout chickens mediated by D10A-Cas9 nickase.}, journal = {FASEB journal : official publication of the Federation of American Societies for Experimental Biology}, volume = {34}, number = {4}, pages = {5688-5696}, doi = {10.1096/fj.201903035R}, pmid = {32100378}, issn = {1530-6860}, mesh = {Animals ; Animals, Genetically Modified/genetics/growth &amp; development/*metabolism ; *CRISPR-Cas Systems ; Chickens ; *Gene Editing ; Germ Cells/cytology/*metabolism ; Muscle, Skeletal/cytology/*metabolism ; Myostatin/antagonists &amp; inhibitors/*physiology ; Phenotype ; }, abstract = {Many studies have been conducted to improve economically important livestock traits such as feed efficiency and muscle growth. Genome editing technologies represent a major advancement for both basic research and agronomic biotechnology development. The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technical platform is a powerful tool used to engineer specific targeted loci. However, the potential occurrence of off-target effects, including the cleavage of unintended targets, limits the practical applications of Cas9-mediated genome editing. In this study, to minimize the off-target effects of this technology, we utilized D10A-Cas9 nickase to generate myostatin-knockout (MSTN KO) chickens via primordial germ cells. D10A-Cas9 nickase (Cas9n)-mediated MSTN KO chickens exhibited significantly larger skeletal muscles in the breast and leg. Degrees of skeletal muscle hypertrophy and hyperplasia induced by myostatin deletion differed by sex and muscle type. The abdominal fat deposition was dramatically lower in MSTN KO chickens than in wild-type chickens. Our results demonstrate that the D10A-Cas9 technical platform can facilitate precise and efficient targeted genome engineering and may broaden the range of applications for genome-edited chickens in practical industrialization and as animal models of human diseases.}, }
@article {pmid31999448, year = {2020}, author = {Siemon, T and Wang, Z and Bian, G and Seitz, T and Ye, Z and Lu, Y and Cheng, S and Ding, Y and Huang, Y and Deng, Z and Liu, T and Christmann, M}, title = {Semisynthesis of Plant-Derived Englerin A Enabled by Microbe Engineering of Guaia-6,10(14)-diene as Building Block.}, journal = {Journal of the American Chemical Society}, volume = {142}, number = {6}, pages = {2760-2765}, doi = {10.1021/jacs.9b12940}, pmid = {31999448}, issn = {1520-5126}, mesh = {CRISPR-Cas Systems ; Escherichia coli/genetics ; *Metabolic Engineering ; Plants/*chemistry ; Saccharomyces cerevisiae/genetics ; Sesquiterpenes, Guaiane/chemical synthesis/*chemistry ; }, abstract = {Herein, we report a short semisynthesis of the potent transient receptor potential canonical (TRPC) channel agonist englerin A (EA) and the related guaianes oxyphyllol and orientalol E. The guaia-6,10(14)-diene starting material was systematically engineered in Escherichia coli and Saccharomyces cerevisiae using the CRISPR/Cas9 system and was produced with high titers. The potentially scalable approach combines the advantages of synthetic biology and chemical synthesis providing an efficient and economical method for producing EA and analogues.}, }
@article {pmid31931564, year = {2020}, author = {Alyami, MZ and Alsaiari, SK and Li, Y and Qutub, SS and Aleisa, FA and Sougrat, R and Merzaban, JS and Khashab, NM}, title = {Cell-Type-Specific CRISPR/Cas9 Delivery by Biomimetic Metal Organic Frameworks.}, journal = {Journal of the American Chemical Society}, volume = {142}, number = {4}, pages = {1715-1720}, doi = {10.1021/jacs.9b11638}, pmid = {31931564}, issn = {1520-5126}, mesh = {Animals ; *Biomimetics ; *CRISPR-Cas Systems ; HeLa Cells ; Heterografts ; Humans ; MCF-7 Cells ; Metal-Organic Frameworks/*chemistry ; Mice ; }, abstract = {Effective and cell-type-specific delivery of CRISPR/Cas9 gene editing elements remains a challenging open problem. Here we report the development of biomimetic cancer cell coated zeolitic imidazolate frameworks (ZIFs) for targeted and cell-specific delivery of this genome editing machinery. Coating ZIF-8 that is encapsulating CRISPR/Cas9 (CC-ZIF) with a cancer cell membrane resulted in the uniformly covered C3-ZIF(cell membrane type). Incubation of C3-ZIFMCF with MCF-7, HeLa, HDFn, and aTC cell lines showed the highest uptake by MCF-7 cells and negligible uptake by the healthy cells (i.e., HDFn and aTC). As to genome editing, a 3-fold repression in the EGFP expression was observed when MCF-7 were transfected with C3-ZIFMCF compared to 1-fold repression in the EGFP expression when MCF-7 were transfected with C3-ZIFHELA. In vivo testing confirmed the selectivity of C3-ZIFMCF to accumulate in MCF-7 tumor cells. This supports the ability of this biomimetic approach to match the needs of cell-specific targeting, which is unquestionably the most critical step in the future translation of genome editing technologies.}, }
@article {pmid31893458, year = {2020}, author = {Bao, A and Tran, LP and Cao, D}, title = {CRISPR/Cas9-Based Gene Editing in Soybean.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2107}, number = {}, pages = {349-364}, doi = {10.1007/978-1-0716-0235-5_19}, pmid = {31893458}, issn = {1940-6029}, mesh = {Agrobacterium/genetics ; CRISPR-Cas Systems ; Cotyledon/genetics/*growth &amp; development ; Gene Editing/*methods ; Mutation ; Plant Breeding ; Soybeans/genetics/*growth &amp; development ; Tissue Culture Techniques ; Transformation, Genetic ; }, abstract = {CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat/CRISPR associated Cas9)-based gene editing is a robust tool for functional genomics research and breeding programs in various crops. In soybean, a number of laboratories have obtained mutants by CRISPR/Cas9 system; however, there has been not yet a detailed method for the CRISPR/Cas9-based gene editing in soybean. Here, we describe the procedures for constructing the CRISPR/Cas9 plasmid suitable for soybean gene editing and the modified protocols for Agrobacterium-mediated soybean transformation and regeneration from cotyledonary node explants containing the Cas9/sgRNA (single guide RNA) transgenes.}, }
@article {pmid31882399, year = {2020}, author = {Ianiri, G and Fang, YF and Dahlmann, TA and Clancey, SA and Janbon, G and Kück, U and Heitman, J}, title = {Mating-Type-Specific Ribosomal Proteins Control Aspects of Sexual Reproduction in Cryptococcus neoformans.}, journal = {Genetics}, volume = {214}, number = {3}, pages = {635-649}, pmid = {31882399}, issn = {1943-2631}, support = {R01 AI039115/AI/NIAID NIH HHS/United States ; R01 AI050113/AI/NIAID NIH HHS/United States ; R37 AI039115/AI/NIAID NIH HHS/United States ; }, mesh = {Alleles ; CRISPR-Cas Systems/genetics ; Cryptococcus neoformans/*genetics/growth &amp; development ; Fungal Proteins/genetics ; Genes, Mating Type, Fungal/*genetics ; Haploidy ; Phenotype ; Reproduction/*genetics ; Ribosomal Proteins/*genetics ; }, abstract = {The MAT locus of Cryptococcus neoformans has a bipolar organization characterized by an unusually large structure, spanning over 100 kb. MAT genes have been characterized by functional genetics as being involved in sexual reproduction and virulence. However, classical gene replacement failed to achieve mutants for five MAT genes (RPL22, RPO41, MYO2, PRT1, and RPL39), indicating that they are likely essential. In the present study, targeted gene replacement was performed in a diploid strain for both the α and a alleles of the ribosomal genes RPL22 and RPL39 Mendelian analysis of the progeny confirmed that both RPL22 and RPL39 are essential for viability. Ectopic integration of the RPL22 allele of opposite MAT identity in the heterozygous RPL22a/rpl22αΔ or RPL22α/rpl22aΔ mutant strains failed to complement their essential phenotype. Evidence suggests that this is due to differential expression of the RPL22 genes, and an RNAi-dependent mechanism that contributes to control RPL22a expression. Furthermore, via CRISPR/Cas9 technology, the RPL22 alleles were exchanged in haploid MATα and MATa strains of C. neoformans These RPL22 exchange strains displayed morphological and genetic defects during bilateral mating. These results contribute to elucidating functions of C. neoformans essential mating type genes that may constitute a type of imprinting system to promote inheritance of nuclei of both mating types.}, }
@article {pmid33453283, year = {2021}, author = {Koay, TW and Osterhof, C and Orlando, IMC and Keppner, A and Andre, D and Yousefian, S and Alonso, MS and Correia, M and Markworth, R and Schödel, J and Hankeln, T and Hoogewijs, D}, title = {Androglobin gene expression patterns and FOXJ1-dependent regulation indicate its functional association with ciliogenesis.}, journal = {The Journal of biological chemistry}, volume = {}, number = {}, pages = {100291}, doi = {10.1016/j.jbc.2021.100291}, pmid = {33453283}, issn = {1083-351X}, abstract = {Androglobin (ADGB) represents the latest addition to the globin superfamily in metazoans. The chimeric protein comprises a calpain domain and a unique circularly permutated globin domain. ADGB expression levels are most abundant in mammalian testis, but its cell type-specific expression, regulation and function have remained unexplored. Analyzing bulk and single-cell mRNA-Seq data from mammalian tissues, we found that -in addition to testes- ADGB is prominently expressed in the female reproductive tract, lungs and brain, specifically being associated with cell types forming motile cilia. Correlation analysis suggested co-regulation of ADGB with FOXJ1, a crucial transcription factor of ciliogenesis. Investigating the transcriptional regulation of the ADGB gene, we characterized its promoter using epigenomic datasets, exogenous promoter-dependent luciferase assays and CRISPR/dCas9-VPR-mediated activation approaches. Reporter gene assays revealed that FOXJ1 indeed substantially enhanced luciferase activity driven by the ADGB promoter. ChIP assays confirmed binding of FOXJ1 to the endogenous ADGB promoter region. We dissected the minimal sequence required for FOXJ1-dependent regulation and fine mapped the FOXJ1 binding site to two evolutionarily conserved regions within the ADGB promoter. FOXJ1 overexpression significantly increased endogenous ADGB mRNA levels in HEK293 and MCF-7 cells. Similar results were observed upon RFX2 overexpression, another key transcription factor in ciliogenesis. The complex transcriptional regulation of the ADGB locus was illustrated by identifying a distal enhancer, responsible for synergistic regulation by RFX2 and FOXJ1. Finally, cell culture studies indicated an ADGB-dependent increase in the number of ciliated cells upon overexpression of the full-length protein, confirming a ciliogenesis-associated role of ADGB in mammals.}, }
@article {pmid33452819, year = {2021}, author = {Barbour, A and Glogauer, J and Grinfeld, L and Ostadsharif Memar, R and Fine, N and Tenenbaum, H and Glogauer, M}, title = {The role of CRISPR-Cas in advancing precision periodontics.}, journal = {Journal of periodontal research}, volume = {}, number = {}, pages = {}, doi = {10.1111/jre.12846}, pmid = {33452819}, issn = {1600-0765}, abstract = {The significant advancement of molecular biology has revolutionized medicine and provided important technologies to further clinical research development. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) are DNA sequences derived from bacteriophages which have previously infected the bacterial species. The CRISPR-Cas system plays a key role in bacterial defense by detecting and destroying DNA fragments during subsequent bacteriophage invasions. The Cas9 enzyme recognizes and cleaves new invading CRISPR-complementary DNA sequences. Researchers have taken advantage of this biological device to manipulate microbes' genes and develop novel therapeutics to tackle systemic disease. In this review, we discuss the potential of utilizing CRISPR-Cas systems in the periodontal field to develop personalized periodontal care. We summarize promising attempts to bring this technology to the clinical setting. Finally, we provide insights regarding future developments to best utilize the CRISPR-Cas systems to advance precision periodontics. Although further research is imperative to evaluate the safety and potential of using CRISPR-Cas to develop precision periodontics approaches, few studies showed promising data to support the investment into this important technology in the dental sector. CRISPR-Cas9 can be a useful tool to create knockouts in vitro and in vivo as a screening tool to identify cellular pathways involved in the pathogenesis of periodontitis. Alternative CRISPR systems such as CRISPRa, CRISPRi, and Cas13 can be used to modify the transcriptome and gene expression of genes involved in periodontitis progression. CRISPR systems such as Cas3 can be used to target the periodontal biofilm and to develop new strategies to reduce or eliminate periodontal pathogens. Currently, the utility of CRISPR-Cas applications in clinical settings is limited. Through this review, we hope to foster further discussion in the periodontal research and clinical communities with respect to the potential clinical application of novel, CRISPR-Cas based, therapeutics for periodontitis.}, }
@article {pmid33449167, year = {2021}, author = {Brandes, RP and Dueck, A and Engelhardt, S and Kaulich, M and Kupatt, C and De Angelis, MT and Leisegang, MS and le Noble, F and Moretti, A and Müller, OJ and Skryabin, BV and Thum, T and Wurst, W}, title = {DGK and DZHK position paper on genome editing: basic science applications and future perspective.}, journal = {Basic research in cardiology}, volume = {116}, number = {1}, pages = {2}, pmid = {33449167}, issn = {1435-1803}, abstract = {For a long time, gene editing had been a scientific concept, which was limited to a few applications. With recent developments, following the discovery of TALEN zinc-finger endonucleases and in particular the CRISPR/Cas system, gene editing has become a technique applicable in most laboratories. The current gain- and loss-of function models in basic science are revolutionary as they allow unbiased screens of unprecedented depth and complexity and rapid development of transgenic animals. Modifications of CRISPR/Cas have been developed to precisely interrogate epigenetic regulation or to visualize DNA complexes. Moreover, gene editing as a clinical treatment option is rapidly developing with first trials on the way. This article reviews the most recent progress in the field, covering expert opinions gathered during joint conferences on genome editing of the German Cardiac Society (DGK) and the German Center for Cardiovascular Research (DZHK). Particularly focusing on the translational aspect and the combination of cellular and animal applications, the authors aim to provide direction for the development of the field and the most frequent applications with their problems.}, }
@article {pmid33338421, year = {2021}, author = {Hoffmann, HH and Schneider, WM and Rozen-Gagnon, K and Miles, LA and Schuster, F and Razooky, B and Jacobson, E and Wu, X and Yi, S and Rudin, CM and MacDonald, MR and McMullan, LK and Poirier, JT and Rice, CM}, title = {TMEM41B Is a Pan-flavivirus Host Factor.}, journal = {Cell}, volume = {184}, number = {1}, pages = {133-148.e20}, doi = {10.1016/j.cell.2020.12.005}, pmid = {33338421}, issn = {1097-4172}, support = {R01 AI124690/AI/NIAID NIH HHS/United States ; P30 CA008748/CA/NCI NIH HHS/United States ; }, mesh = {Animals ; Asian Continental Ancestry Group/genetics ; Autophagy ; COVID-19/genetics/metabolism/virology ; CRISPR-Cas Systems ; Cell Line ; Flavivirus/*physiology ; Flavivirus Infections/*genetics/immunology/metabolism/virology ; Gene Knockout Techniques ; Genome-Wide Association Study ; Host-Pathogen Interactions ; Humans ; Immunity, Innate ; Membrane Proteins/genetics/*metabolism ; Polymorphism, Single Nucleotide ; SARS-CoV-2/physiology ; Virus Replication ; Yellow fever virus/physiology ; Zika Virus/physiology ; }, abstract = {Flaviviruses pose a constant threat to human health. These RNA viruses are transmitted by the bite of infected mosquitoes and ticks and regularly cause outbreaks. To identify host factors required for flavivirus infection, we performed full-genome loss of function CRISPR-Cas9 screens. Based on these results, we focused our efforts on characterizing the roles that TMEM41B and VMP1 play in the virus replication cycle. Our mechanistic studies on TMEM41B revealed that all members of the Flaviviridae family that we tested require TMEM41B. We tested 12 additional virus families and found that SARS-CoV-2 of the Coronaviridae also required TMEM41B for infection. Remarkably, single nucleotide polymorphisms present at nearly 20% in East Asian populations reduce flavivirus infection. Based on our mechanistic studies, we propose that TMEM41B is recruited to flavivirus RNA replication complexes to facilitate membrane curvature, which creates a protected environment for viral genome replication.}, }
@article {pmid33139551, year = {2020}, author = {Takao, T and Sato, M and Maruyama, T}, title = {Optogenetic regulation of embryo implantation in mice using photoactivatable CRISPR-Cas9.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {117}, number = {46}, pages = {28579-28581}, pmid = {33139551}, issn = {1091-6490}, mesh = {Animals ; CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; *Embryo Implantation ; Fertility ; Leukemia Inhibitory Factor/genetics/*metabolism ; Mice, Inbred ICR ; *Optogenetics ; }, abstract = {Embryo implantation is achieved upon successful interaction between a fertilized egg and receptive endometrium and is mediated by spatiotemporal expression of implantation-associated molecules including leukemia inhibitory factor (LIF). Here we demonstrate, in mice, that LIF knockdown via a photoactivatable CRISPR-Cas9 gene editing system and illumination with a light-emitting diode can spatiotemporally disrupt fertility. This system enables dissection of spatiotemporal molecular mechanisms associated with embryo implantation and provides a therapeutic strategy for temporal control of reproductive functions in vivo.}, }
@article {pmid33075436, year = {2020}, author = {Murphy, ZC and Getman, MR and Myers, JA and Burgos Villar, KN and Leshen, E and Kurita, R and Nakamura, Y and Steiner, LA}, title = {Codanin-1 mutations engineered in human erythroid cells demonstrate role of CDAN1 in terminal erythroid maturation.}, journal = {Experimental hematology}, volume = {91}, number = {}, pages = {32-38.e6}, pmid = {33075436}, issn = {1873-2399}, support = {R01 DK104920/DK/NIDDK NIH HHS/United States ; }, mesh = {Acetylation ; Anemia, Dyserythropoietic, Congenital/blood/*genetics ; CRISPR-Cas Systems ; Cell Line ; Cell Nucleus/ultrastructure ; Cell Survival ; Chromatin/ultrastructure ; Erythroid Cells/*cytology/metabolism ; Erythropoiesis/*genetics/physiology ; Exons/genetics ; Gene Editing ; Glycoproteins/deficiency/*genetics/physiology ; Histone Code ; Humans ; Nuclear Proteins/deficiency/*genetics/physiology ; Phenotype ; Protein Processing, Post-Translational ; }, abstract = {The generation of a functional erythrocyte from a committed progenitor requires significant changes in gene expression during hemoglobin accumulation, rapid cell division, and nuclear condensation. Congenital dyserythropoietic anemia type I (CDA-I) is an autosomal recessive disease that presents with erythroid hyperplasia in the bone marrow. Erythroblasts in patients with CDA-I are frequently binucleate and have chromatin bridging and defective chromatin condensation. CDA-1 is most commonly caused by mutations in Codanin-1 (CDAN1). The function of CDAN1 is poorly understood but it is thought to regulate histone incorporation into nascent DNA during cellular replication. The study of CDA-1 has been limited by the lack of in vitro models that recapitulate key features of the disease, and most studies on CDAN1 function have been done in nonerythroid cells. To model CDA-I we generated HUDEP2 mutant lines with deletion or mutation of R1042 of CDAN1, mirroring mutations found in CDA-1 patients. CDAN1 mutant cell lines had decreased viability and increased intercellular bridges and binucleate cells. Further, they had alterations in histone acetylation associated with prematurely elevated erythroid gene expression, including gamma globin. Together, these data imply a specific functional role for CDAN1, specifically R1042 on exon 24, in the regulation of DNA replication and organization during erythroid maturation. Most importantly, generation of models with specific patient mutations, such as R1042, will provide further mechanistic insights into CDA-I pathology.}, }
@article {pmid33020616, year = {2020}, author = {Matharu, N and Ahituv, N}, title = {Modulating gene regulation to treat genetic disorders.}, journal = {Nature reviews. Drug discovery}, volume = {19}, number = {11}, pages = {757-775}, doi = {10.1038/s41573-020-0083-7}, pmid = {33020616}, issn = {1474-1784}, mesh = {Animals ; CRISPR-Cas Systems/drug effects/genetics ; Gene Expression Regulation/*drug effects/genetics ; Genetic Diseases, Inborn/*drug therapy/genetics ; Humans ; Mutation/drug effects/genetics ; Pharmaceutical Preparations/*administration &amp; dosage ; }, abstract = {Over a thousand diseases are caused by mutations that alter gene expression levels. The potential of nuclease-deficient zinc fingers, TALEs or CRISPR fusion systems to treat these diseases by modulating gene expression has recently emerged. These systems can be applied to modify the activity of gene-regulatory elements - promoters, enhancers, silencers and insulators, subsequently changing their target gene expression levels to achieve therapeutic benefits - an approach termed cis-regulation therapy (CRT). Here, we review emerging CRT technologies and assess their therapeutic potential for treating a wide range of diseases caused by abnormal gene dosage. The challenges facing the translation of CRT into the clinic are discussed.}, }
@article {pmid33020605, year = {2020}, author = {Leech, R and Sampath, K}, title = {A CRISPR cut for messenger RNAs.}, journal = {Lab animal}, volume = {49}, number = {11}, pages = {317-319}, doi = {10.1038/s41684-020-00661-3}, pmid = {33020605}, issn = {1548-4475}, mesh = {Animals ; CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; RNA, Messenger/genetics ; }, }
@article {pmid32601291, year = {2020}, author = {Kamble, PG and Hetty, S and Vranic, M and Almby, K and Castillejo-López, C and Abalo, XM and Pereira, MJ and Eriksson, JW}, title = {Proof-of-concept for CRISPR/Cas9 gene editing in human preadipocytes: Deletion of FKBP5 and PPARG and effects on adipocyte differentiation and metabolism.}, journal = {Scientific reports}, volume = {10}, number = {1}, pages = {10565}, pmid = {32601291}, issn = {2045-2322}, mesh = {Adipocytes/*metabolism ; Adipogenesis/genetics ; Adipose Tissue/metabolism ; Adult ; Aged ; CRISPR-Cas Systems/genetics ; Cell Differentiation/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Female ; Gene Editing/*methods ; Gene Knockout Techniques/methods ; Humans ; Middle Aged ; PPAR gamma/genetics ; Proof of Concept Study ; Tacrolimus Binding Proteins/genetics ; }, abstract = {CRISPR/Cas9 has revolutionized the genome-editing field. So far, successful application in human adipose tissue has not been convincingly shown. We present a method for gene knockout using electroporation in preadipocytes from human adipose tissue that achieved at least 90% efficiency without any need for selection of edited cells or clonal isolation. We knocked out the FKBP5 and PPARG genes in preadipocytes and studied the resulting phenotypes. PPARG knockout prevented differentiation into adipocytes. Conversely, deletion of FKBP51, the protein coded by the FKBP5 gene, did not affect adipogenesis. Instead, it markedly modulated glucocorticoid effects on adipocyte glucose metabolism and, furthermore, we show some evidence of altered transcriptional activity of glucocorticoid receptors. This has potential implications for the development of insulin resistance and type 2 diabetes. The reported method is simple, easy to adapt, and enables the use of human primary preadipocytes instead of animal adipose cell models to assess the role of key genes and their products in adipose tissue development, metabolism and pathobiology.}, }
@article {pmid32576837, year = {2020}, author = {Lamsfus-Calle, A and Daniel-Moreno, A and Antony, JS and Epting, T and Heumos, L and Baskaran, P and Admard, J and Casadei, N and Latifi, N and Siegmund, DM and Kormann, MSD and Handgretinger, R and Mezger, M}, title = {Comparative targeting analysis of KLF1, BCL11A, and HBG1/2 in CD34+ HSPCs by CRISPR/Cas9 for the induction of fetal hemoglobin.}, journal = {Scientific reports}, volume = {10}, number = {1}, pages = {10133}, pmid = {32576837}, issn = {2045-2322}, mesh = {Anemia, Sickle Cell/*genetics/therapy ; Antigens, CD34 ; *CRISPR-Cas Systems ; Cells, Cultured ; Fetal Hemoglobin/*genetics ; Gene Editing/*methods ; Gene Expression/genetics ; Humans ; Kruppel-Like Transcription Factors/*genetics ; Molecular Targeted Therapy ; Mutation ; Repressor Proteins/*genetics ; gamma-Globins/*genetics ; }, abstract = {β-hemoglobinopathies are caused by abnormal or absent production of hemoglobin in the blood due to mutations in the β-globin gene (HBB). Imbalanced expression of adult hemoglobin (HbA) induces strong anemia in patients suffering from the disease. However, individuals with natural-occurring mutations in the HBB cluster or related genes, compensate this disparity through γ-globin expression and subsequent fetal hemoglobin (HbF) production. Several preclinical and clinical studies have been performed in order to induce HbF by knocking-down genes involved in HbF repression (KLF1 and BCL11A) or disrupting the binding sites of several transcription factors in the γ-globin gene (HBG1/2). In this study, we thoroughly compared the different CRISPR/Cas9 gene-disruption strategies by gene editing analysis and assessed their safety profile by RNA-seq and GUIDE-seq. All approaches reached therapeutic levels of HbF after gene editing and showed similar gene expression to the control sample, while no significant off-targets were detected by GUIDE-seq. Likewise, all three gene editing platforms were established in the GMP-grade CliniMACS Prodigy, achieving similar outcome to preclinical devices. Based on this gene editing comparative analysis, we concluded that BCL11A is the most clinically relevant approach while HBG1/2 could represent a promising alternative for the treatment of β-hemoglobinopathies.}, }
@article {pmid32527526, year = {2020}, author = {Yang, Y and Liu, G and Chen, X and Liu, M and Zhan, C and Liu, X and Bai, Z}, title = {High efficiency CRISPR/Cas9 genome editing system with an eliminable episomal sgRNA plasmid in Pichia pastoris.}, journal = {Enzyme and microbial technology}, volume = {138}, number = {}, pages = {109556}, doi = {10.1016/j.enzmictec.2020.109556}, pmid = {32527526}, issn = {1879-0909}, mesh = {CRISPR-Associated Protein 9/genetics/metabolism ; *CRISPR-Cas Systems ; Fungal Proteins/genetics ; Gene Editing/*methods ; Gene Expression Regulation, Fungal ; Genome, Fungal/genetics ; Nucleotide Motifs ; Plasmids/*genetics ; Promoter Regions, Genetic/genetics ; RNA, Guide/chemistry/*genetics ; Saccharomycetales/*genetics/growth &amp; development ; }, abstract = {Pichia pastoris is a methylotrophic yeast in which host heterologous expression of proteins has been developed owing to the strong inducible alcohol oxidase promoter (PAOX1). However, it is difficult to manipulate the genome in P. pastoris. Based on previous attempts to apply the CRISPR/Cas9 system in P. pastoris, a CRISPR/Cas9 system with episomal sgRNA plasmid was developed and 100 % genome editing efficiency, high multicopy gene editing and stable multigene editing were obtained without a sharp decline caused by multi-sgRNA. And 28/34 (∼82 %) sgRNAs tested were effective. The CGG may have a slightly higher and more stable cleavage efficiency than the other three NGG motifs, and a low GC content may be preferable for higher cleavage efficiency. This provides researchers with a stable genome editing tool that shows a high editing efficiency, shortening the experimentation period. Furthermore, we introduced dCas9 into P. pastoris and achieved target gene interference, expanding the CRISPR/Cas9 toolbox in P. pastoris.}, }
@article {pmid32321775, year = {2020}, author = {Kagoya, Y and Guo, T and Yeung, B and Saso, K and Anczurowski, M and Wang, CH and Murata, K and Sugata, K and Saijo, H and Matsunaga, Y and Ohashi, Y and Butler, MO and Hirano, N}, title = {Genetic Ablation of HLA Class I, Class II, and the T-cell Receptor Enables Allogeneic T Cells to Be Used for Adoptive T-cell Therapy.}, journal = {Cancer immunology research}, volume = {8}, number = {7}, pages = {926-936}, doi = {10.1158/2326-6066.CIR-18-0508}, pmid = {32321775}, issn = {2326-6074}, mesh = {Allografts ; Animals ; Antigens, CD19/immunology ; CRISPR-Cas Systems ; Cells, Cultured ; Disease Models, Animal ; Histocompatibility Antigens Class I/*chemistry/genetics ; Histocompatibility Antigens Class II/*chemistry/genetics ; Humans ; Immunotherapy, Adoptive/*methods ; Leukocytes, Mononuclear ; Lymphocyte Activation ; Mice ; Mice, Inbred NOD ; Mice, SCID ; Neoplasms/immunology/metabolism/*therapy ; Receptors, Antigen, T-Cell/antagonists &amp; inhibitors/genetics/*immunology ; Receptors, Chimeric Antigen/*immunology ; }, abstract = {Adoptive immunotherapy can induce sustained therapeutic effects in some cancers. Antitumor T-cell grafts are often individually prepared in vitro from autologous T cells, which requires an intensive workload and increased costs. The quality of the generated T cells can also be variable, which affects the therapy's antitumor efficacy and toxicity. Standardized production of antitumor T-cell grafts from third-party donors will enable widespread use of this modality if allogeneic T-cell responses are effectively controlled. Here, we generated HLA class I, HLA class II, and T-cell receptor (TCR) triple-knockout (tKO) T cells by simultaneous knockout of the B2M, CIITA, and TRAC genes through Cas9/sgRNA ribonucleoprotein electroporation. Although HLA-deficient T cells were targeted by natural killer cells, they persisted better than HLA-sufficient T cells in the presence of allogeneic peripheral blood mononuclear cells (PBMC) in immunodeficient mice. When transduced with a CD19 chimeric antigen receptor (CAR) and stimulated by tumor cells, tKO CAR-T cells persisted better when cultured with allogeneic PBMCs compared with TRAC and B2M double-knockout T cells. The CD19 tKO CAR-T cells did not induce graft-versus-host disease but retained antitumor responses. These results demonstrated the benefit of HLA class I, HLA class II, and TCR deletion in enabling allogeneic-sourced T cells to be used for off-the-shelf adoptive immunotherapy.}, }
@article {pmid33445135, year = {2021}, author = {Zuo, F and Marcotte, H}, title = {Advancing mechanistic understanding and bioengineering of probiotic lactobacilli and bifidobacteria by genome editing.}, journal = {Current opinion in biotechnology}, volume = {70}, number = {}, pages = {75-82}, doi = {10.1016/j.copbio.2020.12.015}, pmid = {33445135}, issn = {1879-0429}, abstract = {Typical traditional probiotics lactobacilli and bifidobacteria are gaining great interest to be developed as living diagnostics and therapeutics for improving human health. However, the mechanistic basis underlying their inherent health beneficial property remain incompletely understood which can slow down the translational pipeline in the functional food and pharmaceutical field. Efficient genome editing will advance the understanding of the molecular mechanism of the probiotics' physiological properties and their interaction with the host and the host microbiota, thereby further promote the development of next-generation designer probiotics with improved robustness and tailored functionalities. With the expansion of genome editing strategies such as CRISPR-Cas-based tools and IPSD assisted genome engineering as well as other synthetic biology technologies, the research and application of these health-promoting bacteria for the food and pharmaceutical industry will be further enhanced.}, }
@article {pmid33444542, year = {2020}, author = {Li, Y and Bondy-Denomy, J}, title = {Anti-CRISPRs go viral: the infection biology of CRISPR-Cas inhibitors.}, journal = {Cell host &amp; microbe}, volume = {}, number = {}, pages = {}, doi = {10.1016/j.chom.2020.12.007}, pmid = {33444542}, issn = {1934-6069}, abstract = {Bacteriophages encode diverse anti-CRISPR (Acr) proteins that inhibit CRISPR-Cas immunity during infection of their bacterial hosts. Although detailed mechanisms have been characterized for multiple Acr proteins, an understanding of their role in phage infection biology is just emerging. Here, we review recent work in this area and propose a framework of &quot;phage autonomy&quot; to evaluate CRISPR-immune evasion strategies. During phage infection, Acr proteins are deployed by a tightly regulated &quot;fast on-fast off&quot; transcriptional burst, which is necessary, but insufficient, for CRISPR-Cas inactivation. Instead of a single phage shutting down CRISPR-Cas immunity, a community of acr-carrying phages cooperate to suppress bacterial immunity, displaying low phage autonomy. Enzymatic Acr proteins with novel mechanisms have been recently revealed and are predicted to enhance phage autonomy, while phage DNA protective measures offer the highest phage autonomy observed. These varied Acr mechanisms and strengths also have unexpected impacts on the bacterial populations and competing phages.}, }
@article {pmid33443157, year = {2021}, author = {Kurtz, S and Lucas-Hahn, A and Schlegelberger, B and Göhring, G and Niemann, H and Mettenleiter, TC and Petersen, B}, title = {Knockout of the HMG domain of the porcine SRY gene causes sex reversal in gene-edited pigs.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {118}, number = {2}, pages = {}, doi = {10.1073/pnas.2008743118}, pmid = {33443157}, issn = {1091-6490}, abstract = {The sex-determining region on the Y chromosome (SRY) is thought to be the central genetic element of male sex development in mammals. Pathogenic modifications within the SRY gene are associated with a male-to-female sex reversal syndrome in humans and other mammalian species, including rabbits and mice. However, the underlying mechanisms are largely unknown. To understand the biological function of the SRY gene, a site-directed mutational analysis is required to investigate associated phenotypic changes at the molecular, cellular, and morphological level. Here, we successfully generated a knockout of the porcine SRY gene by microinjection of two CRISPR-Cas ribonucleoproteins, targeting the centrally located &quot;high mobility group&quot; (HMG), followed by a frameshift mutation of the downstream SRY sequence. This resulted in the development of genetically male (XY) pigs with complete external and internal female genitalia, which, however, were significantly smaller than in 9-mo-old age-matched control females. Quantitative digital PCR analysis revealed a duplication of the SRY locus in Landrace pigs similar to the known palindromic duplication in Duroc breeds. Our study demonstrates the central role of the HMG domain in the SRY gene in male porcine sex determination. This proof-of-principle study could assist in solving the problem of sex preference in agriculture to improve animal welfare. Moreover, it establishes a large animal model that is more comparable to humans with regard to genetics, physiology, and anatomy, which is pivotal for longitudinal studies to unravel mammalian sex determination and relevant for the development of new interventions for human sex development disorders.}, }
@article {pmid33441159, year = {2021}, author = {Scott, TA and Morris, KV}, title = {Designer nucleases to treat malignant cancers driven by viral oncogenes.}, journal = {Virology journal}, volume = {18}, number = {1}, pages = {18}, pmid = {33441159}, issn = {1743-422X}, support = {R01 113407-01/MH/NIMH NIH HHS/United States ; }, abstract = {Viral oncogenic transformation of healthy cells into a malignant state is a well-established phenomenon but took decades from the discovery of tumor-associated viruses to their accepted and established roles in oncogenesis. Viruses cause ~ 15% of know cancers and represents a significant global health burden. Beyond simply causing cellular transformation into a malignant form, a number of these cancers are augmented by a subset of viral factors that significantly enhance the tumor phenotype and, in some cases, are locked in a state of oncogenic addiction, and substantial research has elucidated the mechanisms in these cancers providing a rationale for targeted inactivation of the viral components as a treatment strategy. In many of these virus-associated cancers, the prognosis remains extremely poor, and novel drug approaches are urgently needed. Unlike non-specific small-molecule drug screens or the broad-acting toxic effects of chemo- and radiation therapy, the age of designer nucleases permits a rational approach to inactivating disease-causing targets, allowing for permanent inactivation of viral elements to inhibit tumorigenesis with growing evidence to support their efficacy in this role. Although many challenges remain for the clinical application of designer nucleases towards viral oncogenes; the uniqueness and clear molecular mechanism of these targets, combined with the distinct advantages of specific and permanent inactivation by nucleases, argues for their development as next-generation treatments for this aggressive group of cancers.}, }
@article {pmid32868402, year = {2020}, author = {Guan, J and Bondy-Denomy, J}, title = {Intracellular Organization by Jumbo Bacteriophages.}, journal = {Journal of bacteriology}, volume = {203}, number = {2}, pages = {}, pmid = {32868402}, issn = {1098-5530}, abstract = {Since their discovery more than 100 years ago, the viruses that infect bacteria (bacteriophages) have been widely studied as model systems. Largely overlooked, however, have been &quot;jumbo phages,&quot; with genome sizes ranging from 200 to 500 kbp. Jumbo phages generally have large virions with complex structures and a broad host spectrum. While the majority of jumbo phage genes are poorly functionally characterized, recent work has discovered many unique biological features, including a conserved tubulin homolog that coordinates a proteinaceous nucleus-like compartment that houses and segregates phage DNA. The tubulin spindle displays dynamic instability and centers the phage nucleus within the bacterial host during phage infection for optimal reproduction. The shell provides robust physical protection for the enclosed phage genomes against attack from DNA-targeting bacterial immune systems, thereby endowing jumbo phages with broad resistance. In this review, we focus on the current knowledge of the cytoskeletal elements and the specialized nuclear compartment derived from jumbo phages, and we highlight their importance in facilitating spatial and temporal organization over the viral life cycle. Additionally, we discuss the evolutionary relationships between jumbo phages and eukaryotic viruses, as well as the therapeutic potential and drawbacks of jumbo phages as antimicrobial agents in phage therapy.}, }
@article {pmid32471865, year = {2020}, author = {Das, S and Banerjee, A and Kamran, M and Ejazi, SA and Asad, M and Ali, N and Chakrabarti, S}, title = {A chemical inhibitor of heat shock protein 78 (HSP78) from Leishmania donovani represents a potential antileishmanial drug candidate.}, journal = {The Journal of biological chemistry}, volume = {295}, number = {29}, pages = {9934-9947}, pmid = {32471865}, issn = {1083-351X}, mesh = {Animals ; Antiprotozoal Agents/*pharmacology ; CRISPR-Cas Systems ; Cricetinae ; Dinucleoside Phosphates/*pharmacology ; Gene Knockout Techniques ; Heat-Shock Proteins/*antagonists &amp; inhibitors/genetics/metabolism ; Humans ; Leishmania donovani/genetics/*metabolism ; Leishmaniasis, Visceral/*drug therapy/genetics/metabolism ; Macrophages/metabolism/*parasitology ; Mice ; Protozoan Proteins/*antagonists &amp; inhibitors/genetics/metabolism ; }, abstract = {The emergence of resistance to available antileishmanial drugs advocates identification of new drug targets and their inhibitors for visceral leishmaniasis. Here, we identified Leishmania donovani heat shock protein 78 (LdHSP78), a putative caseinolytic protease, as important for parasite infection of host macrophages and a potential therapeutic target. Enrichment of LdHSP78 in infected humans, hamsters, and parasite amastigotes suggested its importance for disease persistence. Heterozygous knockouts of L. donovani HSP78 (LdHSP78+/-) and Leishmania mexicana HSP78 (LmxHSP78+/-) were generated using a flanking UTR-based multifragment ligation strategy and the CRISPR-Cas9 technique, respectively to investigate the significance of HSP78 for disease manifestation. The LdHSP78+/- parasite burden was dramatically reduced in both murine bone marrow-derived macrophages and hamsters, in association with enrichment of proinflammatory cytokines and NO. This finding implies that LdHSP78+/- parasites cannot suppress immune activation and escape NO-mediated toxicity in macrophages. Furthermore, phosphorylation of the mitogen-activated protein kinase p38 was enhanced and phosphorylation of extracellular signal-regulated kinase 1/2 was decreased in cells infected with LdHSP78+/- parasites, compared with WT parasites. Virulence of the LdHSP78+/- strain was restored by episomal addition of the LdHSP78 gene. Finally, using high-throughput virtual screening, we identified P1,P5-di(adenosine-5')-pentaphosphate (Ap5A) ammonium salt as an LdHSP78 inhibitor. It selectively induced amastigote death at doses similar to amphotericin B doses, while exhibiting much less cytotoxicity to healthy macrophages than amphotericin B. In summary, using both a genetic knockout approach and pharmacological inhibition, we establish LdHSP78 as a drug target and Ap5A as a potential lead for improved antileishmanial agents.}, }
@article {pmid31959747, year = {2020}, author = {Huang, T and Liu, Z and Zheng, Y and Feng, T and Gao, Q and Zeng, W}, title = {YTHDF2 promotes spermagonial adhesion through modulating MMPs decay via m6A/mRNA pathway.}, journal = {Cell death &amp; disease}, volume = {11}, number = {1}, pages = {37}, pmid = {31959747}, issn = {2041-4889}, mesh = {Adenosine/*analogs &amp; derivatives/metabolism ; Animals ; Apoptosis ; CRISPR-Associated Protein 9/metabolism ; CRISPR-Cas Systems/genetics ; Cell Adhesion/genetics ; Cell Cycle ; Cell Line ; Cell Movement ; Cell Proliferation ; Extracellular Matrix/metabolism ; Gene Deletion ; Gene Expression Regulation ; Male ; Matrix Metalloproteinases/*metabolism ; Mice, Knockout ; Phenotype ; RNA, Messenger/genetics/metabolism ; RNA-Binding Proteins/genetics/*metabolism ; Spermatogonia/*cytology/metabolism ; }, abstract = {As the foundation of male fertility, spermatogenesis is a complicated and highly controlled process. YTHDF2 plays regulatory roles in biological processes through accelerating the degradation of target mRNAs. However, the function of YTHDF2 in spermatogenesis remains elusive. Here, we knocked out Ythdf2 in mouse spermatogonia via CRISPR/Cas9, and found that depletion of Ythdf2 mainly downregulated the expression of matrix metallopeptidase (MMPs), thus affecting cell adhesion and proliferation. m6A-IP-PCR and RIP-PCR analysis showed that Mmp3, Mmp13, Adamts1 and Adamts9 were modified with m6A and simultaneously interacted with YTHDF2. Moreover, inhibition of Mmp13 partially rescued the phenotypes in Ythdf2-KO cells. Taken together, YTHDF2 regulates cell-matrix adhesion and proliferation through modulating the expression of Mmps by the m6A/mRNA degradation pathway.}, }
@article {pmid33391497, year = {2021}, author = {Wang, L and Zhou, J and Wang, Q and Wang, Y and Kang, C}, title = {Rapid design and development of CRISPR-Cas13a targeting SARS-CoV-2 spike protein.}, journal = {Theranostics}, volume = {11}, number = {2}, pages = {649-664}, pmid = {33391497}, issn = {1838-7640}, mesh = {Antiviral Agents/*administration &amp; dosage ; COVID-19/*drug therapy/virology ; CRISPR-Cas Systems/genetics ; Computational Biology ; Drug Evaluation, Preclinical ; Genetic Vectors/administration &amp; dosage/genetics ; Hep G2 Cells ; Humans ; Molecular Docking Simulation ; RNA, Guide/*genetics ; SARS-CoV-2/genetics ; Sequence Homology, Amino Acid ; Spike Glycoprotein, Coronavirus/*genetics ; }, abstract = {The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a worldwide epidemic of the lethal respiratory coronavirus disease (COVID-19), necessitating urgent development of specific and effective therapeutic tools. Among several therapeutic targets of coronaviruses, the spike protein is of great significance due to its key role in host invasion. Here, we report a potential anti-SARS-CoV-2 strategy based on the CRISPR-Cas13a system. Methods: A comprehensive set of bioinformatics methods, including sequence alignment, structural comparison, and molecular docking, was utilized to identify a SARS-CoV-2-spike(S)-specific segment. A tiling crRNA library targeting this specific RNA segment was designed, and optimal crRNA candidates were selected using in-silico methods. The efficiencies of the crRNA candidates were tested in human HepG2 and AT2 cells. Results: The most effective crRNA sequence inducing a robust cleavage effect on S and a potent collateral cleavage effect were identified. Conclusions: This study provides a rapid design pipeline for a CRISPR-Cas13a-based antiviral tool against SARS-CoV-2. Moreover, it offers a novel approach for anti-virus study even if the precise structures of viral proteins are indeterminate.}, }
@article {pmid33303323, year = {2021}, author = {Dronina, J and Bubniene, US and Ramanavicius, A}, title = {The application of DNA polymerases and Cas9 as representative of DNA-modifying enzymes group in DNA sensor design (review).}, journal = {Biosensors &amp; bioelectronics}, volume = {175}, number = {}, pages = {112867}, doi = {10.1016/j.bios.2020.112867}, pmid = {33303323}, issn = {1873-4235}, mesh = {*Biosensing Techniques ; COVID-19/*diagnosis/genetics/virology ; CRISPR-Cas Systems/genetics ; Diagnostic Tests, Routine ; Humans ; RNA, Viral/genetics/*isolation &amp; purification ; SARS-CoV-2/*isolation &amp; purification/pathogenicity ; }, abstract = {Rapid detection of nucleic acids (DNA or RNA) by inexpensive, selective, accurate, and highly sensitive methods is very important for biosensors. DNA-sensors based on DNA-modifying enzymes for fast determination and monitoring of pathogenic (Zika, Dengue, SARS-Cov-2 (inducer of COVID-19), human papillomavirus, HIV, etc.) viruses and diagnosis of virus-induced diseases is a key factor of this overview. Recently, DNA-modifying enzymes (Taq DNA polymerase, Phi29 DNA polymerase) have been widely used for the diagnosis of virus or pathogenic disease by gold standard (PCR, qPCR, RT-qPCR) methods, therefore, alternative methods have been reviewed. The main mechanisms of DNA metabolism (replication cycle, amplification) and the genomeediting tool CRISPR-Cas9 are purposefully discussed in order to address strategic possibility to design DNA-sensors based on immobilized DNA-enzymes. However, the immobilization of biologically active proteins on a gold carrier technique with the ability to detect viral or bacterial nucleic acids is individual for each DNA-modifying enzyme group, due to a different number of active sites, C and N terminal locations and arrangement, therefore, individual protocols based on the 'masking' of active sites should be elaborated for each enzyme.}, }
@article {pmid33196851, year = {2020}, author = {Sgro, A and Blancafort, P}, title = {Epigenome engineering: new technologies for precision medicine.}, journal = {Nucleic acids research}, volume = {48}, number = {22}, pages = {12453-12482}, pmid = {33196851}, issn = {1362-4962}, support = {R01 CA170370/CA/NCI NIH HHS/United States ; R01 DA036906/DA/NIDA NIH HHS/United States ; }, mesh = {CRISPR-Cas Systems/genetics ; Chromatin/*genetics ; DNA Methylation/*genetics ; Epigenome/*genetics ; *Gene Editing ; Genetic Engineering ; Humans ; Precision Medicine/trends ; }, abstract = {Chromatin adopts different configurations that are regulated by reversible covalent modifications, referred to as epigenetic marks. Epigenetic inhibitors have been approved for clinical use to restore epigenetic aberrations that result in silencing of tumor-suppressor genes, oncogene addictions, and enhancement of immune responses. However, these drugs suffer from major limitations, such as a lack of locus selectivity and potential toxicities. Technological advances have opened a new era of precision molecular medicine to reprogram cellular physiology. The locus-specificity of CRISPR/dCas9/12a to manipulate the epigenome is rapidly becoming a highly promising strategy for personalized medicine. This review focuses on new state-of-the-art epigenome editing approaches to modify the epigenome of neoplasms and other disease models towards a more 'normal-like state', having characteristics of normal tissue counterparts. We highlight biomolecular engineering methodologies to assemble, regulate, and deliver multiple epigenetic effectors that maximize the longevity of the therapeutic effect, and we discuss limitations of the platforms such as targeting efficiency and intracellular delivery for future clinical applications.}, }
@article {pmid33176167, year = {2020}, author = {Mehta, HM and Corey, SJ}, title = {Getting Back to Normal: Correcting SCN by Universal or Precision Strikes.}, journal = {Molecular therapy : the journal of the American Society of Gene Therapy}, volume = {28}, number = {12}, pages = {2525-2526}, pmid = {33176167}, issn = {1525-0024}, mesh = {*CRISPR-Cas Systems ; Congenital Bone Marrow Failure Syndromes ; *Hematopoietic Stem Cell Transplantation ; Mutation ; Neutropenia/congenital ; }, }
@article {pmid33152068, year = {2020}, author = {Cooper, SE and Schwartzentruber, J and Bello, E and Coomber, EL and Bassett, AR}, title = {Screening for functional transcriptional and splicing regulatory variants with GenIE.}, journal = {Nucleic acids research}, volume = {48}, number = {22}, pages = {e131}, pmid = {33152068}, issn = {1362-4962}, support = {206194/WT_/Wellcome Trust/United Kingdom ; }, mesh = {Alleles ; Alternative Splicing/genetics ; Alzheimer Disease/*genetics/pathology/therapy ; CRISPR-Cas Systems/genetics ; Clusterin/*genetics ; Enhancer Elements, Genetic/*genetics ; Gene Editing ; Genetic Variation/genetics ; Genome-Wide Association Study ; Humans ; Induced Pluripotent Stem Cells/metabolism/transplantation ; Mutation ; Polymorphism, Single Nucleotide/genetics ; Regulatory Sequences, Nucleic Acid/*genetics ; }, abstract = {Genome-wide association studies (GWAS) have identified numerous genetic loci underlying human diseases, but a fundamental challenge remains to accurately identify the underlying causal genes and variants. Here, we describe an arrayed CRISPR screening method, Genome engineering-based Interrogation of Enhancers (GenIE), which assesses the effects of defined alleles on transcription or splicing when introduced in their endogenous genomic locations. We use this sensitive assay to validate the activity of transcriptional enhancers and splice regulatory elements in human induced pluripotent stem cells (hiPSCs), and develop a software package (rgenie) to analyse the data. We screen the 99% credible set of Alzheimer's disease (AD) GWAS variants identified at the clusterin (CLU) locus to identify a subset of likely causal variants, and employ GenIE to understand the impact of specific mutations on splicing efficiency. We thus establish GenIE as an efficient tool to rapidly screen for the role of transcribed variants on gene expression.}, }
@article {pmid33104788, year = {2020}, author = {Xu, H and Wang, J and Liang, Y and Fu, Y and Li, S and Huang, J and Xu, H and Zou, W and Chen, B}, title = {TriTag: an integrative tool to correlate chromatin dynamics and gene expression in living cells.}, journal = {Nucleic acids research}, volume = {48}, number = {22}, pages = {e127}, pmid = {33104788}, issn = {1362-4962}, mesh = {Alleles ; Aptamers, Nucleotide/genetics ; CRISPR-Cas Systems/genetics ; Cell Cycle/genetics ; Chromatin/*genetics ; Fluorescent Antibody Technique/methods ; Gene Expression Regulation/genetics ; Gene Regulatory Networks/*genetics ; Humans ; *Molecular Imaging ; *Single-Cell Analysis ; Transcription, Genetic ; }, abstract = {A wealth of single-cell imaging studies have contributed novel insights into chromatin organization and gene regulation. However, a comprehensive understanding of spatiotemporal gene regulation requires developing tools to combine multiple monitoring systems in a single study. Here, we report a versatile tag, termed TriTag, which integrates the functional capabilities of CRISPR-Tag (DNA labeling), MS2 aptamer (RNA imaging) and fluorescent protein (protein tracking). Using this tag, we correlate changes in chromatin dynamics with the progression of endogenous gene expression, by recording both transcriptional bursting and protein production. This strategy allows precise measurements of gene expression at single-allele resolution across the cell cycle or in response to stress. TriTag enables capturing an integrated picture of gene expression, thus providing a powerful tool to study transcriptional heterogeneity and regulation.}, }
@article {pmid32534122, year = {2020}, author = {Li, G and Zhang, X and Wang, H and Liu, D and Li, Z and Wu, Z and Yang, H}, title = {Increasing CRISPR/Cas9-mediated homology-directed DNA repair by histone deacetylase inhibitors.}, journal = {The international journal of biochemistry &amp; cell biology}, volume = {125}, number = {}, pages = {105790}, doi = {10.1016/j.biocel.2020.105790}, pmid = {32534122}, issn = {1878-5875}, mesh = {Acetylation ; Animals ; Benzofurans/*pharmacology ; CRISPR-Cas Systems ; Cell Cycle/drug effects ; Cells, Cultured ; Chromatin Immunoprecipitation ; DNA End-Joining Repair/*drug effects ; DNA Repair/drug effects ; Gene Editing/*methods ; HEK293 Cells ; Histone Deacetylase Inhibitors/*pharmacology ; Humans ; Hydroxamic Acids/*pharmacology ; Ku Autoantigen/metabolism ; Poly (ADP-Ribose) Polymerase-1/metabolism ; Rad51 Recombinase/genetics/metabolism ; Rad52 DNA Repair and Recombination Protein/genetics/metabolism ; Recombinational DNA Repair/*drug effects ; Swine ; }, abstract = {Histone deacetylase inhibitors (HDACis) affect DNA repair pathways by modulating multiple cellular machineries, including chromatin state, DNA repair factor modification, and the cell cycle. These machineries can differentially affect DNA repair outcomes. With the aim to investigate the impacts of HDACis on DNA repair following CRISPR/Cas9 cleavage from the mixed actions, we used two pan-HDACis, trichostatin A (TSA) and PCI-24781, to treat animal immortalized and primary cells, and studied CRISPR/Cas9-mediated genome editing results by nonhomologous end joining (NHEJ) and homology-directed repair (HDR) pathways. We first found that TSA and PCI-24781 increased NHEJ efficiency. However, further analysis of the total NHEJ events demonstrated that alternative end joining (alt-EJ) mainly contributed to the enhanced total NHEJ by HDACis. We then analyzed HDR efficiency with HDACi treatment and found that multiple HDR pathways, including homologous recombination, single strand annealing and single-stranded oligonucleotide (ssODN)-mediated HDR, were all increased with HDACi treatment. TSA also increased CRISPR-induced ssODN-mediated HDR rate in pig parthenogenetic embryos. Analyzing acetylation status of DNA repair factors showed that acetylation levels of classical NHEJ (c-NHEJ) factors KU70 and KU80 and alt-EJ factor PARP1 were significantly enhanced, but alt-EJ factor LIG3 and HDR factors Rad51 and Rad52 were not affected greatly, implying a differential impact on these repair pathways by HDACis. In addition, TSA and PCI-24781 can enrich cells in G2/M phase of the cell cycle which is beneficial for occurrence of HDR. These findings show that HDACis can effectively promote CRISPR-mediated homology-involved DNA repair, including HDR and alt-EJ pathways, through concerted action of multiple cellular machineries.}, }
@article {pmid32470463, year = {2020}, author = {Strezoska, Ž and Dickerson, SM and Maksimova, E and Chou, E and Gross, MM and Hemphill, K and Hardcastle, T and Perkett, M and Stombaugh, J and Miller, GW and Anderson, EM and Vermeulen, A and Smith, AVB}, title = {CRISPR-mediated transcriptional activation with synthetic guide RNA.}, journal = {Journal of biotechnology}, volume = {319}, number = {}, pages = {25-35}, doi = {10.1016/j.jbiotec.2020.05.005}, pmid = {32470463}, issn = {1873-4863}, mesh = {Animals ; Aptamers, Nucleotide/genetics ; *CRISPR-Cas Systems ; Gene Editing/*methods ; HEK293 Cells ; Humans ; Mice ; NIH 3T3 Cells ; *RNA, Guide ; Transcriptional Activation/*genetics ; }, abstract = {The CRISPR-Cas9 system has been adapted for transcriptional activation (CRISPRa) and several second-generation CRISPRa systems (including VPR, SunTag, and SAM) have been developed to recruit different transcriptional activators to a deactivated Cas9, which is guided to a transcriptional start site via base complementarity with a target guide RNA. Multiple studies have shown the benefit of CRISPRa using plasmid or lentiviral expressed guide RNA, but the use of synthetic guide RNA has not been reported. Here we demonstrate the effective use of synthetic guide RNA for gene activation via CRISPRa. CRISPRa crRNA may be used with a canonical tracrRNA using the VPR or SunTag activation systems or with an extended tracrRNA containing an aptamer sequence for the SAM system. Transcriptional activation with synthetic crRNA:tracrRNA is comparable to activation achieved with expression vectors and combining several crRNA sequences targeting the same gene can enhance transcriptional activation. The use of synthetic crRNA is also ideal for simultaneous activation of multiple genes or use with dCas9-VPR mRNA when viral transduction is not feasible. Here, we perform a proof-of-principle arrayed screen using a CRISPRa crRNA library consisting of 153 cytokine receptor targets to identify regulators of IL-6 cytokine secretion. Together, these results demonstrate the suitability of synthetic CRISPRa guide RNA for high throughput, arrayed screening applications which allow for more complex phenotypic readouts to complement viability and drug resistance assays typically used in a pooled screening format.}, }
@article {pmid32460957, year = {2020}, author = {Zhang, Q and Fu, Y and Thakur, C and Bi, Z and Wadgaonkar, P and Qiu, Y and Xu, L and Rice, M and Zhang, W and Almutairy, B and Chen, F}, title = {CRISPR-Cas9 gene editing causes alternative splicing of the targeting mRNA.}, journal = {Biochemical and biophysical research communications}, volume = {528}, number = {1}, pages = {54-61}, pmid = {32460957}, issn = {1090-2104}, support = {P30 ES020957/ES/NIEHS NIH HHS/United States ; R01 ES028263/ES/NIEHS NIH HHS/United States ; R01 ES028335/ES/NIEHS NIH HHS/United States ; }, mesh = {Alternative Splicing/*genetics ; Base Sequence ; CRISPR-Associated Protein 9/*metabolism ; CRISPR-Cas Systems/*genetics ; Cell Line ; Dioxygenases/genetics ; Exons/genetics ; *Gene Editing ; Histone Demethylases/genetics ; Humans ; Nuclear Proteins/genetics ; Polymorphism, Single Nucleotide/genetics ; RNA, Guide/genetics ; RNA, Messenger/genetics/metabolism ; Sequence Deletion ; }, abstract = {The technique of CRISPR-Cas9 gene editing has been widely used to specifically delete the selected target genes through generating double strand breaks (DSBs) and inducing insertion and/or deletion (indel) of the genomic DNAs in the cells. We recently applied this technique to disrupt mineral dust-induced gene (mdig), a potential oncogene as previously reported, by single guide RNA (sgRNA) targeting the third exon of mdig gene in several cell types, including human bronchial epithelial cell line BEAS-2B, lung cancer cell line A549, and human triple negative breast cancer cell line MDA-MB-231 cells. In addition to the successful knockout of mdig gene in these cells, we unexpectedly noted generation of several alternatively spliced mdig mRNAs. Amplification of the mdig mRNAs during the screening of knockout clones by reverse transcription-polymerase chain reaction (RT-PCR) and the subsequent sanger sequencing of DNA revealed deletion and alternative splicing of mdig mRNAs induced by CRISPR-Cas9 gene editing. The most common deletions include nine and twenty-four nucleotides deletion around the DSBs. In addition, interestingly, some mdig mRNAs showed skipping of the entire exon 3, or alternative splicing between exon 2 and exon 8 using the new donor and accept splicing sites, leading to deletion of exons 3, 4, 5, 6, and 7. Accordingly, cautions should be taken when using CRISPR-Cas9 strategy to edit human genes due to the unintended alterative splicing of the target mRNAs. It is very likely that new proteins, some of which may be highly oncogenic, may be generated from CRISPR-Cas9 gene editing.}, }
@article {pmid32446977, year = {2020}, author = {Tyagi, S and Kumar, R and Das, A and Won, SY and Shukla, P}, title = {CRISPR-Cas9 system: A genome-editing tool with endless possibilities.}, journal = {Journal of biotechnology}, volume = {319}, number = {}, pages = {36-53}, doi = {10.1016/j.jbiotec.2020.05.008}, pmid = {32446977}, issn = {1873-4863}, mesh = {Animals ; Biomedical Research ; *CRISPR-Cas Systems ; *Gene Editing/ethics/methods/standards ; Humans ; Plants, Genetically Modified ; }, abstract = {The discovery of CRISPR: Cas9 and its application as a powerful gene-editing tool has transformed the world of basic and applied science, especially the molecular biology dome. Also, the smooth, quick, flexible, and very efficient nature of this technology has enabled the biologists to alter the genome of prokaryotes to complex eukaryotic systems, including plants and animals. Using CRISPR and associated tools, investigation, control, and modification of significant biological events have been more accessible than before. These biological scissors are now being used to accelerate breeding programs of crop and livestock, engineer new antimicrobials, and control disease-carrying pathogens. However, like other techniques, these cutters emerged as a double-edged sword and put several challenges to the scientific society. Here in this review article, we summarized the beneficial application of the CRISPR: Cas9 system and unsafe perception to the society if handled carelessly. We also discussed the limitations and ethical issues related to CRISPR: Cas9 technology.}, }
@article {pmid32408895, year = {2020}, author = {Thom, CS and Jobaliya, CD and Lorenz, K and Maguire, JA and Gagne, A and Gadue, P and French, DL and Voight, BF}, title = {Tropomyosin 1 genetically constrains in vitro hematopoiesis.}, journal = {BMC biology}, volume = {18}, number = {1}, pages = {52}, pmid = {32408895}, issn = {1741-7007}, support = {R56 DK101478/DK/NIDDK NIH HHS/United States ; R01 HL130698/HL/NHLBI NIH HHS/United States ; T32HD043021//National Institute of Child Health and Human Development/International ; R01 DK101478/DK/NIDDK NIH HHS/United States ; R01DK101478/DK/NIDDK NIH HHS/United States ; T32 HD043021/HD/NICHD NIH HHS/United States ; }, mesh = {Blood Platelets/*metabolism ; CRISPR-Cas Systems ; Genome-Wide Association Study ; Hematopoiesis/*genetics ; Hematopoietic Stem Cells/*metabolism ; Humans ; In Vitro Techniques ; Tropomyosin/deficiency/*metabolism ; }, abstract = {BACKGROUND: Identifying causal variants and genes from human genetic studies of hematopoietic traits is important to enumerate basic regulatory mechanisms underlying these traits, and could ultimately augment translational efforts to generate platelets and/or red blood cells in vitro. To identify putative causal genes from these data, we performed computational modeling using available genome-wide association datasets for platelet and red blood cell traits.<br><br>RESULTS: Our model identified a joint collection of genomic features enriched at established trait associations and plausible candidate variants. Additional studies associating variation at these loci with change in gene expression highlighted Tropomyosin 1 (TPM1) among our top-ranked candidate genes. CRISPR/Cas9-mediated TPM1 knockout in human induced pluripotent stem cells (iPSCs) enhanced hematopoietic progenitor development, increasing total megakaryocyte and erythroid cell yields.<br><br>CONCLUSIONS: Our findings may help explain human genetic associations and identify a novel genetic strategy to enhance in vitro hematopoiesis. A similar trait-specific gene prioritization strategy could be employed to help streamline functional validation experiments for virtually any human trait.}, }
@article {pmid32354746, year = {2020}, author = {Chen, X and Gao, YQ and Zheng, YY and Wang, W and Wang, P and Liang, J and Zhao, W and Tao, T and Sun, J and Wei, L and Li, Y and Zhou, Y and Gan, Z and Zhang, X and Chen, HQ and Zhu, MS}, title = {The intragenic microRNA miR199A1 in the dynamin 2 gene contributes to the pathology of X-linked centronuclear myopathy.}, journal = {The Journal of biological chemistry}, volume = {295}, number = {26}, pages = {8656-8667}, pmid = {32354746}, issn = {1083-351X}, mesh = {Animals ; CRISPR-Cas Systems ; Dynamin II/analysis/*genetics ; Female ; Longevity ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; MicroRNAs/analysis/*genetics ; Muscle Strength ; Muscle, Skeletal/metabolism/pathology ; Myopathies, Structural, Congenital/*genetics/pathology ; }, abstract = {Mutations in the myotubularin 1 (MTM1) gene can cause the fatal disease X-linked centronuclear myopathy (XLCNM), but the underlying mechanism is incompletely understood. In this report, using an Mtm1-/y disease model, we found that expression of the intragenic microRNA miR-199a-1 is up-regulated along with that of its host gene, dynamin 2 (Dnm2), in XLCNM skeletal muscle. To assess the role of miR-199a-1 in XLCNM, we crossed miR-199a-1-/- with Mtm1-/y mice and found that the resultant miR-199a-1-Mtm1 double-knockout mice display markers of improved health, as evidenced by lifespans prolonged by 30% and improved muscle strength and histology. Mechanistic analyses showed that miR-199a-1 directly targets nonmuscle myosin IIA (NM IIA) expression and, hence, inhibits muscle postnatal development as well as muscle maturation. Further analysis revealed that increased expression and phosphorylation of signal transducer and activator of transcription 3 (STAT3) up-regulates Dnm2/miR-199a-1 expression in XLCNM muscle. Our results suggest that miR-199a-1 has a critical role in XLCNM pathology and imply that this microRNA could be targeted in therapies to manage XLCNM.}, }
@article {pmid32245271, year = {2020}, author = {Vidyanti, AN and Hsieh, JY and Lin, KJ and Fang, YC and Setyopranoto, I and Hu, CJ}, title = {Role of HMGB1 in an Animal Model of Vascular Cognitive Impairment Induced by Chronic Cerebral Hypoperfusion.}, journal = {International journal of molecular sciences}, volume = {21}, number = {6}, pages = {}, pmid = {32245271}, issn = {1422-0067}, support = {MOST 106-2314-B-038-038-MY2//Ministry of Science and Technology, Taiwan/ ; }, mesh = {Amyloid beta-Peptides/metabolism ; Animals ; Behavior Rating Scale ; Brain Ischemia/diagnostic imaging/genetics/*metabolism/physiopathology ; CRISPR-Cas Systems ; Carotid Stenosis ; *Cerebrovascular Circulation ; Chronic Disease ; Dementia, Vascular/physiopathology ; Disease Models, Animal ; Gene Knockout Techniques ; HMGB1 Protein/genetics/*metabolism ; Hippocampus/diagnostic imaging/pathology/physiopathology ; Interleukin-1beta/metabolism ; Interleukin-6/metabolism ; Magnetic Resonance Imaging ; Male ; Mice ; Mice, Inbred C57BL ; Psychomotor Performance ; Tumor Necrosis Factor-alpha/metabolism ; }, abstract = {The pathophysiology of vascular cognitive impairment (VCI) is associated with chronic cerebral hypoperfusion (CCH). Increased high-mobility group box protein 1 (HMGB1), a nonhistone protein involved in injury and inflammation, has been established in the acute phase of CCH. However, the role of HMGB1 in the chronic phase of CCH remains unclear. We developed a novel animal model of CCH with a modified bilateral common carotid artery occlusion (BCCAO) in C57BL/6 mice. Cerebral blood flow (CBF) reduction, the expression of HMGB1 and its proinflammatory cytokines (tumor necrosis factor-alpha [TNF-α], interleukin [IL]-1β, and IL-6), and brain pathology were assessed. Furthermore, we evaluated the effect of HMGB1 suppression through bilateral intrahippocampus injection with the CRISPR/Cas9 knockout plasmid. Three months after CCH induction, CBF decreased to 30-50% with significant cognitive decline in BCCAO mice. The 7T-aMRI showed hippocampal atrophy, but amyloid positron imaging tomography showed nonsignificant amyloid-beta accumulation. Increased levels of HMGB1, TNF-α, IL-1β, and IL-6 were observed 3 months after BCCAO. HMGB1 suppression with CRISPR/Cas9 knockout plasmid restored TNF-α, IL-1β, and IL-6 and attenuated hippocampal atrophy and cognitive decline. We believe that HMGB1 plays a pivotal role in CCH-induced VCI pathophysiology and can be a potential therapeutic target of VCI.}, }
@article {pmid31859532, year = {2020}, author = {Martin, J and Free, T}, title = {A look back at 2019 in BioTechniques.}, journal = {BioTechniques}, volume = {68}, number = {1}, pages = {2-3}, doi = {10.2144/btn-2019-0164}, pmid = {31859532}, issn = {1940-9818}, mesh = {CRISPR-Cas Systems ; Humans ; *Polymerase Chain Reaction ; *Serial Publications ; Social Media ; Urine Specimen Collection/*methods ; }, }
@article {pmid33428981, year = {2021}, author = {Safari, F and Afarid, M and Rastegari, B and Haghighi, AB and Barekati-Mowahed, M and Behbahani, AB}, title = {CRISPR systems: Novel approaches for detection and combating COVID-19.}, journal = {Virus research}, volume = {}, number = {}, pages = {198282}, doi = {10.1016/j.virusres.2020.198282}, pmid = {33428981}, issn = {1872-7492}, abstract = {Type V and VI CRISPR enzymes are RNA-guided, DNA and RNA-targeting effectors that allow specific gene knockdown. Cas12 and Cas13 are CRISPR proteins that are efficient agents for diagnosis and combating single-stranded RNA (ssRNA) viruses. The programmability of these proteins paves the way for the detection and degradation of RNA viruses by targeting RNAs complementary to its CRISPR RNA (crRNA). Approximately two-thirds of viruses causing diseases contain ssRNA genomes. The Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) has caused the outbreak of the coronavirus disease 2019 (COVID-19), which has infected more than fifty million people worldwide with near 1.3 million deaths since December 2019. Thus, accurate and rapid diagnostic and therapeutic tools are essential for early detection and treatment of this widespread infectious disease. For us, the CRISPR based platforms seem to be a plausible new approach for an accurate detection and treatment of SARS-CoV-2. In this review, we talk about Cas12 and Cas13 CRISPR systems and their applications in diagnosis and treatment of RNA virus mediated diseases. In continue, the SARS-CoV-2 pathogenicity, and its conventional diagnostics and antivirals will be discussed. Moreover, we highlight novel CRISPR based diagnostic platforms and therapies for COVID-19. We also discuss the challenges of diagnostic CRISPR based platforms as well as clarifying the proposed solution for high efficient selective in vivo delivery of CRISPR components into SARS-CoV-2-infected cells.}, }
@article {pmid33428900, year = {2021}, author = {Palaz, F and Kerem Kalkan, A and Tozluyurt, A and Ozsoz, M}, title = {CRISPR-based tools: alternative methods for the diagnosis of COVID-19.}, journal = {Clinical biochemistry}, volume = {}, number = {}, pages = {}, doi = {10.1016/j.clinbiochem.2020.12.011}, pmid = {33428900}, issn = {1873-2933}, abstract = {The recently emerged severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) spread all over the world rapidly and caused a global pandemic. To prevent the virus from spreading to more individuals, it is of great importance to identify and isolate infected individuals through testing. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is the gold standard method for the diagnosis of coronavirus disease (COVID-19) worldwide. However, performing RT-qPCR is limited to centralized laboratories because of the need for sophisticated laboratory equipment and skilled personnel. Further, it can sometimes give false negative or uncertain results. Recently, new methods have been developed for nucleic acid detection and pathogen diagnosis using CRISPR-Cas systems. These methods present rapid and cost-effective diagnostic platforms that provide high sensitivity and specificity without the need for complex instrumentation. Using the CRISPR-based SARS-CoV-2 detection methods, it is possible to increase the number of daily tests in existing laboratories, reduce false negative or uncertain result rates obtained with RT-qPCR, and perform testing in resource-limited settings or at points of need where performing RT-qPCR is not feasible. Here, we briefly describe the RT-qPCR method, and discuss its limitations in meeting the current diagnostic needs. We explain how the unique properties of various CRISPR-associated enzymes are utilized for nucleic acid detection and pathogen diagnosis. Then, we highlight the important features of CRISPR-based diagnostic methods developed for SARS-CoV-2 detection. Finally, we examine the advantages and limitations of these methods, and discuss how they can contribute to improving the efficiency of the current testing systems for combating SARS-CoV-2.}, }
@article {pmid33425987, year = {2020}, author = {Srivastava, S and Upadhyay, DJ and Srivastava, A}, title = {Next-Generation Molecular Diagnostics Development by CRISPR/Cas Tool: Rapid Detection and Surveillance of Viral Disease Outbreaks.}, journal = {Frontiers in molecular biosciences}, volume = {7}, number = {}, pages = {582499}, doi = {10.3389/fmolb.2020.582499}, pmid = {33425987}, issn = {2296-889X}, abstract = {Virus disease spreads effortlessly mechanically or through minute insect vectors that are extremely challenging to avoid. Emergence and reemergence of new viruses such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), H1N1 influenza virus, avian influenza virus, dengue virus, Citrus tristeza virus, and Tomato yellow leaf curl virus have paralyzed the economy of many countries. The cure for major viral diseases is not feasible; however, early detection and surveillance of the disease can obstruct their spread. Therefore, advances in the field of virus diagnosis and the development of new point-of-care testing kits become necessary globally. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) is an emerging technology for gene editing and diagnostics development. Several rapid nucleic acid diagnostic kits have been developed and validated using Cas9, Cas12, and Cas13 proteins. This review summarizes the CRISPR/Cas-based next-generation molecular diagnostic techniques and portability of devices for field-based utilization.}, }
@article {pmid33332350, year = {2020}, author = {House, NCM and Parasuram, R and Layer, JV and Price, BD}, title = {Site-specific targeting of a light activated dCas9-KillerRed fusion protein generates transient, localized regions of oxidative DNA damage.}, journal = {PloS one}, volume = {15}, number = {12}, pages = {e0237759}, pmid = {33332350}, issn = {1932-6203}, mesh = {CRISPR-Cas Systems/*genetics ; Cell Line ; Chromatin/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; DNA/*genetics ; DNA Breaks, Double-Stranded ; DNA Damage/*genetics ; DNA Repair/*genetics ; Endonucleases/genetics ; Genome/genetics ; HEK293 Cells ; Humans ; Light ; Oxidative Stress/*genetics ; RNA, Guide/genetics ; }, abstract = {DNA repair requires reorganization of the local chromatin structure to facilitate access to and repair of the DNA. Studying DNA double-strand break (DSB) repair in specific chromatin domains has been aided by the use of sequence-specific endonucleases to generate targeted breaks. Here, we describe a new approach that combines KillerRed, a photosensitizer that generates reactive oxygen species (ROS) when exposed to light, and the genome-targeting properties of the CRISPR/Cas9 system. Fusing KillerRed to catalytically inactive Cas9 (dCas9) generates dCas9-KR, which can then be targeted to any desired genomic region with an appropriate guide RNA. Activation of dCas9-KR with green light generates a local increase in reactive oxygen species, resulting in &quot;clustered&quot; oxidative damage, including both DNA breaks and base damage. Activation of dCas9-KR rapidly (within minutes) increases both γH2AX and recruitment of the KU70/80 complex. Importantly, this damage is repaired within 10 minutes of termination of light exposure, indicating that the DNA damage generated by dCas9-KR is both rapid and transient. Further, repair is carried out exclusively through NHEJ, with no detectable contribution from HR-based mechanisms. Surprisingly, sequencing of repaired DNA damage regions did not reveal any increase in either mutations or INDELs in the targeted region, implying that NHEJ has high fidelity under the conditions of low level, limited damage. The dCas9-KR approach for creating targeted damage has significant advantages over the use of endonucleases, since the duration and intensity of DNA damage can be controlled in &quot;real time&quot; by controlling light exposure. In addition, unlike endonucleases that carry out multiple cut-repair cycles, dCas9-KR produces a single burst of damage, more closely resembling the type of damage experienced during acute exposure to reactive oxygen species or environmental toxins. dCas9-KR is a promising system to induce DNA damage and measure site-specific repair kinetics at clustered DNA lesions.}, }
@article {pmid33046744, year = {2020}, author = {Kang, D and Shin, W and Yoo, H and Kim, S and Lee, S and Rhee, K}, title = {Cep215 is essential for morphological differentiation of astrocytes.}, journal = {Scientific reports}, volume = {10}, number = {1}, pages = {17000}, pmid = {33046744}, issn = {2045-2322}, mesh = {Animals ; Astrocytes/*physiology ; CRISPR-Cas Systems ; Cell Cycle Proteins/genetics/*metabolism ; Cell Differentiation ; Cell Line ; Mice ; Microtubules/*metabolism ; Nerve Tissue Proteins/genetics/*metabolism ; Neuroglia/*physiology ; RNA, Small Interfering/genetics ; }, abstract = {Cep215 (also known as Cdk5rap2) is a centrosome protein which is involved in microtubule organization. Cep215 is also placed at specific subcellular locations and organizes microtubules outside the centrosome. Here, we report that Cep215 is involved in morphological differentiation of astrocytes. Cep215 was specifically localized at the glial processes as well as centrosomes in developing astrocytes. Morphological differentiation of astrocytes was suppressed in the Cep215-deleted P19 cells and in the Cep215-depleted embryonic hippocampal culture. We confirm that the microtubule organizing function of Cep215 is critical for the glial process formation. However, Cep215 is not involved in the regulation of cell proliferation nor cell specification. Based on the results, we propose that Cep215 organizes microtubules for glial process formation during astrocyte differentiation.}, }
@article {pmid32876764, year = {2020}, author = {Zhao, N and Li, L and Luo, G and Xie, S and Lin, Y and Han, S and Huang, Y and Zheng, S}, title = {Multiplex gene editing and large DNA fragment deletion by the CRISPR/Cpf1-RecE/T system in Corynebacterium glutamicum.}, journal = {Journal of industrial microbiology &amp; biotechnology}, volume = {47}, number = {8}, pages = {599-608}, doi = {10.1007/s10295-020-02304-5}, pmid = {32876764}, issn = {1476-5535}, support = {31671840//National Natural Science Foundation of China/ ; 2018YFA0901700//National Key R&amp;D Program of China/ ; 2019M661676//Postdoctoral Research Foundation of China/ ; }, mesh = {CRISPR-Associated Proteins/*genetics ; CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Corynebacterium glutamicum/enzymology/*genetics/metabolism ; DNA, Bacterial/*genetics ; *Gene Deletion ; Gene Editing/*methods ; Metabolic Engineering/methods ; }, abstract = {Corynebacterium glutamicum is an essential industrial strain that has been widely harnessed for the production of all kinds of value-added products. Efficient multiplex gene editing and large DNA fragment deletion are essential strategies for industrial biotechnological research. Cpf1 is a robust and simple genome editing tool for simultaneous editing of multiplex genes. However, no studies on effective multiplex gene editing and large DNA fragment deletion by the CRISPR/Cpf1 system in C. glutamicum have been reported. Here, we developed a multiplex gene editing method by optimizing the CRISPR/Cpf1-RecT system and a large chromosomal fragment deletion strategy using the CRISPR/Cpf1-RecET system in C. glutamicum ATCC 14067. The CRISPR/Cpf1-RecT system exhibited a precise editing efficiency of more than 91.6% with the PAM sequences TTTC, TTTG, GTTG or CTTC. The sites that could be edited were limited due to the PAM region and the 1-7 nt at the 5' end of the protospacer region. Mutations in the PAM region increased the editing efficiency of the - 6 nt region from 0 to 96.7%. Using a crRNA array, two and three genes could be simultaneously edited in one step via the CRISPR/Cpf1-RecT system, and the efficiency of simultaneously editing two genes was 91.6%, but the efficiency of simultaneously editing three genes was below 10%. The editing efficiency for a deletion of 1 kb was 79.6%, and the editing efficiencies for 5- and 20 kb length DNA fragment deletions reached 91.3% and 36.4%, respectively, via the CRISPR/Cpf1-RecET system. This research provides an efficient and simple tool for C. glutamicum genome editing that can further accelerate metabolic engineering efforts and genome evolution.}, }
@article {pmid32788672, year = {2020}, author = {Akkaya, M and Bansal, A and Sheehan, PW and Pena, M and Cimperman, CK and Qi, CF and Yazew, T and Otto, TD and Billker, O and Miller, LH and Pierce, SK}, title = {Testing the impact of a single nucleotide polymorphism in a Plasmodium berghei ApiAP2 transcription factor on experimental cerebral malaria in mice.}, journal = {Scientific reports}, volume = {10}, number = {1}, pages = {13630}, pmid = {32788672}, issn = {2045-2322}, mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Extracellular Matrix/*parasitology ; Female ; Malaria, Cerebral/*parasitology ; Mice ; Mice, Inbred C57BL ; Plasmodium berghei/*genetics/growth &amp; development/physiology ; *Polymorphism, Single Nucleotide ; Protozoan Proteins/antagonists &amp; inhibitors/*genetics ; Virulence Factors/antagonists &amp; inhibitors/*genetics ; }, abstract = {Cerebral malaria (CM) is the deadliest form of severe Plasmodium infections. Currently, we have limited understanding of the mechanisms by which Plasmodium parasites induce CM. The mouse model of CM, experimental CM (ECM), induced by infection with the rodent parasite, Plasmodium berghei ANKA (PbANKA) has been extensively used to study the pathophysiology of CM. Recent genomic analyses revealed that the coding regions of PbANKA and the closely related Plasmodium berghei NK65 (PbNK65), that does not cause ECM, differ in only 21 single nucleotide polymorphysims (SNPs). Thus, the SNP-containing genes might contribute to the pathogenesis of ECM. Although the majority of these SNPs are located in genes of unknown function, one SNP is located in the DNA binding site of a member of the Plasmodium ApiAP2 transcription factor family, that we recently showed functions as a virulence factor alternating the host's immune response to the parasite. Here, we investigated the impact of this SNP on the development of ECM. Our results using CRISPR-Cas9 engineered parasites indicate that despite its immune modulatory function, the SNP is neither necessary nor sufficient to induce ECM and thus cannot account for parasite strain-specific differences in ECM phenotypes.}, }
@article {pmid32651405, year = {2020}, author = {Gans, I and Hartig, EI and Zhu, S and Tilden, AR and Hutchins, LN and Maki, NJ and Graber, JH and Coffman, JA}, title = {Klf9 is a key feedforward regulator of the transcriptomic response to glucocorticoid receptor activity.}, journal = {Scientific reports}, volume = {10}, number = {1}, pages = {11415}, pmid = {32651405}, issn = {2045-2322}, support = {R03 HD099468/HD/NICHD NIH HHS/United States ; P20 GM104318/GM/NIGMS NIH HHS/United States ; P20 GM103423/GM/NIGMS NIH HHS/United States ; }, mesh = {Animals ; *CRISPR-Cas Systems ; Exons ; *Frameshift Mutation ; Gene Deletion ; Gene Expression Regulation ; Homozygote ; Humans ; Hydrocortisone/metabolism ; Inflammation ; Kruppel-Like Transcription Factors/*metabolism ; Larva ; Mutation ; RNA-Seq ; Receptors, Glucocorticoid/*metabolism ; Receptors, Mineralocorticoid/metabolism ; Signal Transduction ; *Transcriptome ; Up-Regulation ; Zebrafish/genetics ; Zebrafish Proteins/*metabolism ; }, abstract = {The zebrafish has recently emerged as a model system for investigating the developmental roles of glucocorticoid signaling and the mechanisms underlying glucocorticoid-induced developmental programming. To assess the role of the Glucocorticoid Receptor (GR) in such programming, we used CRISPR-Cas9 to produce a new frameshift mutation, GR369-, which eliminates all potential in-frame initiation codons upstream of the DNA binding domain. Using RNA-seq to ask how this mutation affects the larval transcriptome under both normal conditions and with chronic cortisol treatment, we find that GR mediates most of the effects of the treatment, and paradoxically, that the transcriptome of cortisol-treated larvae is more like that of larvae lacking a GR than that of larvae with a GR, suggesting that the cortisol-treated larvae develop GR resistance. The one transcriptional regulator that was both underexpressed in GR369- larvae and consistently overexpressed in cortisol-treated larvae was klf9. We therefore used CRISPR-Cas9-mediated mutation of klf9 and RNA-seq to assess Klf9-dependent gene expression in both normal and cortisol-treated larvae. Our results indicate that Klf9 contributes significantly to the transcriptomic response to chronic cortisol exposure, mediating the upregulation of proinflammatory genes that we reported previously.}, }
@article {pmid32403926, year = {2020}, author = {Modell, AE and Siriwardena, SU and Choudhary, A}, title = {A Jumbo Phage Forms a Nucleus-like Compartment to Evade Bacterial Defense Systems.}, journal = {Biochemistry}, volume = {59}, number = {20}, pages = {1869-1870}, doi = {10.1021/acs.biochem.0c00273}, pmid = {32403926}, issn = {1520-4995}, mesh = {*Bacteriophages/genetics ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Genome, Viral ; Immune System ; }, }
@article {pmid32347717, year = {2020}, author = {Lu, M and Tokuyasu, TA}, title = {CRISPR-Cas13-Based RNA-Interacting Protein Detection in Living Cells.}, journal = {Biochemistry}, volume = {59}, number = {19}, pages = {1791-1792}, doi = {10.1021/acs.biochem.0c00281}, pmid = {32347717}, issn = {1520-4995}, mesh = {CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *RNA/genetics ; }, }
@article {pmid32250930, year = {2020}, author = {Wu, H and Qian, C and Wu, C and Wang, Z and Wang, D and Ye, Z and Ping, J and Wu, J and Ji, F}, title = {End-point dual specific detection of nucleic acids using CRISPR/Cas12a based portable biosensor.}, journal = {Biosensors &amp; bioelectronics}, volume = {157}, number = {}, pages = {112153}, doi = {10.1016/j.bios.2020.112153}, pmid = {32250930}, issn = {1873-4235}, mesh = {Biosensing Techniques/instrumentation/*methods ; *CRISPR-Cas Systems ; Equipment Design ; Food Safety ; Lectins/genetics ; Molecular Diagnostic Techniques/instrumentation/methods ; Nucleic Acid Amplification Techniques/instrumentation/methods ; Plants, Genetically Modified/*genetics ; Polymerase Chain Reaction/instrumentation/methods ; Soybeans/*genetics ; Zea mays/*genetics ; }, abstract = {A CRISPR/Cas12a based portable biosensor (Cas12a-PB) was developed to simultaneously visually detect CaMV35S promoter and Lectin gene from genetically modified (GM) soybean powders (Roundup Ready@). The Cas12a-PB, mainly made of polymethylmethacrylate (PMMA) and PMMA tape, has a connection structure, three channels and three detection chambers. The CRISPR/Cas12a detection reagents were preloaded in detection chambers and the reaction tube was connected to the connection structure by screw threads. After amplification, the amplicons were gone into three detection chambers by swinging the Cas12a-PB to conduct dual detection. Positive samples would produce green fluorescence while negative samples were black under the irradiation of 490 nm LED light. In this study, the Cas12a-PB successively combined with ordinary PCR, rapid PCR and loop-mediated isothermal amplification (LAMP) to achieve dual detection, which made detection process more convenient and portable. As low as 0.1% transgenic ingredients in soybean powders could be detected and the specificity of Cas12a-PB was confirmed with GM maize powders (MON810, GA21), GM soybean powders (DP305423), non-GM peanut and rice as targets. In the end, an amplification chamber combining with Cas12a-PB on a PMMA chip was further designed to eliminate the use of reaction tube and mineral oil, which made operation simpler. The established Cas12a-PB would provide a new reliable solution for multiple targets detection in clinic diagnostics, food safety, etc.}, }
@article {pmid32208677, year = {2020}, author = {Ramesh, A and Ong, T and Garcia, JA and Adams, J and Wheeldon, I}, title = {Guide RNA Engineering Enables Dual Purpose CRISPR-Cpf1 for Simultaneous Gene Editing and Gene Regulation in Yarrowia lipolytica.}, journal = {ACS synthetic biology}, volume = {9}, number = {4}, pages = {967-971}, doi = {10.1021/acssynbio.9b00498}, pmid = {32208677}, issn = {2161-5063}, mesh = {Bacterial Proteins/genetics ; CRISPR-Cas Systems/*genetics ; Endonucleases/genetics ; Gene Editing/*methods ; Gene Expression Regulation, Fungal/*genetics ; Genome, Fungal/genetics ; RNA, Guide/*genetics ; Yarrowia/*genetics ; }, abstract = {Yarrowia lipolytica has fast become a biotechnologically significant yeast for its ability to accumulate lipids to high levels. While there exists a suite of synthetic biology tools for genetic engineering in this yeast, there is a need for multipurposed tools for rapid strain generation. Here, we describe a dual purpose CRISPR-Cpf1 system that is capable of simultaneous gene disruption and gene regulation. Truncating guide RNA spacer length to 16 nt inhibited nuclease activity but not binding to the target loci, enabling gene activation and repression with Cpf1-fused transcriptional regulators. Gene repression was demonstrated using a Cpf1-Mxi1 fusion achieving a 7-fold reduction in mRNA, while CRISPR-activation with Cpf1-VPR increased hrGFP expression by 10-fold. High efficiency disruptions were achieved with gRNAs 23-25 bp in length, and efficiency and repression levels were maintained with multiplexed expression of truncated and full-length gRNAs. The developed CRISPR-Cpf1 system should prove useful in metabolic engineering, genome wide screening, and functional genomics studies.}, }
@article {pmid32160465, year = {2020}, author = {Liow, LT and Go, MK and Chang, MW and Yew, WS}, title = {Toolkit Development for Cyanogenic and Gold Biorecovery Chassis Chromobacterium violaceum.}, journal = {ACS synthetic biology}, volume = {9}, number = {4}, pages = {953-961}, doi = {10.1021/acssynbio.0c00064}, pmid = {32160465}, issn = {2161-5063}, mesh = {CRISPR-Cas Systems/genetics ; *Chromobacterium/genetics/metabolism ; Cyanides/isolation &amp; purification/metabolism ; Drug Resistance, Bacterial/genetics ; Electronic Waste ; Genetic Engineering/*methods ; Gold/isolation &amp; purification/metabolism ; Promoter Regions, Genetic/genetics ; Synthetic Biology/*methods ; }, abstract = {Chromobacterium violaceum has been of interest recently due to its cyanogenic ability and its potential role in environmental sustainability via the biorecovery of gold from electronic waste. However, as with many nonmodel bacteria, there are limited genetic tools to implement the use of this Gram-negative chassis in synthetic biology. We propose a system that involves assaying spontaneous antibiotic resistances and using broad host range vectors to develop episomal vectors for nonmodel Gram-negative bacteria. These developed vectors can subsequently be used to characterize inducible promoters for gene expressions and implementing CRISPRi to inhibit endogenous gene expression for further studies. Here, we developed the first episomal genetic toolkit for C. violaceum consisting of two origins of replication, three antibiotic resistance genes, and four inducible promoter systems. We examined the occurrences of spontaneous resistances of the bacterium to the chosen selection markers to prevent incidences of false positives. We also tested broad host range vectors from four different incompatibility groups and characterized four inducible promoter systems, which potentially can be applied in other Gram-negative nonmodel bacteria. CRISPRi was also implemented to inhibit violacein pigment production in C. violaceum. This systematic toolkit will aid future genetic circuitry building in this chassis and other nonmodel bacteria for synthetic biology and biotechnological applications.}, }
@article {pmid32142605, year = {2020}, author = {Wrist, A and Sun, W and Summers, RM}, title = {The Theophylline Aptamer: 25 Years as an Important Tool in Cellular Engineering Research.}, journal = {ACS synthetic biology}, volume = {9}, number = {4}, pages = {682-697}, doi = {10.1021/acssynbio.9b00475}, pmid = {32142605}, issn = {2161-5063}, mesh = {*Aptamers, Nucleotide ; CRISPR-Cas Systems ; *Cell Engineering ; Cell Line ; Humans ; RNA Interference ; *Riboswitch ; Synthetic Biology ; *Theophylline ; }, abstract = {The theophylline aptamer was isolated from an oligonucleotide library in 1994. Since that time, the aptamer has found wide utility, particularly in synthetic biology, cellular engineering, and diagnostic applications. The primary application of the theophylline aptamer is in the construction and characterization of synthetic riboswitches for regulation of gene expression. These riboswitches have been used to control cellular motility, regulate carbon metabolism, construct logic gates, screen for mutant enzymes, and control apoptosis. Other applications of the theophylline aptamer in cellular engineering include regulation of RNA interference and genome editing through CRISPR systems. Here we describe the uses of the theophylline aptamer for cellular engineering over the past 25 years. In so doing, we also highlight important synthetic biology applications to control gene expression in a ligand-dependent manner.}, }
@article {pmid31953347, year = {2020}, author = {Zhang, L and He, A and Chen, B and Bi, J and Chen, J and Guo, D and Qian, Y and Wang, W and Shi, T and Zhao, Z and Shi, J and An, W and Attenello, F and Lu, W}, title = {A HOTAIR regulatory element modulates glioma cell sensitivity to temozolomide through long-range regulation of multiple target genes.}, journal = {Genome research}, volume = {30}, number = {2}, pages = {155-163}, pmid = {31953347}, issn = {1549-5469}, mesh = {Antineoplastic Agents, Alkylating/pharmacology ; Base Sequence ; CRISPR-Cas Systems/genetics ; Calcium-Binding Proteins/*genetics ; Carrier Proteins/*genetics ; Cell Line, Tumor ; Cell Proliferation/drug effects ; Cell Survival/drug effects ; Drug Resistance, Neoplasm/genetics ; Gene Expression Regulation, Neoplastic/drug effects ; Gene Regulatory Networks/genetics ; Glioma/*drug therapy/genetics/pathology ; Humans ; Neoplasm Proteins/genetics ; RNA, Long Noncoding/*genetics ; Temozolomide/*pharmacology ; Transcription Factors/*genetics ; }, abstract = {Temozolomide (TMZ) is a frequently used chemotherapy for glioma; however, chemoresistance is a major problem limiting its effectiveness. Thus, knowledge of mechanisms underlying this outcome could improve patient prognosis. Here, we report that deletion of a regulatory element in the HOTAIR locus increases glioma cell sensitivity to TMZ and alters transcription of multiple genes. Analysis of a combination of RNA-seq, Capture Hi-C, and patient survival data suggests that CALCOCO1 and ZC3H10 are target genes repressed by the HOTAIR regulatory element and that both function in regulating glioma cell sensitivity to TMZ. Rescue experiments and 3C data confirmed this hypothesis. We propose a new regulatory mechanism governing glioma cell TMZ sensitivity.}, }
@article {pmid31944664, year = {2020}, author = {Ou, B and Jiang, B and Jin, D and Yang, Y and Zhang, M and Zhang, D and Zhao, H and Xu, M and Song, H and Wu, W and Chen, M and Lu, T and Huang, J and Seo, H and Garcia, C and Zheng, W and Guo, W and Lu, Y and Jiang, Y and Yang, S and Kaushik, RS and Li, X and Zhang, W and Zhu, G}, title = {Engineered Recombinant Escherichia coli Probiotic Strains Integrated with F4 and F18 Fimbriae Cluster Genes in the Chromosome and Their Assessment of Immunogenic Efficacy in Vivo.}, journal = {ACS synthetic biology}, volume = {9}, number = {2}, pages = {412-426}, doi = {10.1021/acssynbio.9b00430}, pmid = {31944664}, issn = {2161-5063}, mesh = {Adhesins, Escherichia coli/immunology ; Animals ; Antibodies, Bacterial/blood/immunology ; Bacterial Adhesion ; CRISPR-Cas Systems/*genetics ; Cell Line ; *Chromosomes, Bacterial ; Enterotoxigenic Escherichia coli/*genetics/immunology ; Epithelial Cells/cytology/metabolism ; Escherichia coli Proteins/genetics/immunology ; Female ; Immunoglobulin G/immunology/metabolism ; Mice ; Mice, Inbred BALB C ; Multigene Family ; Swine ; }, abstract = {F4 (K88) and F18 fimbriaed enterotoxigenic Escherichia coli (ETEC) are the predominant causes of porcine postweaning diarrhea (PWD), and vaccines are considered the most effective preventive approach against PWD. Since heterologous DNA integrated into bacterial chromosomes could be effectively expressed with stable inheritance, we chose probiotic EcNc (E. coli Nissle 1917 prototype cured of cryptic plasmids) as a delivery vector to express the heterologous F4 or both F4 and F18 fimbriae and sequentially assessed their immune efficacy of anti-F4 and F18 fimbriae in both murine and piglet models. Employing the CRISPR-cas9 technology, yjcS, pcadA, lacZ, yieN/trkD, maeB, and nth/tppB sites in the chromosome of an EcNc strain were targeted as integration sites to integrate F4 or F18 fimbriae cluster genes under the Ptet promotor to construct two recombinant integration probiotic strains (RIPSs), i.e., nth integration strain (EcNcΔnth/tppB::PtetF4) and multiple integration strain (EcNc::PtetF18x4::PtetF4x2). Expression of F4, both F4 and F18 fimbriae on the surfaces of two RIPSs, was verified with combined methods of agglutination assay, Western blot, and immunofluorescence microscopy. The recombinant strains have improved adherence to porcine intestinal epithelial cell lines. Mice and piglets immunized with the nth integration strain and multiple integration strain through gavage developed anti-F4 and both anti-F4 and anti-F18 IgG immune responses. Moreover, the serum antibodies from the immunized mice and piglets significantly inhibited the adherence of F4+ or both F4+ and F18+ ETEC wild-type strains to porcine intestinal cell lines in vitro, indicating the potential of RIPSs as promising probiotic strains plus vaccine candidates against F4+/F18+ ETEC infection.}, }
@article {pmid31943709, year = {2020}, author = {}, title = {Mucopolysaccharidosis Type I Phenotypically Corrected with Edited Hematopoietic Stem Cells: Instead of altering the IDUA gene, a protein was inserted in a repurposable place in the genome known as a &quot;safe harbor locus&quot;.}, journal = {American journal of medical genetics. Part A}, volume = {182}, number = {2}, pages = {275-276}, doi = {10.1002/ajmg.a.61223}, pmid = {31943709}, issn = {1552-4833}, mesh = {Animals ; Antigens, CD34/genetics ; CRISPR-Cas Systems/genetics ; Gene Editing/methods ; *Hematopoietic Stem Cell Transplantation ; Hematopoietic Stem Cells/metabolism ; Humans ; Iduronidase/*genetics ; Mice ; Mucopolysaccharidosis I/*genetics/pathology/therapy ; Phenotype ; Receptors, CCR5/*genetics ; }, }
@article {pmid31891494, year = {2020}, author = {Fan, J and Liu, Y and Liu, L and Huang, Y and Li, X and Huang, W}, title = {A Multifunction Lipid-Based CRISPR-Cas13a Genetic Circuit Delivery System for Bladder Cancer Gene Therapy.}, journal = {ACS synthetic biology}, volume = {9}, number = {2}, pages = {343-355}, doi = {10.1021/acssynbio.9b00349}, pmid = {31891494}, issn = {2161-5063}, mesh = {Animals ; Apoptosis ; CRISPR-Cas Systems/*genetics ; Cell Line, Tumor ; Disease Models, Animal ; Gene Editing ; *Genetic Therapy ; Humans ; Infrared Rays ; Liposomes/chemistry/*metabolism ; Mice ; Mice, Nude ; Photothermal Therapy ; Plasmids/genetics/metabolism ; Transfection ; Urinary Bladder Neoplasms/pathology/therapy ; Vascular Endothelial Growth Factor Receptor-2/genetics/metabolism ; }, abstract = {The treatment of bladder cancer has recently shown minimal progress. Gene therapy mediated by CRISPR provides a new option for bladder cancer treatment. In this study, we developed a versatile liposome system to deliver the CRISPR-Cas13a gene circuits into bladder cancer cells. After in vitro studies and intravesical perfusion studies in mice, this system showed five advantages: (1) CRISPR-Cas13a, a transcriptional targeting and cleavage tool for gene expression editing, did not affect the stability of the cell genome; (2) the prepared liposome systems were targeted to hVEGFR2, which is always highly expressed in bladder cancer cells; (3) the CRISPR-Cas13a sequence was driven by an artificial tumor specific promoter to achieve further targeting; (4) a near-infrared photosensitizer released using near-infrared light was introduced to control the delivery system; and (5) the plasmids were constructed with three crRNA tandem sequences to achieve multiple targeting and wider therapeutic results. This tumor cell targeting lipid delivery system with near-infrared laser-controlled ability provided a versatile strategy for CRISPR-Cas13a based gene therapy of bladder cancer.}, }
@article {pmid31874862, year = {2020}, author = {Katayama, S and Sato, K and Nakazawa, T}, title = {In vivo and in vitro knockout system labelled using fluorescent protein via microhomology-mediated end joining.}, journal = {Life science alliance}, volume = {3}, number = {1}, pages = {}, pmid = {31874862}, issn = {2575-1077}, mesh = {Animals ; CRISPR-Associated Protein 9/metabolism ; CRISPR-Cas Systems ; Carnitine O-Acetyltransferase/genetics ; Cell Line, Tumor ; Cell Survival/genetics ; DNA End-Joining Repair/*genetics ; Dependovirus/genetics ; Gene Editing/*methods ; Gene Knock-In Techniques/*methods ; Gene Knockout Techniques/*methods ; Genetic Vectors ; Green Fluorescent Proteins/*genetics ; Kelch-Like ECH-Associated Protein 1/genetics/metabolism ; Male ; Mice ; Mice, Inbred C57BL ; NIH 3T3 Cells ; Retinal Ganglion Cells/metabolism ; Staining and Labeling/*methods ; Staphylococcus aureus/enzymology ; Transfection ; }, abstract = {Gene knockout is important for understanding gene function and genetic disorders. The CRISPR/Cas9 system has great potential to achieve this purpose. However, we cannot distinguish visually whether a gene is knocked out and in how many cells it is knocked out among a population of cells. Here, we developed a new system that enables the labelling of knockout cells with fluorescent protein through microhomology-mediated end joining-based knock-in. Using a combination with recombinant adeno-associated virus, we delivered our system into the retina, where the expression of Staphylococcus aureus Cas9 was driven by a retina ganglion cell (RGC)-specific promoter, and knocked out carnitine acetyltransferase (CAT). We evaluated RGCs and revealed that CAT is required for RGC survival. Furthermore, we applied our system to Keap1 and confirmed that Keap1 is not expressed in fluorescently labelled cells. Our system provides a promising framework for cell type-specific genome editing and fluorescent labelling of gene knockout based on knock-in.}, }
@article {pmid31758450, year = {2020}, author = {Jacot, D and Soldati-Favre, D}, title = {CRISPR/Cas9-Mediated Generation of Tetracycline Repressor-Based Inducible Knockdown in Toxoplasma gondii.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2071}, number = {}, pages = {125-141}, doi = {10.1007/978-1-4939-9857-9_7}, pmid = {31758450}, issn = {1940-6029}, mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Cryptosporidium/genetics/pathogenicity ; Gene Knockout Techniques ; Genome, Protozoan/genetics ; Mutagenesis, Site-Directed ; Protein Stability ; Temperature ; Toxoplasma/genetics/*pathogenicity ; }, abstract = {The phylum Apicomplexa groups numerous pathogenic protozoan parasites including Plasmodium, the causative agent of malaria, Cryptosporidium which can cause severe gastrointestinal infections, as well as Babesia, Eimeria, and Theileria that account for considerable economic burdens to poultry and cattle industry. Toxoplasma gondii is the most ubiquitous and opportunistic member of this phylum able to infect all warm-blooded animals and responsible for severe disease in immunocompromised individuals and unborn fetuses.Due to its ease of cultivation and genetic tractability T. gondii has served as recipient for the transfer and adaptation of multiple genetic tools developed to control gene expression. In these parasites, a collection of tight conditional systems exists to control gene expression at the levels of transcription, RNA degradation or protein stability. The recent implementation of the CRISPR/Cas9 technology considerably reduces time and effort to generate transgenic parasites and at the same time increases to an ultimate level of precision the editing of the parasite genome. Here, we provide a step-by-step protocol for CRISPR/Cas9-mediated generation of tetracycline repressor-based inducible knockdown in T. gondii.}, }
@article {pmid33422414, year = {2021}, author = {Behbahani, RG and Danyaei, A and Teimoori, A and Neisi, N and Tahmasbi, MJ}, title = {Breast cancer radioresistance may be overcome by osteopontin gene knocking out with CRISPR/Cas9 technique.}, journal = {Cancer radiotherapie : journal de la Societe francaise de radiotherapie oncologique}, volume = {}, number = {}, pages = {}, doi = {10.1016/j.canrad.2020.08.048}, pmid = {33422414}, issn = {1769-6658}, abstract = {PURPOSE: Osteopontin (OPN) is a phosphoglycoprotein, with a wide range of physiological and pathological roles. High expression of OPN promotes aggressive behavior, causes poor prognosis in tumor cells, and reduces the survival of patients. Since overexpression of OPN gives rise to radioresistance, the effects of the gene knock out using the CRISPR/Cas9 system in combination with radiation are emphasized.<br><br>MATERIAL AND METHODS: We used the CRISPR/Cas9 technique to knock out the OPN gene in the MDA-MB-231 cell line. After transfection, the cells were irradiated. The changes of the OPN mRNA levels, the apoptosis, and the differences in cell viability were assessed.<br><br>RESULTS: A significant reduction in the OPN expression was observed alone or along with irradiation. The knocked out gene alone increased apoptosis rate. The cell viability decreased to after knocking out of the OPN gene. The gene knocking-out combined with irradiation led to more decline of cell viability.<br><br>CONCLUSION: Our results demonstrated that after knocking out the OPN gene, the MDA-MB-231 cells showed a significant radiosensitivity. Therefore, the OPN knock out in combination with conventional radiotherapy, may become an efficient therapeutic target in the future.}, }
@article {pmid33421768, year = {2021}, author = {Oh, SH and Lee, HJ and Ahn, MK and Jeon, MY and Yoon, JS and Jung, YJ and Kim, GN and Baek, IJ and Kim, I and Kim, KM and Sung, YH}, title = {Multiplex gene targeting in the mouse embryo using a Cas9-Cpf1 hybrid guide RNA.}, journal = {Biochemical and biophysical research communications}, volume = {539}, number = {}, pages = {48-55}, doi = {10.1016/j.bbrc.2020.12.072}, pmid = {33421768}, issn = {1090-2104}, abstract = {CRISPR-Cas systems, including Cas9 and Cpf1 (Cas12a), are promising tools for generating gene knockout mouse models. Unlike Cas9, Cpf1 can generate multiple crRNAs from a single concatemeric crRNA precursor, which is favorable for multiplex gene editing. Recently, a hybrid guide RNA (hgRNA) system employing both Cas9 and Cpf1 was developed for multiplex gene editing. As the crRNA of Cpf1 was linked to the 3' end of the sgRNA for Cas9, it can be split into separate guide RNAs by Cpf1. To examine whether this Cas9-Cpf1 hybrid system is suitable for multiplex gene knockouts in the mouse embryo, we generated an hgRNA that simultaneously targets the mouse Il10ra gene by Cas9 and mouse Dr3 (or Tnfrsf25, death receptor3) gene by Cpf1. The expression of hgRNA from a single promoter induced significant indels at each gene in cultured mouse cells upon the co-expression of both Cas9 and Cpf1. Interestingly, the hgRNA exhibited comparable Cas9-mediated indel activity without Cpf1 expression. Similarly, when the hgRNA was co-microinjected with both Cas9 and Cpf1 mRNAs into mouse zygotes at the pronuclear stage, founder mice were generated harboring mutations in both the Il10ra and Dr3 genes. However, when Cas9 mRNA was used alone without Cpf1 mRNA, the mouse Il10ra gene targeting was significantly decreased. These results indicate that the hgRNA system is a possible tool for multiplex gene targeting in the mouse embryo.}, }
@article {pmid33421620, year = {2021}, author = {Ansari, I and Chaturvedi, A and Chitkara, D and Singh, S}, title = {CRISPR/Cas mediated epigenome editing for cancer therapy.}, journal = {Seminars in cancer biology}, volume = {}, number = {}, pages = {}, doi = {10.1016/j.semcancer.2020.12.018}, pmid = {33421620}, issn = {1096-3650}, abstract = {The understanding of the relationship between epigenetic alterations, their effects on gene expression and the knowledge that these epigenetic alterations are reversible, have opened up new therapeutic pathways for treating various diseases, including cancer. This has led the research for a better understanding of the mechanism and pathways of carcinogenesis and provided the opportunity to develop the therapeutic approaches by targeting such pathways. Epi-drugs, DNA methyl transferase (DNMT) inhibitors and histone deacetylase (HDAC) inhibitors are the best examples of epigenetic therapies with clinical applicability. Moreover, precise genome editing technologies such as CRISPR/Cas has proven their efficacy in epigenome editing, including the alteration of epigenetic markers, such as DNA methylation or histone modification. The main disadvantage with DNA gene editing technologies is off-target DNA sequence alteration, which is not an issue with epigenetic editing. It is known that cancer is linked with epigenetic alteration, and thus CRISPR/Cas system shows potential for cancer therapy via epigenome editing. This review outlines the epigenetic therapeutic approach for cancer therapy using CRISPR/Cas, from the basic understanding of cancer epigenetics to potential applications of CRISPR/Cas in treating cancer.}, }
@article {pmid33421369, year = {2021}, author = {Workman, RE and Pammi, T and Nguyen, BTK and Graeff, LW and Smith, E and Sebald, SM and Stoltzfus, MJ and Euler, CW and Modell, JW}, title = {A natural single-guide RNA repurposes Cas9 to autoregulate CRISPR-Cas expression.}, journal = {Cell}, volume = {}, number = {}, pages = {}, doi = {10.1016/j.cell.2020.12.017}, pmid = {33421369}, issn = {1097-4172}, abstract = {CRISPR-Cas systems provide prokaryotes with acquired immunity against viruses and plasmids, but how these systems are regulated to prevent autoimmunity is poorly understood. Here, we show that in the S. pyogenes CRISPR-Cas system, a long-form transactivating CRISPR RNA (tracr-L) folds into a natural single guide that directs Cas9 to transcriptionally repress its own promoter (Pcas). Further, we demonstrate that Pcas serves as a critical regulatory node. De-repression causes a dramatic 3,000-fold increase in immunization rates against viruses; however, heightened immunity comes at the cost of increased autoimmune toxicity. Using bioinformatic analyses, we provide evidence that tracrRNA-mediated autoregulation is widespread in type II-A CRISPR-Cas systems. Collectively, we unveil a new paradigm for the intrinsic regulation of CRISPR-Cas systems by natural single guides, which may facilitate the frequent horizontal transfer of these systems into new hosts that have not yet evolved their own regulatory strategies.}, }
@article {pmid33420656, year = {2021}, author = {Yuza, K and Nagahashi, M and Ichikawa, H and Hanyu, T and Nakajima, M and Shimada, Y and Ishikawa, T and Sakata, J and Takeuchi, S and Okuda, S and Matsuda, Y and Abe, M and Sakimura, K and Takabe, K and Wakai, T}, title = {Activin a Receptor Type 2A Mutation Affects the Tumor Biology of Microsatellite Instability-High Gastric Cancer.}, journal = {Journal of gastrointestinal surgery : official journal of the Society for Surgery of the Alimentary Tract}, volume = {}, number = {}, pages = {}, pmid = {33420656}, issn = {1873-4626}, support = {17K10579//Japan Society for the Promotion of Science/ ; 19H03714//Japan Society for the Promotion of Science/ ; 18K19576//Japan Society for the Promotion of Science/ ; 19K09212//Japan Society for the Promotion of Science/ ; 19K09165//Japan Society for the Promotion of Science/ ; 17K10692//Japan Society for the Promotion of Science/ ; 18K08612//Japan Society for the Promotion of Science/ ; 17H04093//Japan Society for the Promotion of Science/ ; 18H04123//Japan Society for the Promotion of Science/ ; 19K22651//Japan Society for the Promotion of Science/ ; R01CA160688/CA/NCI NIH HHS/United States ; }, abstract = {BACKGROUND: Activin A receptor type 2A (ACVR2A) is one of the most frequently mutated genes in microsatellite instability-high (MSI-H) gastric cancer. However, the clinical relevance of the ACVR2A mutation in MSI-H gastric cancer patients remains unclear. The aims of this study were to explore the effect of ACVR2A mutation on the tumor behavior and to identify the clinicopathological characteristics of gastric cancer patients with ACVR2A mutations.<br><br>METHODS: An in vitro study was performed to investigate the biological role of ACVR2A via CRISPR/Cas9-mediated ACVR2A knockout MKN74 human gastric cancer cells. One hundred twenty-four patients with gastric cancer were retrospectively analyzed, and relations between MSI status, ACVR2A mutations, and clinicopathological factors were evaluated.<br><br>RESULTS: ACVR2A knockout cells showed less aggressive tumor biology than mock-transfected cells, displaying reduced proliferation, migration, and invasion (P < 0.05). MSI mutations were found in 10% (13/124) of gastric cancer patients, and ACVR2A mutations were found in 8.1% (10/124) of patients. All ACVR2A mutations were accompanied by MSI. The 5-year overall survival rates of ACVR2A wild-type patients and ACVR2A-mutated patients were 57% and 90%, respectively (P = 0.048). Multivariate analysis revealed that older age (P = 0.015), distant metastasis (P < 0.001), and ACVR2A wild-type status (P = 0.040) were independent prognostic factors for overall survival.<br><br>CONCLUSIONS: Our study demonstrated that gastric cancer patients with ACVR2A mutation have a significantly better prognosis than those without. Dysfunction of ACVR2A in MKN74 human gastric cancer cells caused less aggressive tumor biology, indicating the importance of ACVR2A in the progression of MSI-H tumors.}, }
@article {pmid33418105, year = {2020}, author = {Das Mukhopadhyay, C and Sharma, P and Sinha, K and Rajarshi, K}, title = {Recent trends in analytical and digital techniques for the detection of the SARS-Cov-2.}, journal = {Biophysical chemistry}, volume = {270}, number = {}, pages = {106538}, doi = {10.1016/j.bpc.2020.106538}, pmid = {33418105}, issn = {1873-4200}, abstract = {The current global outbreak of COVID-19 due to SARS-CoV-2 is an unprecedented humanitarian crisis. Considering the gravity of its impact there is an immediate need to develop a detection technique that is sensitive, specific, fast, and affordable for the clinical diagnosis of the disease. Real time Polymerase Chain Reaction (RT-PCR)-based detection platforms are contemplated to be the gold standard to detect viral RNA. However, that may be susceptible to errors, and there is a risk of obtaining false results, which ultimately compromises the strategy of efficient disease management. Several modern techniques exhibiting assured results with enhanced sensitivity and specificity against the SARS-CoV-2 associated viral components or immune response against it have been developed and may be implemented. The review deals with the conventional RT-PCR detection techniques and compares them to other detection platforms viz., biosensor based detection of antigens, fluorescent or colorimetric detection systems including CRISPR-Cas 13 based SHERLOCK kit, CRISPR Cas-9 based FELUDA test kit, CRISPR DETECTR kit, Next Generation Sequencing or microarray-based kits. These modern techniques are great as a point of care detection methods but should be followed by RT PCR based detection for the confirmation of COVID-19 status.}, }
@article {pmid33412762, year = {2020}, author = {Park, YK and Yoon, BH and Park, SJ and Kim, BK and Kim, SY}, title = {BaSDAS: a web-based pooled CRISPR-Cas9 knockout screening data analysis system.}, journal = {Genomics &amp; informatics}, volume = {18}, number = {4}, pages = {e46}, doi = {10.5808/GI.2020.18.4.e46}, pmid = {33412762}, issn = {1598-866X}, support = {2014M3C9A3068554//National Research Foundation of Korea/ ; 2017MBA9B5060884//National Research Foundation of Korea/ ; //KRIBB Research Initiative Program/ ; }, abstract = {We developed the BaSDAS (Barcode-Seq Data Analysis System), a GUI-based pooled knockout screening data analysis system, to facilitate the analysis of pooled knockout screen data easily and effectively by researchers with limited bioinformatics skills. The BaSDAS supports the analysis of various pooled screening libraries, including yeast, human, and mouse libraries, and provides many useful statistical and visualization functions with a user-friendly web interface for convenience. We expect that BaSDAS will be a useful tool for the analysis of genome-wide screening data and will support the development of novel drugs based on functional genomics information.}, }
@article {pmid32860838, year = {2020}, author = {Das, S and Chandrasekaran, AP and Jo, KS and Ko, NR and Oh, SJ and Kim, KS and Ramakrishna, S}, title = {HAUSP stabilizes Cdc25A and protects cervical cancer cells from DNA damage response.}, journal = {Biochimica et biophysica acta. Molecular cell research}, volume = {1867}, number = {12}, pages = {118835}, doi = {10.1016/j.bbamcr.2020.118835}, pmid = {32860838}, issn = {1879-2596}, mesh = {Animals ; CRISPR-Cas Systems ; DNA Damage/genetics ; Drug Resistance, Neoplasm/*genetics ; Female ; Flow Cytometry ; Gene Expression Regulation, Neoplastic/genetics ; Gene Knockout Techniques ; HEK293 Cells ; HeLa Cells ; Heterografts ; Humans ; Mice ; Ubiquitin-Specific Peptidase 7/*genetics ; Uterine Cervical Neoplasms/drug therapy/*genetics/pathology ; cdc25 Phosphatases/*genetics ; }, abstract = {Resistance to DNA-damaging agents is one of the main reasons for the low survival of cervical cancer patients. Previous reports have suggested that the Cdc25A oncoprotein significantly affects the level of susceptibility to DNA-damaging agents, but the molecular mechanism remains unclear. In this study, we used Western blot and flow cytometry analyses to demonstrate that the deubiquitinating enzyme HAUSP stabilizes Cdc25A protein level. Furthermore, in a co-immunoprecipitation assay, we found that HAUSP interacts with and deubiquitinates Cdc25A both exogenously and endogenously. HAUSP extends the half-life of the Cdc25A protein by circumventing turnover. HAUSP knockout in HeLa cells using the CRISPR/Cas9 system caused a significant delay in Cdc25A-mediated cell cycle progression, cell migration, and colony formation and attenuated tumor progression in a mouse xenograft model. Furthermore, HAUSP-mediated stabilization of the Cdc25A protein produced enhanced resistance to DNA-damaging agents. Overall, our study suggests that targeting Cdc25A and HAUSP could be a promising combinatorial approach to halt progression and minimize antineoplastic resistance in cervical cancer.}, }
@article {pmid32496007, year = {2020}, author = {Zhang, W and Kataoka, M and Yen Doan, H and Wu, FT and Haga, K and Takeda, N and Muramatsu, M and Li, TC}, title = {Isolation and characterization of mammalian orthoreoviruses using a cell line resistant to sapelovirus infection.}, journal = {Transboundary and emerging diseases}, volume = {67}, number = {6}, pages = {2849-2859}, doi = {10.1111/tbed.13655}, pmid = {32496007}, issn = {1865-1682}, support = {(JP18fk0210043, JP19fk0108102)//Japan Agency for Medical Reserach and development (AMED)/ ; 17K08090//Grabt-in-Aid for Scientific Reserarch/ ; }, mesh = {Animals ; Antibodies, Viral/blood ; Blotting, Western/veterinary ; CRISPR-Cas Systems ; Cell Line ; Electrophoresis, Polyacrylamide Gel/veterinary ; Enzyme-Linked Immunosorbent Assay/veterinary ; Feces/virology ; High-Throughput Nucleotide Sequencing/veterinary ; Immunoglobulin G/blood ; Microscopy, Electron/veterinary ; Orthoreovirus, Mammalian/genetics/immunology/*isolation &amp; purification ; Phylogeny ; Picornaviridae/*pathogenicity ; Picornaviridae Infections/*veterinary/virology ; RNA, Viral/genetics ; Reoviridae Infections/*veterinary/virology ; Swine ; Swine Diseases/*virology ; }, abstract = {Porcine sapelovirus (PSV) is a causative agent of acute diarrhoea, pneumonia and reproductive disorders in swine. Since PSV infection interrupts the growth of other viruses due to its high replication capability in cell culture, the prevention of PSV replication is a keystone to the isolation of non-PSV agents from PSV-contaminated samples. In the present study, we established the PSV infection-resistant cell line N1380 and isolated three mammalian orthoreoviruses (MRV) strains, sR1521, sR1677 and sR1590, from swine in Taiwan. These Taiwanese isolates induced an extensive cytopathic effect in N1380 cells upon infection. The complete and empty virus particles were purified from the cell culture supernatants. Next-generation sequencing analyses revealed that the complete virus particles contained 10 segments, including 3 large (L1, L2 and L3), 3 medium (M1, M2 and M3) and 4 small (S1, S2, S3 and S4) segments. In contrast, the empty virus particles without genome were non-infectious. Phylogenetic analyses revealed that the Taiwanese strains belong to serotype 2 MRV (MRV2). We established an ELISA for the detection of IgG antibody against MRV2 by using the empty virus particles as the antigen. A total of 540 swine and 95 wild boar serum samples were collected in Japan, and the positive rates were 100% and 52.6%, respectively. These results demonstrated that MRV infection occurred frequently in both swine and wild boar in Japan. We established a cell line that is efficient for the isolation of MRV, and the ELISA based on the naturally occurring empty particles would be of great value for the surveillance of MRV-related diseases.}, }
@article {pmid32147886, year = {2020}, author = {He, XY and Ren, XH and Peng, Y and Zhang, JP and Ai, SL and Liu, BY and Xu, C and Cheng, SX}, title = {Aptamer/Peptide-Functionalized Genome-Editing System for Effective Immune Restoration through Reversal of PD-L1-Mediated Cancer Immunosuppression.}, journal = {Advanced materials (Deerfield Beach, Fla.)}, volume = {32}, number = {17}, pages = {e2000208}, doi = {10.1002/adma.202000208}, pmid = {32147886}, issn = {1521-4095}, support = {51533006//National Natural Science Foundation of China/ ; 21875169//National Natural Science Foundation of China/ ; }, mesh = {Animals ; Apoptosis ; Aptamers, Nucleotide/*chemistry ; B7-H1 Antigen/*metabolism ; CD8-Positive T-Lymphocytes/cytology/immunology/metabolism ; CRISPR-Cas Systems/*genetics ; Cell Line, Tumor ; Cell Nucleus/metabolism ; Gene Editing/*methods ; Humans ; Hyaluronic Acid/chemistry ; Immunosuppression ; Nanoparticles/chemistry ; Oligodeoxyribonucleotides/chemistry ; Peptides/*chemistry ; Plasmids/chemistry/metabolism ; Programmed Cell Death 1 Receptor/metabolism ; beta Catenin/deficiency/genetics/metabolism ; }, abstract = {Effective reversal of tumor immunosuppression is of critical importance in cancer therapy. A multifunctional delivery vector that can effectively deliver CRISPR-Cas9 plasmid for β-catenin knockout to reverse tumor immunosuppression is constructed. The multi-functionalized delivery vector is decorated with aptamer-conjugated hyaluronic acid and peptide-conjugated hyaluronic acid to combine the tumor cell/nuclear targeting function of AS1411 with the cell penetrating/nuclear translocation function of TAT-NLS. Due to the significantly enhanced plasmid enrichment in malignant cell nuclei, the genome editing system can induce effective β-catenin knockout and suppress Wnt/β-catenin pathway, resulting in notably downregulated proteins involved in tumor progression and immunosuppression. Programmed death-ligand 1 (PD-L1) downregulation in edited tumor cells not only releases the PD-1/PD-L1 brake to improve the cancer killing capability of CD8+ T cells, but also enhances antitumor immune responses of immune cells. This provides a facile strategy to reverse tumor immunosuppression and to restore immunosurveillance and activate anti-tumor immunity.}, }
@article {pmid31981430, year = {2020}, author = {Chen, L and Cai, Y and Qu, M and Wang, L and Sun, H and Jiang, B and Wu, T and Liu, L and Sun, S and Wu, C and Yao, W and Yuan, S and Han, T and Hou, W}, title = {Soybean adaption to high-latitude regions is associated with natural variations of GmFT2b, an ortholog of FLOWERING LOCUS T.}, journal = {Plant, cell &amp; environment}, volume = {43}, number = {4}, pages = {934-944}, pmid = {31981430}, issn = {1365-3040}, mesh = {Adaptation, Physiological/genetics ; Arabidopsis Proteins/genetics/physiology ; CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Cloning, Molecular ; Flowers/growth &amp; development ; Gene Editing ; Gene Expression Regulation, Plant/genetics ; Genetic Variation/genetics/physiology ; Geography ; Photoperiod ; Plant Proteins/*genetics/physiology ; Soybeans/genetics/growth &amp; development/*physiology ; Transcription Factors/*genetics/physiology ; Transcriptome ; }, abstract = {Day length has an important influence on flowering and growth habit in many plant species. In crops such as soybean, photoperiod sensitivity determines the geographical range over which a given cultivar can grow and flower. The soybean genome contains ~10 genes homologous to FT, a central regulator of flowering from Arabidopsis thaliana. However, the precise roles of these soybean FTs are not clearly. Here we show that one such gene, GmFT2b, promotes flowering under long-days (LDs). Overexpression of GmFT2b upregulates expression of flowering-related genes which are important in regulating flowering time. We propose a 'weight' model for soybean flowering under short-day (SD) and LD conditions. Furthermore, we examine GmFT2b sequences in 195 soybean cultivars, as well as flowering phenotypes, geographical distributions and maturity groups. We found that Hap3, a major GmFT2b haplotype, is associated with significantly earlier flowering at higher latitudes. We anticipate our assay to provide important resources for the genetic improvement of soybean, including new germplasm for soybean breeding, and also increase our understanding of functional diversity in the soybean FT gene family.}, }
@article {pmid31882406, year = {2020}, author = {Kandul, NP and Liu, J and Buchman, A and Gantz, VM and Bier, E and Akbari, OS}, title = {Assessment of a Split Homing Based Gene Drive for Efficient Knockout of Multiple Genes.}, journal = {G3 (Bethesda, Md.)}, volume = {10}, number = {2}, pages = {827-837}, pmid = {31882406}, issn = {2160-1836}, support = {DP5 OD023098/OD/NIH HHS/United States ; }, mesh = {CRISPR-Cas Systems ; *Gene Drive Technology ; Gene Editing ; *Gene Knockout Techniques ; Gene Order ; *Gene Targeting/methods ; Genetic Vectors/genetics ; Genotyping Techniques ; Models, Genetic ; Mutation ; RNA, Guide ; Zygote/metabolism ; }, abstract = {Homing based gene drives (HGD) possess the potential to spread linked cargo genes into natural populations and are poised to revolutionize population control of animals. Given that host encoded genes have been identified that are important for pathogen transmission, targeting these genes using guide RNAs as cargo genes linked to drives may provide a robust method to prevent disease transmission. However, effectiveness of the inclusion of additional guide RNAs that target separate genes has not been thoroughly explored. To test this approach, we generated a split-HGD in Drosophila melanogaster that encoded a drive linked effector consisting of a second gRNA engineered to target a separate host-encoded gene, which we term a gRNA-mediated effector (GME). This design enabled us to assess homing and knockout efficiencies of two target genes simultaneously, and also explore the timing and tissue specificity of Cas9 expression on cleavage/homing rates. We demonstrate that inclusion of a GME can result in high efficiency of disruption of both genes during super-Mendelian propagation of split-HGD. Furthermore, both genes were knocked out one generation earlier than expected indicating the robust somatic expression of Cas9 driven by Drosophila germline-limited promoters. We also assess the efficiency of 'shadow drive' generated by maternally deposited Cas9 protein and accumulation of drive-induced resistance alleles along multiple generations, and discuss design principles of HGD that could mitigate the accumulation of resistance alleles while incorporating a GME.}, }
@article {pmid31589320, year = {2020}, author = {Dekkers, JF and Whittle, JR and Vaillant, F and Chen, HR and Dawson, C and Liu, K and Geurts, MH and Herold, MJ and Clevers, H and Lindeman, GJ and Visvader, JE}, title = {Modeling Breast Cancer Using CRISPR-Cas9-Mediated Engineering of Human Breast Organoids.}, journal = {Journal of the National Cancer Institute}, volume = {112}, number = {5}, pages = {540-544}, pmid = {31589320}, issn = {1460-2105}, mesh = {Animals ; Breast/cytology/*physiology ; Breast Neoplasms/*genetics/pathology ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Female ; Gene Editing/methods ; Gene Knockout Techniques ; Genes, p53 ; Heterografts ; Humans ; Mice, Inbred NOD ; Mice, SCID ; Organoids ; PTEN Phosphohydrolase/genetics ; Retinoblastoma Binding Proteins/genetics ; Tissue Engineering/methods ; Tumor Suppressor Protein p53/genetics ; Ubiquitin-Protein Ligases/genetics ; }, abstract = {Breast cancer is characterized by histological and functional heterogeneity, posing a clinical challenge for patient treatment. Emerging evidence suggests that the distinct subtypes reflect the repertoire of genetic alterations and the target cell. However, the precise initiating events that predispose normal epithelium to neoplasia are poorly understood. Here, we demonstrate that breast epithelial organoids can be generated from human reduction mammoplasties (12 out of 12 donors), thus creating a tool to study the clonal evolution of breast cancer. To recapitulate de novo oncogenesis, we exploited clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 for targeted knockout of four breast cancer-associated tumor suppressor genes (P53, PTEN, RB1, NF1) in mammary progenitor cells from six donors. Mutant organoids gained long-term culturing capacity and formed estrogen-receptor positive luminal tumors on transplantation into mice for one out of six P53/PTEN/RB1-mutated and three out of six P53/PTEN/RB1/NF1-mutated lines. These organoids responded to endocrine therapy or chemotherapy, supporting the potential utility of this model to enhance our understanding of the molecular events that culminate in specific subtypes of breast cancer.}, }
@article {pmid33412428, year = {2021}, author = {Liu, PF and Zhao, KR and Liu, ZJ and Wang, L and Ye, SY and Liang, GX}, title = {Cas12a-based electrochemiluminescence biosensor for target amplification-free DNA detection.}, journal = {Biosensors &amp; bioelectronics}, volume = {176}, number = {}, pages = {112954}, doi = {10.1016/j.bios.2020.112954}, pmid = {33412428}, issn = {1873-4235}, abstract = {CRISPR/Cas system have drawn increasing attention in accurate and sensitive nucleic acids detection. Herein, we reported a novel Cas12a-based electrochemiluminescence biosensor for target amplification-free human papilloma virus subtype (HPV-16) DNA detection. During this detection process, Cas12a employed its two-part recognition mechanism to improve the specificity and trans-cleavage capability to achieve signal amplification, while L-Methionine stabilized gold nanoclusters (Met-AuNCs) were served as high-efficiency ECL emitters to achieve ECL signal transition. Given the unique combination of Cas12a with ECL technique, the detection limit was determined as 0.48 pM and the whole detection could be completed within 70 min. We also validated the practical application of the proposed biosensor by using undiluted human blood samples, which gives impetus to the design of new generations of CRISPR/Cas detection system beyond the traditional ones with ultimate applications in sensing analysis and diagnostic technologies.}, }
@article {pmid33411765, year = {2021}, author = {Jubair, L and Lam, AK and Fallaha, S and McMillan, NAJ}, title = {CRISPR/Cas9-loaded stealth liposomes effectively cleared established HPV16-driven tumours in syngeneic mice.}, journal = {PloS one}, volume = {16}, number = {1}, pages = {e0223288}, doi = {10.1371/journal.pone.0223288}, pmid = {33411765}, issn = {1932-6203}, abstract = {Gene-editing has raised the possibility of being able to treat or cure cancers, but key challenges remain, including efficient delivery, in vivo efficacy, and its safety profile. Ideal targets for cancer therapy are oncogenes, that when edited, cause cell death. Here, we show, using the human papillomavirus (HPV) type 16 cancer cell line TC1, that CRISPR/Cas9 targeting the E7 oncogene and packaged in PEGylated liposomes cleared established tumours in immunocompetent mice. Treatment caused no significant toxicity in the spleen or liver. An ideal therapeutic outcome would be the induction of an immunogenic cell death (ICD), such that recurrent tumours would be eliminated by the host immune system. We show here for the first time that CRISPR/Cas9-mediated cell death via targeting E7 did not result in ICD. Overall, our data show that in vivo CRISPR/Cas targeting of oncogenes is an effective treatment approach for cancer.}, }
@article {pmid33411517, year = {2021}, author = {Zhou, T and Huang, M and Lin, J and Huang, R and Xing, D}, title = {High-Fidelity CRISPR/Cas13a trans-Cleavage-Triggered Rolling Circle Amplified DNAzyme for Visual Profiling of MicroRNA.}, journal = {Analytical chemistry}, volume = {}, number = {}, pages = {}, doi = {10.1021/acs.analchem.0c03708}, pmid = {33411517}, issn = {1520-6882}, abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) (CRISPR/Cas) system innovates a next-generation biosensor due to its high-fidelity, programmability, and efficient signal amplification ability. Developing a CRISPR/Cas-based visual detection system could contribute to point-of-care biomarker diagnosis. Existing CRISPR/Cas9-mediated visual detection methods are limited by the inherent properties of Cas9. Herein, we explored the trans-cleavage ability of Cas13a on ribonucleotide-bearing DNA oligo, eliminated the unavailability of the trans-cleavage substrate for subsequent polymerization reaction, and developed a homogeneous CRISPR/Cas13a-based visual detection system (termed vCas) for specific and sensitive detection of miRNA. The results indicated that vCas can provide a detection limit of 1 fM for miR-10b with single-base specificity and can be used to analyze miRNA in serum and cell extracts. Conclusively, vCas holds a great application prospective for clinical molecular diagnosis.}, }
@article {pmid33410425, year = {2020}, author = {Sreepadmanabh, M and Sahu, AK and Chande, A}, title = {COVID-19: Advances in diagnostic tools, treatment strategies, and vaccine development.}, journal = {Journal of biosciences}, volume = {45}, number = {}, pages = {}, pmid = {33410425}, issn = {0973-7138}, abstract = {An unprecedented worldwide spread of the SARS-CoV-2 has imposed severe challenges on healthcare facilities and medical infrastructure. The global research community faces urgent calls for the development of rapid diagnostic tools, effective treatment protocols, and most importantly, vaccines against the pathogen. Pooling together expertise across broad domains to innovate effective solutions is the need of the hour. With these requirements in mind, in this review, we provide detailed critical accounts on the leading efforts at developing diagnostics tools, therapeutic agents, and vaccine candidates. Importantly, we furnish the reader with a multidisciplinary perspective on how conventional methods like serology and RT-PCR, as well as cutting-edge technologies like CRISPR/Cas and artificial intelligence/machine learning, are being employed to inform and guide such investigations. We expect this narrative to serve a broad audience of both active and aspiring researchers in the field of biomedical sciences and engineering and help inspire radical new approaches towards effective detection, treatment, and prevention of this global pandemic.}, }
@article {pmid33402545, year = {2020}, author = {Konishi, CT and Long, C}, title = {Progress and challenges in CRISPR-mediated therapeutic genome editing for monogenic diseases.}, journal = {Journal of biomedical research}, volume = {}, number = {}, pages = {1-15}, doi = {10.7555/JBR.34.20200105}, pmid = {33402545}, issn = {1674-8301}, abstract = {There are an estimated 10 000 monogenic diseases affecting tens of millions of individuals worldwide. The application of CRISPR/Cas genome editing tools to treat monogenic diseases is an emerging strategy with the potential to generate personalized treatment approaches for these patients. CRISPR/Cas-based systems are programmable and sequence-specific genome editing tools with the capacity to generate base pair resolution manipulations to DNA or RNA. The complexity of genomic insults resulting in heritable disease requires patient-specific genome editing strategies with consideration of DNA repair pathways, and CRISPR/Cas systems of different types, species, and those with additional enzymatic capacity and/or delivery methods. In this review we aim to discuss broad and multifaceted therapeutic applications of CRISPR/Cas gene editing systems including in harnessing of homology directed repair, non-homologous end joining, microhomology-mediated end joining, and base editing to permanently correct diverse monogenic diseases.}, }
@article {pmid33402270, year = {2020}, author = {Zhang, Y and Xi, H and Juhas, M}, title = {Biosensing Detection of the SARS-CoV-2 D614G Mutation.}, journal = {Trends in genetics : TIG}, volume = {}, number = {}, pages = {}, doi = {10.1016/j.tig.2020.12.004}, pmid = {33402270}, issn = {0168-9525}, abstract = {The emergence of a mutant strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with an amino acid change from aspartate to a glycine residue at position 614 (D614G) has been reported and this mutant appears to be now dominant in the pandemic. Efficient detection of the SARS-CoV-2 D614G mutant by biosensing technologies is therefore crucial for the control of the pandemic.}, }
@article {pmid33401751, year = {2021}, author = {Taliansky, M and Samarskaya, V and Zavriev, SK and Fesenko, I and Kalinina, NO and Love, AJ}, title = {RNA-Based Technologies for Engineering Plant Virus Resistance.}, journal = {Plants (Basel, Switzerland)}, volume = {10}, number = {1}, pages = {}, doi = {10.3390/plants10010082}, pmid = {33401751}, issn = {2223-7747}, support = {14.W03.31.0003//Government of Russian Federation/ ; }, abstract = {In recent years, non-coding RNAs (ncRNAs) have gained unprecedented attention as new and crucial players in the regulation of numerous cellular processes and disease responses. In this review, we describe how diverse ncRNAs, including both small RNAs and long ncRNAs, may be used to engineer resistance against plant viruses. We discuss how double-stranded RNAs and small RNAs, such as artificial microRNAs and trans-acting small interfering RNAs, either produced in transgenic plants or delivered exogenously to non-transgenic plants, may constitute powerful RNA interference (RNAi)-based technology that can be exploited to control plant viruses. Additionally, we describe how RNA guided CRISPR-CAS gene-editing systems have been deployed to inhibit plant virus infections, and we provide a comparative analysis of RNAi approaches and CRISPR-Cas technology. The two main strategies for engineering virus resistance are also discussed, including direct targeting of viral DNA or RNA, or inactivation of plant host susceptibility genes. We also elaborate on the challenges that need to be overcome before such technologies can be broadly exploited for crop protection against viruses.}, }
@article {pmid33293555, year = {2020}, author = {Carlson-Stevermer, J and Das, A and Abdeen, AA and Fiflis, D and Grindel, BI and Saxena, S and Akcan, T and Alam, T and Kletzien, H and Kohlenberg, L and Goedland, M and Dombroe, MJ and Saha, K}, title = {Design of efficacious somatic cell genome editing strategies for recessive and polygenic diseases.}, journal = {Nature communications}, volume = {11}, number = {1}, pages = {6277}, pmid = {33293555}, issn = {2041-1723}, support = {R35 GM119644/GM/NIGMS NIH HHS/United States ; P30 CA014520/CA/NCI NIH HHS/United States ; }, mesh = {Alleles ; CRISPR-Cas Systems/*genetics ; Cells, Cultured ; Computer Simulation ; Gene Editing/*methods ; Gene Transfer Techniques ; Genetic Therapy/*methods ; Glycogen Storage Disease Type II/genetics/*therapy ; Humans ; Induced Pluripotent Stem Cells ; Infant ; Inheritance Patterns ; Liver/cytology ; Male ; Models, Genetic ; Mutation ; Primary Cell Culture ; }, abstract = {Compound heterozygous recessive or polygenic diseases could be addressed through gene correction of multiple alleles. However, targeting of multiple alleles using genome editors could lead to mixed genotypes and adverse events that amplify during tissue morphogenesis. Here we demonstrate that Cas9-ribonucleoprotein-based genome editors can correct two distinct mutant alleles within a single human cell precisely. Gene-corrected cells in an induced pluripotent stem cell model of Pompe disease expressed the corrected transcript from both corrected alleles, leading to enzymatic cross-correction of diseased cells. Using a quantitative in silico model for the in vivo delivery of genome editors into the developing human infant liver, we identify progenitor targeting, delivery efficiencies, and suppression of imprecise editing outcomes at the on-target site as key design parameters that control the efficacy of various therapeutic strategies. This work establishes that precise gene editing to correct multiple distinct gene variants could be highly efficacious if designed appropriately.}, }
@article {pmid33229326, year = {2020}, author = {Lin, MT and Lai, LL and Zhao, M and Lin, BW and Yao, XP}, title = {[Construction of a striatum-specific Slc20a2 gene knockout mice model by CRISPR/Cas9 AAV system].}, journal = {Yi chuan = Hereditas}, volume = {42}, number = {10}, pages = {1017-1027}, doi = {10.16288/j.yczz.20-138}, pmid = {33229326}, issn = {0253-9772}, mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Gene Knockout Techniques ; Mice ; Mice, Knockout ; *Models, Animal ; RNA, Guide/genetics ; *Sodium-Phosphate Cotransporter Proteins, Type III/genetics ; }, abstract = {Primary familial brain calcification (PFBC) is a chronic progressive neurogenetic disorder. Its clinical symptoms mainly include dyskinesia, cognitive disorder and mental impairment; and the pathogenesis remains unclear. Studies have shown that SLC20A2 is the most common pathogenic gene of the disease. Since the Slc20a2 gene knockout mouse model could result in fetal growth restriction, in order to better understand the pathogenesis of PFBC, the present study used the CRISPR/Cas9 technology to construct a conditional knockout model of Slc20a2 gene in the striatum of mice. First, three sgRNAs (single guide RNAs) were designed to target the exon3 of Slc20a2 gene. The activity of the respective sgRNA was verified by constructing expression plasmids, transfecting cells and Surveyor assay. Second, the SgRNA with the highest activity was selected to generate the recombinant AAV-Cre virus, which was injected into the striatum of mice by stereotactic method. In vitro experiments showed that the three sgRNAs could effectively mediate Cas9 cleavage of the respective target DNA. The activity of Cre recombinase of the AAV-Cre was confirmed by immunofluorescence assay. Immunohistochemistry, TA clone, high-throughput sequencing and Western blot were used to detect and evaluate the efficiency of Slc20a2 gene knockout. The results showed that the Slc20a2 expression in the striatum of mice in the experimental group decreased significantly. In this study, three sgRNAs capable of knockout of Slc20a2 were successfully designed, and the conditional knockout of the Slc20a2 gene in the striatum of mouse was successfully established by the CRISPR/Cas9 technology, thereby providing an effective animal model for studying the pathogenesis of PFBC.}, }
@article {pmid33229321, year = {2020}, author = {Bao, LW and Zhou, YY and Zeng, FY}, title = {[Advances in gene therapy for β-thalassemia and hemophilia based on the CRISPR/Cas9 technology].}, journal = {Yi chuan = Hereditas}, volume = {42}, number = {10}, pages = {949-964}, doi = {10.16288/j.yczz.20-110}, pmid = {33229321}, issn = {0253-9772}, mesh = {CRISPR-Cas Systems/genetics ; Clinical Trials as Topic ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; *Genetic Therapy ; *Hemophilia A/genetics/therapy ; Humans ; Research/trends ; Technology ; *beta-Thalassemia/genetics/therapy ; }, abstract = {Thalassemia and hemophilia are common inherited blood disorders caused by genetic abnormalities. These diseases are difficult to cure and can be inherited to the next generation, causing severe family and social burden. The emergence of gene therapy provides a new treatment for genetic diseases. However, since its first clinical trial in 1990, the development of gene therapy has not been as optimistic in the past three decades as one could hope. The development of gene-editing technology, particularly the third generation gene-editing technology CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9), has given hope in such therapeutic approach for having advantages in high editing efficiency, simple operation, and low cost. Gene editing-mediated gene therapy has thus received increasing attention from the biomedical community. It has shown promises for the treatment of inherited blood disorders, such as thalassemia and hemophilia. This paper reviews the fundamental research progress of gene therapy for β-thalassemia and hemophilia based on CRISPR/Cas9 technology in the past six years. It also summarizes the CRISPR/Cas9-based clinical trials of gene therapy. The problems and possible solutions to this technology for gene therapy are also discussed, thereby providing a reference for the research on gene therapy of inherited blood disorders based on CRISPR/Cas9 technology.}, }
@article {pmid33170892, year = {2020}, author = {Alghamdi, A and Aldossary, W and Albahkali, S and Alotaibi, B and Alrfaei, BM}, title = {The loss of microglia activities facilitates glaucoma progression in association with CYP1B1 gene mutation (p.Gly61Glu).}, journal = {PloS one}, volume = {15}, number = {11}, pages = {e0241902}, pmid = {33170892}, issn = {1932-6203}, mesh = {Amino Acid Substitution ; Animals ; Apoptosis ; Astrocytes/*cytology/metabolism ; CRISPR-Cas Systems ; Cell Proliferation ; Cells, Cultured ; Cytochrome P-450 CYP1B1/*genetics ; Cytokines/metabolism ; Glaucoma/*congenital/genetics ; Humans ; Male ; Mesenchymal Stem Cells/*cytology/metabolism ; Microglia/*cytology/metabolism ; Models, Animal ; NADP/metabolism ; *Point Mutation ; Rats ; }, abstract = {BACKGROUND: Glaucoma represents the second main cause of irreversible loss of eyesight worldwide. Progression of the disease is due to changes around the optic nerve, eye structure and optic nerve environment. Focusing on primary congenital glaucoma, which is not completely understood, we report an evaluation of an untested mutation (c.182G>A, p.Gly61Glu) within the CYP1B1 gene in the context of microglia, astrocytes and mesenchymal stem cells. We investigated the behaviours of these cells, which are needed to maintain eye homeostasis, in response to the CYP1B1 mutation.<br><br>METHODS AND RESULTS: CRISPR technology was used to edit normal CYP1B1 genes within normal astrocytes, microglia and stem cells in vitro. Increased metabolic activities were found in microglia and astrocytes 24 hours after CYP1B1 manipulation. However, these activities dropped by 40% after 72 hrs. In addition, the nicotinamide adenine dinucleotide phosphate (NADP)/NADPH reducing equivalent process decreased by 50% on average after 72 hrs of manipulation. The cytokines measured in mutated microglia showed progressive activation leading to apoptosis, which was confirmed with annexin-V. The cytokines evaluated in mutant astrocytes were abnormal in comparison to those in the control.<br><br>CONCLUSIONS: The results suggest a progressive inflammation that was induced by mutations (p.Gly61Glu) on CYP1B1. Furthermore, the mutations enhanced the microglia's loss of activity. We are the first to show the direct impact of the mutation on microglia. This progressive inflammation might be responsible for primary congenital glaucoma complications, which could be avoided via an anti-inflammatory regimen. This finding also reveals that progressive inflammation affects recovery failure after surgeries to relieve glaucoma. Moreover, microglia are important for the survival of ganglion cells, along with the clearing of pathogens and inflammation. The reduction of their activities may jeopardise homeostasis within the optic nerve environment and complicate the protection of optic nerve components (such as retinal ganglion and glial cells).}, }
@article {pmid33147260, year = {2020}, author = {Liu, H and Robinson, DS and Wu, ZY and Kuo, R and Yoshikuni, Y and Blaby, IK and Cheng, JF}, title = {Bacterial genome editing by coupling Cre-lox and CRISPR-Cas9 systems.}, journal = {PloS one}, volume = {15}, number = {11}, pages = {e0241867}, pmid = {33147260}, issn = {1932-6203}, mesh = {CRISPR-Cas Systems ; Gene Editing/*methods ; Genome, Bacterial ; Integrases/*metabolism ; Photorhabdus/*genetics ; }, abstract = {The past decade has been a golden age for microbiology, marked by the discovery of an unprecedented increase in the number of novel bacterial species. Yet gaining biological knowledge of those organisms has not kept pace with sequencing efforts. To unlock this genetic potential there is an urgent need for generic (i.e. non-species specific) genetic toolboxes. Recently, we developed a method, termed chassis-independent recombinase-assisted genome engineering (CRAGE), enabling the integration and expression of large complex gene clusters directly into the chromosomes of diverse bacteria. Here we expand upon this technology by incorporating CRISPR-Cas9 allowing precise genome editing across multiple bacterial species. To do that we have developed a landing pad that carries one wild-type and two mutant lox sites to allow integration of foreign DNA at two locations through Cre-lox recombinase-mediated cassette exchange (RMCE). The first RMCE event is to integrate the Cas9 and the DNA repair protein genes RecET, and the second RMCE event enables the integration of customized sgRNA and a repair template. Following this workflow, we achieved precise genome editing in four different gammaproteobacterial species. We also show that the inserted landing pad and the entire editing machinery can be removed scarlessly after editing. We report here the construction of a single landing pad transposon and demonstrate its functionality across multiple species. The modular design of the landing pad and accessory vectors allows design and assembly of genome editing platforms for other organisms in a similar way. We believe this approach will greatly expand the list of bacteria amenable to genetic manipulation and provides the means to advance our understanding of the microbial world.}, }
@article {pmid33137164, year = {2020}, author = {Schleicher, EM and Dhoonmoon, A and Jackson, LM and Clements, KE and Stump, CL and Nicolae, CM and Moldovan, GL}, title = {Dual genome-wide CRISPR knockout and CRISPR activation screens identify mechanisms that regulate the resistance to multiple ATR inhibitors.}, journal = {PLoS genetics}, volume = {16}, number = {11}, pages = {e1009176}, pmid = {33137164}, issn = {1553-7404}, support = {R01 ES026184/ES/NIEHS NIH HHS/United States ; R01 GM134681/GM/NIGMS NIH HHS/United States ; F31 CA243301/CA/NCI NIH HHS/United States ; }, mesh = {Apoptosis/drug effects/genetics ; Ataxia Telangiectasia Mutated Proteins/*antagonists &amp; inhibitors ; Biomarkers, Tumor/*genetics/metabolism ; CRISPR-Cas Systems/genetics ; DNA Replication/drug effects/genetics ; Drug Resistance, Neoplasm/*genetics ; Drug Screening Assays, Antitumor ; Gene Knockdown Techniques ; HeLa Cells ; Humans ; Mediator Complex/genetics/metabolism ; Neoplasms/*drug therapy/genetics/pathology ; Protein Kinase Inhibitors/*pharmacology/therapeutic use ; Pyrimidines/pharmacology/therapeutic use ; Signal Transduction/drug effects/genetics ; Sulfoxides/pharmacology/therapeutic use ; Transforming Growth Factor beta/metabolism ; }, abstract = {The ataxia telangiectasia and Rad3-related (ATR) protein kinase is a key regulator of the cellular response to DNA damage. Due to increased amount of replication stress, cancer cells heavily rely on ATR to complete DNA replication and cell cycle progression. Thus, ATR inhibition is an emerging target in cancer therapy, with multiple ATR inhibitors currently undergoing clinical trials. Here, we describe dual genome-wide CRISPR knockout and CRISPR activation screens employed to comprehensively identify genes that regulate the cellular resistance to ATR inhibitors. Specifically, we investigated two different ATR inhibitors, namely VE822 and AZD6738, in both HeLa and MCF10A cells. We identified and validated multiple genes that alter the resistance to ATR inhibitors. Importantly, we show that the mechanisms of resistance employed by these genes are varied, and include restoring DNA replication fork progression, and prevention of ATR inhibitor-induced apoptosis. In particular, we describe a role for MED12-mediated inhibition of the TGFβ signaling pathway in regulating replication fork stability and cellular survival upon ATR inhibition. Our dual genome-wide screen findings pave the way for personalized medicine by identifying potential biomarkers for ATR inhibitor resistance.}, }
@article {pmid33137136, year = {2020}, author = {Chen, K and Yu, Y and Yang, D and Yang, X and Tang, L and Liu, Y and Luo, X and R Walter, J and Liu, Z and Xu, J and Huang, Y}, title = {Gtsf1 is essential for proper female sex determination and transposon silencing in the silkworm, Bombyx mori.}, journal = {PLoS genetics}, volume = {16}, number = {11}, pages = {e1009194}, pmid = {33137136}, issn = {1553-7404}, mesh = {Animals ; Animals, Genetically Modified ; Bombyx/*physiology ; CRISPR-Cas Systems/genetics ; DNA Transposable Elements/*genetics ; Female ; Gene Expression Regulation, Developmental ; Insect Proteins/genetics/*metabolism ; Male ; Mutation ; Nuclear Proteins/genetics/*metabolism ; RNA Interference ; RNA, Small Interfering/metabolism ; Sex Determination Processes/*genetics ; }, abstract = {Sex determination pathways are astoundingly diverse in insects. For instance, the silk moth Bombyx mori uniquely use various components of the piRNA pathway to produce the Fem signal for specification of the female fate. In this study, we identified BmGTSF1 as a novel piRNA factor which participates in B. mori sex determination. We found that BmGtsf1 has a distinct expression pattern compared to Drosophila and mouse. CRISPR/Cas9 induced mutation in BmGtsf1 resulted in partial sex reversal in genotypically female animals by shifting expression of the downstream targets BmMasc and Bmdsx to the male pattern. As levels of Fem piRNAs were substantially reduced in female mutants, we concluded that BmGtsf1 plays a critical role in the biogenesis of the feminizing signal. We also demonstrated that BmGTSF1 physically interacted with BmSIWI, a protein previously reported to be involved in female sex determination, indicating BmGTSF1 function as the cofactor of BmSIWI. BmGtsf1 mutation resulted in piRNA pathway dysregulation, including piRNA biogenesis defects and transposon derepression, suggesting BmGtsf1 is also a piRNA factor in the silkworm. Furthermore, we found that BmGtsf1 mutation leads to gametogenesis defects in both male and female. Our data suggested that BmGtsf1 is a new component involved in the sex determination pathway in B. mori.}, }
@article {pmid33087559, year = {2020}, author = {Lin, RJ and Kuo, MW and Yang, BC and Tsai, HH and Chen, K and Huang, JR and Lee, YS and Yu, AL and Yu, J}, title = {B3GALT5 knockout alters gycosphingolipid profile and facilitates transition to human naïve pluripotency.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {117}, number = {44}, pages = {27435-27444}, doi = {10.1073/pnas.2003155117}, pmid = {33087559}, issn = {1091-6490}, mesh = {CRISPR-Cas Systems/genetics ; *Cell Differentiation ; Cell Line ; Embryonic Stem Cells ; Galactosyltransferases/genetics/*metabolism ; Gene Knockdown Techniques ; Glycosphingolipids/*metabolism ; Humans ; Pluripotent Stem Cells/*metabolism ; Stage-Specific Embryonic Antigens/*metabolism ; }, abstract = {Conversion of human pluripotent stem cells from primed to naïve state is accompanied by altered transcriptome and methylome, but glycosphingolipid (GSL) profiles in naïve human embryonic stem cells (hESCs) have not been systematically characterized. Here we showed a switch from globo-(SSEA-3, SSEA-4, and Globo H) and lacto-series (fucosyl-Lc4Cer) to neolacto-series GSLs (SSEA-1 and H type 2 antigen), along with marked down-regulation of β-1,3-galactosyltransferase (B3GALT5) upon conversion to naïve state. CRISPR/Cas9-generated B3GALT5-knockout (KO) hESCs displayed an altered GSL profile, increased cloning efficiency and intracellular Ca2+, reminiscent of the naïve state, while retaining differentiation ability. The altered GSLs could be rescued through overexpression of B3GALT5. B3GALT5-KO cells cultured with 2iLAF exhibited naïve-like transcriptome, global DNA hypomethylation, and X-chromosome reactivation. In addition, B3GALT5-KO rendered hESCs more resistant to calcium chelator in blocking entry into naïve state. Thus, loss of B3GALT5 induces a distinctive state of hESCs displaying unique GSL profiling with expression of neolacto-glycans, increased Ca2+, and conducive for transition to naïve pluripotency.}, }
@article {pmid32865499, year = {2020}, author = {Müller, M and Schneider, M and Salathé, M and Vayena, E}, title = {Assessing Public Opinion on CRISPR-Cas9: Combining Crowdsourcing and Deep Learning.}, journal = {Journal of medical Internet research}, volume = {22}, number = {8}, pages = {e17830}, pmid = {32865499}, issn = {1438-8871}, mesh = {CRISPR-Cas Systems/*physiology ; Crowdsourcing/*methods ; Deep Learning/*standards ; Humans ; *Public Opinion ; }, abstract = {BACKGROUND: The discovery of the CRISPR-Cas9-based gene editing method has opened unprecedented new potential for biological and medical engineering, sparking a growing public debate on both the potential and dangers of CRISPR applications. Given the speed of technology development and the almost instantaneous global spread of news, it is important to follow evolving debates without much delay and in sufficient detail, as certain events may have a major long-term impact on public opinion and later influence policy decisions.<br><br>OBJECTIVE: Social media networks such as Twitter have shown to be major drivers of news dissemination and public discourse. They provide a vast amount of semistructured data in almost real-time and give direct access to the content of the conversations. We can now mine and analyze such data quickly because of recent developments in machine learning and natural language processing.<br><br>METHODS: Here, we used Bidirectional Encoder Representations from Transformers (BERT), an attention-based transformer model, in combination with statistical methods to analyze the entirety of all tweets ever published on CRISPR since the publication of the first gene editing application in 2013.<br><br>RESULTS: We show that the mean sentiment of tweets was initially very positive, but began to decrease over time, and that this decline was driven by rare peaks of strong negative sentiments. Due to the high temporal resolution of the data, we were able to associate these peaks with specific events and to observe how trending topics changed over time.<br><br>CONCLUSIONS: Overall, this type of analysis can provide valuable and complementary insights into ongoing public debates, extending the traditional empirical bioethics toolset.}, }
@article {pmid32811847, year = {2020}, author = {Yokouchi, Y and Suzuki, S and Ohtsuki, N and Yamamoto, K and Noguchi, S and Soejima, Y and Goto, M and Ishioka, K and Nakamura, I and Suzuki, S and Takenoshita, S and Era, T}, title = {Rapid repair of human disease-specific single-nucleotide variants by One-SHOT genome editing.}, journal = {Scientific reports}, volume = {10}, number = {1}, pages = {13927}, pmid = {32811847}, issn = {2045-2322}, mesh = {Alleles ; Bacterial Proteins/genetics/*metabolism ; CRISPR-Associated Proteins/genetics/*metabolism ; CRISPR-Cas Systems/genetics ; Collagen Type VII/genetics/metabolism ; Endodeoxyribonucleases/genetics/*metabolism ; Endonucleases/genetics ; Gene Editing/*methods ; Genome, Human/genetics ; Humans ; Induced Pluripotent Stem Cells/physiology ; Mutation/genetics ; Nucleotides/genetics ; Pluripotent Stem Cells/physiology ; Polymorphism, Single Nucleotide/*genetics ; Proto-Oncogene Proteins c-ret/genetics/metabolism ; }, abstract = {Many human diseases ranging from cancer to hereditary disorders are caused by single-nucleotide mutations in critical genes. Repairing these mutations would significantly improve the quality of life for patients with hereditary diseases. However, current procedures for repairing deleterious single-nucleotide mutations are not straightforward, requiring multiple steps and taking several months to complete. In the current study, we aimed to repair pathogenic allele-specific single-nucleotide mutations using a single round of genome editing. Using high-fidelity, site-specific nuclease AsCas12a/Cpf1, we attempted to repair pathogenic single-nucleotide variants (SNVs) in disease-specific induced pluripotent stem cells. As a result, we achieved repair of the Met918Thr SNV in human oncogene RET with the inclusion of a single-nucleotide marker, followed by absolute markerless, scarless repair of the RET SNV with no detected off-target effects. The markerless method was then confirmed in human type VII collagen-encoding gene COL7A1. Thus, using this One-SHOT method, we successfully reduced the number of genetic manipulations required for genome repair from two consecutive events to one, resulting in allele-specific repair that can be completed within 3 weeks, with or without a single-nucleotide marker. Our findings suggest that One-SHOT can be used to repair other types of mutations, with potential beyond human medicine.}, }
@article {pmid32745724, year = {2020}, author = {D'Amore, C and Moro, E and Borgo, C and Itami, K and Hirota, T and Pinna, LA and Salvi, M}, title = {&quot;Janus&quot; efficacy of CX-5011: CK2 inhibition and methuosis induction by independent mechanisms.}, journal = {Biochimica et biophysica acta. Molecular cell research}, volume = {1867}, number = {11}, pages = {118807}, doi = {10.1016/j.bbamcr.2020.118807}, pmid = {32745724}, issn = {1879-2596}, mesh = {CRISPR-Cas Systems/genetics ; Casein Kinase II/antagonists &amp; inhibitors/*genetics ; Cell Death/drug effects/*genetics ; Gene Editing ; Hep G2 Cells ; Humans ; Indoles/pharmacology ; Neoplasms/*drug therapy ; Pinocytosis/drug effects/genetics ; Pyrimidines/pharmacology ; Quinolines/pharmacology ; Vacuoles/drug effects/genetics ; rac1 GTP-Binding Protein/antagonists &amp; inhibitors/*genetics ; }, abstract = {Methuosis has been described as a distinctive form of cell death characterized by the displacement of large fluid-filled vacuoles derived from uncontrolled macropinocytosis. Its induction has been proposed as a new strategy against cancer cells. Small molecules, such as indole-based calchones, have been identified as methuosis inducers and, recently, the CK2 inhibitor CX-4945 has been shown to have a similar effect on different cell types. However, the contribution of protein kinase CK2 to methuosis signalling is still controversial. Here we show that methuosis is not related to CK2 activity since it is not affected by structurally unrelated CK2 inhibitors and genetic reduction/ablation of CK2 subunits. Interestingly, CX-5011, a CK2 inhibitor related to CX-4945, behaves as a CK2-independent methuosis inducer, four times more powerful than its parental compound and capable to promote the formation on enlarged cytosolic vacuoles at low micromolar concentrations. We show that pharmacological inhibition of the small GTPase Rac-1, its downregulation by siRNA treatment, or the over-expression of the dominant-negative mutated form of Rac-1 (Rac-1 T17N), impairs CX-5011 ability to induce methuosis. Furthermore, cell treatment with CX-5011 induces a durable activation of Rac-1 that persists for at least 24 h. Worthy of note, CX-5011 is able to promote macropinocytosis not only in mammalian cells, but also in an in-vivo zebrafish model. Based on these evidences, CX-5011 is, therefore, proposed as a potential promising compound for cancer therapies for its dual efficacy as an inhibitor of the pro-survival kinase CK2 and inducer of methuosis.}, }
@article {pmid32658961, year = {2020}, author = {Domènech, EB and Andrés, R and López-Iniesta, MJ and Mirra, S and García-Arroyo, R and Milla, S and Sava, F and Andilla, J and Loza-Álvarez, P and de la Villa, P and Gonzàlez-Duarte, R and Marfany, G}, title = {A New Cerkl Mouse Model Generated by CRISPR-Cas9 Shows Progressive Retinal Degeneration and Altered Morphological and Electrophysiological Phenotype.}, journal = {Investigative ophthalmology &amp; visual science}, volume = {61}, number = {8}, pages = {14}, pmid = {32658961}, issn = {1552-5783}, mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Cells, Cultured ; DNA/*genetics ; DNA Mutational Analysis ; Disease Models, Animal ; Mice ; Mice, Inbred C57BL ; *Mutation ; Phenotype ; Phosphotransferases (Alcohol Group Acceptor)/*genetics/metabolism ; Retinal Cone Photoreceptor Cells/*metabolism/pathology ; Retinal Degeneration/*genetics/metabolism/pathology ; Retinal Pigment Epithelium/*metabolism/pathology ; }, abstract = {Purpose: Close to 100 genes cause retinitis pigmentosa, a Mendelian rare disease that affects 1 out of 4000 people worldwide. Mutations in the ceramide kinase-like gene (CERKL) are a prevalent cause of autosomal recessive cause retinitis pigmentosa and cone-rod dystrophy, but the functional role of this gene in the retina has yet to be fully determined. We aimed to generate a mouse model that resembles the phenotypic traits of patients carrying CERKL mutations to undertake functional studies and assay therapeutic approaches.<br><br>Methods: The Cerkl locus has been deleted (around 97 kb of genomic DNA) by gene editing using the CRISPR-Cas9 D10A nickase. Because the deletion of the Cerkl locus is lethal in mice in homozygosis, a double heterozygote mouse model with less than 10% residual Cerkl expression has been generated. The phenotypic alterations of the retina of this new model have been characterized at the morphological and electrophysiological levels.<br><br>Results: This CerklKD/KO model shows retinal degeneration, with a decreased number of cones and progressive photoreceptor loss, poorly stacked photoreceptor outer segment membranes, defective retinal pigment epithelium phagocytosis, and altered electrophysiological recordings in aged retinas.<br><br>Conclusions: To our knowledge, this is the first Cerkl mouse model to mimic many of the phenotypic traits, including the slow but progressive retinal degeneration, shown by human patients carrying CERKL mutations. This useful model will provide unprecedented insights into the retinal molecular pathways altered in these patients and will contribute to the design of effective treatments.}, }
@article {pmid32422169, year = {2020}, author = {Przewrocka, J and Rowan, A and Rosenthal, R and Kanu, N and Swanton, C}, title = {Unintended on-target chromosomal instability following CRISPR/Cas9 single gene targeting.}, journal = {Annals of oncology : official journal of the European Society for Medical Oncology}, volume = {31}, number = {9}, pages = {1270-1273}, pmid = {32422169}, issn = {1569-8041}, support = {FC001169/MRC_/Medical Research Council/United Kingdom ; FC001169/WT_/Wellcome Trust/United Kingdom ; FC001169/CRUK_/Cancer Research UK/United Kingdom ; }, mesh = {*CRISPR-Cas Systems ; Chromosomal Instability ; *Gene Targeting ; Humans ; }, }
@article {pmid32394708, year = {2020}, author = {Xie, T and Chen, X and Guo, T and Rong, H and Chen, Z and Sun, Q and Batley, J and Jiang, J and Wang, Y}, title = {Targeted Knockout of BnTT2 Homologues for Yellow-Seeded Brassica napus with Reduced Flavonoids and Improved Fatty Acid Composition.}, journal = {Journal of agricultural and food chemistry}, volume = {68}, number = {20}, pages = {5676-5690}, doi = {10.1021/acs.jafc.0c01126}, pmid = {32394708}, issn = {1520-5118}, mesh = {Brassica napus/chemistry/*genetics/metabolism ; CRISPR-Cas Systems ; Color ; Fatty Acids/*chemistry/metabolism ; Flavonoids/*chemistry/metabolism ; Gene Expression Regulation, Plant ; Gene Knockout Techniques ; Plant Proteins/*genetics/metabolism ; Plants, Genetically Modified/chemistry/genetics/metabolism ; Seeds/chemistry/genetics/*metabolism ; }, abstract = {Brassica napus is one of the important oil crops grown worldwide, and oil quality improvement is a major goal in rapeseed breeding. Yellow seed is an excellent trait, which has great potential in improving seed quality and economic value. In this study, we created stable yellow seed mutants using a CRISPR/Cas9 system and obtained the yellow seed phenotype only when the four alleles of two BnTT2 homologues were knocked out, indicating that the two BnTT2 homologues had conserved but redundant functions in regulating seed color. Histochemical staining and flavonoid metabolic analysis proved that the BnTT2 mutation hindered the synthesis and accumulation of proanthocyanidins. Transcriptome analysis also showed that the BnTT2 mutation inhibited the expression of genes in the phenylpropanoid and flavonoid biosynthetic pathway, which might be regulated by the complex of BnTT2, BnTT8 and BnTTG1. In addition, the homozygous mutants of BnTT2 homologues increased oil content and improved fatty acid composition with higher linoleic acid (C18:2) and linolenic acid (C18:3), which could be used for the genetic improvement of rapeseed. Overall, this research showed that the BnTT2 mutation can be used for yellow seed breeding and oil improvement, which is of great significance in improving the economic value of rapeseeds.}, }
@article {pmid32372640, year = {2020}, author = {Shu, P and Li, Z and Min, D and Zhang, X and Ai, W and Li, J and Zhou, J and Li, Z and Li, F and Li, X}, title = {CRISPR/Cas9-Mediated SlMYC2 Mutagenesis Adverse to Tomato Plant Growth and MeJA-Induced Fruit Resistance to Botrytis cinerea.}, journal = {Journal of agricultural and food chemistry}, volume = {68}, number = {20}, pages = {5529-5538}, doi = {10.1021/acs.jafc.9b08069}, pmid = {32372640}, issn = {1520-5118}, mesh = {Acetates/*adverse effects/pharmacology ; Botrytis/*physiology ; CRISPR-Cas Systems ; Cyclopentanes/*adverse effects/pharmacology ; Disease Resistance ; Flowers/genetics/growth &amp; development/immunology ; Fruit/drug effects/genetics/immunology/microbiology ; Gene Expression Regulation, Plant ; Lycopersicon esculentum/drug effects/*genetics/immunology/microbiology ; Mutagenesis ; Oxylipins/*adverse effects/pharmacology ; Plant Diseases/immunology/*microbiology ; Plant Growth Regulators/*pharmacology ; Plant Proteins/*genetics/immunology ; Plants, Genetically Modified/genetics/growth &amp; development/*immunology/microbiology ; }, abstract = {Methyl jasmonate (MeJA), a natural phytohormone, played a critical role not only in plant growth but also in plant defense response to biotic and abiotic stresses. MYC2, a basic helix-loop-helix transcription factor, is a master regulator in MeJA signaling pathway. In the present work, slmyc2 mutants were generated by the clustered regularly interspaced short palindromic repeats and associated Cas9 protein (CRISPR/Cas9) system to investigate the role of SlMYC2 in tomato plant growth and fruit disease resistance induced by exogenous MeJA. The results showed that slmyc2 mutants possessed a higher number of flowers and a lower fruit setting rate in comparison with wild-type plants. In addition, the fruit shape of slmyc2 mutant was prolate, while the control fruits were oblate. Knockout of SlMYC2 significantly decreased the activities of disease defensive and antioxidant enzymes, as well as the expression levels of pathogen-related (PR) genes (SlPR-1 and SlPR-STH2) and the key genes related to jasmonic acid (JA) biosynthesis and signaling pathway including allene oxide cyclase (SlAOC), lipoxygenase D (SlLOXD), SlMYC2, and coronatine insensitive 1 (SlCOI1), and consequently aggravated the disease symptoms. By contrast, the disease symptoms were largely reduced in MeJA-treated fruit that possessed higher activities of these enzymes and expression levels of genes. However, the induction effects of MeJA on fruit disease resistance and these enzymes' activities and genes' expressions were significantly attenuated by knockout of SlMYC2. Therefore, the results indicated that SlMYC2 played positive regulatory roles not only in the growth of tomato plants but also in MeJA-induced disease resistance and the antioxidant process in tomato fruits.}, }
@article {pmid32366884, year = {2020}, author = {Pinzon-Arteaga, C and Snyder, MD and Lazzarotto, CR and Moreno, NF and Juras, R and Raudsepp, T and Golding, MC and Varner, DD and Long, CR}, title = {Efficient correction of a deleterious point mutation in primary horse fibroblasts with CRISPR-Cas9.}, journal = {Scientific reports}, volume = {10}, number = {1}, pages = {7411}, pmid = {32366884}, issn = {2045-2322}, mesh = {Animals ; Apoptosis ; Biotechnology/methods ; *CRISPR-Cas Systems ; Cell Line ; *Exons ; Fibroblasts/*metabolism ; *Gene Editing ; Genetic Engineering/methods ; Glycogen Storage Disease Type IV/*genetics/therapy/veterinary ; Homologous Recombination ; Horses ; Karyotyping ; Phenotype ; *Point Mutation ; RNA, Guide/genetics ; Skin/metabolism ; }, abstract = {Phenotypic selection during animal domestication has resulted in unwanted incorporation of deleterious mutations. In horses, the autosomal recessive condition known as Glycogen Branching Enzyme Deficiency (GBED) is the result of one of these deleterious mutations (102C > A), in the first exon of the GBE1 gene (GBE1102C>A). With recent advances in genome editing, this type of genetic mutation can be precisely repaired. In this study, we used the RNA-guided nuclease CRISPR-Cas9 (clustered regularly-interspaced short palindromic repeats/CRISPR-associated protein 9) to correct the GBE1102C>A mutation in a primary fibroblast cell line derived from a high genetic merit heterozygous stallion. To correct this mutation by homologous recombination (HR), we designed a series of single guide RNAs (sgRNAs) flanking the mutation and provided different single-stranded donor DNA templates. The distance between the Cas9-mediated double-stranded break (DSB) to the mutation site, rather than DSB efficiency, was the primary determinant for successful HR. This framework can be used for targeting other harmful diseases in animal populations.}, }
@article {pmid32355272, year = {2020}, author = {Audebert, C and Bonardi, F and Caboche, S and Guyot, K and Touzet, H and Merlin, S and Gantois, N and Creusy, C and Meloni, D and Mouray, A and Viscogliosi, E and Certad, G and Benamrouz-Vanneste, S and Chabé, M}, title = {Genetic basis for virulence differences of various Cryptosporidium parvum carcinogenic isolates.}, journal = {Scientific reports}, volume = {10}, number = {1}, pages = {7316}, pmid = {32355272}, issn = {2045-2322}, mesh = {Animals ; CRISPR-Cas Systems ; Carcinogenesis/genetics ; Computational Biology ; Cryptosporidiosis/*parasitology ; Cryptosporidium parvum/*genetics/pathogenicity ; Feces ; Female ; Genome ; Genome, Protozoan ; Humans ; Male ; Mice ; Mice, SCID ; Middle Aged ; Oocysts ; Phenotype ; Virulence/*genetics ; Virulence Factors/*genetics ; Young Adult ; }, abstract = {Cryptosporidium parvum is known to cause life-threatening diarrhea in immunocompromised hosts and was also reported to be capable of inducing digestive adenocarcinoma in a rodent model. Interestingly, three carcinogenic isolates of C. parvum, called DID, TUM1 and CHR, obtained from fecal samples of naturally infected animals or humans, showed higher virulence than the commercially available C. parvum IOWA isolate in our animal model in terms of clinical manifestations, mortality rate and time of onset of neoplastic lesions. In order to discover the potential genetic basis of the differential virulence observed between C. parvum isolates and to contribute to the understanding of Cryptosporidium virulence, entire genomes of the isolates DID, TUM1 and CHR were sequenced then compared to the C. parvum IOWA reference genome. 125 common SNVs corresponding to 90 CDSs were found in the C. parvum genome that could explain this differential virulence. In particular variants in several membrane and secreted proteins were identified. Besides the genes already known to be involved in parasite virulence, this study identified potential new virulence factors whose functional characterization can be achieved through CRISPR/Cas9 technology applied to this parasite.}, }
@article {pmid32346083, year = {2020}, author = {Soliman, SHA and Stark, AE and Gardner, ML and Harshman, SW and Breece, CC and Amari, F and Orlacchio, A and Chen, M and Tessari, A and Martin, JA and Visone, R and Freitas, MA and La Perle, KMD and Palmieri, D and Coppola, V}, title = {Tagging enhances histochemical and biochemical detection of Ran Binding Protein 9 in vivo and reveals its interaction with Nucleolin.}, journal = {Scientific reports}, volume = {10}, number = {1}, pages = {7138}, pmid = {32346083}, issn = {2045-2322}, support = {P30 CA016058/CA/NCI NIH HHS/United States ; }, mesh = {Adaptor Proteins, Signal Transducing/genetics/*metabolism ; Animals ; CRISPR-Cas Systems ; Cytoskeletal Proteins/genetics/*metabolism ; Immunohistochemistry ; Mice ; Mice, Knockout ; Nuclear Proteins/genetics/*metabolism ; Phosphoproteins/*metabolism ; Protein Binding ; RNA, Messenger/metabolism ; RNA-Binding Proteins/*metabolism ; }, abstract = {The lack of tools to reliably detect RanBP9 in vivo has significantly hampered progress in understanding the biological functions of this scaffold protein. We report here the generation of a novel mouse strain, RanBP9-TT, in which the endogenous protein is fused with a double (V5-HA) epitope tag at the C-terminus. We show that the double tag does not interfere with the essential functions of RanBP9. In contrast to RanBP9 constitutive knock-out animals, RanBP9-TT mice are viable, fertile and do not show any obvious phenotype. The V5-HA tag allows unequivocal detection of RanBP9 both by IHC and WB. Importantly, immunoprecipitation and mass spectrometry analyses reveal that the tagged protein pulls down known interactors of wild type RanBP9. Thanks to the increased detection power, we are also unveiling a previously unknown interaction with Nucleolin, a protein proposed as an ideal target for cancer treatment. In summary, we report the generation of a new mouse line in which RanBP9 expression and interactions can be reliably studied by the use of commercially available αtag antibodies. The use of this line will help to overcome some of the existing limitations in the study of RanBP9 and potentially unveil unknown functions of this protein in vivo such as those linked to Nucleolin.}, }
@article {pmid32300998, year = {2020}, author = {Wu, C and Chen, Y and Qiu, Y and Niu, X and Zhu, N and Chen, J and Yao, H and Wang, W and Ma, Y}, title = {A simple approach to mediate genome editing in the filamentous fungus Trichoderma reesei by CRISPR/Cas9-coupled in vivo gRNA transcription.}, journal = {Biotechnology letters}, volume = {42}, number = {7}, pages = {1203-1210}, doi = {10.1007/s10529-020-02887-0}, pmid = {32300998}, issn = {1573-6776}, support = {2019M661402//Postdoctoral Research Foundation of China/ ; 222201714053//the Fundamental Research Funds for the Central Universities/ ; }, mesh = {CRISPR-Cas Systems/*genetics ; Gene Editing/*methods ; Genome, Fungal/*genetics ; Hypocreales/*genetics ; Promoter Regions, Genetic/genetics ; RNA, Guide/*genetics ; Transcription, Genetic/genetics ; }, abstract = {OBJECTIVE: To simplify CRISPR/Cas9 genome editing in the industrial filamentous fungus Trichoderma reesei based on in vivo guide RNA (gRNA) transcription.<br><br>RESULTS: Two putative RNA polymerase III U6 snRNA genes were identified in the genome of T. reesei QM6a by BLASTN using Myceliophthora. thermophila U6 snRNA gene as the template. The regions approximately 500 bp upstream of two U6 genes were efficient promoters for the in vivo expression of gRNA. The CRISPR system consisting of Cas9 and in vivo synthesized gRNA under control of the T. reesei U6 snRNA promoters was sufficient to cause a frameshift mutation in the ura5 gene via non-homologous end-joining-mediated events.<br><br>CONCLUSIONS: We report a simple gene editing method using a CRISPR/Cas9-coupled in vivo gRNA transcription system in T. reesei.}, }
@article {pmid32295026, year = {2020}, author = {Anwar, A and Kim, JK}, title = {Transgenic Breeding Approaches for Improving Abiotic Stress Tolerance: Recent Progress and Future Perspectives.}, journal = {International journal of molecular sciences}, volume = {21}, number = {8}, pages = {}, pmid = {32295026}, issn = {1422-0067}, support = {PJ013666//Rural Development Administration/ ; NRF-2017R1A2B4007457//National Research Foundation of Korea, Ministry of Education/ ; }, mesh = {*Adaptation, Biological ; Agriculture ; CRISPR-Cas Systems ; Crops, Agricultural/*genetics/*metabolism ; Gene Editing ; Gene Expression Regulation, Plant ; Genetic Engineering ; MicroRNAs ; *Plant Breeding ; *Plants, Genetically Modified ; Quantitative Trait Loci ; *Stress, Physiological/genetics ; }, abstract = {The recent rapid climate changes and increasing global population have led to an increased incidence of abiotic stress and decreased crop productivity. Environmental stresses, such as temperature, drought, nutrient deficiency, salinity, and heavy metal stresses, are major challenges for agriculture, and they lead to a significant reduction in crop growth and productivity. Abiotic stress is a very complex phenomenon, involving a variety of physiological and biochemical changes in plant cells. Plants exposed to abiotic stress exhibit enhanced levels of ROS (reactive oxygen species), which are highly reactive and toxic and affect the biosynthesis of chlorophyll, photosynthetic capacity, and carbohydrate, protein, lipid, and antioxidant enzyme activities. Transgenic breeding offers a suitable alternative to conventional breeding to achieve plant genetic improvements. Over the last two decades, genetic engineering/transgenic breeding techniques demonstrated remarkable developments in manipulations of the genes for the induction of desired characteristics into transgenic plants. Transgenic approaches provide us with access to identify the candidate genes, miRNAs, and transcription factors (TFs) that are involved in specific plant processes, thus enabling an integrated knowledge of the molecular and physiological mechanisms influencing the plant tolerance and productivity. The accuracy and precision of this phenomenon assures great success in the future of plant improvements. Hence, transgenic breeding has proven to be a promising tool for abiotic stress improvement in crops. This review focuses on the potential and successful applications, recent progress, and future perspectives of transgenic breeding for improving abiotic stress tolerance and productivity in plants.}, }
@article {pmid32198422, year = {2020}, author = {Hanzawa, N and Hashimoto, K and Yuan, X and Kawahori, K and Tsujimoto, K and Hamaguchi, M and Tanaka, T and Nagaoka, Y and Nishina, H and Morita, S and Hatada, I and Yamada, T and Ogawa, Y}, title = {Targeted DNA demethylation of the Fgf21 promoter by CRISPR/dCas9-mediated epigenome editing.}, journal = {Scientific reports}, volume = {10}, number = {1}, pages = {5181}, pmid = {32198422}, issn = {2045-2322}, mesh = {Animals ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; *DNA Demethylation ; DNA Methylation ; Epigenesis, Genetic ; Epigenome ; Fibroblast Growth Factors/*genetics/metabolism ; Gene Editing/methods ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; PPAR alpha/genetics/metabolism ; Promoter Regions, Genetic/genetics ; }, abstract = {Recently, we reported PPARα-dependent DNA demethylation of the Fgf21 promoter in the postnatal mouse liver, where reduced DNA methylation is associated with enhanced gene expression after PPARα activation. However, there is no direct evidence for the effect of site-specific DNA methylation on gene expression. We employed the dCas9-SunTag and single-chain variable fragment (scFv)-TET1 catalytic domain (TET1CD) system to induce targeted DNA methylation of the Fgf21 promoter both in vitro and in vivo. We succeeded in targeted DNA demethylation of the Fgf 21 promoter both in Hepa1-6 cells and PPARα-deficient mice, with increased gene expression response to PPARα synthetic ligand administration and fasting, respectively. This study provides direct evidence that the DNA methylation status of a particular gene may determine the magnitude of the gene expression response to activation cues.}, }
@article {pmid32192326, year = {2020}, author = {Tu, K and Deng, H and Kong, L and Wang, Y and Yang, T and Hu, Q and Hu, M and Yang, C and Zhang, Z}, title = {Reshaping Tumor Immune Microenvironment through Acidity-Responsive Nanoparticles Featured with CRISPR/Cas9-Mediated Programmed Death-Ligand 1 Attenuation and Chemotherapeutics-Induced Immunogenic Cell Death.}, journal = {ACS applied materials &amp; interfaces}, volume = {12}, number = {14}, pages = {16018-16030}, doi = {10.1021/acsami.9b23084}, pmid = {32192326}, issn = {1944-8252}, mesh = {Acids/chemistry ; Animals ; B7-H1 Antigen/genetics/immunology ; CRISPR-Cas Systems/genetics/immunology ; Cell Line, Tumor ; Cell Proliferation/*drug effects ; Cyclin-Dependent Kinase 5/genetics ; Humans ; Immunogenic Cell Death/drug effects/genetics ; Melanoma, Experimental/genetics/*immunology/pathology/therapy ; Mice ; Nanoparticles/*chemistry/therapeutic use ; Paclitaxel/chemistry/immunology/*pharmacology ; T-Lymphocytes/drug effects/immunology ; Transfection ; Tumor Microenvironment/drug effects ; }, abstract = {Blocking immune checkpoints with monoclonal antibody has been verified to achieve potential clinical successes for cancer immunotherapy. However, its application has been impeded by the &quot;cold&quot; tumor microenvironment. Here, weak acidity-responsive nanoparticles co-loaded with CRISPR/Cas9 and paclitaxel (PTX) with the ability to convert &quot;cold&quot; tumor into &quot;hot&quot; tumor are reported. The nanoparticles exhibited high cargo packaging capacity, superior transfection efficiency, well biocompatibility, and effective tumor accumulation. The CRISPR/Cas9 encapsulated in nanoparticles could specifically knock out cyclin-dependent kinase 5 gene to significantly attenuate the expression of programmed death-ligand 1 on tumor cells. More importantly, PTX co-delivered in nanoparticles could significantly induce immunogenic cell death, reduce regulatory T lymphocytes, repolarize tumor-associated macrophages, and enhance antitumor immunity. Therefore, the nanoparticles could effectively convert cold tumor into hot tumor, achieve effective tumor growth inhibition, and prolong overall survival from 16 to 36 days. This research provided a referable strategy for the development of combinatorial immunotherapy and chemotherapy.}, }
@article {pmid32090297, year = {2020}, author = {Zhang, XK and Wang, DN and Chen, J and Liu, ZJ and Wei, LJ and Hua, Q}, title = {Metabolic engineering of β-carotene biosynthesis in Yarrowia lipolytica.}, journal = {Biotechnology letters}, volume = {42}, number = {6}, pages = {945-956}, doi = {10.1007/s10529-020-02844-x}, pmid = {32090297}, issn = {1573-6776}, support = {21576089, 21776081//National Natural Science Foundation of China/ ; B18022//the 111 Project and Research Program of State Key Laboratory of Bioreactor Engineering./ ; }, mesh = {Bioreactors ; CRISPR-Cas Systems/genetics ; Fermentation ; Glucose/metabolism ; Metabolic Engineering/*methods ; Yarrowia/*genetics/metabolism ; beta Carotene/analysis/*metabolism ; }, abstract = {OBJECTIVE: Carotenoids, as potent antioxidant compounds, have gained extensive attention, especially in human health. In this study, the combination of CRISPR/Cas9 integration strategy and fermenter cultivation was utilized to obtain efficient β-carotene-producing Yarrowia lipolytica cell factories for potential industrial application.<br><br>RESULTS: The introduction of the genes of Mucor circinelloides, encoding phytoene dehydrogenase (carB) and bifunctional phytoene synthase/lycopene cyclase (carRP), contributed to the heterologous production of β-carotene in Y. lipolytica XK2. Furthermore, β-carotene production was efficiently enhanced by increasing the copy numbers of the carB and carRP genes and overexpressing of GGS1, ERG13, and HMG, the genes related to the mevalonate (MVA) pathway. Thus, the optimized strain overexpressed a total of eight genes, including three copies of carRP, two copies of carB, and single copies of GGS1, HMG, and ERG13. As a consequence, strain Y. lipolytica XK19 accumulated approximately 408 mg/L β-carotene in shake flask cultures, a twenty-four-fold increase compared to the parental strain Y. lipolytica XK2.<br><br>CONCLUSIONS: 4.5 g/L β-carotene was obtained in a 5-L fermenter through a combination of genetic engineering and culture optimization, suggesting a great capacity and flexibility of Y. lipolytica in the production of carotenoids.}, }
@article {pmid31953182, year = {2020}, author = {Rojas-Sánchez, U and López-Calleja, AC and Millán-Chiu, BE and Fernández, F and Loske, AM and Gómez-Lim, MA}, title = {Enhancing the yield of human erythropoietin in Aspergillus niger by introns and CRISPR-Cas9.}, journal = {Protein expression and purification}, volume = {168}, number = {}, pages = {105570}, doi = {10.1016/j.pep.2020.105570}, pmid = {31953182}, issn = {1096-0279}, mesh = {Aspergillus niger/*genetics/metabolism ; CRISPR-Cas Systems ; Cloning, Molecular ; Erythropoietin/*biosynthesis/genetics ; Fructose-Bisphosphatase/chemistry/genetics ; Gene Expression ; Gene Knockdown Techniques ; *Genes, Fungal ; Genetic Vectors/chemistry/metabolism ; Glycosylation ; Humans ; *Introns ; Plasmids/chemistry/*metabolism ; Promoter Regions, Genetic ; Protein Stability ; Proteolysis ; RNA, Messenger/*genetics/metabolism ; Recombinant Proteins/biosynthesis/genetics ; }, abstract = {Aspergillus niger has been employed to produce heterologous proteins due to its high capacity for expression and secretion; nevertheless, expression levels of human proteins have been modest. We were interested in investigating whether A. niger can express and secret human erythropoietin (HuEPO) at high yields. Our strategy was to combine the presence of introns with CRISPR-Cas9 to increase the yield of the recombinant protein. The epo gene was codon-optimized and its expression driven by the PmbfA promoter. Another version of epo contained introns from the fructose-1,6-bisphosphatase (fbp) gene. Two recombinant clones, uME12 (no introns) and uME23 (with introns), were selected based on the resistance to the antibiotic and because they showed a protein profile different from that of the parental strain, as shown by SDS-PAGE. Expression of epo was confirmed by RT-PCR in both colonies but the recombinant EPO protein (rHUEPO) was detected by Western blot only in uME23. The rHuEPO yield from uME23 was estimated at about 1.8 mg L-1 by ELISA, demonstrating that the presence of introns resulted in higher yield, possibly by conferring more stability to mRNA. On the other hand, as part of our strategy we decided to inactivate in the strain uME23 the following genes vps, prtT, algC and och1 which are involved in protein secretion, regulating of protease expression and protein glycosylation in A. niger, with CRISPR-Cas9, yielding the muPS20 transformant. muPS20 is a protease-free strain and its rHuEPO production level was increased 41.1-fold. Moreover, its molecular weight was ≈27 kDa showing that mutations in the above mentioned genes improved secretion, prevented proteolytic degradation and hyperglycosylation of heterologous protein.}, }
@article {pmid31914394, year = {2020}, author = {Kondo, S and Takahashi, T and Yamagata, N and Imanishi, Y and Katow, H and Hiramatsu, S and Lynn, K and Abe, A and Kumaraswamy, A and Tanimoto, H}, title = {Neurochemical Organization of the Drosophila Brain Visualized by Endogenously Tagged Neurotransmitter Receptors.}, journal = {Cell reports}, volume = {30}, number = {1}, pages = {284-297.e5}, doi = {10.1016/j.celrep.2019.12.018}, pmid = {31914394}, issn = {2211-1247}, mesh = {Animals ; Brain/*diagnostic imaging ; CRISPR-Associated Protein 9/metabolism ; CRISPR-Cas Systems/genetics ; Drosophila Proteins/*metabolism ; Drosophila melanogaster/genetics/*metabolism ; Ethanol/adverse effects ; Gene Expression Regulation ; Genes, Reporter ; Receptors, Dopamine/metabolism ; Receptors, Neurotransmitter/*metabolism ; }, abstract = {Neurotransmitters often have multiple receptors that induce distinct responses in receiving cells. Expression and localization of neurotransmitter receptors in individual neurons are therefore critical for understanding the operation of neural circuits. Here we describe a comprehensive library of reporter strains in which a convertible T2A-GAL4 cassette is inserted into endogenous neurotransmitter receptor genes of Drosophila. Using this library, we profile the expression of 75 neurotransmitter receptors in the brain. Cluster analysis reveals neurochemical segmentation of the brain, distinguishing higher brain centers from the rest. By recombinase-mediated cassette exchange, we convert T2A-GAL4 into split-GFP and Tango to visualize subcellular localization and activation of dopamine receptors in specific cell types. This reveals striking differences in their subcellular localization, which may underlie the distinct cellular responses to dopamine in different behavioral contexts. Our resources thus provide a versatile toolkit for dissecting the cellular organization and function of neurotransmitter systems in the fly brain.}, }
@article {pmid31827259, year = {2020}, author = {Schuh, RS and Gonzalez, EA and Tavares, AMV and Seolin, BG and Elias, LS and Vera, LNP and Kubaski, F and Poletto, E and Giugliani, R and Teixeira, HF and Matte, U and Baldo, G}, title = {Neonatal nonviral gene editing with the CRISPR/Cas9 system improves some cardiovascular, respiratory, and bone disease features of the mucopolysaccharidosis I phenotype in mice.}, journal = {Gene therapy}, volume = {27}, number = {1-2}, pages = {74-84}, pmid = {31827259}, issn = {1476-5462}, mesh = {Animals ; Animals, Newborn ; Bone Diseases/genetics ; CRISPR-Cas Systems/genetics ; Cardiovascular System/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Disease Models, Animal ; Female ; Gene Editing/methods ; Genetic Therapy/methods ; Glycosaminoglycans/metabolism ; Iduronidase/*genetics/metabolism ; Male ; Mice ; Mice, Inbred C57BL ; Mucopolysaccharidosis I/genetics/physiopathology/*therapy ; Phenotype ; RNA, Untranslated/*genetics/metabolism ; }, abstract = {Mucopolysaccharidosis type I (MPS I) is caused by deficiency of alpha-L-iduronidase (IDUA), leading to multisystemic accumulation of glycosaminoglycans (GAG). Untreated MPS I patients may die in the first decades of life, mostly due to cardiovascular and respiratory complications. We previously reported that the treatment of newborn MPS I mice with intravenous administration of lipossomal CRISPR/Cas9 complexes carrying the murine Idua gene aiming at the ROSA26 locus resulted in long-lasting IDUA activity and GAG reduction in various tissues. Following this, the present study reports the effects of gene editing in cardiovascular, respiratory, bone, and neurologic functions in MPS I mice. Bone morphology, specifically the width of zygomatic and femoral bones, showed partial improvement. Although heart valves were still thickened, cardiac mass and aortic elastin breaks were reduced, with normalization of aortic diameter. Pulmonary resistance was normalized, suggesting improvement in respiratory function. In contrast, behavioral abnormalities and neuroinflammation still persisted, suggesting deterioration of the neurological functions. The set of results shows that gene editing performed in newborn animals improved some manifestations of the MPS I disorder in bone, respiratory, and cardiovascular systems. However, further studies will be imperative to find better delivery strategies to reach &quot;hard-to-treat&quot; tissues to ensure better systemic and neurological effects.}, }
@article {pmid31776930, year = {2020}, author = {Zhang, J and Späth, SS and Katz, SG}, title = {Genome-Wide CRISPRi/a Screening in an In Vitro Coculture Assay of Human Immune Cells with Tumor Cells.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2097}, number = {}, pages = {231-252}, pmid = {31776930}, issn = {1940-6029}, support = {R01 HL131793/HL/NHLBI NIH HHS/United States ; R21 AI121993/AI/NIAID NIH HHS/United States ; R21 CA198561/CA/NCI NIH HHS/United States ; }, mesh = {CRISPR-Cas Systems/*genetics ; Coculture Techniques/*methods ; *Genetic Testing ; *Genome ; HEK293 Cells ; High-Throughput Nucleotide Sequencing ; Humans ; Lentivirus/metabolism ; Leukocytes/*immunology ; Neoplasms/*pathology ; Oligonucleotides/metabolism ; Plasmids/metabolism ; RNA, Guide/metabolism ; Transformation, Genetic ; Virion/metabolism ; }, abstract = {Cell-based immunotherapy has achieved preclinical success in certain types of cancer patients, with a few approved cell-based products for clinical use. These achievements revitalized the field of cell engineering/ immunotherapy and brought attention to the opportunities that cell-based immunotherapeutics can offer to patients. On the other hand, obvious indications emphasize the need for a better understanding of the biological mechanisms involved in the immune response. This knowledge may not only ameliorate safety and efficacy, but also determine the possibilities and limitations in use of immune cell engineering for cancer treatment, and facilitate developing novel immunotherapeutic strategies. Recently developed technology based on CRISPR-dCas9 has an immense potential to systematically uncover genetic mechanisms by identifying subsets of essential genes involved in interactions of cancer cells with the immune system. This chapter will present a reliable and reproducible general protocol for the application of genome-wide sgRNA gene-editing tools in the recently established two-cell type co-culture, consisting of immune cells as effectors and cancer cells as targets, utilizing CRISPRi/a-dCas9-based technology.}, }
@article {pmid31776929, year = {2020}, author = {Bailis, W}, title = {CRISPR/Cas9 Gene Targeting in Primary Mouse Bone Marrow-Derived Macrophages.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2097}, number = {}, pages = {223-230}, doi = {10.1007/978-1-0716-0203-4_14}, pmid = {31776929}, issn = {1940-6029}, mesh = {Animals ; CRISPR-Associated Protein 9/*metabolism ; CRISPR-Cas Systems/*genetics ; Cells, Cultured ; Gene Targeting/*methods ; HEK293 Cells ; Humans ; Macrophages/*metabolism ; Mice ; Retroviridae/metabolism ; Transduction, Genetic ; }, abstract = {CRISPR-Cas9 technology allows for rapid, targeted genome editing at nearly any loci with limited off-target effects. Here, we describe a method for using retroviral transduction to deliver single-guide RNA to primary bone marrow-derived macrophages. This protocol allows for high-throughput reverse genetics assays in primary immune cells and is also compatible with retroviral systems for transgene expression.}, }
@article {pmid31702097, year = {2020}, author = {Niu, Q and Wu, S and Li, Y and Yang, X and Liu, P and Xu, Y and Lang, Z}, title = {Expanding the scope of CRISPR/Cas9-mediated genome editing in plants using an xCas9 and Cas9-NG hybrid.}, journal = {Journal of integrative plant biology}, volume = {62}, number = {4}, pages = {398-402}, doi = {10.1111/jipb.12886}, pmid = {31702097}, issn = {1744-7909}, support = {2018YFD1000200//National Key Research and Development Program/ ; 2019ZX08010003-002-006//Major Project of Transgenic Crops of China/ ; XDB27040000//Strategic Priority Research Program of the Chinese Academy of Sciences/ ; }, mesh = {Arabidopsis/*genetics ; CRISPR-Associated Protein 9/*metabolism ; CRISPR-Cas Systems/*genetics ; *Gene Editing ; *Genome, Plant ; Lycopersicon esculentum/*genetics ; Protoplasts/metabolism ; }, abstract = {The widely used Streptococcus pyogenes Cas9 (SpCas9) requires NGG as a protospacer adjacent motif (PAM) for genome editing. Although SpCas9 is a powerful genome-editing tool, its use has been limited on the targetable genomic locus lacking NGG PAM. The SpCas9 variants xCas9 and Cas9-NG have been developed to recognize NG, GAA, and GAT PAMs in human cells. Here, we show that xCas9 cannot recognize NG PAMs in tomato, and Cas9-NG can recognize some of our tested NG PAMs in the tomato and Arabidopsis genomes. In addition, we engineered SpCas9 (XNG-Cas9) based on mutations from both xCas9 and Cas9-NG, and found that XNG-Cas9 can efficiently mutagenize endogenous target sites with NG, GAG, GAA, and GAT PAMs in the tomato or Arabidopsis genomes. The PAM compatibility of XNG-Cas9 is the broadest reported to date among Cas9s (SpCas9 and Cas9-NG) active in plant.}, }
@article {pmid31570788, year = {2020}, author = {Nicolai, S and Mahen, R and Raschellà, G and Marini, A and Pieraccioli, M and Malewicz, M and Venkitaraman, AR and Melino, G}, title = {ZNF281 is recruited on DNA breaks to facilitate DNA repair by non-homologous end joining.}, journal = {Oncogene}, volume = {39}, number = {4}, pages = {754-766}, doi = {10.1038/s41388-019-1028-7}, pmid = {31570788}, issn = {1476-5594}, support = {MC_U132670600/MRC_/Medical Research Council/United Kingdom ; MC_UU_00025/2/MRC_/Medical Research Council/United Kingdom ; 100090/12/Z/WT_/Wellcome Trust/United Kingdom ; }, mesh = {CRISPR-Cas Systems ; Cell Line, Tumor ; *DNA Breaks, Double-Stranded ; *DNA End-Joining Repair ; DNA-Binding Proteins/metabolism ; Databases, Genetic ; Humans ; Neoplasms/*genetics/metabolism/*pathology ; Poly (ADP-Ribose) Polymerase-1/genetics/metabolism ; Prognosis ; Repressor Proteins/genetics/*metabolism ; Survival Rate ; }, abstract = {Efficient repair of DNA double-strand breaks (DSBs) is of critical importance for cell survival. Although non-homologous end joining (NHEJ) is the most used DSBs repair pathway in the cells, how NHEJ factors are sequentially recruited to damaged chromatin remains unclear. Here, we identify a novel role for the zinc-finger protein ZNF281 in participating in the ordered recruitment of the NHEJ repair factor XRCC4 at damage sites. ZNF281 is recruited to DNA lesions within seconds after DNA damage through a mechanism dependent on its DNA binding domain and, at least in part, on poly-ADP ribose polymerase (PARP) activity. ZNF281 binds XRCC4 through its zinc-finger domain and facilitates its recruitment to damaged sites. Consequently, depletion of ZNF281 impairs the efficiency of the NHEJ repair pathway and decreases cell viability upon DNA damage. Survival analyses from datasets of commonly occurring human cancers show that higher levels of ZNF281 correlate with poor prognosis of patients treated with DNA-damaging therapies. Thus, our results define a late ZNF281-dependent regulatory step of NHEJ complex assembly at DNA lesions and suggest additional possibilities for cancer patients' stratification and for the development of personalised therapeutic strategies.}, }
@article {pmid31548614, year = {2020}, author = {Ko, T and Sharma, R and Li, S}, title = {Genome-wide screening identifies novel genes implicated in cellular sensitivity to BRAFV600E expression.}, journal = {Oncogene}, volume = {39}, number = {4}, pages = {723-738}, doi = {10.1038/s41388-019-1022-0}, pmid = {31548614}, issn = {1476-5594}, mesh = {3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)/genetics/*metabolism ; Apoptosis ; CRISPR-Cas Systems ; Cellular Senescence ; Cyclin-Dependent Kinase Inhibitor p21/genetics/metabolism ; Fibroblasts/metabolism ; *Genome, Human ; Humans ; MAP Kinase Signaling System ; Melanocytes/metabolism ; Mitogen-Activated Protein Kinase 1/genetics/metabolism ; *Mutation ; Neoplasms/genetics/metabolism/*pathology ; Proto-Oncogene Proteins B-raf/*genetics/*metabolism ; Receptors, G-Protein-Coupled/genetics/*metabolism ; Signal Transduction ; }, abstract = {The V600E mutation of BRAF (BRAFV600E), which constitutively activates the ERK/MAPK signaling pathway, is frequently found in melanoma and other cancers. Like most other oncogenes, BRAFV600E causes oncogenic stress to normal cells, leading to growth arrest (senescence) or apoptosis. Through genome-wide screening, we identified genes implicated in sensitivity of human skin melanocytes and fibroblasts to BRAFV600E overexpression. Among the identified genes shared by the two cell types are proto-oncogenes ERK2, a component of the ERK/MAPK pathway, and VAV1, a guanine nucleotide exchange factor for Rho family GTPases that also activates the ERK/MAPK pathway. CDKN1A, which has been known to promote senescence of fibroblasts but not melanocytes, is implicated in sensitivity of the fibroblasts but not the melanocytes to BRAFV600E overexpression. Disruptions of GPR4, a pH-sensing G-protein coupled receptor, and DBT, a subunit of the branched chain α-keto acid dehydrogenase that is required for the second and rate-limiting step of branched amino acid catabolism and implicated in maple syrup urine disease, are the most highly selected in the melanocytes upon BRAFV600E overexpression. Disruption of DBT severely attenuates ERK/MAPK signaling, p53 activation, and apoptosis in melanocytes, at least in part due to accumulation of branched chain α-keto acids. The expression level of BRAF positively correlates with that of DBT in all cancer types and with that of GPR4 in most cancer types. Overexpression of DBT kills all four melanoma cell lines tested regardless of the presence of BRAFV600E mutation. Our findings shed new lights on regulations of oncogenic stress signaling and may be informative for development of novel cancer treatment strategies.}, }
@article {pmid33401227, year = {2021}, author = {Pan, C and Sretenovic, S and Qi, Y}, title = {CRISPR/dCas-mediated transcriptional and epigenetic regulation in plants.}, journal = {Current opinion in plant biology}, volume = {60}, number = {}, pages = {101980}, doi = {10.1016/j.pbi.2020.101980}, pmid = {33401227}, issn = {1879-0356}, abstract = {The CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR Associated) system-mediated precise genome editing has revolutionized genome engineering due to ease of use and versatility of multiplexing. Catalytically inactivated Cas variants (dCas) further expand the usefulness of the CRISPR/Cas system for genetics studies and translational research without inducing DNA double-strand breaks. Fusion of diverse effector domains to dCas proteins empowers the CRISPR/dCas system as a multifunctional platform for gene expression regulation, epigenetic regulation and sequence-specific imaging. In this short review, we summarize the recent advances of CRISPR/dCas-mediated transcriptional activation and repression, and epigenetic modifications. We also highlight the future directions and broader applications of the CRISPR/dCas systems in plants.}, }
@article {pmid33399467, year = {2021}, author = {Qin, Z and Yang, Y and Yu, S and Liu, L and Chen, Y and Chen, J and Zhou, J}, title = {Repurposing the Endogenous Type I-E CRISPR/Cas System for Gene Repression in Gluconobacter oxydans WSH-003.}, journal = {ACS synthetic biology}, volume = {}, number = {}, pages = {}, doi = {10.1021/acssynbio.0c00456}, pmid = {33399467}, issn = {2161-5063}, abstract = {Gluconobacter oxydans is well-known for its incomplete oxidizing capacity and has been widely applied in industrial production. However, genetic tools in G. oxydans are still scarce compared with model microorganisms, limiting its metabolic engineering. This study aimed to develop a clustered regularly interspaced short palindromic repeats interference (CRISPRi) system based on the typical type I-E endogenous CRISPR/CRISPR-associated proteins (Cas) system in G. oxydans WSH-003. The nuclease Cas3 in this system was inactivated naturally and hence did not need to be knocked out. Subsequently, the CRISPRi effect was verified by repressing the expression of fluorescent proteins, revealing effective multiplex gene repression. Finally, the endogenous CRISPRi system was used to study the role of the central carbon metabolism pathway, including the pentose phosphate pathway (PPP) and Entner-Doudoroff pathway (EDP), in G. oxydans WSH-003. This was done to demonstrate a metabolic engineering application. The PPP was found to be important for cell growth and the substrate conversion rate. The development of the CRISPRi system enriched the gene regulation tools in G. oxydans and promoted the metabolic engineering modification of G. oxydans to improve its performance. In addition, it might have implications for metabolic engineering modification of other genetically recalcitrant strains.}, }
@article {pmid33398095, year = {2021}, author = {Smith, LM and Jackson, SA and Malone, LM and Ussher, JE and Gardner, PP and Fineran, PC}, title = {The Rcs stress response inversely controls surface and CRISPR-Cas adaptive immunity to discriminate plasmids and phages.}, journal = {Nature microbiology}, volume = {}, number = {}, pages = {}, pmid = {33398095}, issn = {2058-5276}, abstract = {Bacteria harbour multiple innate defences and adaptive CRISPR-Cas systems that provide immunity against bacteriophages and mobile genetic elements. Although some bacteria modulate defences in response to population density, stress and metabolic state, a lack of high-throughput methods to systematically reveal regulators has hampered efforts to understand when and how immune strategies are deployed. We developed a robust approach called SorTn-seq, which combines saturation transposon mutagenesis, fluorescence-activated cell sorting and deep sequencing to characterize regulatory networks controlling CRISPR-Cas immunity in Serratia sp. ATCC 39006. We applied our technology to assess csm gene expression for ~300,000 mutants and uncovered multiple pathways regulating type III-A CRISPR-Cas expression. Mutation of igaA or mdoG activated the Rcs outer-membrane stress response, eliciting cell-surface-based innate immunity against diverse phages via the transcriptional regulators RcsB and RcsA. Activation of this Rcs phosphorelay concomitantly attenuated adaptive immunity by three distinct type I and III CRISPR-Cas systems. Rcs-mediated repression of CRISPR-Cas defence enabled increased acquisition and retention of plasmids. Dual downregulation of cell-surface receptors and adaptive immunity in response to stress by the Rcs pathway enables protection from phage infection without preventing the uptake of plasmids that may harbour beneficial traits.}, }
@article {pmid33397707, year = {2021}, author = {Goh, YJ and Barrangou, R}, title = {A portable CRISPR-Cas9N system for flexible genome engineering in Lactobacillus acidophilus, Lactobacillus gasseri and Lactobacillus paracasei.}, journal = {Applied and environmental microbiology}, volume = {}, number = {}, pages = {}, doi = {10.1128/AEM.02669-20}, pmid = {33397707}, issn = {1098-5336}, abstract = {Diverse Lactobacillus strains are widely used as probiotic cultures in the dairy and dietary supplements industries, and specific strains such as Lactobacillus acidophilus NCFM have been engineered for the development of biotherapeutics. To expand the Lactobacillus manipulation toolbox with enhanced efficiency and ease, we present here a CRISPR-SpyCas9D10A nickase (Cas9N)-based system for programmable engineering of L. acidophilus NCFM, a model probiotic bacterium. Successful single-plasmid delivery system was achieved with the engineered pLbCas9N vector harboring cas9N under the regulation of a Lactobacillus promoter and a cloning region for customized sgRNA and editing template. The functionality of the pLbCas9N system was validated in NCFM with targeted chromosomal deletions ranging between 300 bp and 1.9 kb at various loci (rafE, lacS and ltaS), yielding 35-100% mutant recovery rates. Genome analysis of the mutants confirmed precision and specificity of the pLbCas9N system. To showcase the versatility of this system, we also inserted a mCherry fluorescent protein gene downstream of the pgm gene to create a polycistronic transcript. The pLbCas9N system was further deployed in other species to generate concurrent single base substitution and gene deletion in Lactobacillus gasseri ATCC 33323, and an in-frame gene deletion in Lactobacillus paracasei Lpc-37, highlighting the portability of the system in phylogenetically distant Lactobacillus species, where its targeting activity was not interfered by endogenous CRISPR-Cas systems. Collectively, these editing outcomes illustrate the robustness and versatility of the pLbCas9N system for genome manipulations in diverse lactobacilli, and open new avenues for the engineering of health-promoting lactic acid bacteria.Importance This work describes the development of a broad-host range CRISPR-based editing system for genome manipulations in three Lactobacillus species, which belong to lactic acid bacteria (LAB) commonly known for their long history of use in food fermentations and as indigenous members of healthy microbiota, and their emerging roles in human and