@article {pmid39365820,
year = {2024},
author = {Stransky, LA and Gao, W and Schmidt, LS and Bi, K and Ricketts, CJ and Ramesh, V and James, A and Difilippantonio, S and Ileva, L and Kalen, JD and Karim, B and Jeon, A and Morgan, T and Warner, AC and Turan, S and Unite, J and Tran, B and Choudhari, S and Zhao, Y and Linn, DE and Yun, C and Dhandapani, S and Parab, V and Pinheiro, EM and Morris, N and He, L and Vigeant, SM and Pignon, JC and Sticco-Ivins, M and Signoretti, S and Van Allen, EM and Linehan, WM and Kaelin, WG},
title = {Toward a CRISPR-based mouse model of Vhl-deficient clear cell kidney cancer: Initial experience and lessons learned.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {121},
number = {41},
pages = {e2408549121},
doi = {10.1073/pnas.2408549121},
pmid = {39365820},
issn = {1091-6490},
support = {R35CA100068; P50CA101942;//HHS | NIH | National Cancer Institute (NCI)/ ; U01CA236489//HHS | NIH | National Cancer Institute (NCI)/ ; T32CA236754//HHS | NIH | National Cancer Institute (NCI)/ ; Contract No.HHSN2612015000031//in part with Federal Funds from the National Cancer Institute, National Institutes of Health/ ; },
mesh = {Animals ; *Carcinoma, Renal Cell/genetics/pathology ; *Von Hippel-Lindau Tumor Suppressor Protein/genetics/metabolism ; *Kidney Neoplasms/genetics/pathology/metabolism ; Mice ; *Disease Models, Animal ; Humans ; CRISPR-Cas Systems ; Gene Editing/methods ; Mice, Inbred C57BL ; Axitinib ; Indazoles/pharmacology ; },
abstract = {CRISPR is revolutionizing the ability to do somatic gene editing in mice for the purpose of creating new cancer models. Inactivation of the VHL tumor suppressor gene is the signature initiating event in the most common form of kidney cancer, clear cell renal cell carcinoma (ccRCC). Such tumors are usually driven by the excessive HIF2 activity that arises when the VHL gene product, pVHL, is defective. Given the pressing need for a robust immunocompetent mouse model of human ccRCC, we directly injected adenovirus-associated viruses (AAVs) encoding sgRNAs against VHL and other known/suspected ccRCC tumor suppressor genes into the kidneys of C57BL/6 mice under conditions where Cas9 was under the control of one of two different kidney-specific promoters (Cdh16 or Pax8) to induce kidney tumors. An AAV targeting Vhl, Pbrm1, Keap1, and Tsc1 reproducibly caused macroscopic ccRCCs that partially resembled human ccRCC tumors with respect to transcriptome and cell of origin and responded to a ccRCC standard-of-care agent, axitinib. Unfortunately, these tumors, like those produced by earlier genetically engineered mouse ccRCCs, are HIF2 independent.},
}

Animals
*Carcinoma, Renal Cell/genetics/pathology
*Von Hippel-Lindau Tumor Suppressor Protein/genetics/metabolism
*Kidney Neoplasms/genetics/pathology/metabolism
Mice
*Disease Models, Animal
Humans
CRISPR-Cas Systems
Gene Editing/methods
Mice, Inbred C57BL
Axitinib
Indazoles/pharmacology

@article {pmid39365784,
year = {2024},
author = {Eichmeier, AA and Xenos, MA},
title = {CRISPR is easy: Exposure to Last Week Tonight enhances knowledge about gene editing.},
journal = {PloS one},
volume = {19},
number = {10},
pages = {e0306563},
doi = {10.1371/journal.pone.0306563},
pmid = {39365784},
issn = {1932-6203},
mesh = {Humans ; *Gene Editing/methods ; Female ; Male ; Adult ; Middle Aged ; Young Adult ; Surveys and Questionnaires ; Wit and Humor as Topic ; Adolescent ; Clustered Regularly Interspaced Short Palindromic Repeats ; Knowledge ; CRISPR-Cas Systems ; },
abstract = {Experts have called for public engagement with the governance of controversial scientific research and discoveries, including CRISPR, the technology that enables gene editing. Though engaging and informing citizens who are not interested in the issue is a challenge, recent studies suggest humor has potential to close interest and knowledge gaps. We tested this potential by exposing individuals (N = 303) to one of three videos (an edited clip from Last Week Tonight, an edited clip from 60 Minutes, or control) that contained broadly overlapping facts about gene editing in an online survey. Results show that while exposure to the Last Week Tonight clip did not increase attentiveness to the issue of human gene editing among individuals with lower levels of interest in science, exposure to the humorous clip caused a modest improvement in issue knowledge. Positive main effects on perceived knowledge were found for both treatments. More research is needed but findings suggest that the use of humor in science communication offers potential, though perhaps limited, for broadening public engagement with emerging areas of science.},
}

Humans
*Gene Editing/methods
Female
Male
Adult
Middle Aged
Young Adult
Surveys and Questionnaires
Wit and Humor as Topic
Adolescent
Clustered Regularly Interspaced Short Palindromic Repeats
Knowledge
CRISPR-Cas Systems

@article {pmid39365728,
year = {2024},
author = {Chen, MM and Zhao, Y and Yu, K and Xu, XL and Zhang, XS and Zhang, JL and Wu, SJ and Liu, ZM and Yuan, YM and Guo, XF and Qi, SY and Yi, G and Wang, SQ and Li, HX and Wu, AW and Liu, GS and Deng, SL and Han, HB and Lv, FH and Lian, D and Lian, ZX},
title = {A MSTN[Del73C] mutation with FGF5 knockout sheep by CRISPR/Cas9 promotes skeletal muscle myofiber hyperplasia.},
journal = {eLife},
volume = {12},
number = {},
pages = {},
pmid = {39365728},
issn = {2050-084X},
support = {2022ZD04014//Major Agricultural Biological Breeding Project/ ; 32072722//National Natural Science Foundation of China/ ; 32272853//National Natural Science Foundation of China/ ; 2021YFF1000704//National Key Research and Development Program of China/ ; 2021YFD1200902//National Key Research and Development Program of China/ ; },
mesh = {Animals ; *Myostatin/genetics/metabolism ; Sheep ; *CRISPR-Cas Systems ; *Fibroblast Growth Factor 5/genetics/metabolism ; Muscle Fibers, Skeletal/metabolism/pathology ; Mutation ; Gene Knockout Techniques ; Hyperplasia/genetics ; Muscle, Skeletal/metabolism/pathology ; },
abstract = {Mutations in the well-known Myostatin (MSTN) produce a 'double-muscle' phenotype, which makes it commercially invaluable for improving livestock meat production and providing high-quality protein for humans. However, mutations at different loci of the MSTN often produce a variety of different phenotypes. In the current study, we increased the delivery ratio of Cas9 mRNA to sgRNA from the traditional 1:2 to 1:10, which improves the efficiency of the homozygous mutation of biallelic gene. Here, a MSTN[Del73C] mutation with FGF5 knockout sheep, in which the MSTN and FGF5 dual-gene biallelic homozygous mutations were produced via the deletion of 3-base pairs of AGC in the third exon of MSTN, resulting in cysteine-depleted at amino acid position 73, and the FGF5 double allele mutation led to inactivation of FGF5 gene. The MSTN[Del73C] mutation with FGF5 knockout sheep highlights a dominant 'double-muscle' phenotype, which can be stably inherited. Both F0 and F1 generation mutants highlight the excellent trait of high-yield meat with a smaller cross-sectional area and higher number of muscle fibers per unit area. Mechanistically, the MSTN[Del73C] mutation with FGF5 knockout mediated the activation of FOSL1 via the MEK-ERK-FOSL1 axis. The activated FOSL1 promotes skeletal muscle satellite cell proliferation and inhibits myogenic differentiation by inhibiting the expression of MyoD1, and resulting in smaller myotubes. In addition, activated ERK1/2 may inhibit the secondary fusion of myotubes by Ca[2+]-dependent CaMKII activation pathway, leading to myoblasts fusion to form smaller myotubes.},
}

Animals
*Myostatin/genetics/metabolism
Sheep
*CRISPR-Cas Systems
*Fibroblast Growth Factor 5/genetics/metabolism
Muscle Fibers, Skeletal/metabolism/pathology
Mutation
Gene Knockout Techniques
Hyperplasia/genetics
Muscle, Skeletal/metabolism/pathology

@article {pmid39363082,
year = {2025},
author = {Stukenberg, D and Hoff, J and Faber, A and Becker, A},
title = {Protocol for NT-CRISPR: A Method for Efficient Genome Engineering in Vibrio natriegens.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2850},
number = {},
pages = {365-375},
pmid = {39363082},
issn = {1940-6029},
mesh = {*Vibrio/genetics ; *CRISPR-Cas Systems ; *Genome, Bacterial ; *Gene Editing/methods ; Genetic Engineering/methods ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {Vibrio natriegens is a gram-negative bacterium, which has received increasing attention due to its very fast growth with a doubling time of under 10 min under optimal conditions. To enable a wide range of projects spanning from basic research to biotechnological applications, we developed NT-CRISPR as a new method for genome engineering. This book chapter provides a step-by-step protocol for the use of this previously published tool. NT-CRISPR combines natural transformation with counterselection through CRISPR-Cas9. Thereby, genomic regions can be deleted, foreign sequences can be integrated, and point mutations can be introduced. Furthermore, up to three simultaneous modifications are possible.},
}

*Vibrio/genetics
*CRISPR-Cas Systems
*Genome, Bacterial
*Gene Editing/methods
Genetic Engineering/methods
Clustered Regularly Interspaced Short Palindromic Repeats/genetics

@article {pmid39363080,
year = {2025},
author = {Pozhydaieva, N and Höfer, K},
title = {Utilizing Golden Gate Assembly to Streamline CRISPR-Cas/NgTET-Based Phage Mutagenesis.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2850},
number = {},
pages = {329-343},
pmid = {39363080},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems ; *Mutagenesis ; *Bacteriophages/genetics ; Cytosine/metabolism ; Gene Editing/methods ; Genetic Vectors/genetics ; },
abstract = {Phage engineering is an emerging technology due to the promising potential application of phages in medical and biotechnological settings. Targeted phage mutagenesis tools are required to customize the phages for a specific application and generate, in addition to that, so-called designer phages. CRISPR-Cas technique is used in various organisms to perform targeted mutagenesis. Yet, its efficacy is notably limited for phage mutagenesis due to the highly abundant phage DNA modifications. Addressing this challenge, we have developed a novel approach that involves the temporal removal of phage DNA cytosine modifications, allowing for effective CRISPR-Cas targeting and subsequent introduction of mutations into the phage genome. The removal of cytosine modification relies on the catalytic activity of a eukaryotic ten-eleven translocation methylcytosine (TET) dioxygenase. TET enzymes iteratively de-modify methylated or hydroxymethylated cytosines on phage DNA. The temporal removal of cytosine modification ultimately enables efficient DNA cleavage by Cas enzymes and facilitates mutagenesis. To streamline the application of the coupled TET-CRISPR-Cas system, we use Golden Gate cloning for fast and efficient assembly of a vector that comprises a TET oxidase and a donor DNA required for scarless site-specific phage mutagenesis. Our approach significantly advances the engineering of modified phage genomes, enabling the efficient generation of customized phages for specific applications.},
}

*CRISPR-Cas Systems
*Mutagenesis
*Bacteriophages/genetics
Cytosine/metabolism
Gene Editing/methods
Genetic Vectors/genetics

@article {pmid39363078,
year = {2025},
author = {Rust, S and Randau, L},
title = {Golden Gate Cloning of Synthetic CRISPR RNA Spacer Sequences.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2850},
number = {},
pages = {297-306},
pmid = {39363078},
issn = {1940-6029},
mesh = {*Cloning, Molecular/methods ; *CRISPR-Cas Systems ; *Plasmids/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Escherichia coli/genetics ; RNA/genetics ; },
abstract = {Prokaryotes use CRISPR-Cas systems to interfere with viruses and other mobile genetic elements. CRISPR arrays comprise repeated DNA elements and spacer sequences that can be engineered for custom target sites. These arrays are transcribed into precursor CRISPR RNAs (pre-crRNAs) that undergo maturation steps to form individual CRISPR RNAs (crRNAs). Each crRNA contains a single spacer that identifies the target cleavage site for a large variety of Cas protein effectors. Precise manipulation of spacer sequences within CRISPR arrays is crucial for advancing the functionality of CRISPR-based technologies. Here, we describe a protocol for the design and creation of a minimal, plasmid-based CRISPR array to enable the expression of specific, synthetic crRNAs. Plasmids contain entry spacer sequences with two type IIS restriction sites and Golden Gate cloning enables the efficient exchange of these spacer sequences. Factors that influence the compatibility of the CRISPR arrays with native or recombinant Cas proteins are discussed.},
}

*Cloning, Molecular/methods
*CRISPR-Cas Systems
*Plasmids/genetics
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Escherichia coli/genetics
RNA/genetics

@article {pmid39299233,
year = {2024},
author = {Olvera-León, R and Zhang, F and Offord, V and Zhao, Y and Tan, HK and Gupta, P and Pal, T and Robles-Espinoza, CD and Arriaga-González, FG and Matsuyama, LSAS and Delage, E and Dicks, E and Ezquina, S and Rowlands, CF and Turnbull, C and Pharoah, P and Perry, JRB and Jasin, M and Waters, AJ and Adams, DJ},
title = {High-resolution functional mapping of RAD51C by saturation genome editing.},
journal = {Cell},
volume = {187},
number = {20},
pages = {5719-5734.e19},
doi = {10.1016/j.cell.2024.08.039},
pmid = {39299233},
issn = {1097-4172},
mesh = {Humans ; Female ; *Gene Editing/methods ; *DNA-Binding Proteins/metabolism/genetics ; *Ovarian Neoplasms/genetics ; Breast Neoplasms/genetics ; Alleles ; CRISPR-Cas Systems/genetics ; },
abstract = {Pathogenic variants in RAD51C confer an elevated risk of breast and ovarian cancer, while individuals homozygous for specific RAD51C alleles may develop Fanconi anemia. Using saturation genome editing (SGE), we functionally assess 9,188 unique variants, including >99.5% of all possible coding sequence single-nucleotide alterations. By computing changes in variant abundance and Gaussian mixture modeling (GMM), we functionally classify 3,094 variants to be disruptive and use clinical truth sets to reveal an accuracy/concordance of variant classification >99.9%. Cell fitness was the primary assay readout allowing us to observe a phenomenon where specific missense variants exhibit distinct depletion kinetics potentially suggesting that they represent hypomorphic alleles. We further explored our exhaustive functional map, revealing critical residues on the RAD51C structure and resolving variants found in cancer-segregating kindred. Furthermore, through interrogation of UK Biobank and a large multi-center ovarian cancer cohort, we find significant associations between SGE-depleted variants and cancer diagnoses.},
}

Humans
Female
*Gene Editing/methods
*DNA-Binding Proteins/metabolism/genetics
*Ovarian Neoplasms/genetics
Breast Neoplasms/genetics
Alleles
CRISPR-Cas Systems/genetics

@article {pmid39189759,
year = {2024},
author = {Gong, J and Jiang, Y and Zhang, D and Li, T and Fu, L and Dou, X},
title = {One-tube detection of Salmonella Typhimurium using LAMP and CRISPR-Cas12b.},
journal = {Microbiology spectrum},
volume = {12},
number = {10},
pages = {e0127124},
pmid = {39189759},
issn = {2165-0497},
support = {CX(23)3005//Jiangsu Agricultural Science and Technology Innovation Fund (JASTIF)/ ; },
mesh = {*Salmonella typhimurium/genetics/isolation & purification ; *Nucleic Acid Amplification Techniques/methods ; *CRISPR-Cas Systems ; *Molecular Diagnostic Techniques/methods ; Humans ; Sensitivity and Specificity ; Limit of Detection ; Salmonella Infections/microbiology/diagnosis ; Feces/microbiology ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Bacterial Proteins/genetics ; },
abstract = {UNLABELLED: Salmonella enterica serovar Typhimurium (ST) is a predominant serovar causing foodborne illnesses worldwide. Traditional detection methods often face challenges, including the need for specialized equipment, skilled operators, and lengthy procedures. To address these limitations, we developed a rapid, sensitive, and specific ST detection method by integrating loop-mediated isothermal amplification (LAMP) with the clustered regularly interspaced short palindromic repeats and associated protein 12b (CRISPR/Cas12b) system, all within a single tube. Our results indicate that the LAMP-CRISPR/Cas12b reaction can be completed isothermally in under 1 h without requiring specialized instruments. The platform's limit of detection (LoD) is 12.5 copies per reaction. Additionally, the system demonstrated 100% inclusivity and exclusivity when tested against 30 reference strains, highlighting its specificity. In practical applications, the LoDs for ST in pure nucleic acid and contaminated fecal samples were 2.32 and 23.2 CFU/mL, respectively, with higher sensitivity observed in pure nucleic acid samples. Overall, our findings underscore the potential of the one-tube LAMP-CRISPR/Cas12b platform as a rapid, sensitive, and specific tool for ST detection, particularly in resource-limited settings.

IMPORTANCE: Here, we have provided a novel one-step method for Salmonella Typhimurium detection in one pot by integrating the LAMP assay with the CRISPR/Cas12b system, offering significant advantages in terms of simplicity, speed, and accuracy.},
}

*Salmonella typhimurium/genetics/isolation & purification
*Nucleic Acid Amplification Techniques/methods
*CRISPR-Cas Systems
*Molecular Diagnostic Techniques/methods
Humans
Sensitivity and Specificity
Limit of Detection
Salmonella Infections/microbiology/diagnosis
Feces/microbiology
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Bacterial Proteins/genetics

@article {pmid39091030,
year = {2024},
author = {Li, YJ and Chien, SH and Huang, R and Herrmann, A and Zhao, Q and Li, PC and Zhang, C and Martincuks, A and Santiago, NL and Zong, K and Swiderski, P and Okimoto, RA and Song, M and Rodriguez, L and Forman, SJ and Wang, X and Yu, H},
title = {A platform to deliver single and bi-specific Cas9/guide RNA to perturb genes in vitro and in vivo.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {32},
number = {10},
pages = {3629-3649},
doi = {10.1016/j.ymthe.2024.07.025},
pmid = {39091030},
issn = {1525-0024},
mesh = {Humans ; Animals ; Mice ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *CRISPR-Cas Systems ; *Xenograft Model Antitumor Assays ; Female ; Cell Line, Tumor ; Ovarian Neoplasms/genetics/therapy/pathology ; Receptor, ErbB-2/genetics/metabolism ; Gene Editing/methods ; Gene Knockout Techniques ; CRISPR-Associated Protein 9/genetics/metabolism ; },
abstract = {Although CRISPR-Cas9 technology is poised to revolutionize the treatment of diseases with underlying genetic mutations, it faces some significant issues limiting clinical entry. They include low-efficiency in vivo systemic delivery and undesired off-target effects. Here, we demonstrate, by modifying Cas9 with phosphorothioate-DNA oligos (PSs), that one can efficiently deliver single and bi-specific CRISPR-Cas9/guide RNA (gRNA) dimers in vitro and in vivo with reduced off-target effects. We show that PS-Cas9/gRNA-mediated gene knockout preserves chimeric antigen receptor T cell viability and expansion in vitro and in vivo. PS-Cas9/gRNA mediates gene perturbation in patient-derived tumor organoids and mouse xenograft tumors, leading to potent tumor antitumor effects. Further, HER2 antibody-PS-Cas9/gRNA conjugate selectively perturbs targeted genes in HER2[+] ovarian cancer xenografts in vivo. Moreover, we created bi-specific PS-Cas9 with two gRNAs to target two adjacent sequences of the same gene, leading to efficient targeted gene disruption ex vivo and in vivo with markedly reduced unintended gene perturbation. Thus, the cell-penetrating PS-Cas9/gRNA can achieve efficient systemic delivery and precision in gene disruption.},
}

Humans
Animals
Mice
*RNA, Guide, CRISPR-Cas Systems/genetics
*CRISPR-Cas Systems
*Xenograft Model Antitumor Assays
Female
Cell Line, Tumor
Ovarian Neoplasms/genetics/therapy/pathology
Receptor, ErbB-2/genetics/metabolism
Gene Editing/methods
Gene Knockout Techniques
CRISPR-Associated Protein 9/genetics/metabolism

@article {pmid39086133,
year = {2024},
author = {Katta, V and O'Keefe, K and Li, Y and Mayuranathan, T and Lazzarotto, CR and Wood, RK and Levine, RM and Powers, A and Mayberry, K and Manquen, G and Yao, Y and Zhang, J and Jang, Y and Nimmagadda, N and Dempsey, EA and Lee, G and Uchida, N and Cheng, Y and Fazio, F and Lockey, T and Meagher, M and Sharma, A and Tisdale, JF and Zhou, S and Yen, JS and Weiss, MJ and Tsai, SQ},
title = {Development and IND-enabling studies of a novel Cas9 genome-edited autologous CD34[+] cell therapy to induce fetal hemoglobin for sickle cell disease.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {32},
number = {10},
pages = {3433-3452},
doi = {10.1016/j.ymthe.2024.07.022},
pmid = {39086133},
issn = {1525-0024},
support = {P01 HL053749/HL/NHLBI NIH HHS/United States ; },
mesh = {*Anemia, Sickle Cell/therapy/genetics ; *Fetal Hemoglobin/genetics ; Humans ; *Gene Editing/methods ; *Antigens, CD34/metabolism ; *Hematopoietic Stem Cells/metabolism ; *CRISPR-Cas Systems ; gamma-Globins/genetics ; Promoter Regions, Genetic ; Hematopoietic Stem Cell Transplantation/methods ; Animals ; Genetic Therapy/methods ; },
abstract = {Sickle cell disease (SCD) is a common, severe genetic blood disorder. Current pharmacotherapies are partially effective and allogeneic hematopoietic stem cell transplantation is associated with immune toxicities. Genome editing of patient hematopoietic stem cells (HSCs) to reactivate fetal hemoglobin (HbF) in erythroid progeny offers an alternative potentially curative approach to treat SCD. Although the FDA released guidelines for evaluating genome editing risks, it remains unclear how best to approach pre-clinical assessment of genome-edited cell products. Here, we describe rigorous pre-clinical development of a therapeutic γ-globin gene promoter editing strategy that supported an investigational new drug application cleared by the FDA. We compared γ-globin promoter and BCL11A enhancer targets, identified a potent HbF-inducing lead candidate, and tested our approach in mobilized CD34[+] hematopoietic stem progenitor cells (HSPCs) from SCD patients. We observed efficient editing, HbF induction to predicted therapeutic levels, and reduced sickling. With single-cell analyses, we defined the heterogeneity of HbF induction and HBG1/HBG2 transcription. With CHANGE-seq for sensitive and unbiased off-target discovery followed by targeted sequencing, we did not detect off-target activity in edited HSPCs. Our study provides a blueprint for translating new ex vivo HSC genome editing strategies toward clinical trials for treating SCD and other blood disorders.},
}

*Anemia, Sickle Cell/therapy/genetics
*Fetal Hemoglobin/genetics
Humans
*Gene Editing/methods
*Antigens, CD34/metabolism
*Hematopoietic Stem Cells/metabolism
*CRISPR-Cas Systems
gamma-Globins/genetics
Promoter Regions, Genetic
Hematopoietic Stem Cell Transplantation/methods
Animals
Genetic Therapy/methods

@article {pmid39162514,
year = {2024},
author = {Miah, R and Johannessen, M and Kjos, M and Lentz, CS},
title = {Development of an inducer-free, virulence gene promoter-controlled, and fluorescent reporter-labeled CRISPR interference system in Staphylococcus aureus.},
journal = {Microbiology spectrum},
volume = {12},
number = {10},
pages = {e0060224},
pmid = {39162514},
issn = {2165-0497},
support = {296906//Joint Programming Initiative on Antimicrobial Resistance (JPIAMR)/ ; Start-up grant//Centre for New Antibacterial Strategies (CANS)/ ; },
mesh = {*Promoter Regions, Genetic ; *Staphylococcus aureus/genetics/pathogenicity ; *CRISPR-Cas Systems ; Virulence/genetics ; *Genes, Reporter ; *Virulence Factors/genetics ; *Gene Expression Regulation, Bacterial ; *Bacterial Proteins/genetics/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Humans ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {The dCas9-based Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) interference (CRISPRi) gene regulation technique requires two components: a catalytically inactive Cas9 protein (dCas9) and a single-guide RNA that targets the gene of interest. This system is commonly activated by expressing dCas9 through an inducible gene promoter, but these inducers may affect cellular physiology, and accessibility and permeability of the inducer are limited in relevant model systems. Here, we have developed an alternative approach for CRISPRi activation in the clinical isolate Staphylococcus aureus USA300 LAC, where dCas9 was expressed through endogenous virulence gene promoters (vgp); coagulase, autolysin, or fibronectin-binding protein A. Additionally, we integrated a fluorescent reporter gene into the vgp-CRISPRi system to monitor the activity of the dcas9-controlling promoter. Testing the efficacy of vgp-CRISPRi by inducing growth arrest (when targeting penicillin-binding protein 1), downregulating target gene expression, or blocking coagulase-dependent coagulation of blood plasma, we provide a proof-of-concept demonstration that the virulence gene promoter-driven CRISPRi system is functional in S. aureus.IMPORTANCEThe presented inducer-free, endogenous virulence gene promoter-induced, dCas9-based Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) interference (CRISPRi system addresses several shortcomings related to the use of inducer-dependent systems such as effects on cell physiology or limitations in permeability, and it avoids the high, putatively toxic levels of dCas9 in CRISPRi systems controlled by strong, constitutive promoters.},
}

*Promoter Regions, Genetic
*Staphylococcus aureus/genetics/pathogenicity
*CRISPR-Cas Systems
Virulence/genetics
*Genes, Reporter
*Virulence Factors/genetics
*Gene Expression Regulation, Bacterial
*Bacterial Proteins/genetics/metabolism
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Humans
RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid38819218,
year = {2024},
author = {Waarts, MR and Mowla, S and Boileau, M and Martinez Benitez, AR and Sango, J and Bagish, M and Fernández-Maestre, I and Shan, Y and Eisman, SE and Park, YC and Wereski, M and Csete, I and O'Connor, K and Romero-Vega, AC and Miles, LA and Xiao, W and Wu, X and Koche, RP and Armstrong, SA and Shih, AH and Papapetrou, EP and Butler, JM and Cai, SF and Bowman, RL and Levine, RL},
title = {CRISPR Dependency Screens in Primary Hematopoietic Stem Cells Identify KDM3B as a Genotype-specific Vulnerability in IDH2- and TET2-mutant Cells.},
journal = {Cancer discovery},
volume = {14},
number = {10},
pages = {1860-1878},
doi = {10.1158/2159-8290.CD-23-1092},
pmid = {38819218},
issn = {2159-8290},
support = {P01 CA066996/CA/NCI NIH HHS/United States ; R01 CA176745/CA/NCI NIH HHS/United States ; R01 CA259273/CA/NCI NIH HHS/United States ; U01 AG077925/AG/NIA NIH HHS/United States ; //Geoffrey Beene Foundation (GBF)/ ; F31 CA257367/CA/NCI NIH HHS/United States ; //Fonds de Recherche du Québec - Santé (FRQS)/ ; //'la Caixa' Foundation ('la Caixa')/ ; K08 CA267058/CA/NCI NIH HHS/United States ; P50 CA254838/CA/NCI NIH HHS/United States ; R01 CA176745/CA/NCI NIH HHS/United States ; //Leukemia Research Foundation (LRF)/ ; K08 CA241371/CA/NCI NIH HHS/United States ; R35 CA197594/CA/NCI NIH HHS/United States ; },
mesh = {*Jumonji Domain-Containing Histone Demethylases/genetics/metabolism ; *Hematopoietic Stem Cells/metabolism ; Humans ; *Dioxygenases ; *Isocitrate Dehydrogenase/genetics ; Mutation ; DNA-Binding Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Proto-Oncogene Proteins/genetics/metabolism ; Genotype ; Mice ; Animals ; },
abstract = {Clonal hematopoiesis (CH) is a common premalignant state in the blood and confers an increased risk of blood cancers and all-cause mortality. Identification of therapeutic targets in CH has been hindered by the lack of an ex vivo platform amenable for studying primary hematopoietic stem and progenitor cells (HSPCs). Here, we utilize an ex vivo co-culture system of HSPCs with bone marrow endothelial cells to perform CRISPR/Cas9 screens in mutant HSPCs. Our data reveal that loss of the histone demethylase family members Kdm3b and Jmjd1c specifically reduces the fitness of Idh2- and Tet2-mutant HSPCs. Kdm3b loss in mutant cells leads to decreased expression of critical cytokine receptors including Mpl, rendering mutant HSPCs preferentially susceptible to inhibition of downstream JAK2 signaling. Our study nominates an epigenetic regulator and an epigenetically regulated receptor signaling pathway as genotype-specific therapeutic targets and provides a scalable platform to identify genetic dependencies in mutant HSPCs. Significance: Given the broad prevalence, comorbidities, and risk of malignant transformation associated with CH, there is an unmet need to identify therapeutic targets. We develop an ex vivo platform to perform CRISPR/Cas9 screens in primary HSPCs. We identify KDM3B and downstream signaling components as genotype-specific dependencies in CH and myeloid malignancies. See related commentary by Khabusheva and Goodell, p. 1768.},
}

*Jumonji Domain-Containing Histone Demethylases/genetics/metabolism
*Hematopoietic Stem Cells/metabolism
Humans
*Dioxygenases
*Isocitrate Dehydrogenase/genetics
Mutation
DNA-Binding Proteins/genetics/metabolism
CRISPR-Cas Systems
Proto-Oncogene Proteins/genetics/metabolism
Genotype
Mice
Animals

@article {pmid39363077,
year = {2025},
author = {Valero, AM and Prins, RC and de Vroet, T and Billerbeck, S},
title = {Combining Oligo Pools and Golden Gate Cloning to Create Protein Variant Libraries or Guide RNA Libraries for CRISPR Applications.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2850},
number = {},
pages = {265-295},
pmid = {39363077},
issn = {1940-6029},
mesh = {*Cloning, Molecular/methods ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *CRISPR-Cas Systems ; *Gene Library ; Oligonucleotides/genetics ; Gene Editing/methods ; Proteins/genetics ; },
abstract = {Oligo pools are array-synthesized, user-defined mixtures of single-stranded oligonucleotides that can be used as a source of synthetic DNA for library cloning. While currently offering the most affordable source of synthetic DNA, oligo pools also come with limitations such as a maximum synthesis length (approximately 350 bases), a higher error rate compared to alternative synthesis methods, and the presence of truncated molecules in the pool due to incomplete synthesis. Here, we provide users with a comprehensive protocol that details how oligo pools can be used in combination with Golden Gate cloning to create user-defined protein mutant libraries, as well as single-guide RNA libraries for CRISPR applications. Our methods are optimized to work within the Yeast Toolkit Golden Gate scheme, but are in principle compatible with any other Golden Gate-based modular cloning toolkit and extendable to other restriction enzyme-based cloning methods beyond Golden Gate. Our methods yield high-quality, affordable, in-house variant libraries.},
}

*Cloning, Molecular/methods
*RNA, Guide, CRISPR-Cas Systems/genetics
*CRISPR-Cas Systems
*Gene Library
Oligonucleotides/genetics
Gene Editing/methods
Proteins/genetics

@article {pmid39363066,
year = {2025},
author = {Vegh, P and Chapman, E and Gilmour, C and Fragkoudis, R},
title = {Modular DNA Construct Design for High-Throughput Golden Gate Assembly.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2850},
number = {},
pages = {61-77},
pmid = {39363066},
issn = {1940-6029},
mesh = {*DNA/genetics/chemistry ; *Gene Editing/methods ; *Cloning, Molecular/methods ; CRISPR-Cas Systems ; Software ; Synthetic Biology/methods ; Computational Biology/methods ; High-Throughput Nucleotide Sequencing/methods ; },
abstract = {Golden Gate cloning enables the modular assembly of DNA parts into desired synthetic genetic constructs. The "one-pot" nature of Golden Gate reactions makes them particularly amenable to high-throughput automation, facilitating the generation of thousands of constructs in a massively parallel manner. One potential bottleneck in this process is the design of these constructs. There are multiple parameters that must be considered during the design of an assembly process, and the final design should also be checked and verified before implementation. Doing this by hand for large numbers of constructs is neither practical nor feasible and increases the likelihood of introducing potentially costly errors. In this chapter we describe a design workflow that utilizes bespoke computational tools to automate the key phases of the construct design process and perform sequence editing in batches.},
}

*DNA/genetics/chemistry
*Gene Editing/methods
*Cloning, Molecular/methods
CRISPR-Cas Systems
Software
Synthetic Biology/methods
Computational Biology/methods
High-Throughput Nucleotide Sequencing/methods

@article {pmid39363033,
year = {2024},
author = {Wei, Z and Wang, Y and Zheng, K and Wang, Z and Liu, R and Wang, P and Li, Y and Gao, P and Akbari, OS and Yang, X},
title = {Loss-of-function in testis-specific serine/threonine protein kinase triggers male infertility in an invasive moth.},
journal = {Communications biology},
volume = {7},
number = {1},
pages = {1256},
pmid = {39363033},
issn = {2399-3642},
mesh = {Male ; Animals ; *Moths/genetics ; *Infertility, Male/genetics ; *Testis/metabolism ; *Protein Serine-Threonine Kinases/genetics/metabolism ; Insect Proteins/genetics/metabolism ; Introduced Species ; Loss of Function Mutation ; Spermatogenesis/genetics ; CRISPR-Cas Systems ; },
abstract = {Genetic biocontrol technologies present promising and eco-friendly strategies for the management of pest and insect-transmitted diseases. Although considerable advancements achieve in gene drive applications targeting mosquitoes, endeavors to combat agricultural pests have been somewhat restricted. Here, we identify that the testis-specific serine/threonine kinases (TSSKs) family is uniquely expressed in the testes of Cydia pomonella, a prominent global invasive species. We further generated male moths with disrupted the expression of TSSKs and those with TSSKs disrupted using RNA interference and CRISPR/Cas9 genetic editing techniques, resulting in significant disruptions in spermiogenesis, decreased sperm motility, and hindered development of eggs. Further explorations into the underlying post-transcriptional regulatory mechanisms reveales the involvement of lnc117962 as a competing endogenous RNA (ceRNA) for miR-3960, thereby regulating TSSKs. Notably, orchard trials demonstrates that the release of male strains can effectively suppress population growth. Our findings indicate that targeting TSSKs could serve as a feasible avenue for managing C. pomonella populations, offering significant insights and potential strategies for controlling invasive pests through genetic sterile insect technique (gSIT) technology.},
}

Male
Animals
*Moths/genetics
*Infertility, Male/genetics
*Testis/metabolism
*Protein Serine-Threonine Kinases/genetics/metabolism
Insect Proteins/genetics/metabolism
Introduced Species
Loss of Function Mutation
Spermatogenesis/genetics
CRISPR-Cas Systems

@article {pmid39362874,
year = {2024},
author = {Guo, Y and Zhou, Y and Duan, H and Xu, D and Wei, M and Wu, Y and Xiong, Y and Chen, X and Wang, S and Liu, D and Huang, X and Xin, H and Xiong, Y and Tang, BZ},
title = {CRISPR/Cas-mediated "one to more" lighting-up nucleic acid detection using aggregation-induced emission luminogens.},
journal = {Nature communications},
volume = {15},
number = {1},
pages = {8560},
pmid = {39362874},
issn = {2041-1723},
mesh = {*CRISPR-Cas Systems ; Humans ; *SARS-CoV-2/genetics ; Norovirus/genetics ; COVID-19/virology ; DNA/genetics ; Fluorescent Dyes/chemistry ; },
abstract = {CRISPR diagnostics are effective but suffer from low signal transduction efficiency, limited sensitivity, and poor stability due to their reliance on the trans-cleavage of single-stranded nucleic acid fluorescent reporters. Here, we present CrisprAIE, which integrates CRISPR/Cas reactions with "one to more" aggregation-induced emission luminogen (AIEgen) lighting-up fluorescence generated by the trans-cleavage of Cas proteins to AIEgen-incorporated double-stranded DNA labeled with single-stranded nucleic acid linkers and Black Hole Quencher groups at both ends (Q-dsDNA/AIEgens-Q). CrisprAIE demonstrates superior performance in the clinical nucleic acid detection of norovirus and SARS-CoV-2 regardless of amplification. Moreover, the diagnostic potential of CrisprAIE is further enhanced by integrating it with spherical nucleic acid-modified AIEgens (SNA/AIEgens) and a portable cellphone-based readout device. The improved CrisprAIE system, utilizing Q-dsDNA/AIEgen-Q and SNA/AIEgen reporters, exhibits approximately 80- and 270-fold improvements in sensitivity, respectively, compared to conventional CRISPR-based diagnostics. We believe CrisprAIE can be readily extended as a universal signal generation strategy to significantly enhance the detection efficiency of almost all existing CRISPR-based diagnostics.},
}

*CRISPR-Cas Systems
Humans
*SARS-CoV-2/genetics
Norovirus/genetics
COVID-19/virology
DNA/genetics
Fluorescent Dyes/chemistry

@article {pmid39362812,
year = {2024},
author = {Li, L and Zhang, D and Zhang, Z and Zhang, B},
title = {CRISPR/Cas: a powerful tool for designing and improving oil crops.},
journal = {Trends in biotechnology},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.tibtech.2024.09.007},
pmid = {39362812},
issn = {1879-3096},
abstract = {Improving oil yield and quality is a major goal for crop breeding, and CRISPR/Cas-mediated genome editing has opened a new era for designing oil crops with enhanced yield and quality. CRISPR/Cas technology can not only increase oil production but also enhance oil quality, including enhancing pharmaceutical and health components, improving oil nutrients, and removing allergic and toxic components. As new molecular targets for oil biosynthesis are discovered and the CRISPR/Cas system is further improved, CRISPR/Cas will become a better molecular tool for designing new oil crops with higher oil production, enhanced nutrients, and improved health components. 'CRISPRized' oil crops will have broad applications both in industry (e.g., as biofuels) and in daily human life.},
}

@article {pmid39362215,
year = {2024},
author = {Smalakyte, D and Ruksenaite, A and Sasnauskas, G and Tamulaitiene, G and Tamulaitis, G},
title = {Filament formation activates protease and ring nuclease activities of CRISPR Lon-SAVED.},
journal = {Molecular cell},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.molcel.2024.09.002},
pmid = {39362215},
issn = {1097-4164},
abstract = {To combat phage infection, type III CRISPR-Cas systems utilize cyclic oligoadenylates (cAn) signaling to activate various auxiliary effectors, including the CRISPR-associated Lon-SAVED protease CalpL, which forms a tripartite effector system together with an anti-σ factor, CalpT, and an ECF-like σ factor, CalpS. Here, we report the characterization of the Candidatus Cloacimonas acidaminovorans CalpL-CalpT-CalpS. We demonstrate that cA4 binding triggers CalpL filament formation and activates it to cleave CalpT within the CalpT-CalpS dimer. This cleavage exposes the CalpT C-degron, which targets it for further degradation by cellular proteases. Consequently, CalpS is released to bind to RNA polymerase, causing growth arrest in E. coli. Furthermore, the CalpL-CalpT-CalpS system is regulated by the SAVED domain of CalpL, which is a ring nuclease that cleaves cA4 in a sequential three-step mechanism. These findings provide key mechanistic details for the activation, proteolytic events, and regulation of the signaling cascade in the type III CRISPR-Cas immunity.},
}

@article {pmid39361498,
year = {2024},
author = {Jiang, Y and Chen, X and Miao, P and Feng, N},
title = {Self-Powered FEN1 Biosensor Based on Accelerated CRISPR/Cas Trans-Cleavage around Porous Fe3O4 Nanoparticles.},
journal = {ACS applied materials & interfaces},
volume = {},
number = {},
pages = {},
doi = {10.1021/acsami.4c14192},
pmid = {39361498},
issn = {1944-8252},
abstract = {Flap endonuclease 1 (FEN1) is a structure-specific endonuclease that plays a critical role in the maintenance of genome integrity. In this work, we demonstrate a novel self-powered electrochemical FEN1 biosensor for potential applications in molecular diagnosis. Porous Fe3O4 nanoparticles are first prepared, and single-strand DNA probes are absorbed on the surface of the nanoparticles. Thus, electrochemical species of [Fe(CN)6][3-] can be encapsulated inside the porous nanoparticles with the molecular gate of negatively charged DNA. On the other hand, a dumbbell structured DNA probe with 5' flap is designed. FEN1 is able to cleave the flap and activate the CRISPR/Cas system for the digestion of single-stranded DNA around Fe3O4 nanoparticles. As a result, the leakage of [Fe(CN)6][3-] contributes to an enhanced electrochemical response, which can be used to reveal the level of FEN1. The high sensitivity of this biosensor is due to the application of porous nanomaterials and Mn[2+] accelerated CRISPR/Cas cleavage. It succeeds in detection of biological samples and screening of FEN1 inhibitors. Therefore, this proposed method has potential applications in the early diagnosis of diseases and drug discovery.},
}

@article {pmid39361061,
year = {2024},
author = {Behnam Rad, M and Hakimian, F and Mohebbi, SR and Yadegar, A and Ghourchian, H},
title = {LAMP-CRISPR/Cas12a-based impedimetric biosensor powered by Fe3O4@Au-(S-polyA-S)-Au for detection of SARS-CoV-2.},
journal = {Mikrochimica acta},
volume = {191},
number = {11},
pages = {644},
pmid = {39361061},
issn = {1436-5073},
mesh = {*SARS-CoV-2/genetics/isolation & purification ; *Biosensing Techniques/methods ; *Gold/chemistry ; *Nucleic Acid Amplification Techniques/methods ; Humans ; *CRISPR-Cas Systems ; *RNA, Viral/analysis ; COVID-19/diagnosis/virology ; Limit of Detection ; Electrodes ; Poly A/chemistry ; CRISPR-Associated Proteins ; Magnetite Nanoparticles/chemistry ; Endodeoxyribonucleases/chemistry ; Metal Nanoparticles/chemistry ; Bacterial Proteins ; Molecular Diagnostic Techniques ; },
abstract = {A low-cost, lab-made polytetrafluoroethylene micro-cell, equipped with three electrodes, wasd eveloped for the impedimetric detection of SARS-CoV-2. The gold working electrode was modified with a double-ended thiolated poly-adenine probe, which was conjugated with magnetic Fe3O4@Au nanoparticles (Fe3O4@Au-(S-polyA-S)-Au). After the loop-mediated isothermal amplification (LAMP) of viral RNA, the single-guide RNA (sgRNA), specifically bound to the SARS-CoV-2 target sequence, activates Cas12a. Cas12a then cleaved the immobilized probe. As a result, the magnetic Fe3O4@Au nanoparticles were released and adsorbed onto the gold electrode surface, using an external magnet. This process increased the physical surface area of the gold electrode, facilitating redox ion ([Fe[III/II](CN)6][3-/4-]) electron transfer. The decrease in the charge transfer resistance was utilized for SARS-CoV-2 detection. Our LAMP-CRISPR/Cas12a-based impedimetric biosensor, powered by Fe3O4@Au-(S-polyA-S)-Au, demonstrated impressive capabilities, including a remarkable detection limit of 0.8 aM (0.48 copies/µL) and a linear range of 0.01 to 36.06 fM.},
}

*SARS-CoV-2/genetics/isolation & purification
*Biosensing Techniques/methods
*Gold/chemistry
*Nucleic Acid Amplification Techniques/methods
Humans
*CRISPR-Cas Systems
*RNA, Viral/analysis
COVID-19/diagnosis/virology
Limit of Detection
Electrodes
Poly A/chemistry
CRISPR-Associated Proteins
Magnetite Nanoparticles/chemistry
Endodeoxyribonucleases/chemistry
Metal Nanoparticles/chemistry
Bacterial Proteins
Molecular Diagnostic Techniques

@article {pmid39360998,
year = {2024},
author = {Campbell, ST},
title = {Approval of the First CRISPR-Cas9 Gene Editing Therapy for Sickle Cell Disease.},
journal = {Clinical chemistry},
volume = {70},
number = {10},
pages = {1298},
doi = {10.1093/clinchem/hvae038},
pmid = {39360998},
issn = {1530-8561},
mesh = {*Anemia, Sickle Cell/therapy/genetics ; Humans ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Genetic Therapy/methods ; United States ; United States Food and Drug Administration ; },
}

*Anemia, Sickle Cell/therapy/genetics
Humans
*CRISPR-Cas Systems
*Gene Editing/methods
*Genetic Therapy/methods
United States
United States Food and Drug Administration

@article {pmid39360804,
year = {2024},
author = {Chen, Y and Wu, J and Gao, EB and Lu, Y and Qiu, H},
title = {A rapid visualization method for detecting rotavirus A by combining nuclear acid sequence-based amplification with the CRISPR-Cas12a assay.},
journal = {Journal of medical microbiology},
volume = {73},
number = {10},
pages = {},
doi = {10.1099/jmm.0.001892},
pmid = {39360804},
issn = {1473-5644},
mesh = {Humans ; *Rotavirus/genetics/isolation & purification ; *Rotavirus Infections/diagnosis/virology ; *CRISPR-Cas Systems ; *Sensitivity and Specificity ; Self-Sustained Sequence Replication/methods ; Nucleic Acid Amplification Techniques/methods ; },
abstract = {Introduction. Rotavirus A is the most common pathogen causing diarrhoea in children less than 5 years, leading to severe complications such as dehydration, electrolyte imbalances, acidosis, myocarditis, convulsions, pneumonia, and other life-threatening conditions.Gap statement. There is an urgent need for a rapid and efficient nucleic acid detection strategy to enable early diagnosis and treatment, preventing rotavirus transmission and associated complications.Aim. This article aimed to develop a nuclear acid sequence-based amplification (NASBA)-Cas12a system for detecting rotavirus A using fluorescence intensity or lateral flow strips.Methodology. The NASBA technology was combined with the clustered regularly interspaced short palindromic repeats-Cas12a system to establish a NASBA-Cas12a system for detecting rotavirus A.Results. The NASBA-Cas12a system could detect rotavirus A at 37 ℃ within 70 min and had no cross-reactivity with other viruses, achieving a limit of detection of 1.2 copies μl[-1]. This system demonstrated a sensitivity of 100%, specificity of 90%, positive predictive value of 97.22% and negative predictive value of 100%. The kappa value was 0.933, indicating that the NASBA-Cas12a system was highly consistent with reverse transcription-PCR.Conclusion. The NASBA-Cas12a system exhibited high sensitivity and specificity for detecting rotavirus A, showing great potential for clinical application.},
}

Humans
*Rotavirus/genetics/isolation & purification
*Rotavirus Infections/diagnosis/virology
*CRISPR-Cas Systems
*Sensitivity and Specificity
Self-Sustained Sequence Replication/methods
Nucleic Acid Amplification Techniques/methods

@article {pmid39359675,
year = {2024},
author = {Ma, S and Zhang, S and Liu, K and Hu, T and Hu, C},
title = {Efficient, compact, and versatile: Type I-F2 CRISPR-Cas system.},
journal = {mLife},
volume = {3},
number = {3},
pages = {384-386},
pmid = {39359675},
issn = {2770-100X},
}

@article {pmid39358558,
year = {2024},
author = {Tronik-Le Roux, D and Daouya, M and Poras, I and Desgrandchamps, F and Carosella, ED},
title = {HLA-G neo-expression modifies genetic programs governing tumor cell lines.},
journal = {Cancer immunology, immunotherapy : CII},
volume = {73},
number = {12},
pages = {247},
pmid = {39358558},
issn = {1432-0851},
mesh = {Humans ; *HLA-G Antigens/genetics/metabolism/immunology ; Cell Line, Tumor ; Gene Expression Regulation, Neoplastic ; CRISPR-Cas Systems ; Neuropilin-1/genetics/metabolism ; Immunotherapy/methods ; },
abstract = {The development of immunotherapies has proved to be clinically encouraging to re-establish the immune function modified by the expression of immune inhibitory molecules in tumors. However, there are still patients with poor survival rates following treatment. The elucidation of molecular mechanisms triggered by the neo-expression of particular IC in tumors would constitute a major step toward better understanding tumor evolution and would help to design future clinical protocols. To this end, we investigate the modifications triggered by the neo-expression of the immune checkpoints HLA-G in ccRCC tumor cells. We demonstrate, for the first time, that HLA-G modifies key genes implicated mainly in tumor development, angiogenesis, calcium flow and mitochondria dynamics. The involvement of HLA-G on the expression of genes belonging to these pathways such as ADAM-12, NCAM1 and NRP1 was confirmed by the CRISPR/Cas9-mediated edition of HLA-G. The data reveal multifaceted roles of HLA-G in tumor cells which are far beyond the well-known function of HLA-G in the immune anti-tumor response. This warrants further investigation of HLA-G and these new partners in tumors of different origin so as to propose future new treatments to improve health patient's outcome.},
}

Humans
*HLA-G Antigens/genetics/metabolism/immunology
Cell Line, Tumor
Gene Expression Regulation, Neoplastic
CRISPR-Cas Systems
Neuropilin-1/genetics/metabolism
Immunotherapy/methods

@article {pmid39356718,
year = {2024},
author = {Chen, B and Wang, J and Huang, M and Gui, Y and Wei, Q and Wang, L and Tan, BC},
title = {C1-FDX is required for the assembly of mitochondrial complex I and subcomplexes of complex V in Arabidopsis.},
journal = {PLoS genetics},
volume = {20},
number = {10},
pages = {e1011419},
pmid = {39356718},
issn = {1553-7404},
mesh = {*Arabidopsis/genetics/metabolism/growth & development ; *Arabidopsis Proteins/genetics/metabolism ; *Electron Transport Complex I/metabolism/genetics ; *Mitochondria/metabolism/genetics ; Germination/genetics ; Ferredoxins/metabolism/genetics ; Mutation ; Seeds/genetics/growth & development/metabolism ; Gene Expression Regulation, Plant ; CRISPR-Cas Systems ; Mitochondrial Proteins/genetics/metabolism ; Plants, Genetically Modified ; },
abstract = {C1-FDX (Complex I-ferredoxin) has been defined as a component of CI in a ferredoxin bridge in Arabidopsis mitochondria. However, its full function remains to be addressed. We created two c1-fdx mutants in Arabidopsis using the CRISPR-Cas9 methodology. The mutants show delayed seed germination. Over-expression of C1-FDX rescues the phenotype. Molecular analyses showed that loss of the C1-FDX function decreases the abundance and activity of both CI and subcomplexes of CV. In contrast, the over-expression of C1-FDX-GFP enhances the CI* (a sub-complex of CI) and CV assembly. Immunodetection reveals that the stoichiometric ratio of the α:β subunits in the F1 module of CV is altered in the c1-fdx mutant. In the complemented mutants, C1-FDX-GFP was found to be associated with the F' and α/β sub-complexes of CV. Protein interaction assays showed that C1-FDX could interact with the β, γ, δ, and ε subunits of the F1 module, indicating that C1-FDX, a structural component of CI, also functions as an assembly factor in the assembly of F' and α/β sub-complexes of CV. These results reveal a new role of C1-FDX in the CI and CV assembly and seed germination in Arabidopsis.},
}

*Arabidopsis/genetics/metabolism/growth & development
*Arabidopsis Proteins/genetics/metabolism
*Electron Transport Complex I/metabolism/genetics
*Mitochondria/metabolism/genetics
Germination/genetics
Ferredoxins/metabolism/genetics
Mutation
Seeds/genetics/growth & development/metabolism
Gene Expression Regulation, Plant
CRISPR-Cas Systems
Mitochondrial Proteins/genetics/metabolism
Plants, Genetically Modified

@article {pmid39355887,
year = {2024},
author = {Marfina, SV and Mikhaleva, EA and Akulenko, NV and Ryazansky, SS},
title = {[Method of Inducible Knockdown of Essential Genes in OSC Cell Culture of Drosophila melanogaster].},
journal = {Molekuliarnaia biologiia},
volume = {58},
number = {2},
pages = {305-313},
pmid = {39355887},
issn = {0026-8984},
mesh = {Animals ; *Drosophila melanogaster/genetics ; *Drosophila Proteins/genetics/metabolism ; *Gene Knockdown Techniques ; Female ; Cullin Proteins/genetics/metabolism ; Transcription Factors/genetics/metabolism ; RNA, Small Interfering/genetics/metabolism ; Ovary/metabolism/cytology ; Oogenesis/genetics ; RNA Interference ; Genes, Essential ; CRISPR-Cas Systems ; DNA-Binding Proteins/genetics/metabolism ; },
abstract = {An RNA interference-based method was proposed to achieve an inducible knockdown of genes essential for cell viability. In the method, a genetic cassette in which a copper ion-dependent inducible metallothionein promoter controls expression of a siRNA precursor is inserted into a genomic pre-integrated transgene by CRIPSR/Cas9 technology. The endogenous siRNA source allows the gene knockdown in cell cultures that are refractory to conventional transfection with exogenous siRNA. The efficiency of the method was demonstrated in Drosophila ovarian somatic cell culture (OSC) for two genes that are essential for oogenesis: Cul3, encoding a component of the multiprotein ubiquitin-ligase complex with versatile functions in proteostasis, and cut, encoding a transcription factor regulating differentiation of ovarian follicular cells.},
}

Animals
*Drosophila melanogaster/genetics
*Drosophila Proteins/genetics/metabolism
*Gene Knockdown Techniques
Female
Cullin Proteins/genetics/metabolism
Transcription Factors/genetics/metabolism
RNA, Small Interfering/genetics/metabolism
Ovary/metabolism/cytology
Oogenesis/genetics
RNA Interference
Genes, Essential
CRISPR-Cas Systems
DNA-Binding Proteins/genetics/metabolism

@article {pmid39236708,
year = {2024},
author = {Gong, F and Li, B},
title = {Hybrid non-viral and viral delivery strategy achieves potent gene editing in growing livers with reduced viral dosage.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {32},
number = {10},
pages = {3215-3216},
doi = {10.1016/j.ymthe.2024.08.021},
pmid = {39236708},
issn = {1525-0024},
mesh = {*Gene Editing/methods ; Animals ; *Liver/metabolism ; *Genetic Vectors/administration & dosage/genetics ; Mice ; *Gene Transfer Techniques ; Humans ; Genetic Therapy/methods ; CRISPR-Cas Systems ; },
}

*Gene Editing/methods
Animals
*Liver/metabolism
*Genetic Vectors/administration & dosage/genetics
Mice
*Gene Transfer Techniques
Humans
Genetic Therapy/methods
CRISPR-Cas Systems

@article {pmid39232121,
year = {2024},
author = {Fieni, C and Sorrentino, C and Ciummo, SL and Fontana, A and Lotti, LV and Scialis, S and Calvo Garcia, D and Caulo, M and Di Carlo, E},
title = {Immunoliposome-based targeted delivery of the CRISPR/Cas9gRNA-IL30 complex inhibits prostate cancer and prolongs survival.},
journal = {Experimental & molecular medicine},
volume = {56},
number = {9},
pages = {2033-2051},
pmid = {39232121},
issn = {2092-6413},
support = {IG 2019 - ID. 23264//Associazione Italiana per la Ricerca sul Cancro (Italian Association for Cancer Research)/ ; 2014-2020 (PON R&I)//Ministero dell'Istruzione, dell'Università e della Ricerca (Ministry of Education, University and Research)/ ; ECS00000041//Ministero dell'Istruzione, dell'Università e della Ricerca (Ministry of Education, University and Research)/ ; },
mesh = {Male ; *Prostatic Neoplasms/therapy/genetics/pathology/immunology ; Animals ; Humans ; Mice ; *CRISPR-Cas Systems ; Cell Line, Tumor ; Liposomes ; Xenograft Model Antitumor Assays ; RNA, Guide, CRISPR-Cas Systems ; Gene Editing ; },
abstract = {The development of selective and nontoxic immunotherapy targeting prostate cancer (PC) is challenging. Interleukin (IL)30 plays immunoinhibitory and oncogenic roles in PC, and its tumor-specific suppression may have significant clinical implications. CRISPR/Cas9-mediated IL30 gene deletion in PC xenografts using anti-PSCA antibody-driven lipid nanocomplexes (Cas9gRNA-hIL30-PSCA NxPs) revealed significant genome editing efficiency and circulation stability without off-target effects or organ toxicity. Biweekly intravenous administration of Cas9gRNA-hIL30-PSCA NxPs to PC-bearing mice inhibited tumor growth and metastasis and improved survival. Mechanistically, Cas9gRNA-hIL30-PSCA NxPs suppressed ANGPTL 1/2/4, IL1β, CCL2, CXCL1/6, SERPINE1-F1, EFNB2, PLG, PF4, VEGFA, VEGFD, ANG, TGFβ1, EGF and HGF expression in human PC cells while upregulated CDH1, DKK3 and PTEN expression, leading to low proliferation and extensive ischemic necrosis. In the syngeneic PC model, IL30-targeting immunoliposomes downregulated NFKB1 expression and prevented intratumoral influx of CD11b[+]Gr-1[+]MDCs, Foxp3[+]Tregs, and NKp46[+]RORγt[+]ILC3, and prolonged host survival by inhibiting tumor progression. This study serves as a proof of principle that immunoliposome-based targeted delivery of Cas9gRNA-IL30 represent a potentially safe and effective strategy for PC treatment.},
}

Male
*Prostatic Neoplasms/therapy/genetics/pathology/immunology
Animals
Humans
Mice
*CRISPR-Cas Systems
Cell Line, Tumor
Liposomes
Xenograft Model Antitumor Assays
RNA, Guide, CRISPR-Cas Systems
Gene Editing

@article {pmid39192583,
year = {2024},
author = {Pitner, RA and Chao, JL and Dahl, NP and Fan, MN and Cai, X and Avery, NG and Roe, K and Spiegel, PC and Miao, CH and Gerner, MY and James, RG and Rawlings, DJ},
title = {Blunting specific T-dependent antibody responses with engineered "decoy" B cells.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {32},
number = {10},
pages = {3453-3469},
doi = {10.1016/j.ymthe.2024.08.023},
pmid = {39192583},
issn = {1525-0024},
mesh = {Animals ; Mice ; *B-Lymphocytes/immunology/metabolism ; *Mice, Knockout ; *Antibody Formation/immunology ; *Positive Regulatory Domain I-Binding Factor 1/genetics/metabolism/immunology ; T-Lymphocytes/immunology/metabolism ; Factor VIII/immunology/genetics ; CRISPR-Cas Systems ; Immunoglobulin G/immunology ; Adoptive Transfer ; Humans ; Germinal Center/immunology/metabolism ; },
abstract = {Antibody inhibitors pose an ongoing challenge to the treatment of subjects with inherited protein deficiency disorders, limiting the efficacy of both protein replacement therapy and corrective gene therapy. Beyond their central role as producers of serum antibody, B cells also exhibit many unique properties that could be exploited in cell therapy applications, notably including antigen-specific recognition and the linked capacity for antigen presentation. Here we employed CRISPR-Cas9 to demonstrate that ex vivo antigen-primed Blimp1-knockout "decoy" B cells, incapable of differentiation into plasma cells, participated in and downregulated host antigen-specific humoral responses after adoptive transfer. Following ex vivo antigen pulse, adoptively transferred high-affinity antigen-specific decoy B cells were diverted into germinal centers en masse, thereby reducing participation by endogenous antigen-specific B cells in T-dependent humoral responses and suppressing both cognate and linked antigen-specific immunoglobulin (Ig)G following immunization with conjugated antigen. This effect was dose-dependent and, importantly, did not impact concurrent unrelated antibody responses. We demonstrated the therapeutic potential of this approach by treating factor VIII (FVIII)-knockout mice with antigen-pulsed decoy B cells prior to immunization with an FVIII conjugate protein, thereby blunting the production of serum FVIII-specific IgG by an order of magnitude as well as reducing the proportion of animals exhibiting functional FVIII inhibition by 6-fold.},
}

Animals
Mice
*B-Lymphocytes/immunology/metabolism
*Mice, Knockout
*Antibody Formation/immunology
*Positive Regulatory Domain I-Binding Factor 1/genetics/metabolism/immunology
T-Lymphocytes/immunology/metabolism
Factor VIII/immunology/genetics
CRISPR-Cas Systems
Immunoglobulin G/immunology
Adoptive Transfer
Humans
Germinal Center/immunology/metabolism

@article {pmid39092550,
year = {2024},
author = {Park, ME and Choi, HA and Lee, KR and Heo, JB and Kim, HU},
title = {Identification of High Linoleic Acid Varieties in Tetraploid perilla through Gamma-ray Irradiation and CRISPR/Cas9.},
journal = {Plant & cell physiology},
volume = {65},
number = {9},
pages = {1461-1473},
doi = {10.1093/pcp/pcae084},
pmid = {39092550},
issn = {1471-9053},
support = {RS-2024-00322277//New Breeding Technologies Development Program/ ; NRF-2020R1A2C2008175//Mid-Career Researcher Program of the National Research Foundation of Korea/ ; },
mesh = {*Tetraploidy ; *CRISPR-Cas Systems ; *Fatty Acid Desaturases/genetics/metabolism ; *Gamma Rays ; *Seeds/genetics/radiation effects/metabolism ; *Linoleic Acid/metabolism ; Perilla/genetics/metabolism ; Plant Proteins/genetics/metabolism ; },
abstract = {Perilla [Perilla frutescens (L.) var frutescens] is a traditional oil crop in Asia, recognized for its seeds abundant in α-linolenic acid (18:3), a key omega-3 fatty acid known for its health benefits. Despite the known nutritional value, the reason behind the higher 18:3 content in tetraploid perilla seeds remained unexplored. Gamma irradiation yielded mutants with altered seed fatty acid composition. Among the mutants, DY-46-5 showed a 27% increase in 18:2 due to the 4-bp deletion of PfrFAD3b, and NC-65-12 displayed a 16% increase in 18:2 due to the loss of function of PfrFAD3a through a large deletion. Knocking out both copies of FATTY ACID DESATURASE3 (PfrFAD3a and PfrFAD3b) simultaneously using CRISPR/Cas9 resulted in an increase in 18:2 by up to 75% and a decrease in 18:3 to as low as 0.3% in seeds, emphasizing the pivotal roles of both genes in 18:3 synthesis in tetraploid perilla. Furthermore, diploid Perilla citriodora, the progenitor of cultivated tetraploid perilla, harbors only PfrFAD3b, with a fatty acid analysis revealing lower 18:3 levels than tetraploid perilla. In conclusion, the enhanced 18:3 content in cultivated tetraploid perilla seeds can be attributed to the acquisition of two FAD3 copies through hybridization with wild-type diploid perilla.},
}

*Tetraploidy
*CRISPR-Cas Systems
*Fatty Acid Desaturases/genetics/metabolism
*Gamma Rays
*Seeds/genetics/radiation effects/metabolism
*Linoleic Acid/metabolism
Perilla/genetics/metabolism
Plant Proteins/genetics/metabolism

@article {pmid39066480,
year = {2024},
author = {Yoon, AR and Lee, S and Kim, JH and Park, Y and Koo, T and Yun, CO},
title = {CRISPR-mediated ablation of TP53 and EGFR mutations enhances gefitinib sensitivity and anti-tumor efficacy in lung cancer.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {32},
number = {10},
pages = {3618-3628},
doi = {10.1016/j.ymthe.2024.07.017},
pmid = {39066480},
issn = {1525-0024},
mesh = {Humans ; *Gefitinib/pharmacology/therapeutic use ; *ErbB Receptors/genetics ; *Lung Neoplasms/genetics/drug therapy/pathology/therapy ; *Tumor Suppressor Protein p53/genetics/metabolism ; Animals ; Mice ; Cell Line, Tumor ; *Drug Resistance, Neoplasm/genetics ; *Polymorphism, Single Nucleotide ; Xenograft Model Antitumor Assays ; Mutation ; CRISPR-Cas Systems ; Antineoplastic Agents/pharmacology/therapeutic use ; Gene Editing ; },
abstract = {Multiple pathogenic single-nucleotide polymorphisms (SNPs) have been identified as contributing factors in the aggravation of cancer prognosis and emergence of drug resistance in various cancers. Here, we targeted mutated EGFR and TP53 oncogenes harboring single-nucleotide missense mutations (EGFR-T790M and TP53-R273H) that are associated with gefitinib resistance. Co-delivery of adenine base editor (ABE) and EGFR- and TP53-SNP specific single-guide RNA via adenovirus (Ad) resulted in precise correction of the oncogenic mutations with high accuracy and efficiency in vitro and in vivo. Importantly, compared with a control group treated only with gefitinib, an EGFR inhibitor, co-treatment with Ad/ABE targeting SNPs in TP53 and EGFR in combination with gefitinib increased drug sensitivity and suppressed abnormal tumor growth more efficiently. Taken together, these results indicate that ABE-mediated correction of dual oncogenic SNPs can be an effective strategy for the treatment of drug-resistant cancers.},
}

Humans
*Gefitinib/pharmacology/therapeutic use
*ErbB Receptors/genetics
*Lung Neoplasms/genetics/drug therapy/pathology/therapy
*Tumor Suppressor Protein p53/genetics/metabolism
Animals
Mice
Cell Line, Tumor
*Drug Resistance, Neoplasm/genetics
*Polymorphism, Single Nucleotide
Xenograft Model Antitumor Assays
Mutation
CRISPR-Cas Systems
Antineoplastic Agents/pharmacology/therapeutic use
Gene Editing

@article {pmid38849110,
year = {2024},
author = {Fu, M and Liu, S and Che, Y and Cui, D and Deng, Z and Li, Y and Zou, X and Kong, X and Chen, G and Zhang, M and Liu, Y and Wang, X and Liu, W and Liu, D and Geng, S and Li, A and Mao, L},
title = {Genome-editing of a circadian clock gene TaPRR95 facilitates wheat peduncle growth and heading date.},
journal = {Journal of genetics and genomics = Yi chuan xue bao},
volume = {51},
number = {10},
pages = {1101-1110},
doi = {10.1016/j.jgg.2024.05.011},
pmid = {38849110},
issn = {1673-8527},
mesh = {*Triticum/genetics/growth & development ; *Plant Proteins/genetics/metabolism ; *Gene Editing ; *Gene Expression Regulation, Plant/genetics ; *Circadian Clocks/genetics ; Haplotypes/genetics ; Genome-Wide Association Study ; Alleles ; CRISPR-Cas Systems/genetics ; Phenotype ; },
abstract = {Plant height and heading date are important agronomic traits in wheat (Triticum aestivum L.) that affect final grain yield. In wheat, knowledge of pseudo-response regulator (PRR) genes on agronomic traits is limited. Here, we identify a wheat TaPRR95 gene by genome-wide association studies to be associated with plant height. Triple allele mutant plants produced by CRISPR/Cas9 show increased plant height, particularly the peduncle, with an earlier heading date. The longer peduncle is mainly caused by the increased cell elongation at its upper section, whilst the early heading date is accompanied by elevated expression of flowering genes, such as TaFT and TaCO1. A peduncle-specific transcriptome analysis reveals up-regulated photosynthesis genes and down-regulated IAA/Aux genes for auxin signaling in prr95[aabbdd] plants that may act as a regulatory mechanism to promote robust plant growth. A haplotype analysis identifies a TaPRR95-B haplotype (Hap2) to be closely associated with reduced plant height and increased thousand-grain weight. Moreover, the Hap2 frequency is higher in cultivars than that in landraces, suggesting the artificial selection on the allele during wheat breeding. These findings suggest that TaPRR95 is a regulator for plant height and heading date, thereby providing an important target for wheat yield improvement.},
}

*Triticum/genetics/growth & development
*Plant Proteins/genetics/metabolism
*Gene Editing
*Gene Expression Regulation, Plant/genetics
*Circadian Clocks/genetics
Haplotypes/genetics
Genome-Wide Association Study
Alleles
CRISPR-Cas Systems/genetics
Phenotype

@article {pmid39354597,
year = {2024},
author = {Poyatos-García, J and Soblechero-Martín, P and Liquori, A and López-Martínez, A and Maestre, P and González-Romero, E and Vázquez-Manrique, RP and Muelas, N and García-García, G and Ohana, J and Arechavala-Gomeza, V and Vílchez, JJ},
title = {Deletion of exons 45 to 55 in the DMD gene: from the therapeutic perspective to the in vitro model.},
journal = {Skeletal muscle},
volume = {14},
number = {1},
pages = {21},
pmid = {39354597},
issn = {2044-5040},
support = {2018/0200//Fundación Isabel Gemio/ ; 2018/0200//Fundación Isabel Gemio/ ; CM19/00104//Instituto de Salud Carlos III/ ; PI20/00114//Instituto de Salud Carlos III/ ; CP22/00028//Instituto de Salud Carlos III/ ; PI15/00333//Instituto de Salud Carlos III/ ; CM19/00104//Instituto de Salud Carlos III/ ; APOSTD/2021/212//Conselleria de Cultura, Educación y Ciencia, Generalitat Valenciana/ ; FPU21/00912//Ministerio de Ciencia e Innovación/ ; BC/I/DIV/19/001//Biobizkaia Health Research Institute/ ; 2016111029//Basque Government, Spain/ ; },
mesh = {Humans ; *Muscular Dystrophy, Duchenne/genetics/therapy ; *Dystrophin/genetics ; *Exons ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *Genetic Therapy/methods ; Cell Line ; Sequence Deletion ; Myoblasts/metabolism ; },
abstract = {BACKGROUND: Gene editing therapies in development for correcting out-of-frame DMD mutations in Duchenne muscular dystrophy aim to replicate benign spontaneous deletions. Deletion of 45-55 DMD exons (del45-55) was described in asymptomatic subjects, but recently serious skeletal and cardiac complications have been reported. Uncovering why a single mutation like del45-55 is able to induce diverse phenotypes and grades of severity may impact the strategies of emerging therapies. Cellular models are essential for this purpose, but their availability is compromised by scarce muscle biopsies.

METHODS: We introduced, as a proof-of-concept, using CRISPR-Cas9 edition, a del45-55 mimicking the intronic breakpoints harboured by a subset of patients of this form of dystrophinopathy (designing specific gRNAs), into a Duchenne patient's cell line. The edited cell line was characterized evaluating the dystrophin expression and the myogenic status.

RESULTS: Dystrophin expression was restored, and the myogenic defects were ameliorated in the edited myoblasts harbouring a specific del45-55. Besides confirming the potential of CRISPR-Cas9 to create tailored mutations (despite the low cleavage efficiency of our gRNAs) as a useful approach to generate in vitro models, we also generated an immortalized myoblast line derived from a patient with a specific del45-55.

CONCLUSIONS: Overall, we provide helpful resources to deepen into unknown factors responsible for DMD-pathophysiology.},
}

Humans
*Muscular Dystrophy, Duchenne/genetics/therapy
*Dystrophin/genetics
*Exons
*Gene Editing/methods
*CRISPR-Cas Systems
*Genetic Therapy/methods
Cell Line
Sequence Deletion
Myoblasts/metabolism

@article {pmid39351611,
year = {2024},
author = {Singer, SD and Mukthar, MM and Subedi, U and Poudel, H and Chen, G and Foroud, N and Chatterton, S},
title = {CRISPR/Cas-Mediated Gene Editing in Plant Immunity and Its Potential for the Future Development of Fungal, Oomycete, and Bacterial Pathogen-Resistant Pulse Crops.},
journal = {Plant, cell & environment},
volume = {},
number = {},
pages = {},
doi = {10.1111/pce.15174},
pmid = {39351611},
issn = {1365-3040},
support = {//The authors are grateful for the financial support provided by Agriculture and Agri-Food Canada, the S-CAP ASC-06 Pulse Cluster, Saskatchewan Agriculture Development Fund and Beef Cattle Research Council./ ; },
abstract = {Pulses provide myriad health benefits and are advantageous in an environmental context as a result of their leguminous nature. However, phytopathogenic fungi, oomycetes and bacteria pose a substantial threat to pulse production, at times leading to crop failure. Unfortunately, existing disease management strategies often provide insufficient control, and there is a clear need for the development of new pulse cultivars with durable and broad-spectrum disease resistance. CRISPR/Cas-mediated gene editing has proven its potential for rapidly enhancing disease resistance in many plant species. However, this tool has only very recently been applied in pulse species, and never in the context of plant immunity. In this review, we examine the recent successful utilization of this technology in pulse species for proof-of-concept or the improvement of other traits. In addition, we consider various genes that have been edited in other plant species to reduce susceptibility to pathogens, and discuss current knowledge regarding their roles in pulses. Given the functional conservation of the selected genes across diverse plant species, there is a high likelihood that their editing would elicit similar effects in non-oilseed grain legumes, thus providing a suite of potential targets for CRISPR/Cas-mediated gene editing to promote pulse crop productivity in coming years.},
}

@article {pmid39349793,
year = {2024},
author = {Li, R and Yang, F and Chu, B and Kong, D and Hu, J and Qian, H},
title = {Exploring retinal degenerative diseases through CRISPR-based screening.},
journal = {Molecular biology reports},
volume = {51},
number = {1},
pages = {1029},
pmid = {39349793},
issn = {1573-4978},
support = {12102086//the National Natural Science Foundation of China/ ; 82271276//the National Natural Science Foundation of China/ ; 2023YFC2506100//National Key R&D Program of China/ ; 2024YFHZ0175//Sichuan Science and Technology Program/ ; },
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Retinal Degeneration/genetics/therapy ; Animals ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {The CRISPR (Clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein9) system has emerged as a powerful genetic tool, gaining global recognition as a versatile and efficient gene-editing technique. Its transformation into a high-throughput research platform, CRISPR Screening, has demonstrated wide applicability across various fields such as cancer biology, virology, and drug target discovery, resulting in significant advances. However, its potential in studying retinal degenerative diseases remains largely unexplored, despite the urgent need for effective treatments arising from an incomplete understanding of disease mechanisms. This review aims to present a comprehensive overview of the evolution and current state of CRISPR tools and CRISPR screening methodologies. Noteworthy pioneering studies utilizing these technologies are discussed, alongside experimental design guidelines, including positive and negative selection strategies and delivery methods for sgRNAs (single guide RNAs) and Cas proteins. Furthermore, we explore existing in vitro models appropriate for CRISPR screening in retinal research and identify relevant research questions that could be addressed through this approach. It is anticipated that this review will stimulate innovation in retinal research, facilitating a deeper comprehension of retinal pathophysiology and paving the way for groundbreaking therapeutic interventions and enhanced patient outcomes in the management of retinal degenerative disorders.},
}

Humans
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
*Retinal Degeneration/genetics/therapy
Animals
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid39349521,
year = {2024},
author = {Elrick, H and Peterson, KA and Willis, BJ and Lanza, DG and Acar, EF and Ryder, EJ and Teboul, L and Kasparek, P and Birling, MC and Adams, DJ and Bradley, A and Braun, RE and Brown, SD and Caulder, A and Codner, GF and DeMayo, FJ and Dickinson, ME and Doe, B and Duddy, G and Gertsenstein, M and Goodwin, LO and Hérault, Y and Lintott, LG and Lloyd, KCK and Lorenzo, I and Mackenzie, M and Mallon, AM and McKerlie, C and Parkinson, H and Ramirez-Solis, R and Seavitt, JR and Sedlacek, R and Skarnes, WC and Smedley, D and Wells, S and White, JK and Wood, JA and , and Murray, SA and Heaney, JD and Nutter, LMJ},
title = {Impact of essential genes on the success of genome editing experiments generating 3313 new genetically engineered mouse lines.},
journal = {Scientific reports},
volume = {14},
number = {1},
pages = {22626},
pmid = {39349521},
issn = {2045-2322},
support = {OGI-051//Ontario Genomics/ ; OGI-090//Ontario Genomics/ ; OGI-137//Ontario Genomics/ ; OGI-051//Genome Canada/ ; OGI-090//Genome Canada/ ; OGI-137//Genome Canada/ ; U42OD011185/NH/NIH HHS/United States ; UM1OD023222/NH/NIH HHS/United States ; U42OD011175/NH/NIH HHS/United States ; UM1OD023221/NH/NIH HHS/United States ; U42OD011174/NH/NIH HHS/United States ; UMIHG006348/NH/NIH HHS/United States ; UM1HG006370/NH/NIH HHS/United States ; MC_UP_1502/3/MRC_/Medical Research Council/United Kingdom ; RVO 68378050//Czech Centre for Phenogenomics, Institute of Molecular Genetics of the Czech Academy of Sciences/ ; LM2018126//Ministry of Education, Youth and Sports of the Czech Republic/ ; ANR-10-IDEX-0002-02//Institut National de la Santé et de la Recherche Médicale/ ; ANR-10-LABX-0030-INRT//Institut National de la Santé et de la Recherche Médicale/ ; ANR-10-INBS-07//Institut National de la Santé et de la Recherche Médicale/ ; },
mesh = {Animals ; *Genes, Essential ; Mice ; *Gene Editing/methods ; *Mice, Knockout ; CRISPR-Cas Systems ; Alleles ; Mice, Inbred C57BL ; Male ; Female ; Genetic Engineering/methods ; Phenotype ; },
abstract = {The International Mouse Phenotyping Consortium (IMPC) systematically produces and phenotypes mouse lines with presumptive null mutations to provide insight into gene function. The IMPC now uses the programmable RNA-guided nuclease Cas9 for its increased capacity and flexibility to efficiently generate null alleles in the C57BL/6N strain. In addition to being a valuable novel and accessible research resource, the production of 3313 knockout mouse lines using comparable protocols provides a rich dataset to analyze experimental and biological variables affecting in vivo gene engineering with Cas9. Mouse line production has two critical steps - generation of founders with the desired allele and germline transmission (GLT) of that allele from founders to offspring. A systematic evaluation of the variables impacting success rates identified gene essentiality as the primary factor influencing successful production of null alleles. Collectively, our findings provide best practice recommendations for using Cas9 to generate alleles in mouse essential genes, many of which are orthologs of genes linked to human disease.},
}

Animals
*Genes, Essential
Mice
*Gene Editing/methods
*Mice, Knockout
CRISPR-Cas Systems
Alleles
Mice, Inbred C57BL
Male
Female
Genetic Engineering/methods
Phenotype

@article {pmid39301777,
year = {2024},
author = {Long, X and Zhang, T and Yang, L and Guo, C and Zhao, Q and Cui, Y and Wang, C and Zhang, Y and He, Y},
title = {CRISPR/Cas12a-Based Indirect Competitive Enzyme-Linked Immunosorbent Assay for Sensitive Detection of Ochratoxin A.},
journal = {Journal of agricultural and food chemistry},
volume = {72},
number = {39},
pages = {21912-21921},
doi = {10.1021/acs.jafc.4c06525},
pmid = {39301777},
issn = {1520-5118},
mesh = {*Ochratoxins/analysis ; *Enzyme-Linked Immunosorbent Assay/methods ; *Food Contamination/analysis ; *CRISPR-Cas Systems ; },
abstract = {The high toxicity and widespread contamination of ochratoxin A (OTA) make it urgent to develop a sensitive method to detect trace OTA in complex food matrices. Herein, an indirect competitive enzyme-linked immunosorbent assay (icELISA)-based on the CRISPR/Cas12a system is described. DNA amplicons with multiple activation sequences of the CRISPR/Cas12a system were pre-prepared to improve detection sensitivity. In the absence of OTA, streptavidin-mediated biotinylated DNA amplicons were captured by the biotinylated secondary antibody on the microplate. The captured DNA amplicons activated the CRISPR/Cas12a system, which thereby effectively cleaved the reporter DNA, producing strong fluorescence. The presence of OTA led to a decrease in DNA amplicons on the microplate, resulting in a decrease in activated Cas12a and ultimately a drop in fluorescence intensity. OTA in food matrices at nanogram per milliliter levels can be detected. Therefore, the new method has great potential in monitoring OTA.},
}

*Ochratoxins/analysis
*Enzyme-Linked Immunosorbent Assay/methods
*Food Contamination/analysis
*CRISPR-Cas Systems

@article {pmid39298407,
year = {2024},
author = {Li, J and Song, J and Chen, Y and Zhao, Z and Wang, S and Deng, Y and Lai, S and Yang, H},
title = {CRISPR/Cas12a-Triggered Visible-Light-Driven Photoelectrochemical Assay with Single-Nucleotide Resolution for Drug-Resistant Foodborne Salmonella Detection.},
journal = {Journal of agricultural and food chemistry},
volume = {72},
number = {39},
pages = {21820-21828},
doi = {10.1021/acs.jafc.4c05993},
pmid = {39298407},
issn = {1520-5118},
mesh = {*CRISPR-Cas Systems ; *Light ; *Polymorphism, Single Nucleotide ; *Electrochemical Techniques/methods ; Drug Resistance, Bacterial/genetics ; Biosensing Techniques/methods/instrumentation ; Food Contamination/analysis ; Bacterial Proteins/genetics/metabolism ; Animals ; Salmonella enterica/genetics/radiation effects ; Salmonella/genetics ; Food Microbiology ; },
abstract = {The prevalence of foodborne pathogenic bacteria, especially drug-resistant strains, such as Salmonella enterica, poses serious threats to public health, highlighting the requirement for the development of rapid and precise detection methods. Herein, a CRISPR/Cas12a-triggered visible-light-driven photoelectrochemical (PEC) assay (CasPEC) was developed using a SiO2-quenched BiVO4/MoS2 p/n-type heterojunction as the photoactive material. The CRISPR/Cas12a recognition endowed the CasPEC assay with high specificity capable of resolving single-nucleotide polymorphisms (SNPs) and identifying SNP-involved drug-resistant bacteria. SiO2 was linked to the surface of the BiVO4/MoS2 heterojunction by single-stranded DNA (ssDNA), which would be cleaved by target-activated CRISPR/Cas12a. This cleavage of ssDNA resulted in the detachment of SiO2, thereby achieving a "signal-on" PEC output. Leveraging the multiple-turnover CRISPR cleavage and the outstanding photoactive performance of PEC signaling, the CasPEC assay for S. enterica showed a detection limit of 103 colony-forming units (CFU)/mL and the ability to detect as few as 0.01% drug-resistant strains. The CasPEC assay can accurately sense the S. enterica contamination in complex food matrices, including beef and milk. These findings demonstrated the great potential of the CasPEC assay for detecting pathogenic bacterial contamination in food, particularly concerning food safety related to SNP-involved drug-resistant bacteria.},
}

*CRISPR-Cas Systems
*Light
*Polymorphism, Single Nucleotide
*Electrochemical Techniques/methods
Drug Resistance, Bacterial/genetics
Biosensing Techniques/methods/instrumentation
Food Contamination/analysis
Bacterial Proteins/genetics/metabolism
Animals
Salmonella enterica/genetics/radiation effects
Salmonella/genetics
Food Microbiology

@article {pmid39288439,
year = {2024},
author = {Zheng, Y and Guo, T and Xia, T and Guo, S and Chen, M and Ye, S and Pan, T and Xu, X and Gan, Y and Zhan, Y and Zheng, T and Zheng, Z},
title = {Utility of Arabidopsis KASII Promoter in Development of an Effective CRISPR/Cas9 System for Soybean Genome Editing and Its Application in Engineering of Soybean Seeds Producing Super-High Oleic and Low Saturated Oils.},
journal = {Journal of agricultural and food chemistry},
volume = {72},
number = {39},
pages = {21720-21730},
doi = {10.1021/acs.jafc.4c05840},
pmid = {39288439},
issn = {1520-5118},
mesh = {*Glycine max/genetics/metabolism/chemistry ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Seeds/genetics/metabolism/chemistry ; *Arabidopsis/genetics/metabolism ; *Promoter Regions, Genetic ; *Plants, Genetically Modified/genetics/metabolism/chemistry ; *Fatty Acid Desaturases/genetics/metabolism ; Oleic Acid/metabolism ; Fatty Acids/metabolism/chemistry ; Arabidopsis Proteins/genetics/metabolism ; Plant Oils/metabolism/chemistry ; },
abstract = {This study reports the use of the Arabidopsis KASII promoter (AtKASII) to develop an efficient CRISPR/Cas9 system for soybean genome editing. When this promoter was paired with Arabidopsis U6 promoters to drive Cas9 and single guide RNA expression, respectively, simultaneous editing of the three fatty acid desaturase genes GmFAD2-1A, GmFAD2-1B, and GmFAD3A occurred in more than 60% of transgenic soybean lines at T2 generation, and all the triple mutants possessed desirable high-oleic traits. In sharp contrast, not a single line underwent simultaneous editing of the three target genes when AtKASII was replaced by the widely used AtEC1.2 promoter. Furthermore, our study showed that the stable and inheritable mutations in the high-oleic lines did not alter the overall contents of oil and protein or amino acid composition while increasing the oleic acid content up to 87.6% from approximately 23.8% for wild-type seeds, concomitant with 34.4- and 3.7-fold reductions in linoleic and linolenic acid, respectively. Collectively, this study demonstrates that the AtKASII promoter is highly promising for optimization of the CRISPR/Cas9 system for genome editing in soybean and possibly beyond.},
}

*Glycine max/genetics/metabolism/chemistry
*CRISPR-Cas Systems
*Gene Editing/methods
*Seeds/genetics/metabolism/chemistry
*Arabidopsis/genetics/metabolism
*Promoter Regions, Genetic
*Plants, Genetically Modified/genetics/metabolism/chemistry
*Fatty Acid Desaturases/genetics/metabolism
Oleic Acid/metabolism
Fatty Acids/metabolism/chemistry
Arabidopsis Proteins/genetics/metabolism
Plant Oils/metabolism/chemistry

@article {pmid39183441,
year = {2024},
author = {Yuan, A and Sha, R and Xie, W and Qu, G and Zhang, H and Wang, H and Le, XC and Jiang, G and Peng, H},
title = {RNA-Activated CRISPR/Cas12a Nanorobots Operating in Living Cells.},
journal = {Journal of the American Chemical Society},
volume = {146},
number = {39},
pages = {26657-26666},
doi = {10.1021/jacs.4c02354},
pmid = {39183441},
issn = {1520-5126},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *RNA/metabolism/genetics/chemistry ; CRISPR-Associated Proteins/metabolism/genetics ; Gold/chemistry ; Metal Nanoparticles/chemistry ; Endodeoxyribonucleases/metabolism/genetics ; Bacterial Proteins/metabolism/genetics/chemistry ; },
abstract = {Active clustered regularly interspaced short palindromic repeats (CRISPR/Cas12a) systems possess both cis-cleavage (targeted) and trans-cleavage (collateral) activities, which are useful for genome engineering and diagnostic applications. Both single- and double-stranded DNA can activate crRNA-Cas12a ribonucleoprotein (RNP) to achieve cis- and trans-cleavage enzymatic activities. However, it is not clear whether RNA can activate the CRISPR/Cas12a system and what is critical to the trans-cleavage activity. We report here that RNA can activate the CRISPR/Cas12a system and trigger its trans-cleavage activity. We reveal that the activated crRNA-Cas12a RNP favors the trans-cleavage of longer sequences than commonly used. These new findings of the RNA-activated trans-cleavage capability of Cas12a provided the foundation for the design and construction of CRISPR nanorobots that operate in living cells. We assembled the crRNA-Cas12a RNP and nucleic acid substrates on gold nanoparticles to form CRISPR nanorobots, which dramatically increased the local effective concentration of the substrate in relation to the RNP and the trans-cleavage kinetics. Binding of the target microRNA to the crRNA-Cas12a RNP activated the nanorobots and their trans-cleavage function. The repeated (multiple-turnover) trans-cleavage of the fluorophore-labeled substrates generated amplified fluorescence signals. Sensitive and real-time imaging of specific microRNA in live cells demonstrated the promising potential of the CRISPR nanorobot system for future applications in monitoring and modulating biological functions within living cells.},
}

*CRISPR-Cas Systems/genetics
Humans
*RNA/metabolism/genetics/chemistry
CRISPR-Associated Proteins/metabolism/genetics
Gold/chemistry
Metal Nanoparticles/chemistry
Endodeoxyribonucleases/metabolism/genetics
Bacterial Proteins/metabolism/genetics/chemistry

@article {pmid39155066,
year = {2024},
author = {Guo, Y and Sun, S and Chen, S and Zhang, Y and Wang, W and Deng, Z and Liu, Z and Yu, Z and Xu, H and Guo, J and Zhang, S and Zhong, Y and Zhang, W and Chen, J and Zhou, F and Chang, H and Gao, SJ and Wang, Q},
title = {In vivo induction of sugarcane (Saccharum spp.) haploids by genome editing.},
journal = {Plant physiology},
volume = {196},
number = {2},
pages = {731-734},
pmid = {39155066},
issn = {1532-2548},
support = {2022GDASZH-2022010102//GDAS's Project of Science and Technology Development/ ; Gui Ke AA22117001//Guangxi Science and Technology Major Projects/ ; CARS-17//the Earmarked Fund for China Agriculture Research System/ ; 2021GDASYL-20210301001//the GDAS's Project of Technical Innovation and Incubation Service Platform Construction/ ; 2022-NPY-00-023//the Special Project for Rural Revitalization Strategy in Guangdong Province/ ; 2022KJCX17//Sanya Science and Technology Special Fund/ ; },
mesh = {*Saccharum/genetics ; *Gene Editing/methods ; *Haploidy ; Genome, Plant ; CRISPR-Cas Systems/genetics ; },
abstract = {Mutation of the MATRILINEAL gene in sugarcane induces in vivo maternal haploids and suggests prospects for sugarcane breeding.},
}

*Saccharum/genetics
*Gene Editing/methods
*Haploidy
Genome, Plant
CRISPR-Cas Systems/genetics

@article {pmid38713768,
year = {2024},
author = {Victoria, AJ and Selão, TT and Moreno-Cabezuelo, JÁ and Mills, LA and Gale, GAR and Lea-Smith, DJ and McCormick, AJ},
title = {A toolbox to engineer the highly productive cyanobacterium Synechococcus sp. PCC 11901.},
journal = {Plant physiology},
volume = {196},
number = {2},
pages = {1674-1690},
pmid = {38713768},
issn = {1532-2548},
support = {//Darwin Trust of Edinburgh/ ; //Green Chemicals Beacon of Excellence/ ; //University of Nottingham/ ; //FEBS/ ; //University of Cordoba/ ; //UK Biotechnology and Biological Sciences Research Council/ ; BB/S507404/1//Norwich Research Park Doctoral Training Partnership program/ ; NE/X014428//UK Natural Environmental Research Council/ ; },
mesh = {*Synechococcus/genetics/growth & development ; Promoter Regions, Genetic/genetics ; CRISPR-Cas Systems ; Genetic Engineering/methods ; Gene Editing/methods ; },
abstract = {Synechococcus sp. PCC 11901 (PCC 11901) is a fast-growing marine cyanobacterial strain that has a capacity for sustained biomass accumulation to very high cell densities, comparable to that achieved by commercially relevant heterotrophic organisms. However, genetic tools to engineer PCC 11901 for biotechnology applications are limited. Here we describe a suite of tools based on the CyanoGate MoClo system to unlock the engineering potential of PCC 11901. First, we characterized neutral sites suitable for stable genomic integration that do not affect growth even at high cell densities. Second, we tested a suite of constitutive promoters, terminators, and inducible promoters including a 2,4-diacetylphloroglucinol (DAPG)-inducible PhlF repressor system, which has not previously been demonstrated in cyanobacteria and showed tight regulation and a 228-fold dynamic range of induction. Lastly, we developed a DAPG-inducible dCas9-based CRISPR interference (CRISPRi) system and a modular method to generate markerless mutants using CRISPR-Cas12a. Based on our findings, PCC 11901 is highly responsive to CRISPRi-based repression and showed high efficiencies for single insertion (31% to 81%) and multiplex double insertion (25%) genome editing with Cas12a. We envision that these tools will lay the foundations for the adoption of PCC 11901 as a robust model strain for engineering biology and green biotechnology.},
}

*Synechococcus/genetics/growth & development
Promoter Regions, Genetic/genetics
CRISPR-Cas Systems
Genetic Engineering/methods
Gene Editing/methods

@article {pmid39349459,
year = {2024},
author = {Bexte, T and Albinger, N and Al Ajami, A and Wendel, P and Buchinger, L and Gessner, A and Alzubi, J and Särchen, V and Vogler, M and Rasheed, HM and Jung, BA and Wolf, S and Bhayadia, R and Oellerich, T and Klusmann, JH and Penack, O and Möker, N and Cathomen, T and Rieger, MA and Imkeller, K and Ullrich, E},
title = {CRISPR/Cas9 editing of NKG2A improves the efficacy of primary CD33-directed chimeric antigen receptor natural killer cells.},
journal = {Nature communications},
volume = {15},
number = {1},
pages = {8439},
pmid = {39349459},
issn = {2041-1723},
mesh = {Humans ; *CRISPR-Cas Systems ; *NK Cell Lectin-Like Receptor Subfamily C/genetics/metabolism/immunology ; *Killer Cells, Natural/immunology ; *Receptors, Chimeric Antigen/immunology/genetics ; *Gene Editing/methods ; *Leukemia, Myeloid, Acute/immunology/therapy/genetics ; Cell Line, Tumor ; Animals ; *Sialic Acid Binding Ig-like Lectin 3/genetics/immunology ; Mice ; Immunotherapy, Adoptive/methods ; },
abstract = {Chimeric antigen receptor (CAR)-modified natural killer (NK) cells show antileukemic activity against acute myeloid leukemia (AML) in vivo. However, NK cell-mediated tumor killing is often impaired by the interaction between human leukocyte antigen (HLA)-E and the inhibitory receptor, NKG2A. Here, we describe a strategy that overcomes CAR-NK cell inhibition mediated by the HLA-E-NKG2A immune checkpoint. We generate CD33-specific, AML-targeted CAR-NK cells (CAR33) combined with CRISPR/Cas9-based gene disruption of the NKG2A-encoding KLRC1 gene. Using single-cell multi-omics analyses, we identified transcriptional features of activation and maturation in CAR33-KLRC1[ko]-NK cells, which are preserved following exposure to AML cells. Moreover, CAR33-KLRC1[ko]-NK cells demonstrate potent antileukemic killing activity against AML cell lines and primary blasts in vitro and in vivo. We thus conclude that NKG2A-deficient CAR-NK cells have the potential to bypass immune suppression in AML.},
}

Humans
*CRISPR-Cas Systems
*NK Cell Lectin-Like Receptor Subfamily C/genetics/metabolism/immunology
*Killer Cells, Natural/immunology
*Receptors, Chimeric Antigen/immunology/genetics
*Gene Editing/methods
*Leukemia, Myeloid, Acute/immunology/therapy/genetics
Cell Line, Tumor
Animals
*Sialic Acid Binding Ig-like Lectin 3/genetics/immunology
Mice
Immunotherapy, Adoptive/methods

@article {pmid39297527,
year = {2024},
author = {Zheng, H and Wang, H and Du, C and Zhang, X and Chen, J},
title = {Multifunctional Ethyl Violet@NH2-MIL-88B(Fe) Hybrids: CRISPR-Cas12a-Assisted PEC-FL-CL Triple-Mode Sensitive Detection of HPV-16.},
journal = {Analytical chemistry},
volume = {96},
number = {39},
pages = {15657-15664},
doi = {10.1021/acs.analchem.4c03045},
pmid = {39297527},
issn = {1520-6882},
mesh = {*Human papillomavirus 16/genetics/isolation & purification ; *CRISPR-Cas Systems/genetics ; Electrochemical Techniques/methods ; DNA, Viral/analysis/genetics ; Humans ; Colorimetry/methods ; Biosensing Techniques/methods ; Limit of Detection ; Fluorescent Dyes/chemistry ; },
abstract = {The multimode assay based on multiple response mechanisms has received great attention to effectively improve the accuracy of a sensing platform. However, multifunctional sensing materials for simultaneously satisfying the multiple-mode detections are still in shortage due to the incompatibility of the signal transduction mechanisms in different modes. Here, taking human papillomavirus 16 (HPV-16) DNA (TDNA) as the model due to its important role in cervical cancer, a novel multifunctional material, ethyl violet (EV)@NH2-MIL-88B(Fe) (ENM) hybrids, have been successfully prepared, which could simultaneously satisfy CRISPR-Cas12a-assisted photoelectrochemical (PEC)-fluorescent (FL)-colorimetric (CL) triple-mode detection of TDNA. Based on the TDNA-induced trans-cleavage ability of CRISPR-Cas12a and efficient separation of magnetic beads, ENM was obtained from the single-stranded DNA-surrounded streptavidin-modified magnetic beads-ENM (SMB-ssDNA-ENM) and decomposed by pyrophosphate to get free EV, 2-aminoterephthalic acid (NH2-BDC), and Fe[3+]. Thus, TDNA was sensitively detected based on the EV-enhanced PEC signal of SnS2 nanosheets (PEC mode), fluorescent signal of NH2-BDC (FL mode), and characteristic absorption peak at about 720 nm of Fe[3+]-induced Prussian blue (PB) (CL mode). The designed PEC-FL-CL triple-mode biosensing platform had good performance for the detection of TDNA with a wide linear range (0.1 fM-100 nM) and ultralow detection limits (0.07 fM for PEC, 0.03 fM for FL and 0.09 fM for CL). Additionally, the developed PEC-FL-CL triple-mode biosensing platform has great potential for applications in early disease diagnosis and bioanalysis, as it can be easily extended to other DNA assays through modification of the crRNA sequence within the CRISPR-Cas12a system.},
}

*Human papillomavirus 16/genetics/isolation & purification
*CRISPR-Cas Systems/genetics
Electrochemical Techniques/methods
DNA, Viral/analysis/genetics
Humans
Colorimetry/methods
Biosensing Techniques/methods
Limit of Detection
Fluorescent Dyes/chemistry

@article {pmid39303167,
year = {2024},
author = {Xu, J and Li, Y and Wang, F and Yang, H and Huang, KJ and Cai, R and Tan, W},
title = {A Smartphone-Mediated "All-In-One" Biosensing Chip for Visual and Value-Assisted Detection.},
journal = {Analytical chemistry},
volume = {96},
number = {39},
pages = {15780-15788},
doi = {10.1021/acs.analchem.4c03854},
pmid = {39303167},
issn = {1520-6882},
mesh = {*Biosensing Techniques ; *MicroRNAs/analysis ; *Smartphone ; Humans ; Glucose Oxidase/metabolism/chemistry ; Electrochemical Techniques/methods/instrumentation ; Gold/chemistry ; Limit of Detection ; Palladium/chemistry ; CRISPR-Cas Systems ; },
abstract = {A smartphone-mediated self-powered biosensor is fabricated for miRNA-141 detection based on the CRISPR/Cas12a cross-cutting technique and a highly efficient nanozyme. As a novel nanozyme and a signal-amplified coreaction accelerator, the AuPtPd@GDY nanozyme exhibits an excellent ability to catalyze cascade color reactions and high conductivity to enhance the electrochemical signal for miRNA-141 assays. After CRISPR/Cas12a cross-cutting of S2-glucose oxidase (S2-GOD), the electrochemical signal is weakened, and miRNA-141 is detected by monitoring the decrease in the signal. On the other hand, a cascade reaction among glucose, H2O2, and TMB is catalyzed by GOD and AuPtPd@GDY, respectively, resulting in a color change of the solution, which senses miRNA-141. The self-powered biosensor enables value-assisted and visual detection of miRNA-141 with limits of detection of 3.1 and 15 aM, respectively. Based on the dual-modal self-powered sensing system, a smartphone-mediated "all-in-one" biosensing chip is designed to achieve the real-time and intelligent monitoring of miRNA-141. This work provides a new approach to design multifunctional biosensors to realize the visualization and portable detection of tumor biomarkers.},
}

*Biosensing Techniques
*MicroRNAs/analysis
*Smartphone
Humans
Glucose Oxidase/metabolism/chemistry
Electrochemical Techniques/methods/instrumentation
Gold/chemistry
Limit of Detection
Palladium/chemistry
CRISPR-Cas Systems

@article {pmid39037052,
year = {2024},
author = {Eisenberg, V and Hoogi, S and Katzman, E and Ben Haim, N and Zur-Toledano, R and Radman, M and Reboh, Y and Zadok, O and Kamer, I and Bar, J and Sagi, I and Hendel, A and Cohen, CJ},
title = {Targeting Tumor-Associated Sialic Acids Using Chimeric Switch Receptors Based on Siglec-9 Enhances the Antitumor Efficacy of Engineered T Cells.},
journal = {Cancer immunology research},
volume = {12},
number = {10},
pages = {1380-1391},
doi = {10.1158/2326-6066.CIR-23-0823},
pmid = {39037052},
issn = {2326-6074},
support = {//Adelis Foundation/ ; //Israel Cancer Research Fund (ICRF)/ ; //Sheba Medical Center (SMC)/ ; //Bar-Ilan University/ ; },
mesh = {Humans ; Animals ; *Sialic Acid Binding Immunoglobulin-like Lectins/metabolism ; Mice ; *T-Lymphocytes/immunology/metabolism ; Sialic Acids/metabolism ; Antigens, CD/metabolism ; Cell Line, Tumor ; Xenograft Model Antitumor Assays ; Neoplasms/immunology/therapy/metabolism ; Receptors, Chimeric Antigen/immunology/metabolism/genetics ; CRISPR-Cas Systems ; Tumor Microenvironment/immunology ; },
abstract = {Cancer exploits different mechanisms to escape T-cell immunosurveillance, including overexpression of checkpoint ligands, secretion of immunosuppressive molecules, and aberrant glycosylation. Herein, we report that IFNγ, a potent immunomodulator secreted in the tumor microenvironment, can induce α2,6 hypersialylation in cancer cell lines derived from various histologies. We focused on Siglec-9, a receptor for sialic acid moieties, and demonstrated that the Siglec-9+ T-cell population displayed reduced effector function. We speculated that Siglec-9 in primary human T cells can act as a checkpoint molecule and demonstrated that knocking out Siglec-9 using a clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system enhanced the functionality of primary human T cells. Finally, we aimed to augment cancer-specific T-cell activity by taking advantage of tumor hypersialylation. Thus, we designed several Siglec-9-based chimeric switch receptors (CSR), which included an intracellular moiety derived from costimulatory molecules (CD28/41BB) and different hinge regions. In an antigen-specific context, T cells transduced with Siglec-9 CSRs demonstrated increased cytokine secretions and upregulation of activation markers. Moreover, T cells equipped with specific Siglec-9 CSRs mediated robust antitumor activity in a xenograft model of human tumors. Overall, this work sheds light on tumor evasion mechanisms mediated by sialylated residues and exemplifies an approach to improve engineered T cell-based cancer treatment. See related Spotlight by Abken, p. 1310.},
}

Humans
Animals
*Sialic Acid Binding Immunoglobulin-like Lectins/metabolism
Mice
*T-Lymphocytes/immunology/metabolism
Sialic Acids/metabolism
Antigens, CD/metabolism
Cell Line, Tumor
Xenograft Model Antitumor Assays
Neoplasms/immunology/therapy/metabolism
Receptors, Chimeric Antigen/immunology/metabolism/genetics
CRISPR-Cas Systems
Tumor Microenvironment/immunology

@article {pmid38813729,
year = {2024},
author = {Meyers, S and Gielen, O and Cools, J and Demeyer, S},
title = {Single-cell CRISPR screening characterizes transcriptional deregulation in T-cell acute lymphoblastic leukemia.},
journal = {Haematologica},
volume = {109},
number = {10},
pages = {3167-3181},
doi = {10.3324/haematol.2023.284901},
pmid = {38813729},
issn = {1592-8721},
mesh = {Humans ; *Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/genetics/pathology/metabolism ; *Gene Expression Regulation, Leukemic ; *Single-Cell Analysis ; Signal Transduction ; CRISPR-Cas Systems ; Mutation ; Clustered Regularly Interspaced Short Palindromic Repeats ; Transcription, Genetic ; },
abstract = {T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive type of leukemia caused by accumulation of multiple genetic alterations in T-cell progenitors. However, for many genes it remains unknown how their mutations contribute to disease development. We therefore performed two single-cell CRISPR screens in primary pro-T cells ex vivo to study the transcriptional impact of loss-of-function alterations in T-ALL and correlate this with effects on cell fitness. The various perturbations were clustered based on their effects on E2F/MYC or STAT/NOTCH signatures, which play a defining role in driving T-cell proliferation. Many of the perturbations resulted in positive effects on the STAT and NOTCH signatures and were predicted to behave as haploinsufficient tumor suppressors in T-ALL. Additionally, Spi1 was identified as an essential gene for pro-T-cell survival, associated with deregulation of the MYC signature and epigenetic consequences. In contrast, Bcl11b was identified as a strong tumor suppressor gene in immature T lymphocytes, associated with deregulation of NF-kB and JAK/STAT signaling. We found a correlation between BCL11B expression level and JAK/STAT pathway mutations in T-ALL patients and demonstrated oncogenic cooperation between Bcl11b inactivation and JAK3 hyperactivation in pro-T cells. Altogether, these single-cell CRISPR screens in pro-T cells provide fundamental insights into the mechanisms of transcriptional deregulation caused by genetic alterations in T-ALL.},
}

Humans
*Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/genetics/pathology/metabolism
*Gene Expression Regulation, Leukemic
*Single-Cell Analysis
Signal Transduction
CRISPR-Cas Systems
Mutation
Clustered Regularly Interspaced Short Palindromic Repeats
Transcription, Genetic

@article {pmid39348977,
year = {2024},
author = {Kim, HJ and Kim, DY},
title = {Potential Anti-tumor Properties of PDIA4 in Lung Adenocarcinoma.},
journal = {Anticancer research},
volume = {44},
number = {10},
pages = {4309-4315},
doi = {10.21873/anticanres.17260},
pmid = {39348977},
issn = {1791-7530},
mesh = {Humans ; *Protein Disulfide-Isomerases/metabolism/genetics ; *Adenocarcinoma of Lung/genetics/pathology/metabolism ; *Lung Neoplasms/pathology/genetics/metabolism ; *Apoptosis ; *Cell Proliferation ; A549 Cells ; *Cell Movement ; Cell Line, Tumor ; Gene Expression Regulation, Neoplastic ; Oxidative Stress ; CRISPR-Cas Systems ; },
abstract = {BACKGROUND/AIM: Given the high frequency and mortality rate of lung cancer, diverse molecular studies have been undertaken to understand cancer pathophysiology and develop novel treatment strategies. The PDIA4 gene, which is involved in protein assembly and endoplasmic reticulum homeostasis, is overexpressed in various lung cancer subtypes. However, its exact function in lung adenocarcinoma (LUAD) remains elusive. The study aimed to investigate the role of PDIA4 in LUAD and explore its role as double-agent gene.

MATERIALS AND METHODS: PDIA4 expression was knocked out in A549 and LA-4 lung adenoma cells using the Crispr/Cas9 technology. Cell growth, migration, and apoptosis were analyzed in control and PDIA4-deficient cells.

RESULTS: PDIA4 deficiency resulted in increased cell growth, enhanced migration capacity, and greater resistance to apoptosis in both A549 and LA-4 lung cancer cells. Mechanistically, up-regulation of oxidative stress followed by NF-[Formula: see text]B activation may contribute to tumor-promoting effects observed upon PDIA4 silencing.

CONCLUSION: PDIA4 appears to function as a tumor suppressor in lung adenocarcinoma, suggesting that PDIA4 may act as a double-agent gene, with roles both on tumor suppression and promotion depending on the context.},
}

Humans
*Protein Disulfide-Isomerases/metabolism/genetics
*Adenocarcinoma of Lung/genetics/pathology/metabolism
*Lung Neoplasms/pathology/genetics/metabolism
*Apoptosis
*Cell Proliferation
A549 Cells
*Cell Movement
Cell Line, Tumor
Gene Expression Regulation, Neoplastic
Oxidative Stress
CRISPR-Cas Systems

@article {pmid39348463,
year = {2024},
author = {Wan, Y and Li, S and Xu, W and Wang, K and Guo, W and Yang, C and Li, X and Zhou, J and Wang, J},
title = {Terminal Chemical Modifications of crRNAs Enable Improvement in the Performance of CRISPR-Cas for Point-of-Care Nucleic Acid Detection.},
journal = {Analytical chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.analchem.4c03698},
pmid = {39348463},
issn = {1520-6882},
abstract = {CRISPR-Cas systems, harnessing their precise nucleic acid recognition via CRISPR RNA (crRNA), offer promise for the accurate testing of nucleic acids in the field. However, the inherent susceptibility of crRNA to degradation poses challenges for accurate detection in low-resource settings. Here, we utilized the chemically modified crRNA for the CRISPR-Cas-based assay (CM-CRISPR). We found that the extension and chemical modification to crRNA significantly enhanced the trans-cleavage activity of LbCas12a. The chemically modified crRNA was resistant to degradation, and CM-CRISPR showed superior detection capability in complex environments. CM-CRISPR could be combined with recombinase polymerase amplification (RPA) and applied in a droplet digital platform, enabling attomolar-level sensitivity. We also developed a portable and automated device for a digital CRISPR assay, which is amenable to point-of-care testing (POCT). The extraction-free procedure was integrated with this assay to streamline the workflow, and clinical samples were successfully detected. This work finds a simple and efficient way to improve the performance of CRISPR-Cas and develops a portable platform for POCT, representing a significant advance toward practical applications of CRISPR-based diagnostics.},
}

@article {pmid39347602,
year = {2024},
author = {Kamata, K and Birkholz, N and Ceelen, M and Fagerlund, RD and Jackson, SA and Fineran, PC},
title = {Repurposing an Endogenous CRISPR-Cas System to Generate and Study Subtle Mutations in Bacteriophages.},
journal = {The CRISPR journal},
volume = {},
number = {},
pages = {},
doi = {10.1089/crispr.2024.0047},
pmid = {39347602},
issn = {2573-1602},
abstract = {While bacteriophage applications benefit from effective phage engineering, selecting the desired genotype after subtle modifications remains challenging. Here, we describe a two-phase endogenous CRISPR-Cas-based phage engineering approach that enables selection of small defined edits in Pectobacterium carotovorum phage ZF40. We designed plasmids containing sequences homologous to ZF40 and a mini-CRISPR array. The plasmids allowed genome editing through homologous recombination and counter-selection against non-recombinant phage genomes using an endogenous type I-E CRISPR-Cas system. With this technique, we first deleted target genes and subsequently restored loci with modifications. This two-phase approach circumvented major challenges in subtle phage modifications, including inadequate sequence distinction for CRISPR-Cas counter-selection and the requirement of a protospacer-adjacent motif, limiting sequences that can be modified. Distinct 20-bp barcodes were incorporated through engineering as differential target sites for programmed CRISPR-Cas activity, which allowed quantification of phage variants in mixed populations. This method aids studies and applications that require mixtures of similar phages.},
}

@article {pmid39346764,
year = {2024},
author = {Sallard, E and Fischer, J and Schroeer, K and Dawson, LM and Beaude, N and Affes, A and Ehrke-Schulz, E and Zhang, W and Westhaus, A and Cabanes-Creus, M and Lisowski, L and Ruszics, Z and Ehrhardt, A},
title = {ADEVO: Proof-of-concept of adenovirus-directed EVOlution by random peptide display on the fiber knob.},
journal = {Molecular therapy. Oncology},
volume = {32},
number = {4},
pages = {200867},
pmid = {39346764},
issn = {2950-3299},
abstract = {Directed evolution of viral vectors involves the generation of randomized libraries followed by artificial selection of improved variants. Directed evolution only yielded limited results in adenovirus (AdV) engineering until now, mainly due to insufficient complexities of randomized libraries. Meanwhile, clinical applications of AdVs as gene therapy or oncolytic vectors are still hampered by the predetermined tropism of natural types. To overcome this challenge, we hypothesized that randomized peptide insertions on the capsid surface can be incorporated into the AdV bioengineering toolbox for retargeting. Here we developed AdV-directed EVOlution protocols based on fiber knob peptide display. Human AdV-C5-derived libraries were constructed following three distinct protocols and selected on a panel of cancer cell lines, with the goal of identifying variants able to infect and lyse these tumor cells more efficiently. All protocols enabled the construction of high complexity libraries with up to 9.6 × 10[5] unique variants, an approximate 100-fold improvement compared with previously published AdV libraries. After selection, the most enriched variants, which were robustly selected in various cancer cell lines, did not display enhanced infectivity but rather more efficient replication and cell lysis. Selected inserts also conferred enhanced lysis ability to oncolytic AdVs restricted to telomerase-expressing cell lines.},
}

@article {pmid39345554,
year = {2024},
author = {Goell, JH and Li, J and Mahata, B and Ma, AJ and Kim, S and Shah, S and Shah, S and Contreras, M and Misra, S and Reed, D and Bedford, GC and Escobar, M and Hilton, IB},
title = {Tailoring a CRISPR/Cas-based Epigenome Editor for Programmable Chromatin Acylation and Decreased Cytotoxicity.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
doi = {10.1101/2024.09.22.611000},
pmid = {39345554},
issn = {2692-8205},
abstract = {Engineering histone acylation states can inform mechanistic epigenetics and catalyze therapeutic epigenome editing opportunities. Here, we developed engineered lysine acyltransferases that enable the programmable deposition of acetylation and longer-chain acylations. We show that targeting an engineered lysine crotonyltransferase results in weak levels of endogenous enhancer activation yet retains potency when targeted to promoters. We further identify a single mutation within the catalytic core of human p300 that preserves enzymatic activity while substantially reducing cytotoxicity, enabling improved viral delivery. We leveraged these capabilities to perform single-cell CRISPR activation screening and map enhancers to the genes they regulate in situ. We also discover acylation-specific interactions and find that recruitment of p300, regardless of catalytic activity, to prime editing sites can improve editing efficiency. These new programmable epigenome editing tools and insights expand our ability to understand the mechanistic role of lysine acylation in epigenetic and cellular processes and perform functional genomic screens.},
}

@article {pmid39345221,
year = {2024},
author = {Chen, J and Su, H and Kim, JH and Liu, L and Liu, R},
title = {Recent advances in the CRISPR/Cas system-based visual detection method.},
journal = {Analytical methods : advancing methods and applications},
volume = {},
number = {},
pages = {},
doi = {10.1039/d4ay01147c},
pmid = {39345221},
issn = {1759-9679},
abstract = {Currently, various infectious pathogens and bacterial toxins as well as heavy metal pollution pose severe threats to global environmental health and the socio-economic infrastructure. Therefore, there is a pressing need for rapid, sensitive, and convenient visual molecular detection methods. The rapidly evolving detection approach based on clustered regularly interspaced short palindromic repeats (CRISPR)/associated nucleases (Cas) has opened a new frontier in the field of molecular diagnostics. This paper reviews the development of visual detection methods in recent years based on different Cas and analyzes their advantages and disadvantages as well as the challenges of future research. Firstly, different CRISPR/Cas effectors and their working principles in the diagnosis of various diseases are briefly reviewed. Subsequently, the article focuses on the development of visual readout signals in point-of-care testing using laboratory-based CRISPR/Cas technology, including colorimetric, fluorescence, and lateral flow analysis. Finally, the challenges and prospects of visual detection methods based on CRISPR/Cas technology are discussed.},
}

@article {pmid39344009,
year = {2024},
author = {Wang, Y and Geng, M and Pan, R and Zhang, T and Lu, X and Zhen, X and Che, Y and Li, R and Liu, J and Chen, Y and Guo, J and Yao, Y},
title = {Editing of the MeSWEET10a promoter yields bacterial blight resistance in cassava cultivar SC8.},
journal = {Molecular plant pathology},
volume = {25},
number = {10},
pages = {e70010},
doi = {10.1111/mpp.70010},
pmid = {39344009},
issn = {1364-3703},
support = {32460519//National Natural Science Foundation of China/ ; 322MS135//Hainan Provincial Natural Science Foundation of China/ ; 324MS021//Hainan Provincial Natural Science Foundation of China/ ; CATASCXTD202301//Chinese Academy of Tropical Agricultural Sciences for Science and Technology Innovation Team of National Tropical Agricultural Science Center/ ; CARS-11//the earmarked fund for CARS/ ; B21HJ0303//Hainan Seed Industry Laboratory/ ; },
mesh = {*Manihot/microbiology/genetics ; *Plant Diseases/microbiology/genetics/immunology ; *Disease Resistance/genetics ; *Promoter Regions, Genetic/genetics ; Xanthomonas axonopodis/pathogenicity ; Gene Editing ; Plant Proteins/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Plants, Genetically Modified ; Gene Expression Regulation, Plant ; },
abstract = {Cassava starch is a widely used raw material for industrial production and food source for people. However, cassava bacterial blight (CBB) caused by Xanthomonas axonopodis pv. manihotis (Xam) results in severe yield losses and is the most destructive bacterial disease in all worldwide cassava-growing regions. Xam11 is a highly pathogenic subspecies from China that infects the Chinese local cassava South China No. 8 (SC8) cultivar with marked symptoms. This study showed that the transcription activator-like effector TALE20Xam11 of Xam11 strain regulates the expression of disease-susceptibility gene MeSWEET10a by binding to the EBETALE20 region of the MeSWEET10a promoter in cassava cultivar SC8. CRISPR/Cas9-generated mutations of the EBETALE20 region resulted in a significant reduction in MeSWEET10a expression after infection by Xam11, correlating with reduced disease symptoms, smaller lesion sizes and decreased bacterial proliferation compared with the wild type. Importantly, the edited plants maintained normal growth, development and yield characteristics under greenhouse conditions. The results lay a research foundation for breeding resistant cassava cultivar SC8 to bacterial blight.},
}

*Manihot/microbiology/genetics
*Plant Diseases/microbiology/genetics/immunology
*Disease Resistance/genetics
*Promoter Regions, Genetic/genetics
Xanthomonas axonopodis/pathogenicity
Gene Editing
Plant Proteins/genetics/metabolism
CRISPR-Cas Systems/genetics
Plants, Genetically Modified
Gene Expression Regulation, Plant

@article {pmid39341259,
year = {2024},
author = {Jangra, S and Potts, J and Ghosh, A and Seal, DR},
title = {Genome editing: a novel approach to manage insect vectors of plant viruses.},
journal = {Insect biochemistry and molecular biology},
volume = {},
number = {},
pages = {104189},
doi = {10.1016/j.ibmb.2024.104189},
pmid = {39341259},
issn = {1879-0240},
abstract = {Insect vectors significantly threaten global agriculture by transmitting numerous viruses. Various measures, from conventional insecticides to genetic engineering, are used to mitigate this threat. However, none provide complete resistance. Therefore, researchers are looking for novel control options. In recent years with the advancements in genomic technologies, genomes and transcriptomes of various insect vectors have been generated. However, the lack of knowledge about gene function hinders the development of novel strategies to restrict virus spread. RNA interference (RNAi) is widely used to elucidate gene functions, but its variable efficacy hampers its use in insect vectors. Genome editing has the potential to overcome these challenges and has been extensively used in various insect pest species. This review summarizes the progress and potential of genome editing in plant virus vectors and its application as a functional genomic tool to elucidate virus-vector interactions. We also discuss the major challenges associated with editing insect vectors.},
}

@article {pmid39340568,
year = {2024},
author = {Wang, Y and Cao, J and Du, P and Wang, W and Hu, P and Liu, Y and Ma, Y and Wang, X and Abd El-Aty, AM},
title = {Portable detection of Salmonella in food of animal origin via Cas12a-RAA combined with an LFS/PGM dual-signaling readout biosensor.},
journal = {Mikrochimica acta},
volume = {191},
number = {10},
pages = {631},
pmid = {39340568},
issn = {1436-5073},
support = {2023CXGC010708//the Key R&D Program of Shandong Province, China/ ; SDAIT-10-09//the Modern Agricultural Technology Industry System of Shandong Province/ ; 2022YFD2100500//the National Key R&D Program of China/ ; CARS-38//the China Agriculture Research System/ ; },
mesh = {*Biosensing Techniques/methods/instrumentation ; Animals ; *Salmonella typhimurium/isolation & purification/genetics ; *CRISPR-Cas Systems ; *Bacterial Proteins/genetics ; *Food Microbiology ; Nucleic Acid Amplification Techniques/methods ; CRISPR-Associated Proteins/genetics ; Limit of Detection ; Food Contamination/analysis ; Endodeoxyribonucleases ; Recombinases/metabolism ; Point-of-Care Testing ; },
abstract = {A highly specific and sensitive rapid two-signal assay was developed for the detection of Salmonella typhimurium in foods of animal origin. The invA gene of Salmonella was used as the biorecognition element and recombinase-assisted amplification (RAA) technology for signal amplification. By utilizing the specific recognition and efficient trans-cleavage activity of CRISPR/Cas12a, point-of-care testing (POCT) for S. typhimurium was achieved via lateral flow strips (LFS) and personal glucometer (PGM) biosensors as dual signal readout systems, with sensitivities of 33 CFU/mL and 20 CFU/mL, respectively. Users can select the appropriate test system on the basis of specific application requirements: LFSs are ideal for rapid onsite screening, whereas glucometer biosensors offer precise quantitative determination. This approach simplifies the use of large instruments and overcomes site constraints, demonstrating good accuracy and applicability in animal-derived samples, with significant potential for the detection of other pathogens and for use in restricted environments.},
}

*Biosensing Techniques/methods/instrumentation
Animals
*Salmonella typhimurium/isolation & purification/genetics
*CRISPR-Cas Systems
*Bacterial Proteins/genetics
*Food Microbiology
Nucleic Acid Amplification Techniques/methods
CRISPR-Associated Proteins/genetics
Limit of Detection
Food Contamination/analysis
Endodeoxyribonucleases
Recombinases/metabolism
Point-of-Care Testing

@article {pmid39339951,
year = {2024},
author = {Mattioli, M and Raele, RA and Gautam, G and Borucu, U and Schaffitzel, C and Aulicino, F and Berger, I},
title = {Tuning VSV-G Expression Improves Baculovirus Integrity, Stability and Mammalian Cell Transduction Efficiency.},
journal = {Viruses},
volume = {16},
number = {9},
pages = {},
pmid = {39339951},
issn = {1999-4915},
support = {BB/L01386X/1//BBSRC/EPSRC Research Centre for Synthetic Biology at the University of Bristol/ ; BB/L014181/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; DNA-DOCK, Project No. 834631/ERC_/European Research Council/International ; BB/W013959/1//BBSRC BrisEngBio Proof-of-Concept grant/ ; },
mesh = {Humans ; *Transduction, Genetic ; *Nucleopolyhedroviruses/genetics/physiology ; *Viral Envelope Proteins/genetics/metabolism ; *Genetic Vectors/genetics ; Animals ; *Promoter Regions, Genetic ; Cell Line ; Baculoviridae/genetics ; Gene Editing/methods ; HEK293 Cells ; CRISPR-Cas Systems ; Membrane Glycoproteins ; },
abstract = {Baculoviral vectors (BVs) derived from Autographa californica multiple nucleopolyhedrovirus (AcMNPV) are an attractive tool for multigene delivery in mammalian cells, which is particularly relevant for CRISPR technologies. Most applications in mammalian cells rely on BVs that are pseudotyped with vesicular stomatitis virus G-protein (VSV-G) to promote efficient endosomal release. VSV-G expression typically occurs under the control of the hyperactive polH promoter. In this study, we demonstrate that polH-driven VSV-G expression results in BVs characterised by reduced stability, impaired morphology, and VSV-G induced toxicity at high multiplicities of transduction (MOTs) in target mammalian cells. To overcome these drawbacks, we explored five alternative viral promoters with the aim of optimising VSV-G levels displayed on the pseudotyped BVs. We report that Orf-13 and Orf-81 promoters reduce VSV-G expression to less than 5% of polH, rescuing BV morphology and stability. In a panel of human cell lines, we elucidate that BVs with reduced VSV-G support efficient gene delivery and CRISPR-mediated gene editing, at levels comparable to those obtained previously with polH VSV-G-pseudotyped BVs (polH VSV-G BV). These results demonstrate that VSV-G hyperexpression is not required for efficient transduction of mammalian cells. By contrast, reduced VSV-G expression confers similar transduction dynamics while substantially improving BV integrity, structure, and stability.},
}

Humans
*Transduction, Genetic
*Nucleopolyhedroviruses/genetics/physiology
*Viral Envelope Proteins/genetics/metabolism
*Genetic Vectors/genetics
Animals
*Promoter Regions, Genetic
Cell Line
Baculoviridae/genetics
Gene Editing/methods
HEK293 Cells
CRISPR-Cas Systems
Membrane Glycoproteins

@article {pmid39295522,
year = {2024},
author = {Meng, X and Yan, F and Wang, W and Wang, S and Cong, H and Li, J and Zhao, Y and Wang, T and Li, N and Gao, Y and Wang, J and Feng, N and Xia, X},
title = {A single dose of an ALVAC vector-based RABV virus-like particle candidate vaccine induces a potent immune response in mice, cats and dogs.},
journal = {Emerging microbes & infections},
volume = {13},
number = {1},
pages = {2406280},
doi = {10.1080/22221751.2024.2406280},
pmid = {39295522},
issn = {2222-1751},
mesh = {Animals ; Dogs ; *Rabies Vaccines/immunology/administration & dosage/genetics ; Mice ; *Rabies virus/immunology/genetics ; *Vaccines, Virus-Like Particle/immunology/administration & dosage/genetics ; *Rabies/prevention & control/immunology ; Cats ; *Antibodies, Viral/blood/immunology ; Canarypox virus/immunology/genetics ; Genetic Vectors/genetics ; Female ; Dendritic Cells/immunology ; Immunity, Humoral ; CRISPR-Cas Systems ; Mice, Inbred BALB C ; },
abstract = {Rabies, caused by the Rabies virus (RABV), is a highly fatal zoonotic disease. Existing rabies vaccines have demonstrated good immune efficacy, but the complexity of immunization procedures and high cost has impeded the elimination of RABV, particularly in the post-COVID-19 era. There is a pressing need for safer and more effective rabies vaccines that streamline vaccination protocols and reduce expense. To meet this need, we have developed a potential rabies vaccine candidate called ALVAC-RABV-VLP, utilizing CRISPR/Cas9 gene editing technology. This vaccine employs a canarypox virus vector (ALVAC) to generate RABV virus-like particles (VLPs). In mice, a single dose of ALVAC-RABV-VLP effectively activated dendritic cells (DCs), follicular helper T cells (Tfh), and the germinal centre (GC)/plasma cell axis, resulting in durable and effective humoral immune responses. The survival rate of mice challenged with lethal RABV was 100%. Similarly, in dogs and cats, a single immunization with ALVAC-RABV-VLP elicited a stronger and longer-lasting antibody response. ALVAC-RABV-VLP induced superior cellular and humoral immunity in both mice and beagles compared to the commercial inactivated rabies vaccine. In conclusion, ALVAC-RABV-VLP induced robust protective immune responses in mice, dogs and cats, offering a novel, cost-effective, efficient, and promising approach for herd prevention of rabies.},
}

Animals
Dogs
*Rabies Vaccines/immunology/administration & dosage/genetics
Mice
*Rabies virus/immunology/genetics
*Vaccines, Virus-Like Particle/immunology/administration & dosage/genetics
*Rabies/prevention & control/immunology
Cats
*Antibodies, Viral/blood/immunology
Canarypox virus/immunology/genetics
Genetic Vectors/genetics
Female
Dendritic Cells/immunology
Immunity, Humoral
CRISPR-Cas Systems
Mice, Inbred BALB C

@article {pmid39339886,
year = {2024},
author = {Kolesnik, M and Pavlov, C and Demkina, A and Samolygo, A and Karneyeva, K and Trofimova, A and Sokolova, OS and Moiseenko, AV and Kirsanova, M and Severinov, K},
title = {New Viruses Infecting Hyperthermophilic Bacterium Thermus thermophilus.},
journal = {Viruses},
volume = {16},
number = {9},
pages = {},
doi = {10.3390/v16091410},
pmid = {39339886},
issn = {1999-4915},
support = {24-14-00181//Russian Science Foundation/ ; },
mesh = {*Thermus thermophilus/virology/genetics ; *Bacteriophages/genetics/isolation & purification/classification/physiology ; *Genome, Viral ; *Hot Springs/microbiology/virology ; *Phylogeny ; CRISPR-Cas Systems ; Georgia (Republic) ; Genomics/methods ; },
abstract = {Highly diverse phages infecting thermophilic bacteria of the Thermus genus have been isolated over the years from hot springs around the world. Many of these phages are unique, rely on highly unusual developmental strategies, and encode novel enzymes. The variety of Thermus phages is clearly undersampled, as evidenced, for example, by a paucity of phage-matching spacers in Thermus CRISPR arrays. Using water samples collected from hot springs in the Kunashir Island from the Kuril archipelago and from the Tsaishi and Nokalakevi districts in the Republic of Georgia, we isolated several distinct phages infecting laboratory strains of Thermus thermophilus. Genomic sequence analysis of 11 phages revealed both close relatives of previously described Thermus phages isolated from geographically distant sites, as well as phages with very limited similarity to earlier isolates. Comparative analysis allowed us to predict several accessory phage genes whose products may be involved in host defense/interviral warfare, including a putative Type V CRISPR-cas system.},
}

*Thermus thermophilus/virology/genetics
*Bacteriophages/genetics/isolation & purification/classification/physiology
*Genome, Viral
*Hot Springs/microbiology/virology
*Phylogeny
CRISPR-Cas Systems
Georgia (Republic)
Genomics/methods

@article {pmid39339877,
year = {2024},
author = {Khoo, YW and Wang, Q and Liu, S and Zhan, B and Xu, T and Lv, W and Liu, G and Li, S and Zhang, Z},
title = {Resistance of the CRISPR-Cas13a Gene-Editing System to Potato Spindle Tuber Viroid Infection in Tomato and Nicotiana benthamiana.},
journal = {Viruses},
volume = {16},
number = {9},
pages = {},
doi = {10.3390/v16091401},
pmid = {39339877},
issn = {1999-4915},
support = {2022JBGS0037//'Open bidding for selecting the best candidates' in the Inner Mongolia Autonomous Region/ ; 32072395//National Nature Science Foundations of China/ ; },
mesh = {*Nicotiana/virology/genetics ; *Solanum lycopersicum/virology/genetics ; *Viroids/genetics ; *CRISPR-Cas Systems ; *Plant Diseases/virology/genetics ; *Gene Editing/methods ; *Plants, Genetically Modified/virology ; *Disease Resistance/genetics ; RNA, Viral/genetics/metabolism ; },
abstract = {Gene-editing technology, specifically the CRISPR-Cas13a system, has shown promise in breeding plants resistant to RNA viruses. This system targets RNA and, theoretically, can also combat RNA-based viroids. To test this, the CRISPR-Cas13a system was introduced into tomato plants via transient expression and into Nicotiana benthamiana through transgenic methods, using CRISPR RNAs (crRNAs) targeting the conserved regions of both sense and antisense genomes of potato spindle tuber viroid (PSTVd). In tomato plants, the expression of CRISPR-Cas13a and crRNAs substantially reduced PSTVd accumulation and alleviated disease symptoms. In transgenic N. benthamiana plants, the PSTVd levels were lower as compared to wild-type plants. Several effective crRNAs targeting the PSTVd genomic RNA were also identified. These results demonstrate that the CRISPR-Cas13a system can effectively target and combat viroid RNAs, despite their compact structures.},
}

*Nicotiana/virology/genetics
*Solanum lycopersicum/virology/genetics
*Viroids/genetics
*CRISPR-Cas Systems
*Plant Diseases/virology/genetics
*Gene Editing/methods
*Plants, Genetically Modified/virology
*Disease Resistance/genetics
RNA, Viral/genetics/metabolism

@article {pmid39339858,
year = {2024},
author = {Yuan, X and Xu, K and Yan, F and Liu, Z and Spetz, C and Zhou, H and Wang, X and Jin, H and Wang, X and Liu, Y},
title = {CRISPR/Cas9-Mediated Resistance to Wheat Dwarf Virus in Hexaploid Wheat (Triticum aestivum L.).},
journal = {Viruses},
volume = {16},
number = {9},
pages = {},
doi = {10.3390/v16091382},
pmid = {39339858},
issn = {1999-4915},
support = {31861133020//National Natural Science Foundation of China/ ; },
mesh = {*Triticum/virology/genetics/immunology ; *CRISPR-Cas Systems ; *Geminiviridae/genetics ; *Plant Diseases/virology/genetics/immunology ; *Disease Resistance/genetics ; *Plants, Genetically Modified/virology ; Gene Editing ; Polyploidy ; },
abstract = {Wheat dwarf virus (WDV, genus Mastrevirus, family Geminiviridae) is one of the causal agents of wheat viral disease, which severely impacts wheat production in most wheat-growing regions in the world. Currently, there is little information about natural resistance against WDV in common wheat germplasms. CRISPR/Cas9 technology is being utilized to manufacture transgenic plants resistant to different diseases. In the present study, we used the CRISPR/Cas9 system targeting overlapping regions of coat protein (CP) and movement protein (MP) (referred to as CP/MP) or large intergenic region (LIR) in the wheat variety 'Fielder' to develop resistance against WDV. WDV-inoculated T1 progenies expressing Cas9 and sgRNA for CP/MP and LIR showed complete resistance against WDV and no accumulation of viral DNA compared with control plants. Mutation analysis revealed that the CP/MP and LIR targeting sites have small indels in the corresponding Cas9-positive plants. Additionally, virus inhibition and indel mutations occurred in T2 homozygous lines. Together, our work gives efficient results of the engineering of CRISPR/Cas9-mediated WDV resistance in common wheat plants, and the specific sgRNAs identified in this study can be extended to utilize the CRISPR/Cas9 system to confer resistance to WDV in other cereal crops such as barley, oats, and rye.},
}

*Triticum/virology/genetics/immunology
*CRISPR-Cas Systems
*Geminiviridae/genetics
*Plant Diseases/virology/genetics/immunology
*Disease Resistance/genetics
*Plants, Genetically Modified/virology
Gene Editing
Polyploidy

@article {pmid39338985,
year = {2024},
author = {Sung, K and Nawaz, M and Park, M and Chon, J and Khan, SA and Alotaibi, K and Khan, AA},
title = {Comprehensive Genomic Analysis of Uropathogenic E. coli: Virulence Factors, Antimicrobial Resistance, and Mobile Genetic Elements.},
journal = {Pathogens (Basel, Switzerland)},
volume = {13},
number = {9},
pages = {},
doi = {10.3390/pathogens13090794},
pmid = {39338985},
issn = {2076-0817},
support = {E0770601//United States Food and Drug Administration/ ; },
abstract = {Our whole-genome sequencing analysis of sixteen uropathogenic E. coli isolates revealed a concerning picture of multidrug resistance and potentially virulent bacteria. All isolates belonged to four distinct clonal groups, with the highly prevalent ST131 lineage being associated with extensive antibiotic resistance and virulence factors. Notably, all isolates exhibited multidrug resistance, with some resistant to as many as 12 antibiotics. Fluoroquinolone resistance stemmed primarily from efflux pumps and mutations in gyrase and topoisomerase genes. Additionally, we identified genes encoding resistance to extended-spectrum cephalosporins, trimethoprim/sulfamethoxazole, and various heavy metals. The presence of diverse plasmids and phages suggests the potential for horizontal gene transfer and the dissemination of virulence factors. All isolates harbored genomic islands containing virulence factors associated with adhesion, biofilm formation, and invasion. Genes essential for iron acquisition, flagella biosynthesis, secretion systems, and toxin production were also prevalent. Adding further complexity to understanding the isolates' genetic makeup, we identified CRISPR-Cas systems. This study underscores the need for continued genomic surveillance in understanding the pathogenic mechanisms and resistance profiles of uropathogenic E. coli to aid in developing targeted therapeutic strategies.},
}

@article {pmid39338884,
year = {2024},
author = {Zhang, Y and Miao, P and Wang, J and Sun, Y and Zhang, J and Wang, B and Yan, M},
title = {A Photoelectrochemical Biosensor Mediated by CRISPR/Cas13a for Direct and Specific Detection of MiRNA-21.},
journal = {Sensors (Basel, Switzerland)},
volume = {24},
number = {18},
pages = {},
doi = {10.3390/s24186138},
pmid = {39338884},
issn = {1424-8220},
support = {52173168//National Natural Science Foundation of China/ ; ZR2022MB072//Natural Science Foundation of Shandong Province/ ; TSQN tstp20221131//Taishan Scholars Program project/ ; 2021CXGC010603//Major Scientific and Technological Innovation Project of Shandong Province/ ; XKY22210//Science and Technology Program of University of Jinan/ ; YXH2022ZX02118//Shandong Medical Association Clinical Research Fund - Qilu Special Project/ ; 2022YP21//the second hospital of Shandong University Priya Fund/ ; },
mesh = {*Biosensing Techniques/methods ; *MicroRNAs/blood/analysis/genetics ; *Electrochemical Techniques/methods ; Humans ; *CRISPR-Cas Systems ; Limit of Detection ; Biotin/chemistry ; DNA/chemistry/genetics ; Electrodes ; },
abstract = {Direct detection of miRNA is currently limited by the complex amplification and reverse transcription processes of existing methods, leading to low sensitivity and high operational demands. Herein, we developed a CRISPR/Cas13a-mediated photoelectrochemical (PEC) biosensing platform for direct and sensitive detection of miRNA-21. The direct and specific recognition of target miRNA-21 by crRNA-21 eliminates the need for pre-amplification and reverse transcription of miRNA-21, thereby preventing signal distortion and enhancing the sensitivity and precision of target detection. When crRNA-21 binds to miRNA-21, it activates the trans-cleavage activity of CRISPR/Cas13a, leading to the non-specific cleavage of biotin-modified DNA with uracil bases (biotin-rU-DNA). This cleavage prevents the biotin-rU-DNA from being immobilized on the electrode surface. As a result, streptavidin cannot attach to the electrode via specific biotin binding, reducing spatial resistance and causing a positively correlated increase in the photocurrent response. This Cas-PEC biosensor has good analytical capabilities, linear responses between 10 fM and 10 nM, a minimum detection limit of 9 fM, and an excellent recovery rate in the analysis of real human serum samples. This work presented an innovative solution for detecting other biomarkers in bioanalysis and clinical diagnostics.},
}

*Biosensing Techniques/methods
*MicroRNAs/blood/analysis/genetics
*Electrochemical Techniques/methods
Humans
*CRISPR-Cas Systems
Limit of Detection
Biotin/chemistry
DNA/chemistry/genetics
Electrodes

@article {pmid39338447,
year = {2024},
author = {Turgeman-Grott, I and Shalev, Y and Shemesh, N and Levy, R and Eini, I and Pasmanik-Chor, M and Gophna, U},
title = {A Haloarchaeal Transcriptional Regulator That Represses the Expression of CRISPR-Associated Genes.},
journal = {Microorganisms},
volume = {12},
number = {9},
pages = {},
doi = {10.3390/microorganisms12091772},
pmid = {39338447},
issn = {2076-2607},
support = {ERC-AdG 787514/ERC_/European Research Council/International ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas (CRISPR-associated proteins) systems provide acquired heritable protection to bacteria and archaea against selfish DNA elements, such as viruses. These systems must be tightly regulated because they can capture DNA fragments from foreign selfish elements, and also occasionally from self-chromosomes, resulting in autoimmunity. Most known species from the halophilic archaeal genus Haloferax contain type I-B CRISPR-Cas systems, and the strongest hotspot for self-spacer acquisition by H. mediterranei was a locus that contained a putative transposable element, as well as the gene HFX_2341, which was a very frequent target for self-targeting spacers. To test whether this gene is CRISPR-associated, we investigated it using bioinformatics, deletion, over-expression, and comparative transcriptomics. We show that HFX_2341 is a global transcriptional regulator that can repress diverse genes, since its deletion results in significantly higher expression of multiple genes, especially those involved in nutrient transport. When over-expressed, HFX_2341 strongly repressed the transcript production of all cas genes tested, both those involved in spacer acquisition (cas1, 2 and 4) and those required for destroying selfish genetic elements (cas3 and 5-8). Considering that HFX_2341 is highly conserved in haloarchaea, with homologs that are present in species that do not encode the CRISPR-Cas system, we conclude that it is a global regulator that is also involved in cas gene regulation, either directly or indirectly.},
}

@article {pmid39337500,
year = {2024},
author = {Fizikova, A and Tukhuzheva, Z and Zhokhova, L and Tvorogova, V and Lutova, L},
title = {A New Approach for CRISPR/Cas9 Editing and Selection of Pathogen-Resistant Plant Cells of Wine Grape cv. 'Merlot'.},
journal = {International journal of molecular sciences},
volume = {25},
number = {18},
pages = {},
doi = {10.3390/ijms251810011},
pmid = {39337500},
issn = {1422-0067},
support = {Agreement 075-10-2021-093, Project [GNZH-RD-2008]//Ministry of Science and Higher Education of Russian Federation/ ; },
mesh = {*Vitis/genetics ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Disease Resistance/genetics ; Chitosan/pharmacology ; Plant Diseases/genetics/microbiology ; Plant Cells/metabolism ; Phenols ; Wine ; Plant Proteins/genetics/metabolism ; },
abstract = {Grape is one of the most economically significant berry crops. Owing to the biological characteristics of grapes, such as the long juvenile period (5-8 years), high degree of genome heterozygosity, and the frequent occurrence of inbreeding depression, homozygosity during crossbreeding leads to loss of varietal characteristics and viability. CRISPR/Cas editing has become the tool of choice for improving elite technical grape varieties. This study provides the first evidence of a decrease in the total fraction of phenolic compounds and an increase in the concentration of peroxide compounds in grape callus cells upon the addition of chitosan to the culture medium. These previously unreported metabolic features of the grape response to chitosan have been described and used for the first time to increase the probability of selecting plant cells with MLO7 knockout characterised by an oxidative burst in response to the presence of a pathogen modulated by chitosan in the high-metabolite black grape variety 'Merlot'. This was achieved by using a CRISPR/Cas9 editing vector construction with the peroxide sensor HyPer as a reporter. This research represents the first CRISPR/Cas9 editing of 'Merlot', one of the most economically important elite technical grape varieties.},
}

*Vitis/genetics
*CRISPR-Cas Systems
*Gene Editing/methods
Disease Resistance/genetics
Chitosan/pharmacology
Plant Diseases/genetics/microbiology
Plant Cells/metabolism
Phenols
Wine
Plant Proteins/genetics/metabolism

@article {pmid39337434,
year = {2024},
author = {Yu, XF and Teng, B and Li, JF and Zhang, JV and Su, Z and Ren, PG},
title = {Novel Function of Osteocalcin in Chondrocyte Differentiation and Endochondral Ossification Revealed on a CRISPR/Cas9 bglap-bglap2 Deficiency Mouse Model.},
journal = {International journal of molecular sciences},
volume = {25},
number = {18},
pages = {},
doi = {10.3390/ijms25189945},
pmid = {39337434},
issn = {1422-0067},
support = {2021YFA0719303//National Key R&D Program of China/ ; 32100572//National Natural Science Foundation of China/ ; 32271166//National Natural Science Foundation of China/ ; 2024A1515013017//Guangdong Basic and Applied Basic Research Foundation/ ; JCYJ20200109115441918//Shenzhen Science and Technology Program/ ; JCYJ20210324102013035//Shenzhen Science and Technology Program/ ; SZGSP012//High-level Key Clinical Specialty project of Guangdong Provincial Health Commission (Supporting construction funds of Shenzhen)/ ; LCYJ2022071//Guangdong High-level Hospital Construction Fund and Guangdong High-level Hospital Construction Fund Clinical Research Project of Shenzhen Children's Hospital/ ; },
mesh = {Animals ; *Chondrocytes/metabolism/cytology ; *Osteogenesis/genetics ; Mice ; *Osteocalcin/metabolism/genetics ; *Cell Differentiation/genetics ; *CRISPR-Cas Systems ; *Chondrogenesis/genetics ; Mesenchymal Stem Cells/metabolism/cytology ; Cartilage/metabolism ; Signal Transduction ; Mice, Knockout ; },
abstract = {Endochondral ossification is the process by which cartilage is mineralized into bone, and is essential for the development of long bones. Osteocalcin (OCN), a protein abundant in bone matrix, also exhibits high expression in chondrocytes, especially hypertrophic chondrocytes, while its role in endochondral ossification remains unclear. Utilizing a new CRISPR/Cas9-mediated bglap-bglap2 deficiency (OCN[em]) mouse model generated in our laboratory, we provide the first evidence of OCN's regulatory function in chondrocyte differentiation and endochondral ossification. The OCN[em] mice exhibited significant delays in primary and secondary ossification centers compared to wild-type mice, along with increased cartilage length in growth plates and hypertrophic zones during neonatal and adolescent stages. These anomalies indicated that OCN deficiency disturbed endochondral ossification during embryonic and postnatal periods. Mechanism wise, OCN deficiency was found to increase chondrocyte differentiation and postpone vascularization process. Furthermore, bone marrow mesenchymal stromal cells (BMSCs) from OCN[em] mice demonstrated an increased capacity for chondrogenic differentiation. Transcriptional network analysis implicated that BMP and TGF-β signaling pathways were highly affected in OCN[em] BMSCs, which is closely associated with cartilage development and maintenance. This elucidation of OCN's function in chondrocyte differentiation and endochondral ossification contributes to a more comprehensive understanding of its impact on skeletal development and homeostasis.},
}

Animals
*Chondrocytes/metabolism/cytology
*Osteogenesis/genetics
Mice
*Osteocalcin/metabolism/genetics
*Cell Differentiation/genetics
*CRISPR-Cas Systems
*Chondrogenesis/genetics
Mesenchymal Stem Cells/metabolism/cytology
Cartilage/metabolism
Signal Transduction
Mice, Knockout

@article {pmid39336755,
year = {2024},
author = {Arana, ÁJ and Sánchez, L},
title = {Knockout, Knockdown, and the Schrödinger Paradox: Genetic Immunity to Phenotypic Recapitulation in Zebrafish.},
journal = {Genes},
volume = {15},
number = {9},
pages = {},
doi = {10.3390/genes15091164},
pmid = {39336755},
issn = {2073-4425},
mesh = {*Zebrafish/genetics/immunology ; Animals ; *Gene Knockdown Techniques ; *Phenotype ; Morpholinos/genetics ; Gene Knockout Techniques ; Zebrafish Proteins/genetics ; CRISPR-Cas Systems ; Zygote/metabolism ; Female ; },
abstract = {Previous research has highlighted significant phenotypic discrepancies between knockout and knockdown approaches in zebrafish, raising concerns about the reliability of these methods. However, our study suggests that these differences are not as pronounced as was once believed. By carefully examining the roles of maternal and zygotic gene contributions, we demonstrate that these factors significantly influence phenotypic outcomes, often accounting for the observed discrepancies. Our findings emphasize that morpholinos, despite their potential off-target effects, can be effective tools when used with rigorous controls. We introduce the concept of graded maternal contribution, which explains how the uneven distribution of maternal mRNA and proteins during gametogenesis impacts phenotypic variability. Our research categorizes genes into three types-susceptible, immune, and "Schrödinger" (conditional)-based on their phenotypic expression and interaction with genetic compensation mechanisms. This distinction provides new insights into the paradoxical outcomes observed in genetic studies. Ultimately, our work underscores the importance of considering both maternal and zygotic contributions, alongside rigorous experimental controls, to accurately interpret gene function and the mechanisms underlying disease. This study advocates for the continued use of morpholinos in conjunction with advanced genetic tools like CRISPR/Cas9, stressing the need for a meticulous experimental design to optimize the utility of zebrafish in genetic research and therapeutic development.},
}

*Zebrafish/genetics/immunology
Animals
*Gene Knockdown Techniques
*Phenotype
Morpholinos/genetics
Gene Knockout Techniques
Zebrafish Proteins/genetics
CRISPR-Cas Systems
Zygote/metabolism
Female

@article {pmid39336621,
year = {2024},
author = {Zhao, Y and Li, L and Wei, L and Wang, Y and Han, Z},
title = {Advancements and Future Prospects of CRISPR-Cas-Based Population Replacement Strategies in Insect Pest Management.},
journal = {Insects},
volume = {15},
number = {9},
pages = {},
doi = {10.3390/insects15090653},
pmid = {39336621},
issn = {2075-4450},
support = {GAU-KYQD-2019-26//Scientific and Technological Innovation Foundation of Gansu Agricultural University-PhD research startup foundation/ ; Gaufx-03Y03//Fuxi Youth Talent Project of Gansu Agricultural University/ ; 31900347//National Natural Science Foundation of China/ ; },
abstract = {Population replacement refers to the process by which a wild-type population of insect pests is replaced by a population possessing modified traits or abilities. Effective population replacement necessitates a gene drive system capable of spreading desired genes within natural populations, operating under principles akin to super-Mendelian inheritance. Consequently, releasing a small number of genetically edited insects could potentially achieve population control objectives. Currently, several gene drive approaches are under exploration, including the newly adapted CRISPR-Cas genome editing system. Multiple studies are investigating methods to engineer pests that are incapable of causing crop damage or transmitting vector-borne diseases, with several notable successful examples documented. This review summarizes the recent advancements of the CRISPR-Cas system in the realm of population replacement and provides insights into research methodologies, testing protocols, and implementation strategies for gene drive techniques. The review also discusses emerging trends and prospects for establishing genetic tools in pest management.},
}

@article {pmid39336087,
year = {2024},
author = {Aslam, N and Li, Q and Bashir, S and Yuan, L and Qiao, L and Li, W},
title = {Integrated Review of Transcriptomic and Proteomic Studies to Understand Molecular Mechanisms of Rice's Response to Environmental Stresses.},
journal = {Biology},
volume = {13},
number = {9},
pages = {},
doi = {10.3390/biology13090659},
pmid = {39336087},
issn = {2079-7737},
support = {32070197//Natural Science Foundation of China/ ; },
abstract = {Rice (Oryza sativa L.) is grown nearly worldwide and is a staple food for more than half of the world's population. With the rise in extreme weather and climate events, there is an urgent need to decode the complex mechanisms of rice's response to environmental stress and to breed high-yield, high-quality and stress-resistant varieties. Over the past few decades, significant advancements in molecular biology have led to the widespread use of several omics methodologies to study all aspects of plant growth, development and environmental adaptation. Transcriptomics and proteomics have become the most popular techniques used to investigate plants' stress-responsive mechanisms despite the complexity of the underlying molecular landscapes. This review offers a comprehensive and current summary of how transcriptomics and proteomics together reveal the molecular details of rice's response to environmental stresses. It also provides a catalog of the current applications of omics in comprehending this imperative crop in relation to stress tolerance improvement and breeding. The evaluation of recent advances in CRISPR/Cas-based genome editing and the application of synthetic biology technologies highlights the possibility of expediting the development of rice cultivars that are resistant to stress and suited to various agroecological environments.},
}

@article {pmid39334840,
year = {2024},
author = {Salum, YM and Yin, A and Zaheer, U and Liu, Y and Guo, Y and He, W},
title = {CRISPR/Cas9-Based Genome Editing of Fall Armyworm (Spodoptera frugiperda): Progress and Prospects.},
journal = {Biomolecules},
volume = {14},
number = {9},
pages = {},
doi = {10.3390/biom14091074},
pmid = {39334840},
issn = {2218-273X},
support = {2021YFD1400705//National key R&D program of china/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Animals ; *Spodoptera/genetics ; },
abstract = {The fall armyworm (Spodoptera frugiperda) poses a substantial threat to many important crops worldwide, emphasizing the need to develop and implement advanced technologies for effective pest control. CRISPR/Cas9, derived from the bacterial adaptive immune system, is a prominent tool used for genome editing in living organisms. Due to its high specificity and adaptability, the CRISPR/Cas9 system has been used in various functional gene studies through gene knockout and applied in research to engineer phenotypes that may cause economical losses. The practical application of CRISPR/Cas9 in diverse insect orders has also provided opportunities for developing strategies for genetic pest control, such as gene drive and the precision-guided sterile insect technique (pgSIT). In this review, a comprehensive overview of the recent progress in the application of the CRISPR/Cas9 system for functional gene studies in S. frugiperda is presented. We outline the fundamental principles of applying CRISPR/Cas9 in S. frugiperda through embryonic microinjection and highlight the application of CRISPR/Cas9 in the study of genes associated with diverse biological aspects, including body color, insecticide resistance, olfactory behavior, sex determination, development, and RNAi. The ability of CRISPR/Cas9 technology to induce sterility, disrupt developmental stages, and influence mating behaviors illustrates its comprehensive roles in pest management strategies. Furthermore, this review addresses the limitations of the CRISPR/Cas9 system in studying gene function in S. frugiperda and explores its future potential as a promising tool for controlling this insect pest.},
}

*CRISPR-Cas Systems/genetics
*Gene Editing/methods
Animals
*Spodoptera/genetics

@article {pmid39334101,
year = {2024},
author = {Chey, YCJ and Corbett, MA and Arudkumar, J and Piltz, SG and Thomas, PQ and Adikusuma, F},
title = {CRISPR-mediated megabase-scale transgene de-duplication to generate a functional single-copy full-length humanized DMD mouse model.},
journal = {BMC biology},
volume = {22},
number = {1},
pages = {214},
pmid = {39334101},
issn = {1741-7007},
mesh = {Animals ; *Muscular Dystrophy, Duchenne/genetics/therapy ; Mice ; *Mice, Transgenic ; *Disease Models, Animal ; Humans ; *Transgenes ; *CRISPR-Cas Systems ; Gene Editing/methods ; Dystrophin/genetics ; Gene Duplication ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {BACKGROUND: The development of sequence-specific precision treatments like CRISPR gene editing therapies for Duchenne muscular dystrophy (DMD) requires sequence humanized animal models to enable the direct clinical translation of tested strategies. The current available integrated transgenic mouse model containing the full-length human DMD gene, Tg(DMD)72Thoen/J (hDMDTg), has been found to have two copies of the transgene per locus in a tail-to-tail orientation, which does not accurately simulate the true (single) copy number of the DMD gene. This duplication also complicates analysis when testing CRISPR therapy editing outcomes, as large genetic alterations and rearrangements can occur between the cut sites on the two transgenes.

RESULTS: To address this, we performed long read nanopore sequencing on hDMDTg mice to better understand the structure of the duplicated transgenes. Following that, we performed a megabase-scale deletion of one of the transgenes by CRISPR zygotic microinjection to generate a single-copy, full-length, humanized DMD transgenic mouse model (hDMDTgSc). Functional, molecular, and histological characterisation shows that the single remaining human transgene retains its function and rescues the dystrophic phenotype caused by endogenous murine Dmd knockout.

CONCLUSIONS: Our unique hDMDTgSc mouse model simulates the true copy number of the DMD gene, and can potentially be used for the further generation of DMD disease models that would be better suited for the pre-clinical assessment and development of sequence specific CRISPR therapies.},
}

Animals
*Muscular Dystrophy, Duchenne/genetics/therapy
Mice
*Mice, Transgenic
*Disease Models, Animal
Humans
*Transgenes
*CRISPR-Cas Systems
Gene Editing/methods
Dystrophin/genetics
Gene Duplication
Clustered Regularly Interspaced Short Palindromic Repeats/genetics

@article {pmid39333528,
year = {2024},
author = {Chen, Y and Wang, X and Zhang, J and Jiang, Q and Qiao, B and He, B and Yin, W and Qiao, J and Liu, Y},
title = {Split crRNA with CRISPR-Cas12a enabling highly sensitive and multiplexed detection of RNA and DNA.},
journal = {Nature communications},
volume = {15},
number = {1},
pages = {8342},
pmid = {39333528},
issn = {2041-1723},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Uterine Cervical Neoplasms/genetics/diagnosis/blood ; *MicroRNAs/blood/genetics ; Female ; DNA/genetics ; CRISPR-Associated Proteins/genetics/metabolism ; RNA/genetics/blood ; Endodeoxyribonucleases/genetics/metabolism ; Bacterial Proteins/genetics/metabolism ; Sensitivity and Specificity ; Biomarkers, Tumor/genetics/blood ; },
abstract = {The CRISPR-Cas12a system has revolutionized nucleic acid testing (NAT) with its rapid and precise capabilities, yet it traditionally required RNA pre-amplification. Here we develop rapid fluorescence and lateral flow NAT assays utilizing a split Cas12a system (SCas12a), consisting of a Cas12a enzyme and a split crRNA. The SCas12a assay enables highly sensitive, amplification-free, and multiplexed detection of miRNAs and long RNAs without complex secondary structures. It can differentiate between mature miRNA and its precursor (pre-miRNA), a critical distinction for precise biomarker identification and cancer progression monitoring. The system's specificity is further highlighted by its ability to detect DNA and miRNA point mutations. Notably, the SCas12a system can quantify the miR-21 biomarker in plasma from cervical cancer patients and, when combined with RPA, detect HPV at attomole levels in clinical samples. Together, our work presents a simple and cost-effective SCas12a-based NAT platform for various diagnostic settings.},
}

Humans
*CRISPR-Cas Systems/genetics
*Uterine Cervical Neoplasms/genetics/diagnosis/blood
*MicroRNAs/blood/genetics
Female
DNA/genetics
CRISPR-Associated Proteins/genetics/metabolism
RNA/genetics/blood
Endodeoxyribonucleases/genetics/metabolism
Bacterial Proteins/genetics/metabolism
Sensitivity and Specificity
Biomarkers, Tumor/genetics/blood

@article {pmid39333351,
year = {2024},
author = {Ye, H and Louden, M and Reinders, JAT},
title = {A novel in vivo genome editing doubled haploid system for Zea mays L.},
journal = {Nature plants},
volume = {},
number = {},
pages = {},
pmid = {39333351},
issn = {2055-0278},
abstract = {Doubled haploid (DH) technologies accelerate maize inbred development. Recently, methods using CRISPR-Cas have created gene-edited maize DH populations, albeit with relatively low editing frequencies. Restoring fertility via haploid chromosome doubling remains a critically important production constraint. Thus, improved editing and chromosome doubling outcomes are needed. Here we obtained maternally derived diploid embryos in vivo by ectopically co-expressing Zea mays BABY BOOM and cyclin D-like gene products within unfertilized egg cells. When combined with gene editing, the in vivo method enables the production of mature seed with a maternally derived, gene-edited diploid embryo without requiring in vitro tissue culture methods nor the use of a chemical chromosome doubling agent. In summary, we report a novel approach for creating gene-edited maize DH populations that we expect can accelerate genetic gain in a scalable, cost-effective manner.},
}

@article {pmid39333091,
year = {2024},
author = {O'Neill, MJ and Yang, T and Laudeman, J and Calandranis, ME and Harvey, ML and Solus, JF and Roden, DM and Glazer, AM},
title = {ParSE-seq: a calibrated multiplexed assay to facilitate the clinical classification of putative splice-altering variants.},
journal = {Nature communications},
volume = {15},
number = {1},
pages = {8320},
pmid = {39333091},
issn = {2041-1723},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism ; *Myocytes, Cardiac/metabolism/cytology ; *RNA Splicing/genetics ; *NAV1.5 Voltage-Gated Sodium Channel/genetics/metabolism ; Arrhythmias, Cardiac/genetics ; RNA Splice Sites/genetics ; CRISPR-Cas Systems/genetics ; Calibration ; High-Throughput Nucleotide Sequencing/methods ; Genetic Variation ; Introns/genetics ; HEK293 Cells ; },
abstract = {Interpreting the clinical significance of putative splice-altering variants outside canonical splice sites remains difficult without time-intensive experimental studies. To address this, we introduce Parallel Splice Effect Sequencing (ParSE-seq), a multiplexed assay to quantify variant effects on RNA splicing. We first apply this technique to study hundreds of variants in the arrhythmia-associated gene SCN5A. Variants are studied in 'minigene' plasmids with molecular barcodes to allow pooled variant effect quantification. We perform experiments in two cell types, including disease-relevant induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). The assay strongly separates known control variants from ClinVar, enabling quantitative calibration of the ParSE-seq assay. Using these evidence strengths and experimental data, we reclassify 29 of 34 variants with conflicting interpretations and 11 of 42 variants of uncertain significance. In addition to intronic variants, we show that many synonymous and missense variants disrupted RNA splicing. Two splice-altering variants in the assay also disrupt splicing and sodium current when introduced into iPSC-CMs by CRISPR-Cas9 editing. ParSE-seq provides high-throughput experimental data for RNA-splicing to support precision medicine efforts and can be readily adopted to study other loss-of-function genotype-phenotype relationships.},
}

Humans
*Induced Pluripotent Stem Cells/metabolism
*Myocytes, Cardiac/metabolism/cytology
*RNA Splicing/genetics
*NAV1.5 Voltage-Gated Sodium Channel/genetics/metabolism
Arrhythmias, Cardiac/genetics
RNA Splice Sites/genetics
CRISPR-Cas Systems/genetics
Calibration
High-Throughput Nucleotide Sequencing/methods
Genetic Variation
Introns/genetics
HEK293 Cells

@article {pmid39331467,
year = {2024},
author = {Xu, R and Ma, C and Sheng, J and Zhu, J and Wang, D and Liu, X and Wang, Q and Li, J and Qin, R and Wei, P},
title = {Engineering PE6 prime editors to efficiently insert tags in rice.},
journal = {Plant biotechnology journal},
volume = {},
number = {},
pages = {},
doi = {10.1111/pbi.14456},
pmid = {39331467},
issn = {1467-7652},
support = {32270430//National Natural Science Foundation of China/ ; 32300343//National Natural Science Foundation of China/ ; 2022YFF1002803//National Key Research and Development Program of China/ ; 2208085Y11//Natural Science Foundation of Anhui Province/ ; 2308085Y20//Natural Science Foundation of Anhui Province/ ; 2023M740020//National Postdoctoral Foundation/ ; GZC20230021//National Postdoctoral Foundation/ ; 2022AH010056//Innovative Research Team of Anhui Education/ ; 2023n06020020//Science and Technology Major Project of Anhui Province/ ; },
}

@article {pmid39328406,
year = {2024},
author = {Luo, X and Liang, R and Liang, L and Tang, A and Hou, S and Ding, J and Li, Z and Tang, X},
title = {Advancements, challenges, and future perspectives in developing feline herpesvirus 1 as a vaccine vector.},
journal = {Frontiers in immunology},
volume = {15},
number = {},
pages = {1445387},
pmid = {39328406},
issn = {1664-3224},
mesh = {Animals ; Cats ; *Genetic Vectors ; *Cat Diseases/prevention & control/immunology/virology ; Herpesviridae Infections/prevention & control/veterinary/immunology/virology ; Viral Vaccines/immunology ; Vaccine Development ; Humans ; CRISPR-Cas Systems ; Varicellovirus ; },
abstract = {As the most prevalent companion animal, cats are threatened by numerous infectious diseases and carry zoonotic pathogens such as Toxoplasma gondii and Bartonella henselae, which are the primary causes of human toxoplasmosis and cat-scratch disease. Vaccines play a crucial role in preventing and controlling the spread of diseases in both humans and animals. Currently, there are only three core vaccines available to prevent feline panleukopenia, feline herpesvirus, and feline calicivirus infections, with few vaccines available for other significant feline infectious and zoonotic diseases. Feline herpesvirus, a major component of the core vaccine, offers several advantages and a stable genetic manipulation platform, making it an ideal model for vaccine vector development to prevent and control feline infectious diseases. This paper reviews the technologies involved in the research and development of the feline herpesvirus vaccine vector, including homologous recombination, CRISPR/Cas9, and bacterial artificial chromosomes. It also examines the design and effectiveness of expressing antigens of other pathogens using the feline herpesvirus as a vaccine vector. Additionally, the paper analyzes existing technical bottlenecks and challenges, providing an outlook on its application prospects. The aim of this review is to provide a scientific basis for the research and development of feline herpesvirus as a vaccine vector and to offer new ideas for the prevention and control of significant feline infectious and zoonotic diseases.},
}

Animals
Cats
*Genetic Vectors
*Cat Diseases/prevention & control/immunology/virology
Herpesviridae Infections/prevention & control/veterinary/immunology/virology
Viral Vaccines/immunology
Vaccine Development
Humans
CRISPR-Cas Systems
Varicellovirus

@article {pmid39328243,
year = {2024},
author = {Saleem, MS and Khan, SH and Ahmad, A and Rana, IA and Naveed, ZA and Khan, AI},
title = {The 4Fs of cotton: genome editing of cotton for fiber, food, feed, and fuel to achieve zero hunger.},
journal = {Frontiers in genome editing},
volume = {6},
number = {},
pages = {1401088},
pmid = {39328243},
issn = {2673-3439},
abstract = {Cotton is globally known for its high-priority cellulose-rich natural fiber. In addition to providing fiber for the textile industry, it is an important source material for edible oil, livestock feed, and fuel products. Global warming and the growing population are the major challenges to the world's agriculture and the potential risks to food security. In this context, improving output traits in cotton is necessary to achieve sustainable cotton production. During the last few years, high throughput omics techniques have aided in identifying crucial genes associated with traits of cotton fiber, seed, and plant architecture which could be targeted with more precision and efficiency through the CIRPSR/Cas-mediated genome editing technique. The various CRISPR/Cas systems such as CRISPR/Cas9, CRISPR/nCas9, and CRISPR/Cas12a have been employed to edit cotton genes associated with a wide range of traits including fiber length, flowering, leaf colour, rooting, seed oil, plant architecture, gossypol content, somatic embryogenesis, and biotic and abiotic stresses tolerance, highlighting its effectiveness in editing the cotton genome. Thus, CRISPR/Cas-mediated genome editing has emerged as a technique of choice to tailor crop phenotypes for better yield potential and environmental resilience. The review covers a comprehensive analysis of cotton phenotypic traits and their improvement with the help of the latest genome editing tools to improve fiber, food, feed, and fuel-associated genes of cotton to ensure food security.},
}

@article {pmid39303817,
year = {2024},
author = {Falah, G and Sharvit, L and Atzmon, G},
title = {CRISPR-Cas9 mediated d3GHR knockout in HEK293 cells: Revealing the longevity associated isoform stress resilience.},
journal = {Experimental gerontology},
volume = {196},
number = {},
pages = {112586},
doi = {10.1016/j.exger.2024.112586},
pmid = {39303817},
issn = {1873-6815},
mesh = {Humans ; *CRISPR-Cas Systems ; *Longevity/genetics ; HEK293 Cells ; *Receptors, Somatotropin/genetics/metabolism ; *Protein Isoforms/genetics ; Signal Transduction ; Gene Knockout Techniques ; Cell Survival ; Stress, Physiological ; Gene Editing/methods ; },
abstract = {The Growth Hormone Receptor (GHR) gene encodes a protein that is essential for mediating the biological effects of growth hormone (GH). A series of molecular events are set off when GH binds to its receptor, resulting in a variety of physiological reactions linked to development, growth, and metabolism. Recently a particular genetic variation, within the GHR gene that is labeled as the "d3GHR," which lacks exon 3 was associated with longevity. This specific deletion isoform was connected to changes in the structure of the GHR protein, which may have an impact on the GHR's function. To test in vitro the advantage of the d3 carrier that may link to longevity, we employed the CRISPR/Cas9 technique to produce two isoforms: the homozygotes isoform (d3/d3) and the heterozygotes isoform (d3/fl) using HEK293 cell line. The CRISPR editing effectiveness was >85 %, indicating that we had successfully built the Cas9-gRNA complex that is appropriate for the GHR gene. The viability of the resulted isoform cells was examined under three environmental stressors that mimic some aging processes. In addition, we examined the GHR signaling pathway by selecting potential downstream genes in the GHR signaling cascade. The results show that heterozygotes cells demonstrated higher survival rates under UV radiation compared with the WT cells (87 % compared with 67 % for the WT cells when exposed to 2 min of UV radiation), and in fasting conditions, the d3GHR cells showed a 15 % greater viability than the WT cells. Moreover, the baseline expression levels (without intervention) of the IGF1 and JAK/STAT genes signaling pathways significantly declined in the homozygotes cells compared with the WT (p < 0.05). This noteworthy finding might offer a practical approach to test illness prevention and give the scientific community critical new insights on mechanism associated with lifespan.},
}

Humans
*CRISPR-Cas Systems
*Longevity/genetics
HEK293 Cells
*Receptors, Somatotropin/genetics/metabolism
*Protein Isoforms/genetics
Signal Transduction
Gene Knockout Techniques
Cell Survival
Stress, Physiological
Gene Editing/methods

@article {pmid39327211,
year = {2024},
author = {Morneau, Z and Moineau, S},
title = {The double play of a phage HTH regulator.},
journal = {Trends in microbiology},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.tim.2024.09.005},
pmid = {39327211},
issn = {1878-4380},
abstract = {Bacteriophages use anti-CRISPR (Acr) proteins to inhibit CRISPR-Cas systems. The expression of Acr is regulated by anti-CRISPR-associated (Aca) proteins, which are helix-turn-helix (HTH) repressors that bind DNA. Recently, Birkholz et al. discovered that an Aca can also repress Acr expression by binding RNA, revealing a new function for HTH repressors.},
}

@article {pmid39324814,
year = {2024},
author = {Hall, AN and Hall, BW and Kinney, KJ and Olsen, GG and Banta, AB and Noguera, DR and Donohue, TJ and Peters, JM},
title = {Tools for genetic engineering and gene expression control in Novosphingobium aromaticivorans and Rhodobacter sphaeroides.},
journal = {Applied and environmental microbiology},
volume = {},
number = {},
pages = {e0034824},
doi = {10.1128/aem.00348-24},
pmid = {39324814},
issn = {1098-5336},
abstract = {Alphaproteobacteria have a variety of cellular and metabolic features that provide important insights into biological systems and enable biotechnologies. For example, some species are capable of converting plant biomass into valuable biofuels and bioproducts that have the potential to contribute to the sustainable bioeconomy. Among the Alphaproteobacteria, Novosphingobium aromaticivorans, Rhodobacter sphaeroides, and Zymomonas mobilis show promise as organisms that can be engineered to convert extracted plant lignin or sugars into bioproducts and biofuels. Genetic manipulation of these bacteria is needed to introduce engineered pathways and modulate expression of native genes with the goal of enhancing bioproduct output. Although recent work has expanded the genetic toolkit for Z. mobilis, N. aromaticivorans and R. sphaeroides still need facile, reliable approaches to deliver genetic payloads to the genome and to control gene expression. Here, we expand the platform of genetic tools for N. aromaticivorans and R. sphaeroides to address these issues. We demonstrate that Tn7 transposition is an effective approach for introducing engineered DNA into the chromosome of N. aromaticivorans and R. sphaeroides. We screen a synthetic promoter library to identify isopropyl β-D-1-thiogalactopyranoside-inducible promoters with regulated activity in both organisms (up to ~15-fold induction in N. aromaticivorans and ~5-fold induction in R. sphaeroides). Combining Tn7 integration with promoters from our library, we establish CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) interference systems for N. aromaticivorans and R. sphaeroides (up to ~10-fold knockdown in N. aromaticivorans and R. sphaeroides) that can target essential genes and modulate engineered pathways. We anticipate that these systems will greatly facilitate both genetic engineering and gene function discovery efforts in these species and other Alphaproteobacteria.IMPORTANCEIt is important to increase our understanding of the microbial world to improve health, agriculture, the environment, and biotechnology. For example, building a sustainable bioeconomy depends on the efficient conversion of plant material to valuable biofuels and bioproducts by microbes. One limitation in this conversion process is that microbes with otherwise promising properties for conversion are challenging to genetically engineer. Here we report genetic tools for Novosphingobium aromaticivorans and Rhodobacter sphaeroides that add to the burgeoning set of tools available for genome engineering and gene expression in Alphaproteobacteria. Our approaches allow straightforward insertion of engineered pathways into the N. aromaticivorans or R. sphaeroides genome and control of gene expression by inducing genes with synthetic promoters or repressing genes using CRISPR interference. These tools can be used in future work to gain additional insight into these and other Alphaproteobacteria and to aid in optimizing yield of biofuels and bioproducts.},
}

@article {pmid39324802,
year = {2024},
author = {Tang, Z and Gao, M and Gong, F and Shan, X and Yang, Y and Zhang, Y and Chen, L and Wang, F and Ji, X and Zhou, F and He, Z},
title = {Quantum Dot Reporters Designed for CRISPR-Based Detection of Viral Nucleic Acids.},
journal = {Analytical chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.analchem.4c03541},
pmid = {39324802},
issn = {1520-6882},
abstract = {Diagnostic methods based on CRISPR technology have shown great potential due to their highly specific, efficient, and sensitive detection capabilities. Although the majority of the current studies rely on fluorescent dye-quencher reporters, the limitations of fluorescent dyes, such as poor photostability and small Stokes shifts, urgently necessitate the optimization of reporters. In this study, we developed innovative quantum dot (QD) reporters for the CRISPR/Cas systems, which not only leveraged the advantages of high photoluminescence quantum yield and large Stokes shifts of QDs but were also easily synthesized through a simple one-step hydrothermal method. Based on the trans-cleavage characteristics of Cas12a and Cas13a, two types of QD reporters were designed, the short DNA strand and the hybridization-based QD reporters, achieving the detection of DNA and RNA at the pM level, respectively, and validating the performance in the analysis of clinical samples. Furthermore, based on the unique property of QDs that allowed multicolor emission under one excitation, the application potential for simultaneous detection of diseases was further investigated. Taken together, this work proposed novel QD reporters that could be applied to the various CRISPR/Cas systems, providing a new toolbox to expand the diagnosis of bioanalytical and biomedical fields.},
}

@article {pmid39283984,
year = {2024},
author = {Zhao, R and Xiao, Y and Tang, Y and Lu, B and Li, B},
title = {Label-Free and Universal CRISPR/Cas12a-Based Detection Platform for Nucleic Acid Biomarkers.},
journal = {ACS sensors},
volume = {9},
number = {9},
pages = {4803-4810},
doi = {10.1021/acssensors.4c01233},
pmid = {39283984},
issn = {2379-3694},
mesh = {*CRISPR-Cas Systems/genetics ; *G-Quadruplexes ; Humans ; DNA, Single-Stranded/chemistry ; Biomarkers/analysis ; MicroRNAs/analysis ; CRISPR-Associated Proteins/chemistry ; Biosensing Techniques/methods ; Endodeoxyribonucleases/chemistry ; DNA/chemistry/genetics ; Protoporphyrins/chemistry ; Bacterial Proteins ; },
abstract = {CRISPR/Cas12a has been widely used in molecular diagnostics due to its excellent trans-cleavage activity. However, conventional reporters, such as F/Q-labeled single-stranded DNA (ssDNA) reporters, enzyme-labeled reporters, and spherical nucleic acid reporters, require complex modification or labeling processes. In this study, we have developed a rapid, universal, and label-free CRISPR/Cas12a-based biomarker detection platform via designing a G-quadruplex (G4) containing a hairpin structure as the reporter. The hairpin loop design of hairpin G4 improves the cleavage efficiency of Cas12a and the signal strength of the G4 binding ligand. Meanwhile, the incorporation of a G4 binding dye (protoporphyrin IX) eliminates the need for complex modifications. The CRISPR-hairpin G4 detection platform is capable of detecting ssDNA, double-stranded DNA, genetic RNAs, and miRNAs. Moreover, this platform achieves label-free detection in clinical samples, demonstrating its practical applicability and efficiency.},
}

*CRISPR-Cas Systems/genetics
*G-Quadruplexes
Humans
DNA, Single-Stranded/chemistry
Biomarkers/analysis
MicroRNAs/analysis
CRISPR-Associated Proteins/chemistry
Biosensing Techniques/methods
Endodeoxyribonucleases/chemistry
DNA/chemistry/genetics
Protoporphyrins/chemistry
Bacterial Proteins

@article {pmid39145521,
year = {2024},
author = {Jones-Wonni, B and Kelkar, AH and Achebe, MO},
title = {A Review of Gene Therapies for Hemoglobinopathies.},
journal = {Hemoglobin},
volume = {48},
number = {3},
pages = {141-152},
doi = {10.1080/03630269.2024.2369534},
pmid = {39145521},
issn = {1532-432X},
mesh = {Humans ; *Genetic Therapy/methods ; *Hemoglobinopathies/therapy/genetics ; *Gene Editing ; CRISPR-Cas Systems ; },
abstract = {Due to the significant morbidity and mortality of hemoglobinopathies, curative options have long been pursued. The overall goal of gene therapy is to modify a patient's own hematopoietic stem cells to overcome the deleterious effects of the underlying genetic defect by gene addition, gene editing, or gene silencing. Gene addition incorporates genes with superior function than the abnormal gene; gene editing takes advantage of molecular tools such as zinc finger proteins, Transcription Activator-Like Effector Nucleases and Clustered Regularly Interspaced Short Palindromic Repeats coupled with Cas9 proteins (CRISPR-Cas9) which allow for sequence-specific breaks in DNA that disrupt gene function; and gene silencing suppresses gene expression by interference with mRNA transcription/protein translation or epigenetic modification. The majority of gene therapy strategies for hemoglobinopathies have targeted erythroid-specific BCL11A, a major regulator of fetal hemoglobin repression at the gamma-globin locus, in the normal fetal-to-adult hemoglobin switch that occurs shortly after birth. Other goals have involved the incorporation of anti-sickling globins, such as β[T87Q] or βAS3. Landmark clinical trials of gene therapy in transfusion-dependent thalassemia and sickle cell disease have shown remarkable efficacy and acceptable safety and culminated in recent regulatory approvals of gene therapy for both diseases in Europe and the United States.},
}

Humans
*Genetic Therapy/methods
*Hemoglobinopathies/therapy/genetics
*Gene Editing
CRISPR-Cas Systems

@article {pmid38969862,
year = {2024},
author = {Yan, H and Tang, W},
title = {Programmed RNA editing with an evolved bacterial adenosine deaminase.},
journal = {Nature chemical biology},
volume = {20},
number = {10},
pages = {1361-1370},
pmid = {38969862},
issn = {1552-4469},
mesh = {*Adenosine Deaminase/metabolism/genetics ; *RNA Editing ; Humans ; *Escherichia coli/genetics/metabolism ; Open Reading Frames ; Interferon Regulatory Factors/genetics/metabolism ; Adenosine/analogs & derivatives/metabolism/chemistry ; Inosine/metabolism/genetics ; CRISPR-Cas Systems ; HEK293 Cells ; },
abstract = {Programmed RNA editing presents an attractive therapeutic strategy for genetic disease. In this study, we developed bacterial deaminase-enabled recoding of RNA (DECOR), which employs an evolved Escherichia coli transfer RNA adenosine deaminase, TadA8e, to deposit adenosine-to-inosine editing to CRISPR-specified sites in the human transcriptome. DECOR functions in a variety of cell types, including human lung fibroblasts, and delivers on-target activity similar to ADAR-overexpressing RNA-editing platforms with 88% lower off-target effects. High-fidelity DECOR further reduces off-target effects to basal level. We demonstrate the clinical potential of DECOR by targeting Van der Woude syndrome-causing interferon regulatory factor 6 (IRF6) insufficiency. DECOR-mediated RNA editing removes a pathogenic upstream open reading frame (uORF) from the 5' untranslated region of IRF6 and rescues primary ORF expression from 12.3% to 36.5%, relative to healthy transcripts. DECOR expands the current portfolio of effector proteins and opens new territory in programmed RNA editing.},
}

*Adenosine Deaminase/metabolism/genetics
*RNA Editing
Humans
*Escherichia coli/genetics/metabolism
Open Reading Frames
Interferon Regulatory Factors/genetics/metabolism
Adenosine/analogs & derivatives/metabolism/chemistry
Inosine/metabolism/genetics
CRISPR-Cas Systems
HEK293 Cells

@article {pmid38952047,
year = {2024},
author = {Zhu, X and Luo, H and Yu, X and Lv, H and Su, L and Zhang, K and Wu, J},
title = {Genome-Wide CRISPRi Screening of Key Genes for Recombinant Protein Expression in Bacillus Subtilis.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {11},
number = {33},
pages = {e2404313},
doi = {10.1002/advs.202404313},
pmid = {38952047},
issn = {2198-3844},
support = {2019YFA0706900//National Key Research and Development Program of China/ ; DWKF20210004//Shenzhen Institute of Synthetic Biology Scientific Research Program/ ; JUSRP122012//Fundamental Research Funds for the Central Universities/ ; BE2023686//Natural Science Foundation of Jiangsu Province/ ; 32272264//National Natural Science Foundation of China/ ; },
mesh = {*Bacillus subtilis/genetics/metabolism ; *Recombinant Proteins/genetics/metabolism ; Metabolic Engineering/methods ; CRISPR-Cas Systems/genetics ; Genome, Bacterial/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {Bacillus subtilis is an industrially important microorganism that is often used as a microbial cell factory for the production of recombinant proteins due to its food safety, rapid growth, and powerful secretory capacity. However, the lack of data on functional genes related to recombinant protein production has hindered the further development of B. subtilis cell factories. Here, a strategy combining genome-wide CRISPRi screening and targeted CRISPRa activation to enhance recombinant protein expression is proposed. First, a CRISPRi library covering a total of 4225 coding genes (99.7%) in the B. subtilis genome and built the corresponding high-throughput screening methods is constructed. Twelve key genes for recombinant protein expression are identified, including targets without relevant functional annotations. Meanwhile, the transcription of recombinant protein genes by CRISPRa is up-regulated. These screened or selected genes can be easily applied to metabolic engineering by constructing sgRNA arrays. The relationship between differential pathways and recombinant protein expression in engineered strains by transcriptome analysis is also revealed. High-density fermentation and generalisability validation results prove the reliability of the strategy. This method can be extended to other industrial hosts to support functional gene annotation and the design of novel cell factories.},
}

*Bacillus subtilis/genetics/metabolism
*Recombinant Proteins/genetics/metabolism
Metabolic Engineering/methods
CRISPR-Cas Systems/genetics
Genome, Bacterial/genetics
Clustered Regularly Interspaced Short Palindromic Repeats/genetics

@article {pmid38943253,
year = {2024},
author = {Ruan, W and Xu, S and An, Y and Cui, Y and Liu, Y and Wang, Y and Ismail, M and Liu, Y and Zheng, M},
title = {Brain-Targeted Cas12a Ribonucleoprotein Nanocapsules Enable Synergetic Gene Co-Editing Leading to Potent Inhibition of Orthotopic Glioblastoma.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {11},
number = {33},
pages = {e2402178},
doi = {10.1002/advs.202402178},
pmid = {38943253},
issn = {2198-3844},
support = {52073079//National Natural Science Foundation of China/ ; 52373133//National Natural Science Foundation of China/ ; U20A20338//National Natural Science Foundation of China/ ; K23036Y//Henan Natural Science Foundation/ ; 232300421044//Henan Natural Science Foundation/ ; //Henan University Double First-Class Foundation/ ; 2021C04019//Key Research and Development Program of Zhejiang Province/ ; },
mesh = {*Glioblastoma/genetics/therapy/metabolism ; Animals ; Mice ; *Nanocapsules ; *Gene Editing/methods ; *Brain Neoplasms/genetics/therapy/metabolism ; *CRISPR-Cas Systems/genetics ; Humans ; *Polo-Like Kinase 1 ; Disease Models, Animal ; Cell Line, Tumor ; Protein Serine-Threonine Kinases/genetics/metabolism ; Proto-Oncogene Proteins/genetics/metabolism ; ErbB Receptors/genetics/metabolism ; Cell Cycle Proteins/genetics/metabolism ; Mice, Nude ; CRISPR-Associated Proteins/genetics/metabolism ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {Gene-editing technology shows great potential in glioblastoma (GBM) therapy. Due to the complexity of GBM pathogenesis, a single gene-editing-based therapy is unlikely to be successful; therefore, a multi-gene knockout strategy is preferred for effective GBM inhibition. Here, a non-invasive, biodegradable brain-targeted CRISPR/Cas12a nanocapsule is used that simultaneously targeted dual oncogenes, EGFR and PLK1, for effective GBM therapy. This cargo nanoencapsulation technology enables the CRISPR/Cas12a system to achieve extended blood half-life, efficient blood-brain barrier (BBB) penetration, active tumor targeting, and selective release. In U87MG cells, the combinatorial gene editing system resulted in 61% and 33% knockout of EGFR and PLK1, respectively. Following systemic administration, the CRISPR/Cas12a system demonstrated promising brain tumor accumulation that led to extensive EGFR and PLK1 gene editing in both U87MG and patient-derived GSC xenograft mouse models with negligible off-target gene editing detected through NGS. Additionally, CRISPR/Cas12a nanocapsules that concurrently targeted the EGFR and PLK1 oncogenes showed superior tumor growth suppression and significantly improved the median survival time relative to nanocapsules containing single oncogene knockouts, signifying the potency of the multi-oncogene targeting strategy. The findings indicate that utilization of the CRISPR/Cas12a combinatorial gene editing technique presents a practical option for gene therapy in GBM.},
}

*Glioblastoma/genetics/therapy/metabolism
Animals
Mice
*Nanocapsules
*Gene Editing/methods
*Brain Neoplasms/genetics/therapy/metabolism
*CRISPR-Cas Systems/genetics
Humans
*Polo-Like Kinase 1
Disease Models, Animal
Cell Line, Tumor
Protein Serine-Threonine Kinases/genetics/metabolism
Proto-Oncogene Proteins/genetics/metabolism
ErbB Receptors/genetics/metabolism
Cell Cycle Proteins/genetics/metabolism
Mice, Nude
CRISPR-Associated Proteins/genetics/metabolism
Bacterial Proteins
Endodeoxyribonucleases

@article {pmid37713087,
year = {2023},
author = {Xu, Y and Wang, Y and Liang, FS},
title = {Site-Specific m[6] A Erasing via Conditionally Stabilized CRISPR-Cas13b Editor.},
journal = {Angewandte Chemie (International ed. in English)},
volume = {62},
number = {43},
pages = {e202309291},
pmid = {37713087},
issn = {1521-3773},
support = {R21 CA247638/CA/NCI NIH HHS/United States ; R21CA247638/NH/NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Adenosine/analogs & derivatives/chemistry/metabolism ; Gene Editing ; Humans ; },
abstract = {N6-methyladenosine (m[6] A) on RNAs plays an important role in regulating various biological processes and CRIPSR technology has been employed for programmable m[6] A editing. However, the bulky size of CRISPR protein and constitutively expressed CRISPR/RNA editing enzymes can interfere with the native function of target RNAs and cells. Herein, we reported a conditional m[6] A editing platform (FKBP*-dCas13b-ALK) based on a ligand stabilized dCas13 editor. The inducible expression of this m[6] A editing system was achieved by adding or removing the Shield-1 molecule. We further demonstrated that the targeted recruitment of dCas13b-m[6] A eraser fusion protein and site-specific m[6] A erasing were achieved under the control of Shield-1. Moreover, the release and degradation of dCas13b fusion protein occurred faster than the restoration of m[6] A on the target RNAs after Shield-1 removal, which provides an ideal opportunity to study the m[6] A function with minimal steric interference from bulky dCas13b fusion protein.},
}

*CRISPR-Cas Systems/genetics
*Adenosine/analogs & derivatives/chemistry/metabolism
Gene Editing
Humans

@article {pmid39324412,
year = {2024},
author = {Deng, R and Bai, Y and Liu, Y and Lu, Y and Zhao, Z and Deng, Y and Yang, H},
title = {Correction: DNAzyme-activated CRISPR/Cas assay for sensitive and one-pot detection of lead contamination.},
journal = {Chemical communications (Cambridge, England)},
volume = {},
number = {},
pages = {},
doi = {10.1039/d4cc90326a},
pmid = {39324412},
issn = {1364-548X},
abstract = {Correction for 'DNAzyme-activated CRISPR/Cas assay for sensitive and one-pot detection of lead contamination' by Ruijie Deng et al., Chem. Commun., 2024, 60, 5976-5979, https://doi.org/10.1039/D4CC01852D.},
}

@article {pmid39324194,
year = {2024},
author = {Khmeleva, SA and Ptitsyn, KG and Kurbatov, LK and Timoshenko, OS and Suprun, EV and Radko, SP and Lisitsa, AV},
title = {Biosensing platforms for DNA diagnostics based on CRISPR/Cas nucleases: towards the detection of nucleic acids at the level of single molecules in non-laboratory settings.},
journal = {Biomeditsinskaia khimiia},
volume = {70},
number = {5},
pages = {287-303},
doi = {10.18097/PBMC20247005287},
pmid = {39324194},
issn = {2310-6972},
mesh = {*CRISPR-Cas Systems ; Humans ; *Biosensing Techniques/methods ; *Nucleic Acid Amplification Techniques/methods ; DNA/analysis/genetics ; },
abstract = {The use of CRISPR/Cas nucleases for the development of DNA diagnostic systems in out-of-laboratory conditions (point-of-need testing, PONT) has demonstrated rapid growth in the last few years, starting with the appearance in 2017-2018 of the first diagnostic platforms known as DETECTR and SHERLOCK. The platforms are based on a combination of methods of nucleic acid isothermal amplification with selective CRISPR/Cas detection of target amplicons. This significantly improves the sensitivity and specificity of PONT, making them comparable with or even superior to the sensitivity and specificity of polymerase chain reaction, considered as the "gold standard" of DNA diagnostics. The review considers modern approaches to the coupling of CRISPR/Cas detection using Cas9, Cas12a, Cas12b, Cas13a, Cas14, and Cas3 nucleases to various methods of nucleic acid isothermal amplification, with an emphasis on works in which sensitivity at the level of single molecules (attomolar and subattomolar concentrations of the target) is achieved. The properties of CRISPR/Cas nucleases used for targeted DNA diagnostics and the features of methods of nucleic acid isothermal amplification are briefly considered in the context of the development of diagnostic biosensing platforms. Special attention is paid to the most promising directions for the development of DNA diagnostics using CRISPR/Cas nuclease.},
}

*CRISPR-Cas Systems
Humans
*Biosensing Techniques/methods
*Nucleic Acid Amplification Techniques/methods
DNA/analysis/genetics

@article {pmid39319719,
year = {2024},
author = {Song, Y and Zhou, J and Ni, Y},
title = {[Construction of a Kluyveromyces lactis strain with multi-copy integration for enhanced bovine chymosin production by CRISPR/Cas9 and UV mutagenesis].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {40},
number = {9},
pages = {2983-2997},
doi = {10.13345/j.cjb.240183},
pmid = {39319719},
issn = {1872-2075},
mesh = {*Chymosin/genetics/metabolism/biosynthesis ; *Kluyveromyces/genetics/metabolism ; Animals ; Cattle ; *CRISPR-Cas Systems ; *Ultraviolet Rays ; *Mutagenesis ; Recombinant Proteins/genetics/biosynthesis/metabolism ; },
abstract = {Bovine chymosin is an essential food enzyme widely used in cheese production in the dairy industry. This study used a codon-optimized prochymosin gene to construct an expression cassette for extracellular expression of bovine chymosin in Kluyveromyces lactis. After integration of the prochymosin gene into the host cell genome, the single-copy integration strain KLUcym showed the clotting activity of 40 U/mL in a shake flask. The CRISPR/Cas9 system was employed to delete amdS and construct the double-copy integration strain and triple-copy integration strain, which achieved the clotting activities of 70 U/mL and 78 U/mL in shake flasks, separately. Subsequently, multiple rounds of UV mutagenesis were performed on the double-copy strain KLUcym[D], and a recombinant K. lactis strain with a high yield of bovine chymosin was obtained. This strain achieved the clotting activity of 270 U/mL in a shake flask and 600 U/mL in a 5 L bioreactor after 76 h. In summary, we construct a strain KLUcym[D]-M2 for high production of bovine chymosin, which lays a foundation of industrial fermentation.},
}

*Chymosin/genetics/metabolism/biosynthesis
*Kluyveromyces/genetics/metabolism
Animals
Cattle
*CRISPR-Cas Systems
*Ultraviolet Rays
*Mutagenesis
Recombinant Proteins/genetics/biosynthesis/metabolism

@article {pmid39316448,
year = {2024},
author = {Wang, Y and Shi, YN and Xiang, H and Shi, YM},
title = {Exploring nature's battlefield: organismic interactions in the discovery of bioactive natural products.},
journal = {Natural product reports},
volume = {},
number = {},
pages = {},
doi = {10.1039/d4np00018h},
pmid = {39316448},
issn = {1460-4752},
abstract = {Covering: up to March 2024.Microbial natural products have historically been a cornerstone for the discovery of therapeutic agents. Advanced (meta)genome sequencing technologies have revealed that microbes harbor far greater biosynthetic capabilities than previously anticipated. However, despite the application of CRISPR/Cas-based gene editing and high-throughput technologies to activate silent biosynthetic gene clusters, the rapid identification of new natural products has not led to a proportional increase in the discovery rate of lead compounds or drugs. A crucial issue in this gap may be insufficient knowledge about the inherent biological and physiological functions of microbial natural products. Addressing this gap necessitates recognizing that the generation of functional natural products is deeply rooted in the interactions between the producing microbes and other (micro)organisms within their ecological contexts, an understanding that is essential for harnessing their potential therapeutic benefits. In this review, we highlight the discovery of functional microbial natural products from diverse niches, including those associated with humans, nematodes, insects, fungi, protozoa, plants, and marine animals. Many of these findings result from an organismic-interaction-guided strategy using multi-omic approaches. The current importance of this topic lies in its potential to advance drug discovery in an era marked by increasing antimicrobial resistance.},
}

@article {pmid39292561,
year = {2024},
author = {Langley, J and Baudrier, L and Billon, P},
title = {A protocol for the detection of precision genome editing in human cells using One-pot DTECT.},
journal = {STAR protocols},
volume = {5},
number = {3},
pages = {103307},
pmid = {39292561},
issn = {2666-1667},
mesh = {Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Genome, Human/genetics ; Polymerase Chain Reaction/methods ; },
abstract = {Prime editing is a highly versatile CRISPR-based genome editing technology that allows for the precise installation of desired genetic variants. This protocol describes how to use One-pot DTECT to assess prime editing efficiency in human cells. Key steps include conducting prime editing, extracting genomic DNA, performing AcuI-tagging PCR, capturing genetic signatures, and detecting captured signatures through qualitative, quantitative, and visual methods. One-pot DTECT enables same-day detection of targeted genetic signatures introduced by precision genome editing technologies using off-the-shelf reagents. For complete details on the use and execution of this protocol, please refer to Baudrier et al.[1].},
}

Humans
*Gene Editing/methods
*CRISPR-Cas Systems/genetics
Genome, Human/genetics
Polymerase Chain Reaction/methods

@article {pmid39269899,
year = {2024},
author = {Noguchi, Y and Maruoka, M and Suzuki, J},
title = {Protocol for in vivo CRISPR screening targeting murine testicular cells.},
journal = {STAR protocols},
volume = {5},
number = {3},
pages = {103306},
pmid = {39269899},
issn = {2666-1667},
mesh = {Animals ; Male ; Mice ; *Testis/cytology/metabolism ; *CRISPR-Cas Systems/genetics ; *Spermatogenesis/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {In vivo genome-wide screening elucidates tissue-specific molecular events. Here, we present a protocol for an in vivo genome-wide CRISPR-Cas9 single-guide RNA (sgRNA) library screening technique optimized for mouse testicular cells to investigate spermatogenesis. We describe steps for virus injection, sperm sorting, and primase-based whole-genome amplification. We then detail procedures for library reconstruction using a "revival screening" technique. Our approach reveals intricate spermatogenesis processes and is adaptable for diverse tissue-specific studies. For complete details on the use and execution of this protocol, please refer to Noguchi et al.[1].},
}

Animals
Male
Mice
*Testis/cytology/metabolism
*CRISPR-Cas Systems/genetics
*Spermatogenesis/genetics
RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid39243376,
year = {2024},
author = {Negueruela, J and Vandenbempt, V and Talamantes, S and Ribeiro-Costa, F and Nunes, M and Dias, A and Bansal, M and Gurzov, EN},
title = {Protocol for CRISPR-Cas12a genome editing of protein tyrosine phosphatases in human pluripotent stem cells and functional β-like cell generation.},
journal = {STAR protocols},
volume = {5},
number = {3},
pages = {103297},
pmid = {39243376},
issn = {2666-1667},
mesh = {Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Pluripotent Stem Cells/cytology/metabolism ; *Insulin-Secreting Cells/cytology/metabolism ; *Protein Tyrosine Phosphatases/genetics/metabolism ; Mice ; Animals ; Cell Differentiation/genetics ; Human Embryonic Stem Cells/cytology/metabolism ; },
abstract = {Gene editing of human pluripotent stem cells is a promising approach for developing targeted gene therapies for metabolic diseases. Here, we present a protocol for generating a CRISPR-Cas12a gene knockout of protein tyrosine phosphatases in human embryonic stem cells. We describe steps for differentiating the edited clones into pancreatic islet-like spheroids rich in β-like cells. We then detail procedures for implanting these spheroids under the murine kidney capsule for in vivo maturation.},
}

Humans
*Gene Editing/methods
*CRISPR-Cas Systems/genetics
*Pluripotent Stem Cells/cytology/metabolism
*Insulin-Secreting Cells/cytology/metabolism
*Protein Tyrosine Phosphatases/genetics/metabolism
Mice
Animals
Cell Differentiation/genetics
Human Embryonic Stem Cells/cytology/metabolism

@article {pmid39222044,
year = {2024},
author = {Handgretinger, R and Mezger, M},
title = {An evaluation of exagamglogene autotemcel for the treatment of sickle cell disease and transfusion-dependent beta-thalassaemia.},
journal = {Expert opinion on biological therapy},
volume = {24},
number = {9},
pages = {883-888},
doi = {10.1080/14712598.2024.2399134},
pmid = {39222044},
issn = {1744-7682},
mesh = {Humans ; *beta-Thalassemia/therapy/genetics ; *Anemia, Sickle Cell/therapy/genetics ; *Genetic Therapy/adverse effects ; CRISPR-Cas Systems ; Hematopoietic Stem Cell Transplantation ; Child ; Blood Transfusion ; Adolescent ; Adult ; Young Adult ; Fetal Hemoglobin/genetics ; },
abstract = {INTRODUCTION: Sickle cell disease is the most common hereditary hemoglobinopathy followed by beta-thalassemia. Until recently, allogeneic stem cell transplantation was the only curative approach. Based on the Crispr-Cas9-technology enabling targeting specific genes of interest, fetal hemoglobin which is normally shut-off after birth can be switched on and sufficient levels can alleviate symptoms in sickle cell disease and avoid transfusions in beta-thalassemia. Two first-in-human clinical studies in sickle cell disease and beta-thalassemia aiming to increase the level of fetal hemoglobin by using Crispr-Cas9 to modify autologous hematopoietic stem cells in patients aged 12-35 years have proved safety and efficacy and have shown promising clinical outcomes.

AREAS COVERED: The paper summarizes the outcome of the results of the two recently published clinical studies and compares them with the other available curative approaches.

EXPERT OPINION: Based on the currently available safety and efficacy data of the two published clinical results on gene therapy with Crispr-Cas9 modified autologous stem cells (exagamglogene autotemcel), it can be anticipated that this approach will add significantly to the therapeutic options for patients with sickle cell disease and beta-thalassemia and can be considered for all patients above 12 years of age independent of a suitable allogeneic stem cell donor.},
}

Humans
*beta-Thalassemia/therapy/genetics
*Anemia, Sickle Cell/therapy/genetics
*Genetic Therapy/adverse effects
CRISPR-Cas Systems
Hematopoietic Stem Cell Transplantation
Child
Blood Transfusion
Adolescent
Adult
Young Adult
Fetal Hemoglobin/genetics

@article {pmid39180746,
year = {2024},
author = {Chen, L and Qiao, B and Li, H and Zhang, P and Li, Q},
title = {Protocol for generation of CRISPR-Cas9-mediated specific genomic insertion of P2A-Gal4 to reveal endogenous gene expression in Drosophila.},
journal = {STAR protocols},
volume = {5},
number = {3},
pages = {103184},
pmid = {39180746},
issn = {2666-1667},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Drosophila melanogaster/genetics ; *Drosophila Proteins/genetics/metabolism ; Transcription Factors/genetics/metabolism ; Gene Expression/genetics ; Animals, Genetically Modified ; Mutagenesis, Insertional/methods/genetics ; Homologous Recombination/genetics ; },
abstract = {Generating a transgene with a reporter inserted into the genome helps us study endogenous gene expression patterns in model organisms. Here, using Drosophila melanogaster, we present a protocol for generating a P2A-Gal4 insertion through CRISPR-Cas9-mediated homology recombination. We describe the design strategy, steps for constructing the injection plasmids, and the fly-cross scheme for screening the transformants from the G0 generation. This protocol can also be applied to introduce mutations or various genetic tools into the fly genome. For complete details on the use and execution of this protocol, please refer to Li et al.[1].},
}

Animals
*CRISPR-Cas Systems/genetics
*Drosophila melanogaster/genetics
*Drosophila Proteins/genetics/metabolism
Transcription Factors/genetics/metabolism
Gene Expression/genetics
Animals, Genetically Modified
Mutagenesis, Insertional/methods/genetics
Homologous Recombination/genetics

@article {pmid39178116,
year = {2024},
author = {Zhang, X and Duan, Y and Li, S and Zhang, Z and Peng, L and Ma, X and Wang, T and Xiang, S and Chen, G and Zhou, D and Lu, D and Qian, M and Wang, Z},
title = {CRISPR screening identifies PRMT1 as a key pro-ferroptotic gene via a two-layer regulatory mechanism.},
journal = {Cell reports},
volume = {43},
number = {9},
pages = {114662},
doi = {10.1016/j.celrep.2024.114662},
pmid = {39178116},
issn = {2211-1247},
mesh = {*Protein-Arginine N-Methyltransferases/metabolism/genetics ; Animals ; *Ferroptosis/genetics ; Humans ; Mice ; CRISPR-Cas Systems/genetics ; Mice, Inbred C57BL ; Glutathione/metabolism ; Amino Acid Transport System y+/metabolism/genetics ; Male ; Liver/metabolism ; Repressor Proteins/metabolism/genetics ; HEK293 Cells ; },
abstract = {Ferroptosis is a form of nonapoptotic cell death characterized by iron-dependent peroxidation of polyunsaturated phospholipids. However, much remains unknown about the regulators of ferroptosis. Here, using CRISPR-Cas9-mediated genetic screening, we identify protein arginine methyltransferase 1 (PRMT1) as a crucial promoter of ferroptosis. We find that PRMT1 decreases the expression of solute carrier family 7 member 11 (SLC7A11) to limit the abundance of intracellular glutathione (GSH). Moreover, we show that PRMT1 interacts with ferroptosis suppressor protein 1 (FSP1), a GSH-independent ferroptosis suppressor, to inhibit the membrane localization and enzymatic activity of FSP1 through arginine dimethylation at R316, thus reducing CoQ10H2 content and inducing ferroptosis sensitivity. Importantly, genetic depletion or pharmacological inhibition of PRMT1 in mice prevents ferroptotic events in the liver and improves the overall survival under concanavalin A (ConA) exposure. Hence, our findings suggest that PRMT1 is a key regulator of ferroptosis and a potential target for antiferroptosis therapeutics.},
}

*Protein-Arginine N-Methyltransferases/metabolism/genetics
Animals
*Ferroptosis/genetics
Humans
Mice
CRISPR-Cas Systems/genetics
Mice, Inbred C57BL
Glutathione/metabolism
Amino Acid Transport System y+/metabolism/genetics
Male
Liver/metabolism
Repressor Proteins/metabolism/genetics
HEK293 Cells

@article {pmid39146186,
year = {2024},
author = {Yan, Y and Schetelig, MF},
title = {Protocol for genetic engineering in Drosophila suzukii using microinjection.},
journal = {STAR protocols},
volume = {5},
number = {3},
pages = {103248},
pmid = {39146186},
issn = {2666-1667},
mesh = {Animals ; *Drosophila/genetics ; *Microinjections/methods ; *Genetic Engineering/methods ; *Gene Editing/methods ; CRISPR-Cas Systems/genetics ; DNA Transposable Elements/genetics ; Gene Targeting/methods ; },
abstract = {The spotted wing Drosophila (Drosophila suzukii Matsumura) is recognized globally as a significant economic pest. Here, we present a protocol for genetic engineering in D. suzukii using microinjection. We describe steps for genetic engineering techniques, including transposon-mediated germline transformation, recombinase-mediated genome targeting, and CRISPR-mediated gene editing. This protocol can significantly expand the toolkit for functional genomics and genetic control studies of this pest. For complete details on the use and execution of this protocol, please refer to Schetelig and Handler,[1] Schetelig et al.[2] Yan et al.,[3] and Yan et al.[4].},
}

Animals
*Drosophila/genetics
*Microinjections/methods
*Genetic Engineering/methods
*Gene Editing/methods
CRISPR-Cas Systems/genetics
DNA Transposable Elements/genetics
Gene Targeting/methods

@article {pmid39133612,
year = {2024},
author = {Matsui, S and Shiley, JR and Buckley, M and Lim, HW and Hu, YC and Mayhew, CN and Iwafuchi, M},
title = {Protocol for establishing inducible CRISPRd system for blocking transcription factor-binding sites in human pluripotent stem cells.},
journal = {STAR protocols},
volume = {5},
number = {3},
pages = {103233},
pmid = {39133612},
issn = {2666-1667},
mesh = {Humans ; *Pluripotent Stem Cells/metabolism/cytology ; *Transcription Factors/metabolism/genetics ; *CRISPR-Cas Systems/genetics ; Binding Sites ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; Doxycycline/pharmacology ; Lentivirus/genetics ; },
abstract = {Transcription factor (TF) gene knockout or knockdown experiments provide comprehensive downstream effects on gene regulation. However, distinguishing primary direct effects from secondary effects remains challenging. To assess the direct effect of TF binding events, we present a protocol for establishing a doxycycline (Dox)-inducible CRISPRd system in human pluripotent stem cells (hPSCs). We describe the steps for establishing CRISPRd host hPSCs, designing and preparing single-guide RNA (sgRNA) expression lentivirus vectors, generating CRISPRd hPSCs transduced with sgRNAs, and analyzing CRISPRd TF-block effects by chromatin immunoprecipitation (ChIP)-qPCR. For complete details on the use and execution of this protocol, please refer to Matsui et al.[1].},
}

Humans
*Pluripotent Stem Cells/metabolism/cytology
*Transcription Factors/metabolism/genetics
*CRISPR-Cas Systems/genetics
Binding Sites
RNA, Guide, CRISPR-Cas Systems/genetics/metabolism
Doxycycline/pharmacology
Lentivirus/genetics

@article {pmid39128010,
year = {2024},
author = {Pantina, VD and Verona, F and Turdo, A and Veschi, V and Modica, C and Lo Iacono, M and Gaggianesi, M and Di Bella, S and Todaro, M and Di Franco, S and Stassi, G},
title = {Protocol for generation and engineering of thyroid cell lineages using CRISPR-Cas9 editing to recapitulate thyroid cancer histotype progression.},
journal = {STAR protocols},
volume = {5},
number = {3},
pages = {103263},
pmid = {39128010},
issn = {2666-1667},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Thyroid Neoplasms/genetics/pathology ; *Gene Editing/methods ; *Thyroid Gland/pathology/cytology/metabolism ; *Cell Lineage/genetics ; Human Embryonic Stem Cells/metabolism/cytology ; Disease Progression ; },
abstract = {Thyroid carcinoma represents the first malignancy among the endocrine organs. Investigating the cellular hierarchy and the mechanisms underlying the initiation of thyroid carcinoma is crucial in thyroid cancer research. Here, we present a protocol for deriving thyroid cell lineage from human embryonic stem cells. We also describe steps for engineering thyroid progenitor cells utilizing CRISPR-Cas9 technology, which can be used to perform in vivo studies, thus facilitating the development of representative thyroid tumorigenesis models. For complete details on the use and execution of this protocol, please refer to Veschi et al.[1].},
}

Humans
*CRISPR-Cas Systems/genetics
*Thyroid Neoplasms/genetics/pathology
*Gene Editing/methods
*Thyroid Gland/pathology/cytology/metabolism
*Cell Lineage/genetics
Human Embryonic Stem Cells/metabolism/cytology
Disease Progression