@article {pmid39255283,
year = {2024},
author = {Dort, EN and Hamelin, RC},
title = {Heterogeneity in establishment of polyethylene glycol-mediated plasmid transformations for five forest pathogenic Phytophthora species.},
journal = {PloS one},
volume = {19},
number = {9},
pages = {e0306158},
doi = {10.1371/journal.pone.0306158},
pmid = {39255283},
issn = {1932-6203},
mesh = {*Phytophthora/genetics/pathogenicity ; *Plasmids/genetics ; *Polyethylene Glycols/pharmacology ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *Transformation, Genetic ; Forests ; Plant Diseases/microbiology/parasitology ; },
abstract = {Plasmid-mediated DNA transformation is a foundational molecular technique and the basis for most CRISPR-Cas9 gene editing systems. While plasmid transformations are well established for many agricultural Phytophthora pathogens, development of this technique in forest Phytophthoras is lacking. Given our long-term research objective to develop CRISPR-Cas9 gene editing in a forest pathogenic Phytophthora species, we sought to establish the functionality of polyethylene glycol (PEG)-mediated plasmid transformation in five species: P. cactorum, P. cinnamomi, P. cryptogea, P. ramorum, and P. syringae. We used the agricultural pathogen P. sojae, a species for which PEG-mediated transformations are well-established, as a transformation control. Using a protocol previously optimized for P. sojae, we tested transformations in the five forest Phytophthoras with three different plasmids: two developed for CRISPR-Cas9 gene editing and one developed for fluorescent protein tagging. Out of the five species tested, successful transformation, as indicated by stable growth of transformants on a high concentration of antibiotic selective growth medium and diagnostic PCR, was achieved only with P. cactorum and P. ramorum. However, while transformations in P. cactorum were consistent and stable, transformations in P. ramorum were highly variable and yielded transformants with very weak mycelial growth and abnormal morphology. Our results indicate that P. cactorum is the best candidate to move forward with CRISPR-Cas9 protocol development and provide insight for future optimization of plasmid transformations in forest Phytophthoras.},
}

*Phytophthora/genetics/pathogenicity
*Plasmids/genetics
*Polyethylene Glycols/pharmacology
*Gene Editing/methods
*CRISPR-Cas Systems
*Transformation, Genetic
Forests
Plant Diseases/microbiology/parasitology

@article {pmid39254993,
year = {2024},
author = {Schleske, JM and Hubrich, J and Wirth, JO and D'Este, E and Engelhardt, J and Hell, SW},
title = {MINFLUX reveals dynein stepping in live neurons.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {121},
number = {38},
pages = {e2412241121},
doi = {10.1073/pnas.2412241121},
pmid = {39254993},
issn = {1091-6490},
mesh = {*Dyneins/metabolism ; *Neurons/metabolism ; Animals ; CRISPR-Cas Systems ; Adenosine Triphosphate/metabolism ; Mice ; },
abstract = {Dynein is the primary molecular motor responsible for retrograde intracellular transport of a variety of cargoes, performing successive nanometer-sized steps within milliseconds. Due to the limited spatiotemporal precision of established methods for molecular tracking, current knowledge of dynein stepping is essentially limited to slowed-down measurements in vitro. Here, we use MINFLUX fluorophore localization to directly track CRISPR/Cas9-tagged endogenous dynein with nanometer/millisecond precision in living primary neurons. We show that endogenous dynein primarily takes 8 nm steps, including frequent sideways steps but few backward steps. Strikingly, the majority of direction reversals between retrograde and anterograde movement occurred on the time scale of single steps (16 ms), suggesting a rapid regulatory reversal mechanism. Tug-of-war-like behavior during pauses or reversals was unexpectedly rare. By analyzing the dwell time between steps, we concluded that a single rate-limiting process underlies the dynein stepping mechanism, likely arising from just one adenosine 5'-triphosphate hydrolysis event being required during each step. Our study underscores the power of MINFLUX localization to elucidate the spatiotemporal changes underlying protein function in living cells.},
}

*Dyneins/metabolism
*Neurons/metabolism
Animals
CRISPR-Cas Systems
Adenosine Triphosphate/metabolism
Mice

@article {pmid39227744,
year = {2024},
author = {Su, P and Liu, Y and Chen, T and Xue, Y and Zeng, Y and Zhu, G and Chen, S and Teng, M and Ci, X and Guo, M and He, MY and Hao, J and Chu, V and Xu, W and Wang, S and Mehdipour, P and Xu, X and Marhon, SA and Soares, F and Pham, NA and Wu, BX and Her, PH and Feng, S and Alshamlan, N and Khalil, M and Krishnan, R and Yu, F and Chen, C and Burrows, F and Hakem, R and Lupien, M and Harding, S and Lok, BH and O'Brien, C and Berlin, A and De Carvalho, DD and Brooks, DG and Schramek, D and Tsao, MS and He, HH},
title = {In vivo CRISPR screens identify a dual function of MEN1 in regulating tumor-microenvironment interactions.},
journal = {Nature genetics},
volume = {56},
number = {9},
pages = {1890-1902},
pmid = {39227744},
issn = {1546-1718},
support = {886012001223//Princess Margaret Cancer Foundation (PMCF)/ ; 142246, 152863,152864,159567,438793//Gouvernement du Canada | Canadian Institutes of Health Research (Instituts de Recherche en Santé du Canada)/ ; FDN-148395//Gouvernement du Canada | Canadian Institutes of Health Research (Instituts de Recherche en Santé du Canada)/ ; 1090, 1124//Terry Fox Research Institute (Institut de Recherche Terry Fox)/ ; 31801111//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {*Tumor Microenvironment/genetics ; Animals ; Humans ; Mice ; *Proto-Oncogene Proteins/genetics/metabolism ; *CD8-Positive T-Lymphocytes/immunology/metabolism ; *CRISPR-Cas Systems ; Cell Line, Tumor ; *Histone-Lysine N-Methyltransferase/genetics/metabolism ; Myeloid-Lymphoid Leukemia Protein/genetics ; Neoplasms/genetics/pathology ; Cell Proliferation/genetics ; Gene Expression Regulation, Neoplastic ; Female ; },
abstract = {Functional genomic screens in two-dimensional cell culture models are limited in identifying therapeutic targets that influence the tumor microenvironment. By comparing targeted CRISPR-Cas9 screens in a two-dimensional culture with xenografts derived from the same cell line, we identified MEN1 as the top hit that confers differential dropout effects in vitro and in vivo. MEN1 knockout in multiple solid cancer types does not impact cell proliferation in vitro but significantly promotes or inhibits tumor growth in immunodeficient or immunocompetent mice, respectively. Mechanistically, MEN1 knockout redistributes MLL1 chromatin occupancy, increasing H3K4me3 at repetitive genomic regions, activating double-stranded RNA expression and increasing neutrophil and CD8[+] T cell infiltration in immunodeficient and immunocompetent mice, respectively. Pharmacological inhibition of the menin-MLL interaction reduces tumor growth in a CD8[+] T cell-dependent manner. These findings reveal tumor microenvironment-dependent oncogenic and tumor-suppressive functions of MEN1 and provide a rationale for targeting MEN1 in solid cancers.},
}

*Tumor Microenvironment/genetics
Animals
Humans
Mice
*Proto-Oncogene Proteins/genetics/metabolism
*CD8-Positive T-Lymphocytes/immunology/metabolism
*CRISPR-Cas Systems
Cell Line, Tumor
*Histone-Lysine N-Methyltransferase/genetics/metabolism
Myeloid-Lymphoid Leukemia Protein/genetics
Neoplasms/genetics/pathology
Cell Proliferation/genetics
Gene Expression Regulation, Neoplastic
Female

@article {pmid39217735,
year = {2024},
author = {Wei, B and Wang, J and Dai, L and Zhang, K},
title = {Probing the higher order structure of oligonucleotides through anion exchange chromatography.},
journal = {Journal of chromatography. A},
volume = {1734},
number = {},
pages = {465314},
doi = {10.1016/j.chroma.2024.465314},
pmid = {39217735},
issn = {1873-3778},
mesh = {Chromatography, Ion Exchange/methods ; *Oligonucleotides/chemistry/analysis ; RNA, Guide, CRISPR-Cas Systems/chemistry ; Chromatography, Reverse-Phase/methods ; Hydrophobic and Hydrophilic Interactions ; Anions/chemistry ; },
abstract = {Large synthetic oligonucleotides such as guide ribonucleic acid (gRNA), a critical reagent in clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 genome editing, have complex higher order structures (HOS) inherent in their design. In this study, we first developed a generic anion exchange chromatography (AEX) method for the comprehensive analysis of a 100mer single guide ribonucleic acid (sgRNA) impurity profiling. AEX demonstrated superior resolution compared to other common chromatographic methods employed for sgRNA analysis, such as Ion-Pairing Reversed Phase Liquid Chromatography (IP-RPLC) and Hydrophilic Interaction Chromatography (HILIC). Moreover, we discovered AEX's potential in probing the HOS of RNAs by adjusting the temperature and using organic additives. Our study also highlighted that sgRNA possesses a unique HOS distinctly different from other therapeutic nucleic acids, such as antisense oligonucleotides and messenger RNAs.},
}

Chromatography, Ion Exchange/methods
*Oligonucleotides/chemistry/analysis
RNA, Guide, CRISPR-Cas Systems/chemistry
Chromatography, Reverse-Phase/methods
Hydrophobic and Hydrophilic Interactions
Anions/chemistry

@article {pmid39192723,
year = {2024},
author = {Gu, A and Dong, Y and Li, L and Yu, D and Zhang, J and Chen, Y},
title = {CRISPR/Cas12a and Hybridization Chain Reaction-Coregulated Magnetic Relaxation Switching Biosensor for Sensitive Detection of Viable Salmonella in Animal-Derived Foods.},
journal = {Journal of agricultural and food chemistry},
volume = {72},
number = {36},
pages = {20130-20139},
doi = {10.1021/acs.jafc.4c05540},
pmid = {39192723},
issn = {1520-5118},
mesh = {*Biosensing Techniques/methods/instrumentation ; *Salmonella typhimurium/isolation & purification/genetics ; *CRISPR-Cas Systems ; Animals ; *Food Contamination/analysis ; Food Microbiology/methods ; Bacterial Proteins/genetics/metabolism ; Nucleic Acid Hybridization ; Limit of Detection ; Swine ; },
abstract = {We combined a CRISPR/Cas12a system with a hybridization chain reaction (HCR) to develop an ultrasensitive magnetic relaxation switching (MRS) biosensor for detecting viable Salmonella typhimurium (S. typhimurium). Magnetic nanoparticles of two sizes (30 and 1000 nm: MNP30 and MNP1000, respectively) were coupled through HCR. The S. typhimurium gene-activated CRISPR/Cas12a system released MNP30 from the MNP1000-HCR-MNP30 complex through a trans-cleavage reaction. After magnetic separation, released MNP30 was collected from the supernatant and served as a transverse relaxation time (T2) signal probe. Quantitative detection of S. typhimurium is achieved by establishing a linear relationship between the change in T2 and the target gene. The biosensor's limit of detection was 77 CFU/mL (LOD = 3S/M, S = 22.30, M = 0.87), and the linear range was 10[2]-10[8] CFU/mL. The accuracy for detecting S. typhimurium in real samples is comparable to that of qPCR. Thus, this is a promising method for the rapid and effective detection of foodborne pathogens.},
}

*Biosensing Techniques/methods/instrumentation
*Salmonella typhimurium/isolation & purification/genetics
*CRISPR-Cas Systems
Animals
*Food Contamination/analysis
Food Microbiology/methods
Bacterial Proteins/genetics/metabolism
Nucleic Acid Hybridization
Limit of Detection
Swine

@article {pmid39182792,
year = {2024},
author = {Qian, T and Wei, W and Dong, Y and Zhang, P and Chen, X and Chen, P and Li, M and Ye, BC},
title = {Metabolic engineering of the oleaginous yeast Yarrowia lipolytica for 2-phenylethanol overproduction.},
journal = {Bioresource technology},
volume = {411},
number = {},
pages = {131354},
doi = {10.1016/j.biortech.2024.131354},
pmid = {39182792},
issn = {1873-2976},
mesh = {*Yarrowia/metabolism/genetics ; *Metabolic Engineering/methods ; *Phenylethyl Alcohol/metabolism ; CRISPR-Cas Systems ; Shikimic Acid/metabolism ; },
abstract = {The rose fragrance molecule 2-phenylethanol (2-PE) has huge market demand in the cosmetics, food and pharmaceutical industries. However, current 2-PE synthesis methods do not meet the efficiency market requirement. In this study, CRISPR-Cas9-related metabolic engineering strategies were applied to Yarrowia lipolytica for the de novo biosynthesis of 2-PE. Initially, overexpressing exogenous feedback-resistant EcAROG[fbr] and EcPheA[fbr] increased 2-PE production to 276.3 mg/L. Subsequently, the ylARO10 and ylPAR4 from endogenous genes were enhanced with the multi-copies to increase the titer to 605 mg/L. Knockout of ylTYR1 and enhancement of shikimate pathway by removing the precursor metabolic bottleneck and overexpressing the genes ylTKT, ylARO1, and ylPHA2 resulted in a significant increase of the 2-PE titer to 2.4 g/L at 84 h, with the yield of 0.06 g/gglu, which is the highest yield for de novo synthesis in yeast. This study provides a valuable precedent for the efficient biosynthesis of shikimate pathway derivatives.},
}

*Yarrowia/metabolism/genetics
*Metabolic Engineering/methods
*Phenylethyl Alcohol/metabolism
CRISPR-Cas Systems
Shikimic Acid/metabolism

@article {pmid39136471,
year = {2024},
author = {Fernandes, LGV and Hamond, C and Tibbs-Cortes, BW and Putz, EJ and Olsen, SC and Palmer, MV and Nally, JE},
title = {CRISPR-prime editing, a versatile genetic tool to create specific mutations with a single nucleotide resolution in Leptospira.},
journal = {mBio},
volume = {15},
number = {9},
pages = {e0151624},
doi = {10.1128/mbio.01516-24},
pmid = {39136471},
issn = {2150-7511},
mesh = {*Leptospira/genetics/pathogenicity ; *Gene Editing/methods ; *CRISPR-Cas Systems ; Leptospirosis/microbiology ; Animals ; Mutation ; Humans ; },
abstract = {Leptospirosis, caused by pathogenic bacteria from the genus Leptospira, is a global zoonosis responsible for more than one million human cases and 60,000 deaths annually. The disease also affects many domestic animal species. Historically, genetic manipulation of Leptospira has been difficult to perform, resulting in limited knowledge on pathogenic mechanisms of disease and the identification of virulence factors. The application of CRISPR/Cas9 and its variations have helped fill these gaps but the generation of knockout mutants remains challenging because double-strand breaks (DSBs) inflicted by Cas9 nuclease are lethal to Leptospira cells. The novel CRISPR prime editing (PE) strategy is the first precise genome-editing technology that allows deletions, insertions, and base substitutions without introducing DSBs. This revolutionary technique utilizes a nickase Cas9 that cleaves a single strand of DNA, coupled with an engineered reverse transcriptase and a modified single-guide RNA (termed prime editing guide RNA) containing an extended 3' end with the desired edits. We demonstrate the application of CRISPR-PE in both saprophytic and pathogenic Leptospira from multiple species and serovars by introducing deletions or insertions into target DNA with a remarkable precision of just one nucleotide. Additionally, we demonstrate the ability to genetically manipulate Leptospira borgpetersenii, a prevalent pathogenic species of humans, domestic cattle, and wildlife animals. Rapid plasmid loss by mutated strains in liquid culture allows for the generation of knockout strains without selective markers, which can be readily used to elucidate virulence factors and develop optimized bacterin and/or live vaccines against leptospirosis.IMPORTANCELeptospirosis is a geographically widespread bacterial zoonosis. Genetic manipulation of pathogenic Leptospira spp. has been laborious and difficult to perform, limiting our ability to understand how leptospires cause disease. The application of the CRISPR/Cas9 system to Leptospira enhanced our ability to generate knockdown and knockout mutants; however, the latter remains challenging. Here, we demonstrate the application of the CRISPR prime editing technique in Leptospira, allowing the generation of knockout mutants in several pathogenic species, with mutations comprising just a single nucleotide resolution. Notably, we generated a mutant in the Leptospira borgpetersenii background, a prevalent pathogenic species of humans and cattle. Our application of this method opens new avenues for studying pathogenic mechanisms of Leptospira and the identification of virulence factors across multiple species. These methods can also be used to facilitate the generation of marker-less knockout strains for updated and improved bacterin and/or live vaccines.},
}

*Leptospira/genetics/pathogenicity
*Gene Editing/methods
*CRISPR-Cas Systems
Leptospirosis/microbiology
Animals
Mutation
Humans

@article {pmid39082802,
year = {2024},
author = {Tachibana, Y and Sasai, M and Yamamoto, M},
title = {CRISPR screens identify genes essential for in vivo virulence among proteins of hyperLOPIT-unassigned subcellular localization in Toxoplasma.},
journal = {mBio},
volume = {15},
number = {9},
pages = {e0172824},
doi = {10.1128/mbio.01728-24},
pmid = {39082802},
issn = {2150-7511},
support = {JP20fk0108137//Japan Agency for Medical Research and Development (AMED)/ ; JP23fk0108682//Japan Agency for Medical Research and Development (AMED)/ ; JP223fa627002//Japan Agency for Medical Research and Development (AMED)/ ; 23KJ1469//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; },
mesh = {*Toxoplasma/genetics/pathogenicity/metabolism ; Animals ; *Protozoan Proteins/genetics/metabolism ; Virulence ; Mice ; Clustered Regularly Interspaced Short Palindromic Repeats ; Proteomics ; Virulence Factors/genetics/metabolism ; Female ; CRISPR-Cas Systems ; Toxoplasmosis/parasitology ; Genes, Essential ; },
abstract = {The research field to identify and characterize genes essential for in vivo virulence in Toxoplasma gondii has been dramatically advanced by a series of in vivo clustered regularly interspaced short palindromic repeats (CRISPR) screens. Although subcellular localizations of thousands of proteins were predicted by the spatial proteomic method called hyperLOPIT, those of more than 1,000 proteins remained unassigned, and their essentiality in virulence was also unknown. In this study, we generated two small-scale gRNA libraries targeting approximately 600 hyperLOPIT-unassigned proteins and performed in vivo CRISPR screens. As a result, we identified several genes essential for in vivo virulence that were previously unreported. We further characterized two candidates, TgGTPase and TgRimM, which are localized in the cytoplasm and the apicoplast, respectively. Both genes are essential for parasite virulence and widely conserved in the phylum Apicomplexa. Collectively, our current study provides a resource for estimating the in vivo essentiality of Toxoplasma proteins with previously unknown localizations.IMPORTANCEToxoplasma gondii is a protozoan parasite that causes severe infection in immunocompromised patients or newborns. Toxoplasma possesses more than 8,000 genes; however, the genes essential for in vivo virulence were not fully identified. The apicomplexan parasites, including Toxoplasma, developed unique organelles that do not exist in other model organisms; thus, determining the subcellular location of parasite proteins is important for understanding their functions. Here, we used in vivo genetic screens that enabled us to investigate hundreds of genes in Toxoplasma during mouse infection. We screened approximately 600 parasite proteins with previously unknown subcellular localizations. We identified many novel genes that confer parasite virulence in mice. Among the top hits, we characterized two genes essential for in vivo virulence, TgGTPase and TgRimM, which are widely conserved in the phylum Apicomplexa. Our findings will contribute to understanding how apicomplexans adapt to the host environment and cause disease.},
}

*Toxoplasma/genetics/pathogenicity/metabolism
Animals
*Protozoan Proteins/genetics/metabolism
Virulence
Mice
Clustered Regularly Interspaced Short Palindromic Repeats
Proteomics
Virulence Factors/genetics/metabolism
Female
CRISPR-Cas Systems
Toxoplasmosis/parasitology
Genes, Essential

@article {pmid39254327,
year = {2024},
author = {Fang, M and Zhang, R and Wang, C and Liu, Z and Fei, M and Tang, B and Yang, H and Sun, D},
title = {Engineering probiotic Escherichia coli Nissle 1917 to block transfer of multiple antibiotic resistance genes by exploiting a type I CRISPR-Cas system.},
journal = {Applied and environmental microbiology},
volume = {},
number = {},
pages = {e0081124},
doi = {10.1128/aem.00811-24},
pmid = {39254327},
issn = {1098-5336},
abstract = {UNLABELLED: Many multidrug-resistant (MDR) bacteria have evolved through the accumulation of antibiotic resistance genes (ARGs). Although the potential risk of probiotics as reservoirs of ARGs has been recognized, strategies for blocking the transfer of ARGs while using probiotics have rarely been explored. The probiotic Escherichia coli Nissle 1917 (EcN) has long been used for treating intestinal diseases. Here, we demonstrate frequent transfer of ARGs into EcN both in vitro and in vivo, raising concerns about its potential risk of accumulating antibiotic resistance. Given that no CRISPR-Cas system was found in natural EcN, we integrated the type I-E CRISPR-Cas3 system derived from E. coli BW25113 into EcN. The engineered EcN was able to efficiently cleave multiple ARGs [i.e., mcr-1, blaNDM-1, and tet(X)] encoding enzymes for degrading last-resort antibiotics. Through co-incubation of EcN expressing Cas3-Cascade and that expressing Cas9, we showed that the growth of the former strain outcompeted the latter strain, demonstrating a better clinical application prospect of EcN expressing the type I-E CRISPR-Cas3 system. In the intestine of a model animal (i.e., zebrafish), the engineered EcN exhibited immunity against the transfer of CRISPR-targeted ARGs. Our work equips EcN with immunity against the transfer of multiple ARGs by exploiting the exogenous type I-E CRISPR-Cas3 system, thereby reducing the risk of the spread of ARGs while using it as a probiotic chassis for generating living therapeutics.

IMPORTANCE: To reduce the development of antibiotic resistance, probiotics have been considered as a substitute for antibiotics. However, probiotics themselves are reservoirs of antibiotic resistance genes (ARGs). This study introduces a new strategy for limiting the spread of ARGs by engineering the typical probiotic strain Escherichia coli Nissle 1917 (EcN), which has been used for treating intestinal diseases and developed as living therapeutics. We also demonstrate that the type I CRISPR-Cas system imposes a lower growth burden than the type II CRISPR-Cas system, highlighting its promising clinical application potential. Our work not only provides a new strategy for restricting the transfer of ARGs while using probiotics but also enriches the genetic engineering toolbox of EcN, paving the way for the safe use and development of probiotics as living therapeutics.},
}

@article {pmid39254031,
year = {2024},
author = {Cheng, Y and Lyu, J and Han, J and Feng, L and Li, X and Li, P and Zhang, S and Zang, W},
title = {A specific and ultrasensitive Cas12a/crRNA assay with recombinase polymerase amplification and lateral flow biosensor technology for the rapid detection of Streptococcus pyogenes.},
journal = {Microbiology spectrum},
volume = {},
number = {},
pages = {e0034524},
doi = {10.1128/spectrum.00345-24},
pmid = {39254031},
issn = {2165-0497},
abstract = {UNLABELLED: The potential of CRISPR/Cas systems for nucleic acid detection in novel biosensing applications is remarkable. The current clinical diagnostic detection of Streptococcus pyogenes (S. pyogenes) is based on serological identification, culture, and PCR. We report a rapid, simple, and sensitive method for detecting and screening for S. pyogenes. This novel method is a promising supplemental test. After 10 min of the sample processing and 10 min of recombinase polymerase amplification, followed by 10 min of Cas12 reaction and 3 min of lateral flow biosensor (LFB) readout, a visible outcome can be observed without the need for magnification within 33 min. This platform is robust, inexpensive, and appropriate for on-site testing. A new technique for detection was created using CRISPR-Cas12a technology, which includes two measurements: a fluorescent-CRISPR-S. pyogenes test and a LFB-CRISPR-S. pyogenes test. An approach utilizing CRISPR Cas12a was developed, and the accuracy and precision of this technique were assessed. The LoD for the fluorescence-CRISPR- S. pyogenes assay was 1 copy/μL, and the technique effectively differentiated S. pyogenes from other microorganisms. Moreover, the detection outcomes were presented in a user-friendly manner using lateral flow biosensor strips. Conclusion: A rapid and sensitive Cas12a/crRNA assay using recombinase RPA and LFB was developed to detect S. pyogenes. The Cas12a/crRNA-based assay exhibited high specificity among different bacteria strains and extremely high sensitivity. The accuracy and rapidity of this method make it a promising tool for S. pyogenes detection and screening.

IMPORTANCE: Patients may experience a range of symptoms due to Streptococcus pyogenes infections, including superficial skin infections, pharyngitis, and invasive diseases in subcutaneous tissues like streptococcal toxic shock syndrome. At present, the clinical diagnostic detection of S. pyogenes is based on serological identification, culture, and PCR. These detection methods are time-consuming and require sophisticated equipment, making these methods challenging for routine laboratories. Thus, there is a need for a detection platform that is capable of quickly and accurately identifying S. pyogenes. In this study, a rapid and sensitive Cas12a/crRNA assay using recombinase RPA and LFB was developed to detect S. pyogenes. The Cas12a/crRNA-based assay exhibited high specificity among different bacteria strains and extremely high sensitivity. This method probably plays an important role for S. pyogenes detection and screening.},
}

@article {pmid39253867,
year = {2024},
author = {Acevedo-Rodriguez, JG and Contreras, CA and Ochoa, TJ},
title = {Viral diarrheas - newer advances in diagnosis and management.},
journal = {Current opinion in infectious diseases},
volume = {37},
number = {5},
pages = {385-391},
doi = {10.1097/QCO.0000000000001053},
pmid = {39253867},
issn = {1473-6527},
mesh = {Humans ; *Diarrhea/diagnosis/virology/therapy ; Molecular Diagnostic Techniques/methods ; Virus Diseases/diagnosis/therapy ; Norovirus/genetics/isolation & purification ; Caliciviridae Infections/diagnosis/therapy ; Nucleic Acid Amplification Techniques/methods ; Gastrointestinal Microbiome ; },
abstract = {PURPOSE OF REVIEW: Viruses are the most common etiological agents of diarrhea in children. Despite rotavirus vaccine introduction, rotavirus remains as the leading cause of death globally, followed by norovirus, which represents a diagnostic challenge. Here, we describe new advances in the diagnosis and management of viral diarrheas.

RECENT FINDINGS: Although immunoassays are widely used for their fast turnaround time and low cost, molecular techniques have become the most reliable diagnostic method due to their high sensitivity and capacity to analyze multiple pathogens in gastrointestinal panels. Isothermal nucleic acid amplification assays (LAMP and RPA) are promising techniques since they do not require sophisticated equipment and can be used as point-of-care testing. CRISPR/Cas nucleic acid detection systems are new diagnostic methods with great potential. Several recent published articles describe the role of human intestinal enteroids to characterize norovirus infection, to test new drugs, and for vaccine development. The interaction between the human gut microbiota and gastrointestinal viral infections has been extensively reviewed and offers some innovative mechanisms for therapeutic and preventive measures.

SUMMARY: Although important advances have been made, more research is needed to address remaining challenges and further improve diagnostic capabilities and better management strategies for this critical infectious disease.},
}

Humans
*Diarrhea/diagnosis/virology/therapy
Molecular Diagnostic Techniques/methods
Virus Diseases/diagnosis/therapy
Norovirus/genetics/isolation & purification
Caliciviridae Infections/diagnosis/therapy
Nucleic Acid Amplification Techniques/methods
Gastrointestinal Microbiome

@article {pmid39253640,
year = {2024},
author = {Jia, Y and Horvath, K and Rananaware, SR and Jain, PK and Sampath, J},
title = {Exploring the Thermostability of CRISPR Cas12b using Molecular Dynamics Simulations.},
journal = {ArXiv},
volume = {},
number = {},
pages = {},
pmid = {39253640},
issn = {2331-8422},
abstract = {CRISPR (clustered regularly interspaced short palindromic repeat)- based diagnostics are at the forefront of rapid detection platforms of infectious diseases. The integration of reverse transcription-loop-mediated isothermal amplification (RT-LAMP) with CRISPR-Cas protein systems has led to the creation of advanced one-pot assays. The sensitivity of these assays has been bolstered by the utilization of a thermophilic Cas12 protein, BrCas12b, and its engineered variant, which exhibits enhanced thermal stability and allows for broader operation temperatures of the assay. Here, we perform all-atom molecular dynamics (MD) simulations on wild-type and mutant BrCas12b to reveal the mechanism of stabilization conferred by the mutation. High-temperature simulations reveal a small structural change along with greater flexibility in the PAM-interacting domain of the mutant BrCas12b, with marginal structural and flexibility changes in the other mutated domains. Comparative essential dynamics analysis between the wild-type and mutant BrCas12b at both ambient and elevated temperatures provides insights into the stabilizing effects of the mutations. Our findings not only offer a comprehensive insight into the dynamic alterations induced by mutations but reveal important motions in BrCas12b, important for the rational design of diagnostic and therapeutic platforms of Cas12 proteins.},
}

@article {pmid39253479,
year = {2024},
author = {Chen, DF and Roe, LT and Li, Y and Borges, AL and Zhang, JY and Babbar, P and Maji, S and Stevens, MGV and Correy, GJ and Diolaiti, ME and Smith, DH and Ashworth, A and Stroud, RM and Kelly, MJS and Bondy-Denomy, J and Fraser, JS},
title = {AcrIF11 is a potent CRISPR-specific ADP-ribosyltransferase encoded by phage and plasmid.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
doi = {10.1101/2024.08.26.609590},
pmid = {39253479},
issn = {2692-8205},
abstract = {Phage-encoded anti-CRISPR (Acr) proteins inhibit CRISPR-Cas systems to allow phage replication and lysogeny maintenance. Most of the Acrs characterized to date are stable stoichiometric inhibitors, and while enzymatic Acrs have been characterized biochemically, little is known about their potency, specificity, and reversibility. Here, we examine AcrIF11, a widespread phage and plasmid-encoded ADP-ribosyltransferase (ART) that inhibits the Type I-F CRISPR-Cas system. We present an NMR structure of an AcrIF11 homolog that reveals chemical shift perturbations consistent with NAD (cofactor) binding. In experiments that model both lytic phage replication and MGE/lysogen stability under high targeting pressure, AcrIF11 is a highly potent CRISPR-Cas inhibitor and more robust to Cas protein level fluctuations than stoichiometric inhibitors. Furthermore, we demonstrate that AcrIF11 is remarkably specific, predominantly ADP-ribosylating Csy1 when expressed in P. aeruginosa . Given the reversible nature of ADP-ribosylation, we hypothesized that ADPr eraser enzymes (macrodomains) could remove ADPr from Csy1, a potential limitation of PTM-based CRISPR inhibition. We demonstrate that diverse macrodomains can indeed remove the modification from Csy1 in P. aeruginosa lysate. Together, these experiments connect the in vitro observations of AcrIF11's enzymatic activity to its potent and specific effects in vivo , clarifying the advantages and drawbacks of enzymatic Acrs in the evolutionary arms race between phages and bacteria.},
}

@article {pmid39251884,
year = {2024},
author = {Li, X and Liu, Y and Han, J and Zhang, L and Liu, Z and Wang, L and Zhang, S and Zhang, Q and Fu, P and Yin, H and Zhu, H and Zhang, H},
title = {Structural basis for the type I-F Cas8-HNH system.},
journal = {The EMBO journal},
volume = {},
number = {},
pages = {},
pmid = {39251884},
issn = {1460-2075},
support = {32322040//MOST | National Natural Science Foundation of China (NSFC)/ ; 32300036//MOST | National Natural Science Foundation of China (NSFC)/ ; },
abstract = {The Cas3 nuclease is utilized by canonical type I CRISPR-Cas systems for processive target DNA degradation, while a newly identified type I-F CRISPR variant employs an HNH nuclease domain from the natural fusion Cas8-HNH protein for precise target cleavage both in vitro and in human cells. Here, we report multiple cryo-electron microscopy structures of the type I-F Cas8-HNH system at different functional states. The Cas8-HNH Cascade complex adopts an overall G-shaped architecture, with the HNH domain occupying the C-terminal helical bundle domain (HB) of the Cas8 protein in canonical type I systems. The Linker region connecting Cas8-NTD and HNH domains adopts a rigid conformation and interacts with the Cas7.6 subunit, enabling the HNH domain to be in a functional position. The full R-loop formation displaces the HNH domain away from the Cas6 subunit, thus activating the target DNA cleavage. Importantly, our results demonstrate that precise target cleavage is dictated by a C-terminal helix of the HNH domain. Together, our work not only delineates the structural basis for target recognition and activation of the type I-F Cas8-HNH system, but also guides further developments leveraging this system for precise DNA editing.},
}

@article {pmid39251587,
year = {2024},
author = {Zerbib, J and Ippolito, MR and Eliezer, Y and De Feudis, G and Reuveni, E and Savir Kadmon, A and Martin, S and Viganò, S and Leor, G and Berstler, J and Muenzner, J and Mülleder, M and Campagnolo, EM and Shulman, ED and Chang, T and Rubolino, C and Laue, K and Cohen-Sharir, Y and Scorzoni, S and Taglietti, S and Ratti, A and Stossel, C and Golan, T and Nicassio, F and Ruppin, E and Ralser, M and Vazquez, F and Ben-David, U and Santaguida, S},
title = {Human aneuploid cells depend on the RAF/MEK/ERK pathway for overcoming increased DNA damage.},
journal = {Nature communications},
volume = {15},
number = {1},
pages = {7772},
pmid = {39251587},
issn = {2041-1723},
support = {1805/21//Israel Science Foundation (ISF)/ ; NA//Israel Cancer Research Fund (Israel Cancer Research Fund, Inc.)/ ; 2019228//United States - Israel Binational Science Foundation (BSF)/ ; AIRC-MFAG 2018 - ID. 21665//Associazione Italiana per la Ricerca sul Cancro (Italian Association for Cancer Research)/ ; ERC-SyG-2020 951475//EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 European Research Council (H2020 Excellent Science - European Research Council)/ ; },
mesh = {Humans ; *DNA Damage ; *Aneuploidy ; *MAP Kinase Signaling System/drug effects ; *Phthalazines/pharmacology ; Cell Line, Tumor ; Piperazines/pharmacology ; raf Kinases/metabolism/genetics ; Neoplasms/genetics/metabolism/pathology ; CRISPR-Cas Systems ; Cell Line ; Proto-Oncogene Proteins c-raf/metabolism/genetics ; Drug Resistance, Neoplasm/genetics ; },
abstract = {Aneuploidy is a hallmark of human cancer, yet the molecular mechanisms to cope with aneuploidy-induced cellular stresses remain largely unknown. Here, we induce chromosome mis-segregation in non-transformed RPE1-hTERT cells and derive multiple stable clones with various degrees of aneuploidy. We perform a systematic genomic, transcriptomic and proteomic profiling of 6 isogenic clones, using whole-exome DNA, mRNA and miRNA sequencing, as well as proteomics. Concomitantly, we functionally interrogate their cellular vulnerabilities, using genome-wide CRISPR/Cas9 and large-scale drug screens. Aneuploid clones activate the DNA damage response and are more resistant to further DNA damage induction. Aneuploid cells also exhibit elevated RAF/MEK/ERK pathway activity and are more sensitive to clinically-relevant drugs targeting this pathway, and in particular to CRAF inhibition. Importantly, CRAF and MEK inhibition sensitize aneuploid cells to DNA damage-inducing chemotherapies and to PARP inhibitors. We validate these results in human cancer cell lines. Moreover, resistance of cancer patients to olaparib is associated with high levels of RAF/MEK/ERK signaling, specifically in highly-aneuploid tumors. Overall, our study provides a comprehensive resource for genetically-matched karyotypically-stable cells of various aneuploidy states, and reveals a therapeutically-relevant cellular dependency of aneuploid cells.},
}

Humans
*DNA Damage
*Aneuploidy
*MAP Kinase Signaling System/drug effects
*Phthalazines/pharmacology
Cell Line, Tumor
Piperazines/pharmacology
raf Kinases/metabolism/genetics
Neoplasms/genetics/metabolism/pathology
CRISPR-Cas Systems
Cell Line
Proto-Oncogene Proteins c-raf/metabolism/genetics
Drug Resistance, Neoplasm/genetics

@article {pmid39217761,
year = {2024},
author = {Paenkaew, S and Poommouang, A and Pradit, W and Chomdej, S and Nganvongpanit, K and Siengdee, P and Buddhachat, K},
title = {Feasibility of implementing RPA coupled with CRISPR-Cas12a (RPA-Cas12a) for Hepatozoon canis detection in dogs.},
journal = {Veterinary parasitology},
volume = {331},
number = {},
pages = {110298},
doi = {10.1016/j.vetpar.2024.110298},
pmid = {39217761},
issn = {1873-2550},
mesh = {Animals ; Dogs ; *Dog Diseases/parasitology/diagnosis ; *Coccidiosis/veterinary/diagnosis/parasitology ; *CRISPR-Cas Systems ; *RNA, Ribosomal, 18S/genetics ; Sensitivity and Specificity ; Nucleic Acid Amplification Techniques/veterinary/methods ; Feasibility Studies ; Recombinases/metabolism ; Eucoccidiida/genetics/isolation & purification ; },
abstract = {Hepatozoonosis, caused by the protozoan Hepatozoon canis, is a prevalent blood disease affecting owned and stray dogs and cats. The prevalence of these parasites among companion animals in Thailand remains poorly understood. Diagnosing the old-world form of the disease is challenging due to the wide range of nonspecific clinical signs and the reliance on finding low levels of Hepatozoon gamonts in blood smears for conventional diagnosis. PCR demonstrates high specificity and sensitivity but it requires sophisticated instrumentation. Therefore, we established recombinase polymerase amplification (RPA) coupled with Cas12a for H. canis detection based on 18S rRNA. Our findings showed that RPA-Cas12a using gRNA_H was highly specific to H. canis, without yielding positives for other pathogen species including Babesia species. Even in cases of co-infection, RPA-Cas12a only detected positives in samples containing H. canis. This approach detected minimal amounts of H. canis18S rRNA-harboring plasmid at 10 copies per reaction, whereas plasmid-spiked canine blood enabled detection at a minimal amount of 100 copies per reaction. The performance of RPA-Cas12a was validated by comparing it with quantitative PCR-high resolution melting analysis (qPCR-HRM) and sequencing based on 35 canine blood samples. RPA-Cas12a demonstrated precision and accuracy values of 94 % and 90 %, respectively comparable to qPCR-HRM. Overall, these results indicate that RPA-Cas12a serves as a promising tool for H. canis detection as indicated by comparable performance to qPCR-HRM and is suitable for implementation in small animal hospitals or clinics due to its minimal resource requirements, thereby contributing to effective diagnosis and treatment for infected dogs.},
}

Animals
Dogs
*Dog Diseases/parasitology/diagnosis
*Coccidiosis/veterinary/diagnosis/parasitology
*CRISPR-Cas Systems
*RNA, Ribosomal, 18S/genetics
Sensitivity and Specificity
Nucleic Acid Amplification Techniques/veterinary/methods
Feasibility Studies
Recombinases/metabolism
Eucoccidiida/genetics/isolation & purification

@article {pmid39089176,
year = {2024},
author = {Huang, F and Li, X and Zhou, Y and Tang, W and Dang, Z and Kui, J and Zhang, C and Zhang, X},
title = {Optimization of CRISPR/Cas12a detection assay and its application in the detection of Echinococcus granulosus.},
journal = {Veterinary parasitology},
volume = {331},
number = {},
pages = {110276},
doi = {10.1016/j.vetpar.2024.110276},
pmid = {39089176},
issn = {1873-2550},
mesh = {Animals ; *Echinococcus granulosus/genetics/isolation & purification ; *Echinococcosis/diagnosis/veterinary/parasitology ; *CRISPR-Cas Systems ; Dogs ; *Dog Diseases/parasitology/diagnosis ; *Sensitivity and Specificity ; Feces/parasitology ; China ; },
abstract = {Cystic echinococcosis, resulting from infection with Echinococcus granulosus, poses a significant challenge as a neglected tropical disease owing to the lack of any known effective treatment. Primarily affecting under-resourced, remote, and conflict-ridden regions, the disease is compounded by the limitations of current detection techniques, such as microscopy, physical imaging, ELISA, and qPCR, which are unsuitable for application in these areas. The emergence of CRISPR/Cas12a as a promising tool for nucleic acid detection, characterized by its unparalleled specificity, heightened sensitivity, and rapid detection time, offers a potential solution. In this study, we present a one-pot CRISPR/Cas12a detection method for E. granulosus (genotype G1, sheep strain) integrating recombinase polymerase amplification (RPA) with suboptimal protospacer adjacent motif (PAM) and structured CRISPR RNA (crRNA) to enhance reaction efficiency. The evaluation of the assay's performance using hydatid cyst spiked dog feces and the examination of 62 dog fecal samples collected from various regions of Western China demonstrate its efficacy. The assay permits visual observation of test results about 15 minutes under blue light and displays superior portability and reaction speed relative to qPCR, achieving a sensitivity level of 10 copies of standard plasmids of the target gene. Analytic specificity was verified against four tapeworm species (E. multilocularis, H. taeniaeformis, M. benedeni, and D. caninum) and two other helminths (T. canis and F. hepatica), with negative results also noted for Mesocestoides sp. This study presents a rapid, sensitive, and time-efficient DNA detection method for E. granulosus of hydatid cyst spiked and clinical dog feces, potential serving as an alternative tool for field detection. This novel assay is primarily used to diagnose the definitive host of E. granulosus. Further validation using a larger set of clinical fecal samples is warranted, along with additional exploration of more effective approaches for nucleic acid release.},
}

Animals
*Echinococcus granulosus/genetics/isolation & purification
*Echinococcosis/diagnosis/veterinary/parasitology
*CRISPR-Cas Systems
Dogs
*Dog Diseases/parasitology/diagnosis
*Sensitivity and Specificity
Feces/parasitology
China

@article {pmid38960022,
year = {2024},
author = {Krappinger, JC and Aguilar Gomez, CM and Hoenikl, A and Schusterbauer, V and Hatzl, AM and Feichtinger, J and Glieder, A},
title = {dMAD7 is a promising tool for targeted gene regulation in the methylotrophic yeast Komagataella phaffii.},
journal = {New biotechnology},
volume = {83},
number = {},
pages = {110-120},
doi = {10.1016/j.nbt.2024.06.008},
pmid = {38960022},
issn = {1876-4347},
mesh = {*Saccharomycetales/genetics/metabolism ; *Green Fluorescent Proteins/metabolism/genetics ; CRISPR-Cas Systems ; Gene Expression Regulation, Fungal ; Fungal Proteins/genetics/metabolism ; Plasmids/genetics/metabolism ; },
abstract = {The methylotrophic yeast Komagataella phaffii is a popular host system for the pharmaceutical and biotechnological production of recombinant proteins. CRISPR-Cas9 and its derivative CRISPR interference (CRISPRi) offer a promising avenue to further enhance and exploit the full capabilities of this host. MAD7 and its catalytically inactive variant "dead" MAD7 (dMAD7) represent an interesting alternative to established CRISPR-Cas9 systems and are free to use for industrial and academic research. CRISPRi utilizing dMAD7 does not introduce double-strand breaks but only binds to the DNA to regulate gene expression. Here, we report the first use of dMAD7 in K. phaffii to regulate the expression of the enhanced green fluorescent protein (eGFP). A reduction of eGFP fluorescence level (up to 88 %) was achieved in random integration experiments using dMAD7 plasmids. Integration loci/events of investigated strains were assessed through whole genome sequencing. Additionally, RNA-sequencing experiments corroborated the whole genome sequencing results and showed a significantly reduced expression of eGFP in strains containing a dMAD7 plasmid, among others. Our findings conclusively demonstrate the utility of dMAD7 in K. phaffii through successfully regulating eGFP expression.},
}

*Saccharomycetales/genetics/metabolism
*Green Fluorescent Proteins/metabolism/genetics
CRISPR-Cas Systems
Gene Expression Regulation, Fungal
Fungal Proteins/genetics/metabolism
Plasmids/genetics/metabolism

@article {pmid39246130,
year = {2024},
author = {Andorfer, P and Kahlig, CI and Pakusic, D and Pachlinger, R and John, C and Schrenk, I and Eisenhut, P and Lengler, J and Innthaler, B and Micutkova, L and Kraus, B and Brizzee, C and Crawford, J and Hernandez Bort, JA},
title = {Cas-CLOVER-mediated knockout of STAT1: A novel approach to engineer packaging HEK-293 cell lines used for rAAV production.},
journal = {Biotechnology journal},
volume = {19},
number = {9},
pages = {e2400415},
doi = {10.1002/biot.202400415},
pmid = {39246130},
issn = {1860-7314},
mesh = {Humans ; *Dependovirus/genetics ; *STAT1 Transcription Factor/genetics/metabolism ; HEK293 Cells ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Gene Knockout Techniques/methods ; Genetic Vectors/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {In addressing the limitations of CRISPR-Cas9, including off-target effects and high licensing fees for commercial use, Cas-CLOVER, a dimeric gene editing tool activated by two guide RNAs, was recently developed. This study focused on implementing and evaluating Cas-CLOVER in HEK-293 cells used for recombinant adeno-associated virus (rAAV) production by targeting the signal transducer and activator of transcription 1 (STAT1) locus, which is crucial for cell growth regulation and might influence rAAV production yields. Cas-CLOVER demonstrated impressive efficiency in gene editing, achieving over 90% knockout (KO) success. Thirteen selected HEK-293 STAT1 KO sub-clones were subjected to extensive analytical characterization to assess their genomic stability, crucial for maintaining cell integrity and functionality. Additionally, rAAV9 productivity, Rep protein pattern profile, and potency, among others, were assessed. Clones showed significant variation in capsid and vector genome titers, with capsid titer reductions ranging from 15% to 98% and vector genome titers from 16% to 55%. Interestingly, the Cas-CLOVER-mediated STAT1 KO bulk cell population showed a better ratio of full to empty capsids. Our study also established a comprehensive analytical workflow to detect and evaluate the gene KOs generated by this innovative tool, providing a solid groundwork for future research in precise gene editing technologies.},
}

Humans
*Dependovirus/genetics
*STAT1 Transcription Factor/genetics/metabolism
HEK293 Cells
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
*Gene Knockout Techniques/methods
Genetic Vectors/genetics
RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid39245805,
year = {2024},
author = {Cetin, B and Erendor, F and Eksi, YE and Sanlioglu, AD and Sanlioglu, S},
title = {Gene and cell therapy of human genetic diseases: Recent advances and future directions.},
journal = {Journal of cellular and molecular medicine},
volume = {28},
number = {17},
pages = {e70056},
doi = {10.1111/jcmm.70056},
pmid = {39245805},
issn = {1582-4934},
mesh = {Humans ; *Genetic Therapy/methods ; *Genetic Diseases, Inborn/therapy/genetics ; *Cell- and Tissue-Based Therapy/methods/trends ; *Gene Editing/methods ; *CRISPR-Cas Systems ; Animals ; },
abstract = {Disruptions in normal development and the emergence of health conditions often result from the malfunction of vital genes in the human body. Decades of scientific research have focused on techniques to modify or substitute defective genes with healthy alternatives, marking a new era in disease treatment, prevention and cure. Recent strides in science and technology have reshaped our understanding of disorders, medication development and treatment recommendations, with human gene and cell therapy at the forefront of this transformative shift. Its primary objective is the modification of genes or adjustment of cell behaviour for therapeutic purposes. In this review, we focus on the latest advances in gene and cell therapy for treating human genetic diseases, with a particular emphasis on FDA and EMA-approved therapies and the evolving landscape of genome editing. We examine the current state of innovative gene editing technologies, particularly the CRISPR-Cas systems. As we explore the progress, ethical considerations and prospects of these innovations, we gain insight into their potential to revolutionize the treatment of genetic diseases, along with a discussion of the challenges associated with their regulatory pathways. This review traces the origins and evolution of these therapies, from conceptual ideas to practical clinical applications, marking a significant milestone in the field of medical science.},
}

Humans
*Genetic Therapy/methods
*Genetic Diseases, Inborn/therapy/genetics
*Cell- and Tissue-Based Therapy/methods/trends
*Gene Editing/methods
*CRISPR-Cas Systems
Animals

@article {pmid39244373,
year = {2024},
author = {Wang, Z and Chen, H and Hu, A and Cui, X and Shi, C and Lu, Z and Meng, F and Lv, F and Zhao, H and Bie, X},
title = {Establishment of LAMP-CRISPR/Cas12a for rapid detection of Escherichia coli O157:H7 and one-pot detection.},
journal = {Food microbiology},
volume = {124},
number = {},
pages = {104622},
doi = {10.1016/j.fm.2024.104622},
pmid = {39244373},
issn = {1095-9998},
mesh = {*Escherichia coli O157/genetics/isolation & purification ; *CRISPR-Cas Systems ; *Milk/microbiology ; *Food Microbiology/methods ; *Nucleic Acid Amplification Techniques/methods ; Animals ; Sensitivity and Specificity ; Food Contamination/analysis ; Molecular Diagnostic Techniques/methods ; },
abstract = {Escherichia coli O157:H7 is a pathogenic serotype of Escherichia coli. Consumption of food contaminated with E. coli O157:H7 could cause a range of diseases. Therefore, it is of great importance to establish rapid and accurate detection methods for E. coli O157:H7 in food. In this study, based on LAMP and combined with the CRISPR/cas12a system, a sensitive and specific rapid detection method for E. coli O157:H7 was established, and One-Pot detection method was also constructed. The sensitivity of this method could stably reach 9.2 × 10° CFU/mL in pure culture, and the whole reaction can be completed within 1 h. In milk, E. coli O157:H7 with an initial contamination of 7.4 × 10° CFU/mL only needed to be cultured for 3 h to be detected. The test results can be judged by the fluorescence curve or by visual observation under a UV lamp, eliminating instrument limitations and One-Pot detection can effectively prevent the problem of false positives. In a word, the LAMP-CRISPR/cas12a system is a highly sensitive and convenient method for detecting E. coli O157:H7.},
}

*Escherichia coli O157/genetics/isolation & purification
*CRISPR-Cas Systems
*Milk/microbiology
*Food Microbiology/methods
*Nucleic Acid Amplification Techniques/methods
Animals
Sensitivity and Specificity
Food Contamination/analysis
Molecular Diagnostic Techniques/methods

@article {pmid39087566,
year = {2024},
author = {Zhang, Y and Zhang, S},
title = {CRISPR perfect adaptation for robust control of cellular immune and apoptotic responses.},
journal = {Nucleic acids research},
volume = {52},
number = {16},
pages = {10005-10016},
doi = {10.1093/nar/gkae665},
pmid = {39087566},
issn = {1362-4962},
support = {32171416//National Natural Science Foundation of China/ ; 2023YFA0915601//Ministry of Science and Technology/ ; //Tsinghua University Dushi Plan Foundation/ ; //Beijing Frontier Research Center for Biological Structure/ ; },
mesh = {*Apoptosis/genetics ; Humans ; *CRISPR-Cas Systems ; NF-kappa B/metabolism ; Gene Expression Regulation ; HEK293 Cells ; T-Lymphocytes/immunology ; Immunity, Cellular/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Animals ; },
abstract = {A central challenge in the quest for precise gene regulation within mammalian cells is the development of regulatory networks that can achieve perfect adaptation-where outputs consistently return to a set baseline post-stimulus. Here, we present such a system that leverages the CRISPR activation (CRISPRa) and anti-CRISPR proteins as two antithetic elements to establish perfect adaptation in mammalian cells and dynamically regulate gene expression. We demonstrate that this system can maintain stable expression levels of target genes in the face of external perturbations, thus providing a robust platform for biological applications. The versatility of our system is further showcased through its integration with endogenous regulatory mechanisms in T cells, such as the NF-κB-mediated immune response, and its ability to program apoptosis responses for precise spatial and temporal control of cellular growth and death. This study not only advances our understanding of gene regulation in mammalian cells but also opens new avenues for therapeutic intervention, particularly in diseases characterized by dysregulated gene expression.},
}

*Apoptosis/genetics
Humans
*CRISPR-Cas Systems
NF-kappa B/metabolism
Gene Expression Regulation
HEK293 Cells
T-Lymphocytes/immunology
Immunity, Cellular/genetics
Clustered Regularly Interspaced Short Palindromic Repeats
Animals

@article {pmid38818919,
year = {2024},
author = {Khademi, Z and Mahmoudi, Z and Sukhorukov, VN and Jamialahmadi, T and Sahebkar, A},
title = {CRISPR/Cas9 Technology: A Novel Approach to Obesity Research.},
journal = {Current pharmaceutical design},
volume = {30},
number = {23},
pages = {1791-1803},
pmid = {38818919},
issn = {1873-4286},
mesh = {Humans ; *Obesity/genetics/therapy ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Animals ; },
abstract = {Gene editing technology, particularly Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) has transformed medical research. As a newly developed genome editing technique, CRISPR technology has strongly assisted scientists in enriching their comprehension of the roles of individual genes and their influences on a vast spectrum of human malignancies. Despite considerable progress in elucidating obesity's molecular pathways, current anti-obesity medications fall short in effectiveness. A thorough understanding of the genetic foundations underlying various neurobiological pathways related to obesity, as well as the neuro-molecular mechanisms involved, is crucial for developing effective obesity treatments. Utilizing CRISPR-based technologies enables precise determination of the roles of genes that encode transcription factors or enzymes involved in processes, such as lipogenesis, lipolysis, glucose metabolism, and lipid storage within adipose tissue. This innovative approach allows for the targeted suppression or activation of genes regulating obesity, potentially leading to effective weight management strategies. In this review, we have provided a detailed overview of obesity's molecular genetics, the fundamentals of CRISPR/Cas9 technology, and how this technology contributes to the discovery and therapeutic targeting of new genes associated with obesity.},
}

Humans
*Obesity/genetics/therapy
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
Animals

@article {pmid38416162,
year = {2024},
author = {Tea, M and Pan, YK and Lister, JGR and Perry, SF and Gilmour, KM},
title = {Effects of serta and sertb knockout on aggression in zebrafish (Danio rerio).},
journal = {Journal of comparative physiology. A, Neuroethology, sensory, neural, and behavioral physiology},
volume = {210},
number = {5},
pages = {785-799},
pmid = {38416162},
issn = {1432-1351},
mesh = {Animals ; *Zebrafish/genetics/metabolism ; *Aggression/physiology ; Female ; *Zebrafish Proteins/genetics/metabolism ; Male ; *Serotonin Plasma Membrane Transport Proteins/genetics/metabolism ; Serotonin/metabolism ; Behavior, Animal/physiology ; Gene Knockout Techniques ; Animals, Genetically Modified ; CRISPR-Cas Systems ; Brain/metabolism ; },
abstract = {Zebrafish (Danio rerio) are unusual in having two paralogues of the serotonin re-uptake transporter (Sert), slc6a4a (serta) and slc6a4b (sertb), the transporter that serves in serotonin re-uptake from a synapse into the pre-synaptic cell or in serotonin uptake from the extracellular milieu into cells in the peripheral tissues. To address a knowledge gap concerning the specific roles of these paralogues, we used CRISPR/Cas9 technology to generate zebrafish knockout lines predicted to lack functional expression of Serta or Sertb. The consequences of loss-of-function of Serta or Sertb were assessed at the gene expression level, focusing on the serotonergic signalling pathway, and at the behaviour level, focusing on aggression. Whereas serta mRNA was expressed in all tissues examined, with high expression in the heart, gill and brain, only the brain displayed substantial sertb mRNA expression. In both serta[-/-] and sertb[-/-] fish, changes in transcript abundances of multiple components of the serotonin signalling pathway were detected, including proteins involved in serotonin synthesis (tph1a, tph1b, tph2, ddc), packaging (vmat2) and degradation (mao), and serotonin receptors (htr1aa, htr1ab). Using a mirror aggression test, serta[-/-] male but not female fish exhibited greater aggression than wildtype fish. However, both male and female sertb[-/-] fish displayed less aggression than their wildtype counterparts. These differences in behaviour between serta[-/-] and sertb[-/-] individuals hold promise for increasing our understanding of the neurophysiological basis of aggression in zebrafish.},
}

Animals
*Zebrafish/genetics/metabolism
*Aggression/physiology
Female
*Zebrafish Proteins/genetics/metabolism
Male
*Serotonin Plasma Membrane Transport Proteins/genetics/metabolism
Serotonin/metabolism
Behavior, Animal/physiology
Gene Knockout Techniques
Animals, Genetically Modified
CRISPR-Cas Systems
Brain/metabolism

@article {pmid39242595,
year = {2024},
author = {Dvorakova, T and Finisguerra, V and Formenti, M and Loriot, A and Boudhan, L and Zhu, J and Van den Eynde, BJ},
title = {Enhanced tumor response to adoptive T cell therapy with PHD2/3-deficient CD8 T cells.},
journal = {Nature communications},
volume = {15},
number = {1},
pages = {7789},
pmid = {39242595},
issn = {2041-1723},
support = {T.0091.18//Fonds De La Recherche Scientifique - FNRS (Belgian National Fund for Scientific Research)/ ; WELBIO-CR-2019C-05//Fonds De La Recherche Scientifique - FNRS (Belgian National Fund for Scientific Research)/ ; 2019-094//Stichting Tegen Kanker (Belgian Foundation Against Cancer)/ ; },
mesh = {Animals ; *CD8-Positive T-Lymphocytes/immunology/metabolism ; *Hypoxia-Inducible Factor-Proline Dioxygenases/metabolism/genetics ; *Immunotherapy, Adoptive/methods ; Mice ; *Tumor Microenvironment/immunology ; *Hypoxia-Inducible Factor 1, alpha Subunit/metabolism/genetics ; Mice, Inbred C57BL ; Cell Line, Tumor ; Lymphocyte Activation/immunology ; CRISPR-Cas Systems ; Humans ; Neoplasms/immunology/therapy/genetics ; Female ; Procollagen-Proline Dioxygenase ; },
abstract = {While adoptive cell therapy has shown success in hematological malignancies, its potential against solid tumors is hindered by an immunosuppressive tumor microenvironment (TME). In recent years, members of the hypoxia-inducible factor (HIF) family have gained recognition as important regulators of T-cell metabolism and function. The role of HIF signalling in activated CD8 T cell function in the context of adoptive cell transfer, however, has not been explored in full depth. Here we utilize CRISPR-Cas9 technology to delete prolyl hydroxylase domain-containing enzymes (PHD) 2 and 3, thereby stabilizing HIF-1 signalling, in CD8 T cells that have already undergone differentiation and activation, modelling the T cell phenotype utilized in clinical settings. We observe a significant boost in T-cell activation and effector functions following PHD2/3 deletion, which is dependent on HIF-1α, and is accompanied by an increased glycolytic flux. This improvement in CD8 T cell performance translates into an enhancement in tumor response to adoptive T cell therapy in mice, across various tumor models, even including those reported to be extremely resistant to immunotherapeutic interventions. These findings hold promise for advancing CD8 T-cell based therapies and overcoming the immune suppression barriers within challenging tumor microenvironments.},
}

Animals
*CD8-Positive T-Lymphocytes/immunology/metabolism
*Hypoxia-Inducible Factor-Proline Dioxygenases/metabolism/genetics
*Immunotherapy, Adoptive/methods
Mice
*Tumor Microenvironment/immunology
*Hypoxia-Inducible Factor 1, alpha Subunit/metabolism/genetics
Mice, Inbred C57BL
Cell Line, Tumor
Lymphocyte Activation/immunology
CRISPR-Cas Systems
Humans
Neoplasms/immunology/therapy/genetics
Female
Procollagen-Proline Dioxygenase

@article {pmid39241751,
year = {2024},
author = {Birkholz, N and Fineran, PC},
title = {Anti-CRISPRs deconstruct bacterial defense.},
journal = {Molecular cell},
volume = {84},
number = {17},
pages = {3172-3174},
doi = {10.1016/j.molcel.2024.08.008},
pmid = {39241751},
issn = {1097-4164},
mesh = {*CRISPR-Cas Systems ; *Bacteriophages/genetics ; *Bacteria/genetics/immunology ; Clustered Regularly Interspaced Short Palindromic Repeats ; Bacterial Proteins/genetics/metabolism ; },
abstract = {Deploying anti-CRISPR proteins is a potent strategy used by phages to inhibit bacterial CRISPR-Cas defense. In a new Nature paper, Trost et al.[1] discover and characterize an exciting anti-CRISPR mechanism with possible implications beyond this microscopic arms race.},
}

*CRISPR-Cas Systems
*Bacteriophages/genetics
*Bacteria/genetics/immunology
Clustered Regularly Interspaced Short Palindromic Repeats
Bacterial Proteins/genetics/metabolism

@article {pmid39178617,
year = {2024},
author = {Punetha, M and Saini, S and Choudhary, S and Sharma, S and Bala, R and Kumar, P and Sharma, RK and Yadav, PS and Datta, TK and Kumar, D},
title = {Establishment of CRISPR-Cas9 ribonucleoprotein mediated MSTN gene edited pregnancy in buffalo: Compare cells transfection and zygotes electroporation.},
journal = {Theriogenology},
volume = {229},
number = {},
pages = {158-168},
doi = {10.1016/j.theriogenology.2024.08.027},
pmid = {39178617},
issn = {1879-3231},
mesh = {Animals ; *Buffaloes/genetics ; *Electroporation/veterinary/methods ; Female ; Pregnancy ; *CRISPR-Cas Systems ; *Gene Editing/methods/veterinary ; *Transfection/veterinary/methods ; *Nuclear Transfer Techniques/veterinary ; Myostatin/genetics ; Zygote/metabolism ; },
abstract = {Genome editing is recognized as a powerful tool in agriculture and research, enhancing our understanding of genetic function, diseases, and productivity. However, its progress in buffaloes has lagged behind other mammals due to several challenges, including long gestational periods, single pregnancies, and high raising costs. In this study, we aimed to generate MSTN-edited buffaloes, known for their distinctive double-muscling phenotype, as a proof of concept. To meet our goal, we used somatic cell nuclear transfer (SCNT) and zygotic electroporation (CRISPR-EP) technique. For this, we firstly identified the best transfection method for introduction of RNP complex into fibroblast which was further used for SCNT. For this, we compared the transfection, cleavage efficiency and cell viability of nucleofection and lipofection in adult fibroblasts. The cleavage, transfection efficiency and cell viability of nucleofection group was found to be significantly (P ≤ 0.05) higher than lipofection group. Four MSTN edited colony were generated using nucleofection, out of which three colonies was found to be biallelic and one was monoallelic. Further, we compared the efficacy, embryonic developmental potential and subsequent pregnancy outcome of SCNT and zygotic electroporation. The blastocyst rate of electroporated group was found to be significantly (P ≤ 0.05) higher than SCNT group. However, the zygotic electroporation group resulted into two pregnancies which were confirmed to be MSTN edited. Since, the zygotic electroporation does not require complex micromanipulation techniques associated with SCNT, it has potential for facilitating the genetic modification in large livestock such as buffaloes. The present study lays the basis for inducing genetic alternation with practical or biological significance.},
}

Animals
*Buffaloes/genetics
*Electroporation/veterinary/methods
Female
Pregnancy
*CRISPR-Cas Systems
*Gene Editing/methods/veterinary
*Transfection/veterinary/methods
*Nuclear Transfer Techniques/veterinary
Myostatin/genetics
Zygote/metabolism

@article {pmid39239522,
year = {2024},
author = {Wu, Y and Chen, S and Huang, G and Zhang, L and Zhong, L and Feng, Y and Wen, P and Liu, J},
title = {Transcriptome analysis reveals EBF1 ablation-induced injuries in cardiac system.},
journal = {Theranostics},
volume = {14},
number = {12},
pages = {4894-4915},
pmid = {39239522},
issn = {1838-7640},
mesh = {Animals ; Humans ; Mice ; *Trans-Activators/genetics/metabolism ; *Myocytes, Cardiac/metabolism ; *Gene Expression Profiling ; Cell Differentiation/genetics ; Heart/physiopathology ; Mice, Knockout ; Pluripotent Stem Cells/metabolism ; Transcriptome/genetics ; CRISPR-Cas Systems/genetics ; },
abstract = {Rationale: Regulatory processes of transcription factors (TFs) shape heart development and influence the adult heart's response to stress, contributing to cardiac disorders. Despite their significance, the precise mechanisms underpinning TF-mediated regulation remain elusive. Here, we identify that EBF1, as a TF, is highly expressed in human heart tissues. EBF1 is reported to be associated with human cardiovascular disease, but its roles are unclear in heart. In this study, we investigated EBF1 function in cardiac system. Methods: RNA-seq was utilized to profile EBF1 expression patterns. CRISPR/Cas9 was utilized to knock out EBF1 to investigate its effects. Human pluripotent stem cells (hPSCs) differentiated into cardiac lineages were used to mimic cardiac development. Cardiac function was evaluated on mouse model with Ebf1 knockout by using techniques such as echocardiography. RNA-seq was conducted to analyze transcriptional perturbations. ChIP-seq was employed to elucidate EBF1-bound genes and the underlying regulatory mechanisms. Results: EBF1 was expressed in some human and mouse cardiomyocyte. Knockout of EBF1 inhibited cardiac development. ChIP-seq indicated EBF1's binding on promoters of cardiogenic TFs pivotal to cardiac development, facilitating their transcriptional expression and promoting cardiac development. In mouse, Ebf1 depletion triggered transcriptional perturbations of genes, resulting in cardiac remodeling. Mechanistically, we found that EBF1 directly bound to upstream chromatin regions of cardiac hypertrophy-inducing genes, contributing to cardiac hypertrophy. Conclusions: We uncover the mechanisms underlying EBF1-mediated regulatory processes, shedding light on cardiac development, and the pathogenesis of cardiac remodeling. These findings emphasize EBF1's critical role in orchestrating diverse aspects of cardiac processes and provide a promising therapeutic intervention for cardiomyopathy.},
}

Animals
Humans
Mice
*Trans-Activators/genetics/metabolism
*Myocytes, Cardiac/metabolism
*Gene Expression Profiling
Cell Differentiation/genetics
Heart/physiopathology
Mice, Knockout
Pluripotent Stem Cells/metabolism
Transcriptome/genetics
CRISPR-Cas Systems/genetics

@article {pmid39239521,
year = {2024},
author = {Ryu, JY and Cerecedo-Lopez, C and Yang, H and Ryu, I and Du, R},
title = {Brain-targeted intranasal delivery of protein-based gene therapy for treatment of ischemic stroke.},
journal = {Theranostics},
volume = {14},
number = {12},
pages = {4773-4786},
pmid = {39239521},
issn = {1838-7640},
mesh = {Animals ; *Administration, Intranasal ; Mice ; Humans ; *Ischemic Stroke/therapy/genetics ; *Nanoparticles/administration & dosage ; *Genetic Therapy/methods ; *Sirtuin 1/genetics/metabolism ; *Brain/metabolism ; *Disease Models, Animal ; Male ; Calcium Phosphates ; CRISPR-Cas Systems ; Mice, Inbred C57BL ; Endothelial Cells/metabolism ; Brain Ischemia/therapy/genetics ; Infarction, Middle Cerebral Artery/therapy/genetics ; Epithelial Cells/metabolism ; },
abstract = {Gene therapy using a protein-based CRISPR system in the brain has practical limitations due to current delivery systems, especially in the presence of arterial occlusion. To overcome these obstacles and improve stability, we designed a system for intranasal administration of gene therapy for the treatment of ischemic stroke. Methods: Nanoparticles containing the protein-based CRISPR/dCas9 system targeting Sirt1 were delivered intranasally to the brain in a mouse model of ischemic stroke. The CRISPR/dCas9 system was encapsulated with calcium phosphate (CaP) nanoparticles to prevent them from being degraded. They were then conjugated with β-hydroxybutyrates (bHb) to target monocarboxylic acid transporter 1 (MCT1) in nasal epithelial cells to facilitate their transfer into the brain. Results: Human nasal epithelial cells were shown to uptake and transfer nanoparticles to human brain endothelial cells with high efficiency in vitro. The intranasal administration of the dCas9/CaP/PEI-PEG-bHb nanoparticles in mice effectively upregulated the target gene, Sirt1, in the brain, decreased cerebral edema and increased survival after permanent middle cerebral artery occlusion. Additionally, we observed no significant in vivo toxicity associated with intranasal administration of the nanoparticles, highlighting the safety of this approach. Conclusion: This study demonstrates that the proposed protein-based CRISPR-dCas9 system targeting neuroprotective genes in general, and SIRT1 in particular, can be a potential novel therapy for acute ischemic stroke.},
}

Animals
*Administration, Intranasal
Mice
Humans
*Ischemic Stroke/therapy/genetics
*Nanoparticles/administration & dosage
*Genetic Therapy/methods
*Sirtuin 1/genetics/metabolism
*Brain/metabolism
*Disease Models, Animal
Male
Calcium Phosphates
CRISPR-Cas Systems
Mice, Inbred C57BL
Endothelial Cells/metabolism
Brain Ischemia/therapy/genetics
Infarction, Middle Cerebral Artery/therapy/genetics
Epithelial Cells/metabolism

@article {pmid38816517,
year = {2024},
author = {Wang, B and Xu, Y and Wan, AH and Wan, G and Wang, QP},
title = {Integrating genome-wide CRISPR screens and in silico drug profiling for targeted antidote development.},
journal = {Nature protocols},
volume = {19},
number = {9},
pages = {2739-2770},
pmid = {38816517},
issn = {1750-2799},
mesh = {*Antidotes/pharmacology ; Humans ; *CRISPR-Cas Systems ; *Computer Simulation ; Drug Evaluation, Preclinical/methods ; Animals ; },
abstract = {Numerous toxins threaten humans, but specific antidotes are unavailable for most of them. Although CRISPR screening has aided the discovery of the mechanisms of some toxins, developing targeted antidotes remains a significant challenge. Recently, we established a systematic framework to develop antidotes by combining the identification of novel drug targets by using a genome-wide CRISPR screen with a virtual screen of drugs approved by the US Food and Drug Administration. This approach allows for a comprehensive understanding of toxin mechanisms at the whole-genome level and facilitates the identification of promising antidote drugs targeting specific molecules. Here, we present step-by-step instructions for executing genome-scale CRISPR-Cas9 knockout screens of toxins in HAP1 cells. We also provide detailed guidance for conducting an in silico drug screen and an in vivo drug validation. By using this protocol, it takes ~4 weeks to perform the genome-scale screen, 4 weeks for sequencing and data analysis, 4 weeks to validate candidate genes, 1 week for the virtual screen and 2 weeks for in vitro drug validation. This framework has the potential to accelerate the development of antidotes for a wide range of toxins and can rapidly identify promising drug candidates that are already known to be safe and effective. This could lead to the development of new antidotes much more quickly than traditional methods, protecting lives from diverse toxins and advancing human health.},
}

*Antidotes/pharmacology
Humans
*CRISPR-Cas Systems
*Computer Simulation
Drug Evaluation, Preclinical/methods
Animals

@article {pmid39238167,
year = {2024},
author = {Sánchez-León, S and Marín-Sanz, M and Guzmán-López, MH and Gavilán-Camacho, M and Simón, E and Barro, F},
title = {CRISPR/Cas9-mediated multiplex gene editing of gamma and omega gliadins, paving the way for gliadin-free wheat.},
journal = {Journal of experimental botany},
volume = {},
number = {},
pages = {},
doi = {10.1093/jxb/erae376},
pmid = {39238167},
issn = {1460-2431},
abstract = {Wheat is a staple cereal in the human diet. Despite its significance, an increasing percentage of the population suffers adverse reactions to wheat, which are triggered by wheat gluten, particularly the gliadin fractions. In this study, we employed CRISPR/Cas multiplexing to introduce targeted mutations into γ- and ω-gliadin genes of wheat, to produce lines deficient in one or both immunogenic gliadin fractions simultaneously. For this work, eight single guide RNAs (sgRNAs) were designed and combined into four plasmids to produce 59 modified wheat lines, of which 20 exhibited mutations in the target genes. Characterization of these lines through Sanger or NGS sequencing revealed a complex pattern of InDels, including deletions spanning multiple sgRNAs. The mutations were transmitted to the offspring, and the analysis of homozygous derived lines by RP-HPLC and monoclonal antibodies showed a 97.7% reduction in gluten content. Crossing these lines with other CRISPR/Cas lines deficient in the α-gliadins allowed multiple mutations to be combined. This work represents an important step forward in the use of CRISPR/Cas to develop gluten-free wheat.},
}

@article {pmid39237013,
year = {2024},
author = {Gattani, A and Mandal, S and Agrawal, A and Patel, P and Jain, AK and Singh, P and Garg, A and Mishra, A},
title = {CRISPR-Based Electrochemical Biosensors For Animal Health: Recent Advances.},
journal = {Progress in biophysics and molecular biology},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.pbiomolbio.2024.09.001},
pmid = {39237013},
issn = {1873-1732},
abstract = {Animal diseases are a major concern to animal welfare, human health and the global economy. Early detection, prevention and control of these animal diseases are crucial to ensure sustainability of livestock sector, to reduce farm losses and protecting public health. Points of care (POC) devices are small, portable instruments that provide rapid results thus reduce the risk of disease transmission and enable early intervention. CRISPR based diagnostics offer more accurate and efficient solution for monitoring animal health due to their quick response, can detect very low level of pathogenic organism or disease markers and specificity. These diagnostics are particularly useful in the in area with limited resources or access to common diagnostic methods, especially in developing countries. The ability of electrochemical sensors to detect accurately very low analyte concentration makes them suitable for POC diagnostics and field application. CRISPR base electrochemical biosensors show great potential in revolutionizing disease detection and diagnosis including animal health. However, challenges, such as achieving selectivity and sensitivity, need to be addressed to enhance the competitiveness of these biosensors. Currently, most CRISPR based bioassay research focuses on nucleic acid target detection, but researchers exploring to monitor small organic/ inorganic non-nucleic acid molecules like toxins and proteins. Emerging diagnostics would be centered on CRISPR-Cas system will offer great potential as an accurate, specific and effective means to identify microorganism, virus, toxins, small molecules, peptides and nucleic acid related to various animal health disorders particularly when integrated into electrochemical biosensing platform.},
}

@article {pmid39235647,
year = {2024},
author = {Byiringiro, I and Pan, C and Qi, Y},
title = {Orthogonal genome editing and transcriptional activation in tomato using CRISPR-Combo systems.},
journal = {Plant cell reports},
volume = {43},
number = {9},
pages = {227},
pmid = {39235647},
issn = {1432-203X},
support = {IOS-2029889//Directorate for Biological Sciences/ ; IOS-2132693//Directorate for Biological Sciences/ ; 21010111//Foundation for Food and Agriculture Research/ ; },
mesh = {*Solanum lycopersicum/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *Transcriptional Activation/genetics ; Genome, Plant/genetics ; Plants, Genetically Modified/genetics ; },
abstract = {The CRISPR-Combo systems (Cas9-Combo and CBE-Combo) are designed for comprehensive genetic manipulation, enabling Cas9-based targeted mutagenesis or cytosine base editing with simultaneous gene activation in tomato stable lines.},
}

*Solanum lycopersicum/genetics
*Gene Editing/methods
*CRISPR-Cas Systems
*Transcriptional Activation/genetics
Genome, Plant/genetics
Plants, Genetically Modified/genetics

@article {pmid39009674,
year = {2024},
author = {Pauzaite, T and Wit, N and Seear, RV and Nathan, JA},
title = {Deubiquitinating enzyme mutagenesis screens identify a USP43-dependent HIF-1 transcriptional response.},
journal = {The EMBO journal},
volume = {43},
number = {17},
pages = {3677-3709},
pmid = {39009674},
issn = {1460-2075},
support = {215477/Z/19/Z//Wellcome Trust (WT)/ ; },
mesh = {Humans ; *Hypoxia-Inducible Factor 1, alpha Subunit/metabolism/genetics ; *Deubiquitinating Enzymes/metabolism/genetics ; Mutagenesis ; CRISPR-Cas Systems ; HEK293 Cells ; Signal Transduction ; Ubiquitination ; },
abstract = {The ubiquitination and proteasome-mediated degradation of Hypoxia Inducible Factors (HIFs) is central to metazoan oxygen-sensing, but the involvement of deubiquitinating enzymes (DUBs) in HIF signalling is less clear. Here, using a bespoke DUBs sgRNA library we conduct CRISPR/Cas9 mutagenesis screens to determine how DUBs are involved in HIF signalling. Alongside defining DUBs involved in HIF activation or suppression, we identify USP43 as a DUB required for efficient activation of a HIF response. USP43 is hypoxia regulated and selectively associates with the HIF-1α isoform, and while USP43 does not alter HIF-1α stability, it facilitates HIF-1 nuclear accumulation and binding to its target genes. Mechanistically, USP43 associates with 14-3-3 proteins in a hypoxia and phosphorylation dependent manner to increase the nuclear pool of HIF-1. Together, our results highlight the multifunctionality of DUBs, illustrating that they can provide important signalling functions alongside their catalytic roles.},
}

Humans
*Hypoxia-Inducible Factor 1, alpha Subunit/metabolism/genetics
*Deubiquitinating Enzymes/metabolism/genetics
Mutagenesis
CRISPR-Cas Systems
HEK293 Cells
Signal Transduction
Ubiquitination

@article {pmid38884692,
year = {2024},
author = {Han, U and Han, N and Park, B and Jeon, TJ},
title = {RapB Regulates Cell Adhesion and Migration in Dictyostelium, Similar to RapA.},
journal = {Journal of microbiology (Seoul, Korea)},
volume = {62},
number = {8},
pages = {627-637},
pmid = {38884692},
issn = {1976-3794},
mesh = {*Dictyostelium/genetics/physiology/metabolism/growth & development/cytology ; *Cell Adhesion ; *Chemotaxis ; *Protozoan Proteins/metabolism/genetics ; *Cell Movement ; CRISPR-Cas Systems ; ras Proteins/metabolism/genetics ; Gene Editing ; Signal Transduction ; },
abstract = {Ras small GTPases act as molecular switches in various cellular signaling pathways, including cell migration, proliferation, and differentiation. Three Rap proteins are present in Dictyostelium; RapA, RapB, and RapC. RapA and RapC have been reported to have opposing functions in the control of cell adhesion and migration. Here, we investigated the role of RapB, a member of the Ras GTPase subfamily in Dictyostelium, focusing on its involvement in cell adhesion, migration, and developmental processes. This study revealed that RapB, similar to RapA, played a crucial role in regulating cell morphology, adhesion, and migration. rapB null cells, which were generated by CRISPR/Cas9 gene editing, displayed altered cell size, reduced cell-substrate adhesion, and increased migration speed during chemotaxis. These phenotypes of rapB null cells were restored by the expression of RapB and RapA, but not RapC. Consistent with these results, RapB, similar to RapA, failed to rescue the phenotypes of rapC null cells, spread morphology, increased cell adhesion, and decreased migration speed during chemotaxis. Multicellular development of rapB null cells remained unaffected. These results suggest that RapB is involved in controlling cell morphology and cell adhesion. Importantly, RapB appears to play an inhibitory role in regulating the migration speed during chemotaxis, possibly by controlling cell-substrate adhesion, resembling the functions of RapA. These findings contribute to the understanding of the functional relationships among Ras subfamily proteins.},
}

*Dictyostelium/genetics/physiology/metabolism/growth & development/cytology
*Cell Adhesion
*Chemotaxis
*Protozoan Proteins/metabolism/genetics
*Cell Movement
CRISPR-Cas Systems
ras Proteins/metabolism/genetics
Gene Editing
Signal Transduction

@article {pmid39233906,
year = {2024},
author = {Gan, T and Yu, J and Deng, Z and He, J},
title = {ERA-CRISPR/Cas12a system: a rapid, highly sensitive and specific assay for Mycobacterium tuberculosis.},
journal = {Frontiers in cellular and infection microbiology},
volume = {14},
number = {},
pages = {1454076},
pmid = {39233906},
issn = {2235-2988},
mesh = {*Mycobacterium tuberculosis/genetics/isolation & purification ; *CRISPR-Cas Systems ; *Sensitivity and Specificity ; Humans ; *Nucleic Acid Amplification Techniques/methods ; Tuberculosis/diagnosis/microbiology ; Recombinases/metabolism/genetics ; Molecular Diagnostic Techniques/methods ; Bacterial Proteins/genetics ; DNA, Bacterial/genetics ; CRISPR-Associated Proteins/genetics ; Endodeoxyribonucleases ; },
abstract = {INTRODUCTION: Mycobacterium tuberculosis, the causative agent of human tuberculosis, poses a significant threat to global public health and imposes a considerable burden on the economy. However, existing laboratory diagnostic methods for M. tuberculosis are time-consuming and have limited sensitivity levels.

METHODS: The CRISPR/Cas system, commonly known as the "gene scissors", demonstrates remarkable specificity and efficient signal amplification capabilities. Enzymatic recombinase amplification (ERA) was utilized to rapidly amplify trace DNA fragments at a consistent temperature without relying on thermal cyclers. By integrating of CRISPR/Cas12a with ERA, we successfully developed an ERA-CRISPR/Cas12a detection system that enables rapid identification of M. tuberculosis.

RESULTS: The sensitivity of the ERA-CRISPR/Cas12a fluorescence and lateral flow systems was 9 copies/μL and 90 copies/μL, respectively. Simultaneously, the detection system exhibited no cross-reactivity with various of respiratory pathogens and non-tuberculosis mycobacteria, demonstrating a specificity of 100%. The positive concordance rate between the ERA-CRISPR/Cas12a fluorescence system and commercial qPCR was 100% in 60 clinical samples. Meanwhile, the lateral flow system showed a positive concordance rate of 93.8% when compared to commercial qPCR. Both methods demonstrated a negative concordance rate of 100%, and the test results can be obtained in 50 min at the earliest.

DISCUSSION: The ERA-CRISPR/Cas12a system offers a rapid, sensitive, and specific method that presents a novel approach to laboratory diagnosis of M. tuberculosis.},
}

*Mycobacterium tuberculosis/genetics/isolation & purification
*CRISPR-Cas Systems
*Sensitivity and Specificity
Humans
*Nucleic Acid Amplification Techniques/methods
Tuberculosis/diagnosis/microbiology
Recombinases/metabolism/genetics
Molecular Diagnostic Techniques/methods
Bacterial Proteins/genetics
DNA, Bacterial/genetics
CRISPR-Associated Proteins/genetics
Endodeoxyribonucleases

@article {pmid39232173,
year = {2024},
author = {Katz, MA and Sawyer, EM and Oriolt, L and Kozlova, A and Williams, MC and Margolis, SR and Johnson, M and Bondy-Denomy, J and Meeske, AJ},
title = {Diverse viral cas genes antagonize CRISPR immunity.},
journal = {Nature},
volume = {},
number = {},
pages = {},
pmid = {39232173},
issn = {1476-4687},
abstract = {Prokaryotic CRISPR-Cas immunity is subverted by anti-CRISPRs (Acrs), which inhibit Cas protein activities when expressed during the phage lytic cycle or from resident prophages or plasmids[1]. Acrs often bind to specific cognate Cas proteins, and hence inhibition is typically limited to a single CRISPR-Cas subtype[2]. Furthermore, although acr genes are frequently organized together in phage-associated gene clusters[3], how such inhibitors initially evolve has remained unclear. Here we investigated the Acr content and inhibition specificity of diverse Listeria isolates, which naturally harbour four CRISPR-Cas systems (types I-B, II-A, II-C and VI-A). We observed widespread antagonism of CRISPR, which we traced to 11 previously unknown and 4 known acr gene families encoded by endogenous mobile elements. Among these were two Acrs that possess sequence homology to type I-B Cas proteins, one of which assembles into a defective interference complex. Surprisingly, an additional type I-B Cas homologue did not affect type I immunity, but instead inhibited the RNA-targeting type VI CRISPR system by means of CRISPR RNA (crRNA) degradation. By probing viral sequence databases, we detected abundant orphan cas genes located within putative anti-defence gene clusters. Among them, we verified the activity of a particularly broad-spectrum cas3 homologue that inhibits type I-B, II-A and VI-A CRISPR immunity. Our observations provide direct evidence of Acr evolution by cas gene co-option, and new genes with potential for broad-spectrum control of genome editing technologies.},
}

@article {pmid39232160,
year = {2024},
author = {Chen, J and Jia, Y and Sun, Y and Liu, K and Zhou, C and Liu, C and Li, D and Liu, G and Zhang, C and Yang, T and Huang, L and Zhuang, Y and Wang, D and Xu, D and Zhong, Q and Guo, Y and Li, A and Seim, I and Jiang, L and Wang, L and Lee, SMY and Liu, Y and Wang, D and Zhang, G and Liu, S and Wei, X and Yue, Z and Zheng, S and Shen, X and Wang, S and Qi, C and Chen, J and Ye, C and Zhao, F and Wang, J and Fan, J and Li, B and Sun, J and Jia, X and Xia, Z and Zhang, H and Liu, J and Zheng, Y and Liu, X and Wang, J and Yang, H and Kristiansen, K and Xu, X and Mock, T and Li, S and Zhang, W and Fan, G},
title = {Global marine microbial diversity and its potential in bioprospecting.},
journal = {Nature},
volume = {},
number = {},
pages = {},
pmid = {39232160},
issn = {1476-4687},
abstract = {The past two decades has witnessed a remarkable increase in the number of microbial genomes retrieved from marine systems[1,2]. However, it has remained challenging to translate this marine genomic diversity into biotechnological and biomedical applications[3,4]. Here we recovered 43,191 bacterial and archaeal genomes from publicly available marine metagenomes, encompassing a wide range of diversity with 138 distinct phyla, redefining the upper limit of marine bacterial genome size and revealing complex trade-offs between the occurrence of CRISPR-Cas systems and antibiotic resistance genes. In silico bioprospecting of these marine genomes led to the discovery of a novel CRISPR-Cas9 system, ten antimicrobial peptides, and three enzymes that degrade polyethylene terephthalate. In vitro experiments confirmed their effectiveness and efficacy. This work provides evidence that global-scale sequencing initiatives advance our understanding of how microbial diversity has evolved in the oceans and is maintained, and demonstrates how such initiatives can be sustainably exploited to advance biotechnology and biomedicine.},
}

@article {pmid39190773,
year = {2024},
author = {Kumar, A and Daripa, P and Rasool, K and Chakraborty, D and Jain, N and Maiti, S},
title = {Deciphering the Thermodynamic Landscape of CRISPR/Cas9: Insights into Enhancing Gene Editing Precision and Efficiency.},
journal = {The journal of physical chemistry. B},
volume = {128},
number = {35},
pages = {8409-8422},
doi = {10.1021/acs.jpcb.4c04044},
pmid = {39190773},
issn = {1520-5207},
mesh = {*CRISPR-Cas Systems ; *Thermodynamics ; *Gene Editing/methods ; DNA/chemistry/metabolism/genetics ; Calorimetry ; CRISPR-Associated Protein 9/metabolism/genetics/chemistry ; RNA, Guide, CRISPR-Cas Systems/chemistry/metabolism/genetics ; },
abstract = {The thermodynamic landscape of the CRISPR/Cas9 system plays a crucial role in understanding and optimizing the performance of this revolutionary genome-editing technology. In this research, we utilized isothermal titration calorimetry and microscale thermophoresis techniques to thoroughly investigate the thermodynamic properties governing CRISPR/Cas9 interactions. Our findings revealed that the binding between sgRNA and Cas9 is primarily governed by entropy, which compensates for an unfavorable enthalpy change. Conversely, the interaction between the CRISPR RNP complex and the target DNA is characterized by a favorable enthalpy change, offsetting an unfavorable entropy change. Notably, both interactions displayed negative heat capacity changes, indicative of potential hydration, ionization, or structural rearrangements. However, we noted that the involvement of water molecules and counterions in the interactions is minimal, suggesting that structural rearrangements play a significant role in influencing the binding thermodynamics. These results offer a nuanced understanding of the energetic contributions and structural dynamics underlying CRISPR-mediated gene editing. Such insights are invaluable for optimizing the efficiency and specificity of CRISPR-based genome editing applications, ultimately advancing our ability to precisely manipulate genetic material in various organisms for research, therapeutic, and biotechnological purposes.},
}

*CRISPR-Cas Systems
*Thermodynamics
*Gene Editing/methods
DNA/chemistry/metabolism/genetics
Calorimetry
CRISPR-Associated Protein 9/metabolism/genetics/chemistry
RNA, Guide, CRISPR-Cas Systems/chemistry/metabolism/genetics

@article {pmid39169620,
year = {2024},
author = {Capelletti, S and García Soto, SC and Gonçalves, MAFV},
title = {On RNA-programmable gene modulation as a versatile set of principles targeting muscular dystrophies.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.ymthe.2024.08.016},
pmid = {39169620},
issn = {1525-0024},
abstract = {The repurposing of RNA-programmable CRISPR systems from genome editing into epigenome editing tools is gaining pace, including in research and development efforts directed at tackling human disorders. This momentum stems from the increasing knowledge regarding the epigenetic factors and networks underlying cell physiology and disease etiology and from the growing realization that genome editing principles involving chromosomal breaks generated by programmable nucleases are prone to unpredictable genetic changes and outcomes. Hence, engineered CRISPR systems are serving as versatile DNA-targeting scaffolds for heterologous and synthetic effector domains that, via locally recruiting transcription factors and chromatin remodeling complexes, seek interfering with loss-of-function and gain-of-function processes underlying recessive and dominant disorders, respectively. Here, after providing an overview about epigenetic drugs and CRISPR-Cas-based activation and interference platforms, we cover the testing of these platforms in the context of molecular therapies for muscular dystrophies. Finally, we examine attributes, obstacles, and deployment opportunities for CRISPR-based epigenetic modulating technologies.},
}

@article {pmid39163859,
year = {2024},
author = {Tran, NT and Han, R},
title = {Rapidly evolving genome and epigenome editing technologies.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {32},
number = {9},
pages = {2803-2806},
doi = {10.1016/j.ymthe.2024.08.011},
pmid = {39163859},
issn = {1525-0024},
mesh = {*Gene Editing/methods ; Humans ; *CRISPR-Cas Systems ; Epigenome ; Animals ; Transcription Activator-Like Effector Nucleases/genetics/metabolism ; DNA Breaks, Double-Stranded ; Genetic Therapy/methods ; },
abstract = {Genome editing technologies are rapidly evolving, from the early zinc-finger nucleases, transcription activator-like effector nucleases (TALENs), and CRISPR-Cas9 (Figure 1, initial genome editing technologies), which generate double-strand breaks (DSBs), to base editing, which makes precise nucleobase conversion without inducing DSBs, and prime editing, which can carry out all types of edits without DSBs or donor DNA templates. The emergence of these revolutionary technologies offers us unprecedented opportunities for biomedical research and therapy development.},
}

*Gene Editing/methods
Humans
*CRISPR-Cas Systems
Epigenome
Animals
Transcription Activator-Like Effector Nucleases/genetics/metabolism
DNA Breaks, Double-Stranded
Genetic Therapy/methods

@article {pmid39127296,
year = {2025},
author = {Li, G and Zhou, J and Gao, N and Liu, R and Shen, J},
title = {Establishment of a rapid detection method for Mycoplasma pneumoniae based on RPA-CRISPR-Cas12a technology.},
journal = {Clinica chimica acta; international journal of clinical chemistry},
volume = {564},
number = {},
pages = {119906},
doi = {10.1016/j.cca.2024.119906},
pmid = {39127296},
issn = {1873-3492},
mesh = {*Mycoplasma pneumoniae/genetics/isolation & purification ; Humans ; *CRISPR-Cas Systems/genetics ; Nucleic Acid Amplification Techniques/methods ; Pneumonia, Mycoplasma/diagnosis/microbiology ; },
abstract = {Mycoplasma pneumoniae can cause respiratory infections and pneumonia, posing a serious threat to the health of children and adolescents. Early diagnosis of Mycoplasma pneumoniae infection is crucial for clinical treatment. Currently, diagnostic methods for Mycoplasma pneumoniae infection include pathogen detection, molecular biology techniques, and bacterial culture, all of which have certain limitations. Here, we developed a rapid, simple, and accurate detection method for Mycoplasma pneumoniae that does not rely on large equipment or complex operations. This technology combines the CRISPR-Cas12a system with recombinase polymerase amplification (RPA), allowing the detection results to be observed through fluorescence curves and immunochromatographic lateral flow strips.It has been validated that RPA-CRISPR/Cas12a fluorescence analysis and RPA-CRISPR/Cas12-immunochromatographic exhibit no cross-reactivity with other common pathogens, and The established detection limit was ascertained to be as low as 10[2] copies/µL.Additionally, 49 clinical samples were tested and compared with fluorescence quantitative polymerase chain reaction, demonstrating a sensitivity and specificity of 100%. This platform exhibits promising clinical performance and holds significant potential for clinical application, particularly in settings with limited resources, such as clinical care points or resource-constrained areas.},
}

*Mycoplasma pneumoniae/genetics/isolation & purification
Humans
*CRISPR-Cas Systems/genetics
Nucleic Acid Amplification Techniques/methods
Pneumonia, Mycoplasma/diagnosis/microbiology

@article {pmid39122070,
year = {2024},
author = {Wang, Q and Ma, C and Mao, H and Wang, J},
title = {Epigenome editing by a CRISPR-Cas9-based acetyltransferase activates the ZNF334 gene to inhibit the growth of colorectal cancer.},
journal = {International journal of biological macromolecules},
volume = {277},
number = {Pt 4},
pages = {134580},
doi = {10.1016/j.ijbiomac.2024.134580},
pmid = {39122070},
issn = {1879-0003},
mesh = {Humans ; *Colorectal Neoplasms/genetics/pathology ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Gene Expression Regulation, Neoplastic ; Acetylation ; *Epigenesis, Genetic ; Cell Proliferation/genetics ; Promoter Regions, Genetic/genetics ; Animals ; Cell Line, Tumor ; Histones/metabolism ; Mice ; Acetyltransferases/genetics/metabolism ; },
abstract = {Although therapeutic targets for colorectal cancer (CRC) treatment have been developed, the treatment outcomes are not ideal and survival rates for CRC patients remain low. It is critical to identify a specific target and develop an effective CRC treatment system. The ZNF334 gene is a newly identified member of Zinc-finger proteins (ZNFs), which is essential for key biological processes associated with tumorigenesis. Abnormal epigenetic reprogramming of the ZNF334 gene promoter region decreases its expression in CRC and further induces the occurrence of CRC. Here, we clarified that P300 in CRC can regulate the H3K9/27 ac in the ZNF334 promoter. Furthermore, histone acetylation of the ZNF334 promoter region was increased by dCas9-P300 to normalize the deficiency of ZNF334 expression, thereby inhibiting the growth of CRC. Collectively, our findings enable a facile way to affect gene expression using CRISPR/Cas9-based epigenome editing and further determine the causal link between histone acetylation and gene activation, providing a promising gene therapy strategy for the CRC treatment.},
}

Humans
*Colorectal Neoplasms/genetics/pathology
*CRISPR-Cas Systems
*Gene Editing/methods
*Gene Expression Regulation, Neoplastic
Acetylation
*Epigenesis, Genetic
Cell Proliferation/genetics
Promoter Regions, Genetic/genetics
Animals
Cell Line, Tumor
Histones/metabolism
Mice
Acetyltransferases/genetics/metabolism

@article {pmid39039289,
year = {2024},
author = {Yang, Q and Abebe, JS and Mai, M and Rudy, G and Kim, SY and Devinsky, O and Long, C},
title = {T4 DNA polymerase prevents deleterious on-target DNA damage and enhances precise CRISPR editing.},
journal = {The EMBO journal},
volume = {43},
number = {17},
pages = {3733-3751},
pmid = {39039289},
issn = {1460-2075},
support = {Departmental Start-Up Grant//NYU Langone Health/ ; },
mesh = {*Gene Editing/methods ; Humans ; *CRISPR-Cas Systems ; Animals ; Mice ; *DNA Damage ; DNA-Directed DNA Polymerase/metabolism/genetics ; Myocytes, Cardiac/metabolism ; Dystrophin/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; },
abstract = {Unintended on-target chromosomal alterations induced by CRISPR/Cas9 in mammalian cells are common, particularly large deletions and chromosomal translocations, and present a safety challenge for genome editing. Thus, there is still an unmet need to develop safer and more efficient editing tools. We screened diverse DNA polymerases of distinct origins and identified a T4 DNA polymerase derived from phage T4 that strongly prevents undesired on-target damage while increasing the proportion of precise 1- to 2-base-pair insertions generated during CRISPR/Cas9 editing (termed CasPlus). CasPlus induced substantially fewer on-target large deletions while increasing the efficiency of correcting common frameshift mutations in DMD and restored higher level of dystrophin expression than Cas9-alone in human cardiomyocytes. Moreover, CasPlus greatly reduced the frequency of on-target large deletions during mouse germline editing. In multiplexed guide RNAs mediating gene editing, CasPlus repressed chromosomal translocations while maintaining gene disruption efficiency that was higher or comparable to Cas9 in primary human T cells. Therefore, CasPlus offers a safer and more efficient gene editing strategy to treat pathogenic variants or to introduce genetic modifications in human applications.},
}

*Gene Editing/methods
Humans
*CRISPR-Cas Systems
Animals
Mice
*DNA Damage
DNA-Directed DNA Polymerase/metabolism/genetics
Myocytes, Cardiac/metabolism
Dystrophin/genetics/metabolism
RNA, Guide, CRISPR-Cas Systems/genetics/metabolism

@article {pmid39231976,
year = {2024},
author = {Huang, Y and Mei, H and Deng, C and Wang, W and Yuan, C and Nie, Y and Li, JD and Liu, J},
title = {EXTL3 and NPC1 are mammalian host factors for Autographa californica multiple nucleopolyhedrovirus infection.},
journal = {Nature communications},
volume = {15},
number = {1},
pages = {7711},
pmid = {39231976},
issn = {2041-1723},
mesh = {Animals ; *Nucleopolyhedroviruses/genetics/physiology ; Humans ; Mice ; *Niemann-Pick C1 Protein ; *N-Acetylglucosaminyltransferases/metabolism/genetics ; HEK293 Cells ; Endosomes/metabolism ; Heparitin Sulfate/metabolism ; Virus Internalization ; Transduction, Genetic ; Sf9 Cells ; Liver/metabolism/virology ; CRISPR-Cas Systems ; },
abstract = {Baculovirus is an obligate parasitic virus of the phylum Arthropoda. Baculovirus including Autographa californica multiple nucleopolyhedrovirus (AcMNPV) has been widely used in the laboratory and industrial preparation of proteins or protein complexes. Due to its large packaging capacity and non-replicative and non-integrative natures in mammals, baculovirus has been proposed as a gene therapy vector for transgene delivery. However, the mechanism of baculovirus transduction in mammalian cells has not been fully illustrated. Here, we employed a cell surface protein-focused CRISPR screen to identify host dependency factors for baculovirus transduction in mammalian cells. The screening experiment uncovered a series of baculovirus host factors in human cells, including exostosin-like glycosyltransferase 3 (EXTL3) and NPC intracellular cholesterol transporter 1 (NPC1). Further investigation illustrated that EXTL3 affected baculovirus attachment and entry by participating in heparan sulfate biosynthesis. In addition, NPC1 promoted baculovirus transduction by mediating membrane fusion and endosomal escape. Moreover, in vivo, baculovirus transduction in Npc1[-/+] mice showed that disruption of Npc1 gene significantly reduced baculovirus transduction in mouse liver. In summary, our study revealed the functions of EXTL3 and NPC1 in baculovirus attachment, entry, and endosomal escape in mammalian cells, which is useful for understanding baculovirus transduction in human cells.},
}

Animals
*Nucleopolyhedroviruses/genetics/physiology
Humans
Mice
*Niemann-Pick C1 Protein
*N-Acetylglucosaminyltransferases/metabolism/genetics
HEK293 Cells
Endosomes/metabolism
Heparitin Sulfate/metabolism
Virus Internalization
Transduction, Genetic
Sf9 Cells
Liver/metabolism/virology
CRISPR-Cas Systems

@article {pmid39231962,
year = {2024},
author = {Makar, AN and Boraman, A and Mosen, P and Simpson, JE and Marques, J and Michelberger, T and Aitken, S and Wheeler, AP and Winter, D and von Kriegsheim, A and Gammoh, N},
title = {The V-ATPase complex component RNAseK is required for lysosomal hydrolase delivery and autophagosome degradation.},
journal = {Nature communications},
volume = {15},
number = {1},
pages = {7743},
pmid = {39231962},
issn = {2041-1723},
support = {C52370/A21586//Cancer Research UK (CRUK)/ ; },
mesh = {*Lysosomes/metabolism ; Humans ; *Autophagosomes/metabolism ; *Autophagy ; *Endosomal Sorting Complexes Required for Transport/metabolism/genetics ; CRISPR-Cas Systems ; Vacuolar Proton-Translocating ATPases/metabolism/genetics ; Hydrolases/metabolism/genetics ; HeLa Cells ; HEK293 Cells ; },
abstract = {Autophagy is a finely orchestrated process required for the lysosomal degradation of cytosolic components. The final degradation step is essential for clearing autophagic cargo and recycling macromolecules. Using a CRISPR/Cas9-based screen, we identify RNAseK, a highly conserved transmembrane protein, as a regulator of autophagosome degradation. Analyses of RNAseK knockout cells reveal that, while autophagosome maturation is intact, cargo degradation is severely disrupted. Importantly, lysosomal protease activity and acidification remain intact in the absence of RNAseK suggesting a specificity to autolysosome degradation. Analyses of lysosome fractions show reduced levels of a subset of hydrolases in the absence of RNAseK. Of these, the knockdown of PLD3 leads to a defect in autophagosome clearance. Furthermore, the lysosomal fraction of RNAseK-depleted cells exhibits an accumulation of the ESCRT-III complex component, VPS4a, which is required for the lysosomal targeting of PLD3. Altogether, here we identify a lysosomal hydrolase delivery pathway required for efficient autolysosome degradation.},
}

*Lysosomes/metabolism
Humans
*Autophagosomes/metabolism
*Autophagy
*Endosomal Sorting Complexes Required for Transport/metabolism/genetics
CRISPR-Cas Systems
Vacuolar Proton-Translocating ATPases/metabolism/genetics
Hydrolases/metabolism/genetics
HeLa Cells
HEK293 Cells

@article {pmid39231901,
year = {2024},
author = {Fan, X and Lei, Y and Wang, L and Wu, X and Li, D},
title = {Advancing CRISPR base editing technology through innovative strategies and ideas.},
journal = {Science China. Life sciences},
volume = {},
number = {},
pages = {},
pmid = {39231901},
issn = {1869-1889},
abstract = {The innovation of CRISPR/Cas gene editing technology has developed rapidly in recent years. It is widely used in the fields of disease animal model construction, biological breeding, disease diagnosis and screening, gene therapy, cell localization, cell lineage tracking, synthetic biology, information storage, etc. However, developing idealized editors in various fields is still a goal for future development. This article focuses on the development and innovation of non-DSB editors BE and PE in the platform-based CRISPR system. It first explains the application of ideas for improvement such as "substitution", "combination", "adaptation", and "adjustment" in BE and PE development and then catalogues the ingenious inversions and leaps of thought reflected in the innovations made to CRISPR technology. It will then elaborate on the efforts currently being made to develop small editors to solve the problem of AAV overload and summarize the current application status of editors for in vivo gene modification using AAV as a delivery system. Finally, it summarizes the inspiration brought by CRISPR/Cas innovation and assesses future prospects for development of an idealized editor.},
}

@article {pmid39231196,
year = {2024},
author = {Pozhydaieva, N and Billau, FA and Wolfram-Schauerte, M and Ramírez Rojas, AA and Paczia, N and Schindler, D and Höfer, K},
title = {Temporal epigenome modulation enables efficient bacteriophage engineering and functional analysis of phage DNA modifications.},
journal = {PLoS genetics},
volume = {20},
number = {9},
pages = {e1011384},
doi = {10.1371/journal.pgen.1011384},
pmid = {39231196},
issn = {1553-7404},
abstract = {Lytic bacteriophages hold substantial promise in medical and biotechnological applications. Therefore a comprehensive understanding of phage infection mechanisms is crucial. CRISPR-Cas systems offer a way to explore these mechanisms via site-specific phage mutagenesis. However, phages can resist Cas-mediated cleavage through extensive DNA modifications like cytosine glycosylation, hindering mutagenesis efficiency. Our study utilizes the eukaryotic enzyme NgTET to temporarily reduce phage DNA modifications, facilitating Cas nuclease cleavage and enhancing mutagenesis efficiency. This approach enables precise DNA targeting and seamless point mutation integration, exemplified by deactivating specific ADP-ribosyltransferases crucial for phage infection. Furthermore, by temporally removing DNA modifications, we elucidated the effects of these modifications on T4 phage infections without necessitating gene deletions. Our results present a strategy enabling the investigation of phage epigenome functions and streamlining the engineering of phages with cytosine DNA modifications. The described temporal modulation of the phage epigenome is valuable for synthetic biology and fundamental research to comprehend phage infection mechanisms through the generation of mutants.},
}

@article {pmid39231187,
year = {2024},
author = {Ghieh, F and Passet, B and Poumerol, E and Castille, J and Calvel, P and Vilotte, JL and Sellem, E and Jouneau, L and Mambu-Mambueni, H and Garchon, HJ and Pailhoux, E and Vialard, F and Mandon-Pépin, B},
title = {A partial deletion within the meiosis-specific sporulation domain SPO22 of Tex11 is not associated with infertility in mice.},
journal = {PloS one},
volume = {19},
number = {9},
pages = {e0309974},
doi = {10.1371/journal.pone.0309974},
pmid = {39231187},
issn = {1932-6203},
mesh = {Animals ; Male ; Mice ; *Spermatogenesis/genetics ; *Meiosis/genetics ; *Azoospermia/genetics/pathology ; Infertility, Male/genetics ; Sequence Deletion ; Humans ; Testis/metabolism/pathology ; CRISPR-Cas Systems ; },
abstract = {Azoospermia (the complete absence of spermatozoa in the semen) is a common cause of male infertility. The etiology of azoospermia is poorly understood. Whole-genome analysis of azoospermic men has identified a number of candidate genes, such as the X-linked testis-expressed 11 (TEX11) gene. Using a comparative genomic hybridization array, an exonic deletion (exons 10-12) of TEX11 had previously been identified in two non-apparent azoospermic patients. However, the putative impact of this genetic alteration on spermatogenesis and the azoospermia phenotype had not been validated functionally. We therefore used a CRISPR/Cas9 system to generate a mouse model (Tex11Ex9-11del/Y) with a partial TEX11 deletion that mimicked the human mutation. Surprisingly, the mutant male Tex11Ex9-11del/Y mice were fertile. The sperm concentration, motility, and morphology were normal. Similarly, the mutant mouse line's testis transcriptome was normal, and the expression of spermatogenesis genes was not altered. These results suggest that the mouse equivalent of the partial deletion observed in two infertile male with azoospermia has no impact on spermatogenesis or fertility in mice, at least of a FVB/N genetic background and until 10 months of age. Mimicking a human mutation does not necessarily lead to the same human phenotype in mice, highlighting significant differences species.},
}

Animals
Male
Mice
*Spermatogenesis/genetics
*Meiosis/genetics
*Azoospermia/genetics/pathology
Infertility, Male/genetics
Sequence Deletion
Humans
Testis/metabolism/pathology
CRISPR-Cas Systems

@article {pmid39230778,
year = {2024},
author = {Jesudoss, D and Ponnurangan, V and Kumar, MPR and Kumar, KK and Mannu, J and Sankarasubramanian, H and Duraialagaraja, S and Eswaran, K and Loganathan, A and Shanmugam, V},
title = {Advances in breeding, biotechnology, and nanotechnological approaches to combat sheath blight disease in rice.},
journal = {Molecular biology reports},
volume = {51},
number = {1},
pages = {958},
pmid = {39230778},
issn = {1573-4978},
support = {BT/PR40456/AGIII/103/1248/2020//Department of Biotechnology, New Delhi, India/ ; },
mesh = {*Oryza/genetics/microbiology ; *Plant Diseases/microbiology/genetics ; *Disease Resistance/genetics ; *Rhizoctonia/pathogenicity ; *Plant Breeding/methods ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Biotechnology/methods ; Plants, Genetically Modified/genetics ; Nanotechnology/methods ; },
abstract = {Sheath blight, caused by the fungus Rhizoctonia solani, is a major problem that significantly impacts rice production and can lead to substantial yield losses. The disease has become increasingly problematic in recent years due to the widespread use of high-yielding semi-dwarf rice cultivars, dense planting, and heavy application of nitrogenous fertilizers. The disease has become more challenging to manage due to its diverse host range and the lack of resistant cultivars. Despite utilizing traditional methods, the problem persists without a satisfactory solution. Therefore, modern approaches, including advanced breeding, transgenic methods, genome editing using CRISPR/Cas9 technology, and nanotechnological interventions, are being explored to develop rice plants resistant to sheath blight disease. This review primarily focuses on these recent advancements in combating the sheath blight disease.},
}

*Oryza/genetics/microbiology
*Plant Diseases/microbiology/genetics
*Disease Resistance/genetics
*Rhizoctonia/pathogenicity
*Plant Breeding/methods
*Gene Editing/methods
*CRISPR-Cas Systems/genetics
*Biotechnology/methods
Plants, Genetically Modified/genetics
Nanotechnology/methods

@article {pmid39228326,
year = {2024},
author = {Jansen, PR and van Haelst, M and Wolthuis, RMF},
title = {[Genetic therapy by CRISPR-Cas: paving the way to clinical practice].},
journal = {Nederlands tijdschrift voor geneeskunde},
volume = {168},
number = {},
pages = {},
pmid = {39228326},
issn = {1876-8784},
mesh = {Humans ; *Genetic Therapy/methods/ethics ; *CRISPR-Cas Systems ; Genetic Diseases, Inborn/therapy ; },
abstract = {CRISPR-Cas technology is a revolutionary technology to modify DNA sequences. Owing to its effectiveness and accuracy, CRISPR-Cas holds important promises for 'genetic therapy' for various hereditary disorders. CRISPR-Cas enables researchers to modify specific parts of the genome with unprecedented precision, which, in specific cases, can be applied to correct disease-causing DNA variants. However, several important barriers are complicating the clinical implementation trajectory. There are for example concerns regarding the safety, effectiveness and ethical justification. Nevertheless, as CRISPR-Cas becomes more widely known, doctors and healthcare providers are expected to be well aware of the developments surrounding CRISPR-Cas therapy. Professional expertise and clear communication about the possibilities and limitations to patients with genetic disorders are essential, partly to avoid making promises that are unrealistic in the short term. Altogether, geneticists, medical centers and regulators are now facing the challenges to start translating CRISPR-Cas technology into clinical practice, in an effective and ethical manner.},
}

Humans
*Genetic Therapy/methods/ethics
*CRISPR-Cas Systems
Genetic Diseases, Inborn/therapy

@article {pmid39228116,
year = {2024},
author = {Chokwassanasakulkit, T and McMillan, NAJ},
title = {Merkel Cell Polyomavirus-Pathophysiology and Treatment in the Era of Gene-Targeted Therapies.},
journal = {Reviews in medical virology},
volume = {34},
number = {5},
pages = {e2580},
doi = {10.1002/rmv.2580},
pmid = {39228116},
issn = {1099-1654},
support = {//Griffith University/ ; 2027649//National Health and Medical Research Council/ ; 2027569//National Health and Medical Research Council/ ; },
mesh = {Humans ; *Merkel cell polyomavirus/genetics ; *Carcinoma, Merkel Cell/virology/therapy/genetics ; *Polyomavirus Infections/virology/therapy ; *Genetic Therapy/methods ; Skin Neoplasms/therapy/virology/genetics ; Animals ; Tumor Virus Infections/virology/therapy ; RNA, Small Interfering/genetics ; },
abstract = {Merkel cell polyomavirus (MCPyV) is a significant contributor to the development of Merkel cell carcinoma (MCC), an aggressive skin cancer with high recurrence and a low survival rate. In fact, it is the deadliest skin cancer. The precise routes of transmission for MCPyV-positive MCC remain unclear, but several factors may trigger its development. Conventional treatments for MCC are not highly effective, especially in patients with metastasis, with a clear need for new treatment options. Gene-targeted therapies hold great promise for the treatment of MCC, including the use of siRNA and CRISPR/Cas (C/Cas) but critically none have yet been translated into clinical trials. Validating this approach is the fact that several siRNA products are already FDA licenced, while C/Cas has entered clinical trial, albeit for conditions other than MCC. There are many challenges that must be overcome to move from preclinical research to the clinic. In this review, we provide a comprehensive summary of the current understanding of MCC, with a particular focus on MCPyV-positive MCC, and the status of gene-targeted therapies. Additionally, we discuss the major obstacles that impede MCC research and explore future prospects.},
}

Humans
*Merkel cell polyomavirus/genetics
*Carcinoma, Merkel Cell/virology/therapy/genetics
*Polyomavirus Infections/virology/therapy
*Genetic Therapy/methods
Skin Neoplasms/therapy/virology/genetics
Animals
Tumor Virus Infections/virology/therapy
RNA, Small Interfering/genetics

@article {pmid39227830,
year = {2024},
author = {Du, Q and Wei, Y and Zhang, L and Ren, D and Gao, J and Dong, X and Bai, L and Li, J},
title = {An improved CRISPR and CRISPR interference (CRISPRi) toolkit for engineering the model methanogenic archaeon Methanococcus maripaludis.},
journal = {Microbial cell factories},
volume = {23},
number = {1},
pages = {239},
pmid = {39227830},
issn = {1475-2859},
support = {32393974, 32393972 and 32270054//National Natural Science Foundation of China/ ; 2020YFA0906800 and 2019YFA0905500//National Key Research and Development Program of China/ ; },
mesh = {*Methanococcus/genetics ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Plasmids/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Genome, Archaeal ; Clustered Regularly Interspaced Short Palindromic Repeats ; Genetic Engineering/methods ; },
abstract = {BACKGROUND: The type II based CRISPR-Cas system remains restrictedly utilized in archaea, a featured domain of life that ranks parallelly with Bacteria and Eukaryotes. Methanococcus maripaludis, known for rapid growth and genetic tractability, serves as an exemplary model for studying archaeal biology and exploring CO2-based biotechnological applications. However, tools for controlled gene regulation remain deficient and CRISPR-Cas tools still need improved in this archaeon, limiting its application as an archaeal model cellular factory.

RESULTS: This study not only improved the CRISPR-Cas9 system for optimizing multiplex genome editing and CRISPR plasmid construction efficiencies but also pioneered an effective CRISPR interference (CRISPRi) system for controlled gene regulation in M. maripaludis. We developed two novel strategies for balanced expression of multiple sgRNAs, facilitating efficient multiplex genome editing. We also engineered a strain expressing Cas9 genomically, which simplified the CRISPR plasmid construction and facilitated more efficient genome modifications, including markerless and scarless gene knock-in. Importantly, we established a CRISPRi system using catalytic inactive dCas9, achieving up to 100-fold repression on target gene. Here, sgRNAs targeting near and downstream regions of the transcription start site and the 5'end ORF achieved the highest repression efficacy. Furthermore, we developed an inducible CRISPRi-dCas9 system based on TetR/tetO platform. This facilitated the inducible gene repression, especially for essential genes.

CONCLUSIONS: Therefore, these advancements not only expand the toolkit for genetic manipulation but also bridge methodological gaps for controlled gene regulation, especially for essential genes, in M. maripaludis. The robust toolkit developed here paves the way for applying M. maripaludis as a vital model archaeal cell factory, facilitating fundamental biological studies and applied biotechnology development of archaea.},
}

*Methanococcus/genetics
*CRISPR-Cas Systems
*Gene Editing/methods
Plasmids/genetics
RNA, Guide, CRISPR-Cas Systems/genetics
Genome, Archaeal
Clustered Regularly Interspaced Short Palindromic Repeats
Genetic Engineering/methods

@article {pmid39227671,
year = {2024},
author = {Grüttner, S and Kempken, F},
title = {A user-friendly CRISPR/Cas9 system for mutagenesis of Neurospora crassa.},
journal = {Scientific reports},
volume = {14},
number = {1},
pages = {20469},
pmid = {39227671},
issn = {2045-2322},
mesh = {*Neurospora crassa/genetics ; *CRISPR-Cas Systems ; *Mutagenesis ; *Gene Editing/methods ; Fungal Proteins/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; Genome, Fungal ; },
abstract = {As a widely used eukaryotic model organism, Neurospora crassa offers advantages in genetic studies due to its diverse biology and rapid growth. Traditional genetic manipulation methods, such as homologous recombination, require a considerable amount of time and effort. In this study, we present an easy-to-use CRIPSR/Cas9 system for N. crassa, in which the cas9 sequence is incorporated into the fungal genome and naked guide RNA is introduced via electroporation. Our approach eliminates the need for constructing multiple vectors, speeding up the mutagenesis process. Using cyclosporin-resistant-1 (csr-1) as a selectable marker gene, we achieved 100% editing efficiency under selection conditions. Furthermore, we successfully edited the non-selectable gene N-acylethanolamine amidohydrolase-2 (naa-2), demonstrating the versatility of the system. Combining gRNAs targeting csr-1 and naa-2 simultaneously increased the probability of finding mutants carrying the non-selectable mutation. The system is not only user-friendly but also effective, providing a rapid and efficient method for generating loss-of-function mutants in N. crassa compared to traditional methods.},
}

*Neurospora crassa/genetics
*CRISPR-Cas Systems
*Mutagenesis
*Gene Editing/methods
Fungal Proteins/genetics/metabolism
RNA, Guide, CRISPR-Cas Systems/genetics
Genome, Fungal

@article {pmid39185736,
year = {2024},
author = {Tong, S and Hong, R and Chen, W and Chai, M and Zhang, Y and Sun, Y and Wang, Q and Li, D},
title = {Synchronous Bioproduction of Betanin and Mycoprotein in the Engineered Edible Fungus Fusarium venenatum.},
journal = {Journal of agricultural and food chemistry},
volume = {72},
number = {35},
pages = {19462-19469},
doi = {10.1021/acs.jafc.4c06071},
pmid = {39185736},
issn = {1520-5118},
mesh = {*Fusarium/metabolism/genetics ; *Betacyanins/metabolism/chemistry ; *Fungal Proteins/genetics/metabolism ; *Fermentation ; Metabolic Engineering ; CRISPR-Cas Systems ; Food Coloring Agents/metabolism/chemistry ; },
abstract = {Sustainable production of edible microbial proteins and red food colorants is an important demand for future food. Therefore, creation of a chassis strain that can efficiently synthesize both products is extremely necessary and meaningful. To realize this envision, a CRISPR/Cas9-based visual multicopy integration system was successfully developed in Fusarium venenatum. Subsequently, the de novo synthesis of the red food colorant betanin was achieved in the engineered F. venenatum using the above system. After fermentation optimization, the final yields of betanin and mycoprotein reached 1.91 and 9.53 g/L, respectively, when the constant pH naturally decreased from 6 to 4 without the addition of acid after 48 h of fermentation. These results determine a highly suitable chassis strain for the microbial biomanufacturing of betanin, and the obtained engineered strain here is expected to expand the application prospect and improve economic returns of F. venenatum in the field of future food.},
}

*Fusarium/metabolism/genetics
*Betacyanins/metabolism/chemistry
*Fungal Proteins/genetics/metabolism
*Fermentation
Metabolic Engineering
CRISPR-Cas Systems
Food Coloring Agents/metabolism/chemistry

@article {pmid39039029,
year = {2024},
author = {Crain, AT and Nevil, M and Leatham-Jensen, MP and Reeves, KB and Matera, AG and McKay, DJ and Duronio, RJ},
title = {Redesigning the Drosophila histone gene cluster: an improved genetic platform for spatiotemporal manipulation of histone function.},
journal = {Genetics},
volume = {228},
number = {1},
pages = {},
doi = {10.1093/genetics/iyae117},
pmid = {39039029},
issn = {1943-2631},
support = {R35 GM136435/GM/NIGMS NIH HHS/United States ; T32-GM007092/GF/NIH HHS/United States ; //A.T.C./ ; //M.N./ ; R35-GM136435//R.J.D./ ; R35-GM128851//A.G.M./ ; //D.J.M/ ; },
mesh = {Animals ; *Histones/metabolism/genetics ; *Drosophila melanogaster/genetics ; *Drosophila Proteins/genetics/metabolism ; Gene Editing/methods ; Multigene Family ; CRISPR-Cas Systems ; },
abstract = {Mutating replication-dependent (RD) histone genes is an important tool for understanding chromatin-based epigenetic regulation. Deploying this tool in metazoans is particularly challenging because RD histones in these organisms are typically encoded by many genes, often located at multiple loci. Such gene arrangements make the ability to generate homogenous histone mutant genotypes by site-specific gene editing quite difficult. Drosophila melanogaster provides a solution to this problem because the RD histone genes are organized into a single large tandem array that can be deleted and replaced with transgenes containing mutant histone genes. In the last ∼15 years several different RD histone gene replacement platforms were developed using this simple strategy. However, each platform contains weaknesses that preclude full use of the powerful developmental genetic capabilities available to Drosophila researchers. Here we describe the development of a newly engineered platform that rectifies many of these weaknesses. We used CRISPR to precisely delete the RD histone gene array (HisC), replacing it with a multifunctional cassette that permits site-specific insertion of either one or two synthetic gene arrays using selectable markers. We designed this cassette with the ability to selectively delete each of the integrated gene arrays in specific tissues using site-specific recombinases. We also present a method for rapidly synthesizing histone gene arrays of any genotype using Golden Gate cloning technologies. These improvements facilitate the generation of histone mutant cells in various tissues at different stages of Drosophila development and provide an opportunity to apply forward genetic strategies to interrogate chromatin structure and gene regulation.},
}

Animals
*Histones/metabolism/genetics
*Drosophila melanogaster/genetics
*Drosophila Proteins/genetics/metabolism
Gene Editing/methods
Multigene Family
CRISPR-Cas Systems

@article {pmid39030344,
year = {2024},
author = {Jana, B and Liu, X and Dénéréaz, J and Park, H and Leshchiner, D and Liu, B and Gallay, C and Zhu, J and Veening, JW and van Opijnen, T},
title = {CRISPRi-TnSeq maps genome-wide interactions between essential and non-essential genes in bacteria.},
journal = {Nature microbiology},
volume = {9},
number = {9},
pages = {2395-2409},
pmid = {39030344},
issn = {2058-5276},
support = {AI135737//U.S. Department of Health & Human Services | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ ; AI156203//U.S. Department of Health & Human Services | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ ; AI124302//U.S. Department of Health & Human Services | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ ; AI158076//U.S. Department of Health & Human Services | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ ; AI124402//U.S. Department of Health & Human Services | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ ; 82270012//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {*Streptococcus pneumoniae/genetics/metabolism ; *Genes, Essential/genetics ; *Genome, Bacterial/genetics ; *DNA Transposable Elements/genetics ; *CRISPR-Cas Systems ; Gene Regulatory Networks ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Genes, Bacterial/genetics ; Bacterial Proteins/genetics/metabolism ; Gene Knockout Techniques ; },
abstract = {Genetic interactions identify functional connections between genes and pathways, establishing gene functions or druggable targets. Here we use CRISPRi-TnSeq, CRISPRi-mediated knockdown of essential genes alongside TnSeq-mediated knockout of non-essential genes, to map genome-wide interactions between essential and non-essential genes in Streptococcus pneumoniae. Transposon-mutant libraries constructed in 13 CRISPRi strains enabled screening of ~24,000 gene pairs. This identified 1,334 genetic interactions, including 754 negative and 580 positive interactions. Network analyses show that 17 non-essential genes pleiotropically interact with more than half the essential genes tested. Validation experiments confirmed that a 7-gene subset protects against perturbations. Furthermore, we reveal hidden redundancies that compensate for essential gene loss, relationships between cell wall synthesis, integrity and cell division, and show that CRISPRi-TnSeq identifies synthetic and suppressor-type relationships between both functionally linked and disparate genes and pathways. Importantly, in species where CRISPRi and Tn-Seq are established, CRISPRi-TnSeq should be straightforward to implement.},
}

*Streptococcus pneumoniae/genetics/metabolism
*Genes, Essential/genetics
*Genome, Bacterial/genetics
*DNA Transposable Elements/genetics
*CRISPR-Cas Systems
Gene Regulatory Networks
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Genes, Bacterial/genetics
Bacterial Proteins/genetics/metabolism
Gene Knockout Techniques

@article {pmid39025450,
year = {2024},
author = {Wu, CJ and Livak, F and Ashwell, JD},
title = {The histone methyltransferase KMT2D maintains cellular glucocorticoid responsiveness by shielding the glucocorticoid receptor from degradation.},
journal = {The Journal of biological chemistry},
volume = {300},
number = {8},
pages = {107581},
pmid = {39025450},
issn = {1083-351X},
mesh = {Humans ; *Receptors, Glucocorticoid/metabolism/genetics ; Neoplasm Proteins/metabolism/genetics ; Glucocorticoids/metabolism/pharmacology ; DNA-Binding Proteins/metabolism/genetics ; Jurkat Cells ; Proteolysis ; Histone-Lysine N-Methyltransferase/metabolism/genetics ; Apoptosis ; CRISPR-Cas Systems ; Cell Line, Tumor ; },
abstract = {Because of their ability to induce lymphocyte apoptosis, glucocorticoids (GC) are widely used to treat hematological malignancies such as lymphomas and multiple myeloma. Their effectiveness is often limited, however, due to the development of glucocorticoid resistance by a variety of molecular mechanisms. Here we performed an unbiased genome-wide CRISPR screen with the human T-cell leukemia cell line Jurkat to find previously unidentified genes required for GC-induced apoptosis. One such gene was KMT2D (also known as MLL2 or MLL4), which encodes a histone lysine methyltransferase whose mutations are associated with a variety of cancers, blood malignancies in particular, and are considered markers of poor prognosis. Knockout of KMT2D by CRISPR/Cas9 gene editing in Jurkat and several multiple myeloma cell lines downregulated GR protein expression. Surprisingly, this was not due to a reduction in GR transcripts, but rather to a decrease in the protein's half-life, primarily due to proteasomal degradation. Reconstitution of KMT2D expression restored GR levels. In contrast to the known ability of KMT2D to control gene transcription through covalent histone methylation, KMT2D-mediated upregulation of GR levels did not require its methyltransferase activity. Co-immunoprecipitation and proximity ligation assays found constitutive binding of KMT2D to the GR, which was enhanced in the presence of GC. These observations reveal KMT2D to be essential for the stabilization of cellular GR levels, and suggest a possible mechanism by which KMT2D mutations may lead to GC resistance in some malignancies.},
}

Humans
*Receptors, Glucocorticoid/metabolism/genetics
Neoplasm Proteins/metabolism/genetics
Glucocorticoids/metabolism/pharmacology
DNA-Binding Proteins/metabolism/genetics
Jurkat Cells
Proteolysis
Histone-Lysine N-Methyltransferase/metabolism/genetics
Apoptosis
CRISPR-Cas Systems
Cell Line, Tumor

@article {pmid38820091,
year = {2024},
author = {Petrosky, SJ and Williams, TM and Rebeiz, M},
title = {A genetic screen of transcription factors in the Drosophila melanogaster abdomen identifies novel pigmentation genes.},
journal = {G3 (Bethesda, Md.)},
volume = {14},
number = {9},
pages = {},
doi = {10.1093/g3journal/jkae097},
pmid = {38820091},
issn = {2160-1836},
support = {R35GM14196/GF/NIH HHS/United States ; },
mesh = {Animals ; *Drosophila melanogaster/genetics ; *Pigmentation/genetics ; *Transcription Factors/genetics/metabolism ; *Drosophila Proteins/genetics ; Abdomen ; Phenotype ; CRISPR-Cas Systems ; Genetic Testing ; Animals, Genetically Modified ; Gene Regulatory Networks ; },
abstract = {Gene regulatory networks specify the gene expression patterns needed for traits to develop. Differences in these networks can result in phenotypic differences between organisms. Although loss-of-function genetic screens can identify genes necessary for trait formation, gain-of-function screens can overcome genetic redundancy and identify loci whose expression is sufficient to alter trait formation. Here, we leveraged transgenic lines from the Transgenic RNAi Project at Harvard Medical School to perform both gain- and loss-of-function CRISPR/Cas9 screens for abdominal pigmentation phenotypes. We identified measurable effects on pigmentation patterns in the Drosophila melanogaster abdomen for 21 of 55 transcription factors in gain-of-function experiments and 7 of 16 tested by loss-of-function experiments. These included well-characterized pigmentation genes, such as bab1 and dsx, and transcription factors that had no known role in pigmentation, such as slp2. Finally, this screen was partially conducted by undergraduate students in a Genetics Laboratory course during the spring semesters of 2021 and 2022. We found this screen to be a successful model for student engagement in research in an undergraduate laboratory course that can be readily adapted to evaluate the effect of hundreds of genes on many different Drosophila traits, with minimal resources.},
}

Animals
*Drosophila melanogaster/genetics
*Pigmentation/genetics
*Transcription Factors/genetics/metabolism
*Drosophila Proteins/genetics
Abdomen
Phenotype
CRISPR-Cas Systems
Genetic Testing
Animals, Genetically Modified
Gene Regulatory Networks

@article {pmid39227603,
year = {2024},
author = {Bergman, S and Tuller, T},
title = {Codon usage and expression-based features significantly improve prediction of CRISPR efficiency.},
journal = {NPJ systems biology and applications},
volume = {10},
number = {1},
pages = {100},
pmid = {39227603},
issn = {2056-7189},
mesh = {*Codon Usage/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Humans ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Computational Biology/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; Codon/genetics ; Gene Expression/genetics ; },
abstract = {CRISPR is a precise and effective genome editing technology; but despite several advancements during the last decade, our ability to computationally design gRNAs remains limited. Most predictive models have relatively low predictive power and utilize only the sequence of the target site as input. Here we suggest a new category of features, which incorporate the target site genomic position and the presence of genes close to it. We calculate four features based on gene expression and codon usage bias indices. We show, on CRISPR datasets taken from 3 different cell types, that such features perform comparably with 425 state-of-the-art predictive features, ranking in the top 2-12% of features. We trained new predictive models, showing that adding expression features to them significantly improves their r[2] by up to 0.04 (relative increase of 39%), achieving average correlations of up to 0.38 on their validation sets; and that these features are deemed important by different feature importance metrics. We believe that incorporating the target site's position, in addition to its sequence, in features such as we have generated here will improve our ability to predict, design and understand CRISPR experiments going forward.},
}

*Codon Usage/genetics
*Gene Editing/methods
*CRISPR-Cas Systems/genetics
Humans
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Computational Biology/methods
RNA, Guide, CRISPR-Cas Systems/genetics
Codon/genetics
Gene Expression/genetics

@article {pmid39227468,
year = {2024},
author = {Akhtar, N and Shadab, M and Bhatti, N and Sajid Ansarì, M and Siddiqui, MB},
title = {Biotechnological frontiers in harnessing allelopathy for sustainable crop production.},
journal = {Functional & integrative genomics},
volume = {24},
number = {5},
pages = {155},
pmid = {39227468},
issn = {1438-7948},
mesh = {*Allelopathy ; *Crops, Agricultural/genetics/metabolism ; Biotechnology ; Crop Production/methods ; CRISPR-Cas Systems ; Weed Control/methods ; },
abstract = {Allelopathy, the phenomenon in which plants release biochemical compounds that influence the growth and development of neighbouring plants, presents promising opportunities for revolutionizing agriculture towards sustainability. This abstract explores the role of biotechnological advancements in unlocking the potential of allelopathy for sustainable crop production and its applications in agriculture, ecology, and natural resource management. By combining molecular, genetic, biochemical, and bioinformatic tools, researchers can unravel the complexities of allelopathic interactions and their potential for sustainable crop production and environmental stewardship. The development of novel management methods for weed control is getting a lot of attention with the introduction of new genetic technologies such as Gene drive, Transgene technologies, Gene silencing, Marker-assisted selection (MAS), and Clustered regularly interspaced short palindromic repeats (CRISPR-Cas9). By strengthening competitive characteristics these tools hold great promise for boosting crops' ability to compete with weeds. Considering recent literature, this review highlights the genetic, transcriptomics, and metabolomics approaches to allelopathy. Employing allelopathic properties in agriculture offer sustainable benefits like natural weed management, pest management, and reduced chemical pollution, but challenges include environmental factors, toxicity, regulatory hurdles, and limited resources. Effective integration requires continued research, regulatory support, and farmer education . Also, we aimed to identify the biotechnological domains requiring more investigation and to provide the basis for future advances through this assessment.},
}

*Allelopathy
*Crops, Agricultural/genetics/metabolism
Biotechnology
Crop Production/methods
CRISPR-Cas Systems
Weed Control/methods

@article {pmid39227390,
year = {2024},
author = {Brands, J and Bravo, S and Jürgenliemke, L and Grätz, L and Schihada, H and Frechen, F and Alenfelder, J and Pfeil, C and Ohse, PG and Hiratsuka, S and Kawakami, K and Schmacke, LC and Heycke, N and Inoue, A and König, G and Pfeifer, A and Wachten, D and Schulte, G and Steinmetzer, T and Watts, VJ and Gomeza, J and Simon, K and Kostenis, E},
title = {A molecular mechanism to diversify Ca[2+] signaling downstream of Gs protein-coupled receptors.},
journal = {Nature communications},
volume = {15},
number = {1},
pages = {7684},
pmid = {39227390},
issn = {2041-1723},
support = {214362475/GRK1873/2//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; 494832089 /GRK2873//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; 290847012/FOR2372//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; 214362475/GRK1873/2//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; 494832089 /GRK2873//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; JP21H04791//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; JP21H05113//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; JP21H05037//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; JPJSBP120213501//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; JP22ama121038//Japan Agency for Medical Research and Development (AMED)/ ; JP22zf0127007//Japan Agency for Medical Research and Development (AMED)/ ; JPMJFR215T//MEXT | Japan Science and Technology Agency (JST)/ ; JPMJMS2023//MEXT | Japan Science and Technology Agency (JST)/ ; 22714181//MEXT | Japan Science and Technology Agency (JST)/ ; },
mesh = {Humans ; *Calcium Signaling ; *Phospholipase C beta/metabolism/genetics ; HEK293 Cells ; *Receptors, G-Protein-Coupled/metabolism/genetics ; *Calcium/metabolism ; GTP-Binding Protein alpha Subunits, Gq-G11/metabolism/genetics ; CRISPR-Cas Systems ; GTP-Binding Protein alpha Subunits, Gs/metabolism/genetics ; Cyclic AMP/metabolism ; Animals ; Gene Editing ; Cytosol/metabolism ; GTP-Binding Protein beta Subunits/metabolism/genetics ; Adenylyl Cyclases/metabolism/genetics ; },
abstract = {A long-held tenet in inositol-lipid signaling is that cleavage of membrane phosphoinositides by phospholipase Cβ (PLCβ) isozymes to increase cytosolic Ca[2+] in living cells is exclusive to Gq- and Gi-sensitive G protein-coupled receptors (GPCRs). Here we extend this central tenet and show that Gs-GPCRs also partake in inositol-lipid signaling and thereby increase cytosolic Ca[2+]. By combining CRISPR/Cas9 genome editing to delete Gαs, the adenylyl cyclase isoforms 3 and 6, or the PLCβ1-4 isozymes, with pharmacological and genetic inhibition of Gq and G11, we pin down Gs-derived Gβγ as driver of a PLCβ2/3-mediated cytosolic Ca[2+] release module. This module does not require but crosstalks with Gαs-dependent cAMP, demands Gαq to release PLCβ3 autoinhibition, but becomes Gq-independent with mutational disruption of the PLCβ3 autoinhibited state. Our findings uncover the key steps of a previously unappreciated mechanism utilized by mammalian cells to finetune their calcium signaling regulation through Gs-GPCRs.},
}

Humans
*Calcium Signaling
*Phospholipase C beta/metabolism/genetics
HEK293 Cells
*Receptors, G-Protein-Coupled/metabolism/genetics
*Calcium/metabolism
GTP-Binding Protein alpha Subunits, Gq-G11/metabolism/genetics
CRISPR-Cas Systems
GTP-Binding Protein alpha Subunits, Gs/metabolism/genetics
Cyclic AMP/metabolism
Animals
Gene Editing
Cytosol/metabolism
GTP-Binding Protein beta Subunits/metabolism/genetics
Adenylyl Cyclases/metabolism/genetics

@article {pmid39227100,
year = {2024},
author = {Harris, HC and Warren, FJ},
title = {The impact of Cas9-mediated mutagenesis of genes encoding potato starch-branching enzymes on starch structural properties and in vitro digestibility.},
journal = {Carbohydrate polymers},
volume = {345},
number = {},
pages = {122561},
doi = {10.1016/j.carbpol.2024.122561},
pmid = {39227100},
issn = {1879-1344},
mesh = {*Solanum tuberosum/genetics/chemistry ; *1,4-alpha-Glucan Branching Enzyme/genetics/metabolism/chemistry ; *Starch/chemistry/metabolism ; *Mutagenesis ; Digestion ; CRISPR-Cas Systems/genetics ; Amylopectin/chemistry/metabolism ; Amylose/chemistry/metabolism ; Plant Proteins/genetics/chemistry/metabolism ; },
abstract = {The digestibility of starch is affected by amylose content, and increasing amylopectin chain length which can be manipulated by alterations to genes encoding starch-branching enzymes (SBEs). We investigated the impact of Cas9-mediated mutagenesis of SBEs in potato on starch structural properties and digestibility. Four potato starches with edited SBE genes were tested. One lacked SBE1 and SBE2, two lacked SBE2 and had reduced SBE1, and one had reduced SBE2 only. Starch structure and thermal properties were characterised by DSC and XRD. The impact of different thermal treatments on digestibility was studied using an in vitro digestion protocol. All native potato starches were resistant to digestion, and all gelatinised starches were highly digestible. SBE modified starches had higher gelatinisation temperatures than wild type potatoes and retrograded more rapidly. Gelatinisation and 18 h of retrogradation, increased gelatinisation enthalpy, but this did not translate to differences in digestion. Following 7 days of retrogradation, starch from three modified SBE starch lines was less digestible than starch from wild-type potatoes, likely due to the recrystallisation of the long amylopectin chains. Our results indicate that reductions in SBE in potato may be beneficial to health by increasing the amount of fibre reaching the colon after retrogradation.},
}

*Solanum tuberosum/genetics/chemistry
*1,4-alpha-Glucan Branching Enzyme/genetics/metabolism/chemistry
*Starch/chemistry/metabolism
*Mutagenesis
Digestion
CRISPR-Cas Systems/genetics
Amylopectin/chemistry/metabolism
Amylose/chemistry/metabolism
Plant Proteins/genetics/chemistry/metabolism

@article {pmid39226667,
year = {2024},
author = {Yang, S and Zhang, Y and Li, C and Tan, X},
title = {Repurposing endogenous Type I-D CRISPR-Cas system for genome editing in Synechococcus sp. PCC7002.},
journal = {Microbiological research},
volume = {288},
number = {},
pages = {127884},
doi = {10.1016/j.micres.2024.127884},
pmid = {39226667},
issn = {1618-0623},
abstract = {Synechococcus sp. PCC7002 has been considered as a photosynthetic chassis for the conversion of CO2 into biochemicals through genetic modification. However, conventional genetic manipulation techniques prove inadequate for comprehensive genetic modifications in this strain. Here, we present the development of a genome editing tool tailored for S. PCC7002, leveraging its endogenous type I-D CRISPR-Cas system. Utilizing this novel tool, we successfully deleted the glgA1 gene and iteratively edited the genome to obtain a double mutant of glgA1 and glgA2 genes. Additionally, large DNA fragments encompassing the entire type I-A (∼14 kb) or III-B CRISPR-Cas (∼21 kb) systems were completely knocked-out in S. PCC7002 using our tool. Furthermore, the endogenous pAQ5 plasmid, approximately 38 kb in length, was successfully cured from S. PCC7002. Our work demonstrates the feasibility of harnessing the endogenous CRISPR-Cas system for genome editing in S. PCC7002, thereby enriching the genetic toolkit for this species and providing a foundation for future enhancements in its biosynthetic efficiency.},
}

@article {pmid39225329,
year = {2024},
author = {Feng, S and Zhang, Y and Wang, Y and Gao, Y and Song, Y},
title = {Harnessing Gene Editing Technology for Tumor Microenvironment Modulation: An Emerging Anticancer Strategy.},
journal = {Chemistry (Weinheim an der Bergstrasse, Germany)},
volume = {},
number = {},
pages = {e202402485},
doi = {10.1002/chem.202402485},
pmid = {39225329},
issn = {1521-3765},
abstract = {Cancer is a multifaceted disease influenced by both intrinsic cellular traits and extrinsic factors, with the tumor microenvironment (TME) being crucial in its progression. To satisfy their high proliferation and aggressiveness, cancer cell always plunders large amounts of nutrition and releases various signals to the surrounding, forming a dynamic TME with special metabolic, immune, microbial and physical characteristics. Due to the neglect of interactions between tumor cell and TME, traditional cancer therapies often struggle with challenges such as drug resistance, low efficacy, and recurrence. Importantly, with the development of gene editing technologies, particularly the CRISPR-Cas system, offers promising new strategies for cancer treatment. Combined with nanomaterials strategies, CRISPR-Cas technology exhibits precision, affordability, and user-friendliness with reduced side effects, which holds great promise for profoundly altering the TME at a genetic level, potentially leading to lasting anticancer outcomes. This review will delve into how CRISPR-Cas can be leveraged to manipulate the TME, examining its potential as a transformative anticancer therapy.},
}

@article {pmid39225039,
year = {2024},
author = {Ma, J and Zhang, W and Rahimialiabadi, S and Ganesh, NU and Sun, Z and Parvez, S and Peterson, RT and Yeh, JJ},
title = {Instantaneous visual genotyping and facile site-specific transgenesis via CRISPR-Cas9 and phiC31 integrase.},
journal = {Biology open},
volume = {13},
number = {9},
pages = {},
doi = {10.1242/bio.061666},
pmid = {39225039},
issn = {2046-6390},
support = {R01 GM134069/GF/NIH HHS/United States ; 201808210354//China Scholarship Council/ ; //Harvard Medical School; University of Utah/ ; },
mesh = {*CRISPR-Cas Systems ; *Integrases/genetics/metabolism ; Animals ; *Zebrafish/genetics ; *Genotype ; Gene Transfer Techniques ; Genotyping Techniques ; Transgenes ; Genes, Reporter ; Gene Editing/methods ; Animals, Genetically Modified ; },
abstract = {Here, we introduce 'TICIT', targeted integration by CRISPR-Cas9 and integrase technologies, which utilizes the site-specific DNA recombinase - phiC31 integrase - to insert large DNA fragments into CRISPR-Cas9 target loci. This technique, which relies on first knocking in a 39-basepair phiC31 landing site via CRISPR-Cas9, enables researchers to repeatedly perform site-specific transgenesis at the exact genomic location with high precision and efficiency. We applied this approach to devise a method for the instantaneous determination of a zebrafish's genotype simply by examining its color. When a zebrafish mutant line must be propagated as heterozygotes due to homozygous lethality, employing this method allows facile identification of a population of homozygous mutant embryos even before the mutant phenotypes manifest. Thus, it should facilitate various downstream applications, such as large-scale chemical screens. We demonstrated that TICIT could also create reporter fish driven by an endogenous promoter. Further, we identified a landing site in the tyrosinase gene that could support transgene expression in a broad spectrum of tissue and cell types. In sum, TICIT enables site-specific DNA integration without requiring complex donor DNA construction. It can yield consistent transgene expression, facilitate diverse applications in zebrafish, and may be applicable to cells in culture and other model organisms.},
}

*CRISPR-Cas Systems
*Integrases/genetics/metabolism
Animals
*Zebrafish/genetics
*Genotype
Gene Transfer Techniques
Genotyping Techniques
Transgenes
Genes, Reporter
Gene Editing/methods
Animals, Genetically Modified

@article {pmid39224035,
year = {2024},
author = {Chopra, A and Bhuvanagiri, G and Natu, K and Chopra, A},
title = {Role of CRISPR-Cas systems in periodontal disease pathogenesis and potential for periodontal therapy: A review.},
journal = {Molecular oral microbiology},
volume = {},
number = {},
pages = {},
doi = {10.1111/omi.12483},
pmid = {39224035},
issn = {2041-1014},
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPRs) are DNA sequences capable of editing a host genome sequence. CRISPR and its specific CRISPR-associated (Cas) protein complexes have been adapted for various applications. These include activating or inhibiting specific genetic sequences or acting as molecular scissors to cut and modify the host DNA precisely. CRISPR-Cas systems are also naturally present in many oral bacteria, where they aid in nutrition, biofilm formation, inter- and intraspecies communication (quorum sensing), horizontal gene transfer, virulence, inflammation modulation, coinfection, and immune response evasion. It even functions as an adaptive immune system, defending microbes against invading viruses and foreign genetic elements from other bacteria by targeting and degrading their DNA. Recently, CRISPR-Cas systems have been tested as molecular editing tools to manipulate specific genes linked with periodontal disease (such as periodontitis) and as novel methods of delivering antimicrobial agents to overcome antimicrobial resistance. With the rapidly increasing role of CRISPR in treating inflammatory diseases, its application in periodontal disease is also becoming popular. Therefore, this review aims to discuss the different types of CRISPR-Cas in oral microbes and their role in periodontal disease pathogenesis and precision periodontal therapy.},
}

@article {pmid39223923,
year = {2024},
author = {Shi, S and Ge, Y and Yan, Q and Wan, S and Li, M and Li, M},
title = {Activating UCHL1 through the CRISPR activation system promotes cartilage differentiation mediated by HIF-1α/SOX9.},
journal = {Journal of cellular and molecular medicine},
volume = {28},
number = {17},
pages = {e70051},
doi = {10.1111/jcmm.70051},
pmid = {39223923},
issn = {1582-4934},
support = {2023A04J0427//Basic and Applied Basic Research Foundation of Guangzhou/ ; PY2021028//Science Research Cultivation Program of Stomatological Hospital, Southern Medical University/ ; B2023405//Medical Science and Technology Research Fund of Guangdong Province/ ; },
mesh = {*Ubiquitin Thiolesterase/metabolism/genetics ; *SOX9 Transcription Factor/metabolism/genetics ; *Hypoxia-Inducible Factor 1, alpha Subunit/metabolism/genetics ; *Cell Differentiation/genetics ; *Chondrogenesis/genetics ; Animals ; Humans ; Cartilage/metabolism ; Chondrocytes/metabolism/cytology ; Mesenchymal Stem Cells/metabolism/cytology ; CRISPR-Cas Systems ; Mice ; },
abstract = {Developing strategies to enhance cartilage differentiation in mesenchymal stem cells and preserve the extracellular matrix is crucial for successful cartilage tissue reconstruction. Hypoxia-inducible factor-1α (HIF-1α) plays a pivotal role in maintaining the extracellular matrix and chondrocyte phenotype, thus serving as a key regulator in chondral tissue engineering strategies. Recent studies have shown that Ubiquitin C-terminal hydrolase L1 (UCHL1) is involved in the deubiquitylation of HIF-1α. However, the regulatory role of UCHL1 in chondrogenic differentiation has not been investigated. In the present study, we initially validated the promotive effect of UCHL1 expression on chondrogenesis in adipose-derived stem cells (ADSCs). Subsequently, a hybrid baculovirus system was designed and employed to utilize three CRISPR activation (CRISPRa) systems, employing dead Cas9 (dCas9) from three distinct bacterial sources to target UCHL1. Then UCHL1 and HIF-1α inhibitor and siRNA targeting SRY-box transcription factor 9 (SOX9) were used to block UCHL1, HIF-1α and SOX9, respectively. Cartilage differentiation and chondrogenesis were measured by qRT-PCR, immunofluorescence and histological staining. We observed that the CRISPRa system derived from Staphylococcus aureus exhibited superior efficiency in activating UCHL1 compared to the commonly used the CRISPRa system derived from Streptococcus pyogenes. Furthermore, the duration of activation was extended by utilizing the Cre/loxP-based hybrid baculovirus. Moreover, our findings show that UCHL1 enhances SOX9 expression by regulating the stability and localization of HIF-1α, which promotes cartilage production in ADSCs. These findings suggest that activating UCHL1 using the CRISPRa system holds significant potential for applications in cartilage regeneration.},
}

*Ubiquitin Thiolesterase/metabolism/genetics
*SOX9 Transcription Factor/metabolism/genetics
*Hypoxia-Inducible Factor 1, alpha Subunit/metabolism/genetics
*Cell Differentiation/genetics
*Chondrogenesis/genetics
Animals
Humans
Cartilage/metabolism
Chondrocytes/metabolism/cytology
Mesenchymal Stem Cells/metabolism/cytology
CRISPR-Cas Systems
Mice

@article {pmid39223394,
year = {2024},
author = {Razzaq, MK and Babur, MN and Awan, MJA and Raza, G and Mobeen, M and Aslam, A and Siddique, KHM},
title = {Revolutionizing soybean genomics: How CRISPR and advanced sequencing are unlocking new potential.},
journal = {Functional & integrative genomics},
volume = {24},
number = {5},
pages = {153},
pmid = {39223394},
issn = {1438-7948},
mesh = {*Glycine max/genetics ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Genome, Plant ; Genomics/methods ; Plant Breeding/methods ; Polymorphism, Single Nucleotide ; Genome-Wide Association Study ; },
abstract = {Soybean Glycine max L., paleopolyploid genome, poses challenges to its genetic improvement. However, the development of reference genome assemblies and genome sequencing has completely changed the field of soybean genomics, allowing for more accurate and successful breeding techniques as well as research. During the single-cell revolution, one of the most advanced sequencing tools for examining the transcriptome landscape is single-cell RNA sequencing (scRNA-seq). Comprehensive resources for genetic improvement of soybeans may be found in the SoyBase and other genomics databases. CRISPR-Cas9 genome editing technology provides promising prospects for precise genetic modifications in soybean. This method has enhanced several soybean traits, including as yield, nutritional value, and resistance to both biotic and abiotic stresses. With base editing techniques that allow for precise DNA modifications, the use of CRISPR-Cas9 is further increased. With the availability of the reference genome for soybeans and the following assembly of wild and cultivated soybeans, significant chromosomal rearrangements and gene duplication events have been identified, offering new perspectives on the complex genomic structure of soybeans. Furthermore, major single nucleotide polymorphisms (SNPs) linked to stachyose and sucrose content have been found through genome-wide association studies (GWAS), providing important tools for enhancing soybean carbohydrate profiles. In order to open up new avenues for soybean genetic improvement, future research approaches include investigating transcriptional divergence processes, enhancing genetic resources, and incorporating CRISPR-Cas9 technologies.},
}

*Glycine max/genetics
*CRISPR-Cas Systems
*Gene Editing/methods
*Genome, Plant
Genomics/methods
Plant Breeding/methods
Polymorphism, Single Nucleotide
Genome-Wide Association Study

@article {pmid39222468,
year = {2024},
author = {Inam, S and Muhammad, A and Irum, S and Rehman, N and Riaz, A and Uzair, M and Khan, MR},
title = {Genome editing for improvement of biotic and abiotic stress tolerance in cereals.},
journal = {Functional plant biology : FPB},
volume = {51},
number = {},
pages = {},
doi = {10.1071/FP24092},
pmid = {39222468},
issn = {1445-4416},
mesh = {*Gene Editing ; *Edible Grain/genetics ; *Stress, Physiological/genetics ; *CRISPR-Cas Systems ; Climate Change ; Genome, Plant ; },
abstract = {Global agricultural production must quadruple by 2050 to fulfil the needs of a growing global population, but climate change exacerbates the difficulty. Cereals are a very important source of food for the world population. Improved cultivars are needed, with better resistance to abiotic stresses like drought, salt, and increasing temperatures, and resilience to biotic stressors like bacterial and fungal infections, and pest infestation. A popular, versatile, and helpful method for functional genomics and crop improvement is genome editing. Rapidly developing genome editing techniques including clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein (Cas) are very important. This review focuses on how CRISPR/Cas9 genome editing might enhance cereals' agronomic qualities in the face of climate change, providing important insights for future applications. Genome editing efforts should focus on improving characteristics that confer tolerance to conditions exacerbated by climate change (e.g. drought, salt, rising temperatures). Improved water usage efficiency, salt tolerance, and heat stress resilience are all desirable characteristics. Cultivars that are more resilient to insect infestations and a wide range of biotic stressors, such as bacterial and fungal diseases, should be created. Genome editing can precisely target genes linked to disease resistance pathways to strengthen cereals' natural defensive systems.},
}

*Gene Editing
*Edible Grain/genetics
*Stress, Physiological/genetics
*CRISPR-Cas Systems
Climate Change
Genome, Plant

@article {pmid39171996,
year = {2024},
author = {Wang, Q and Xia, J and Yang, C and Chen, X and Chen, B and Li, Y and Huang, H and Lin, B and Guo, L and Xu, J},
title = {Cross-Priming-Linked Hierarchical Isothermal Amplification Programming Progressive Activating Clustered Regularly Interspaced Short Palindromic Repeats/Cas12a in miRNA Signaling.},
journal = {Analytical chemistry},
volume = {96},
number = {35},
pages = {14205-14214},
doi = {10.1021/acs.analchem.4c02795},
pmid = {39171996},
issn = {1520-6882},
mesh = {*Nucleic Acid Amplification Techniques/methods ; *MicroRNAs/genetics/analysis ; Humans ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; CRISPR-Cas Systems/genetics ; Signal Transduction ; Endodeoxyribonucleases/genetics/metabolism ; Bacterial Proteins ; CRISPR-Associated Proteins ; },
abstract = {Cascade isothermal nucleic acid amplification, which integrates several different amplification protocols to enhance the assay performance, is widely utilized in biosensing, particularly for detecting microRNAs (miRNAs), crucial biomarkers associated with tumor initiation and progression. However, striking a balance between a high amplification efficiency and simplicity in design remains a challenge. Therefore, methods achieving high amplification efficiency without significantly increasing complexity are highly favored. In this study, we propose a novel approach for miRNA detection, employing cross-priming-linked hierarchical isothermal amplification (CP-HIA) to progressively activate the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a system. The CP-HIA method strategically combines nicking-rolling circle amplification (n-RCA) and palindrome-aided circular strand displacement amplification (p-CSDA) for miRNA detection. Remarkably, this method utilizes only two main probes. Its key innovation lies in the interactive cross-priming strategy, wherein the amplification product from n-RCA is recycled to further drive p-CSDA, and vice versa. This interactive process establishes a hierarchical amplification, significantly enriching the activation probes for progressive CRISPR/Cas12a activation and subsequent target signal amplification. Consequently, the method exhibits greatly enhanced analytical performance, including high sensitivity and specificity in detecting low concentrations of miRNA. As low as 1.06 fM miRNA can thus be quantitatively detected, and the linear response of the miRNA is from 10 fM to 10 nM. These features demonstrate its potential for early disease diagnosis and monitoring. We anticipate that the CP-HIA method will serve as a promising platform for developing advanced molecular diagnostic tools for biomedical research.},
}

*Nucleic Acid Amplification Techniques/methods
*MicroRNAs/genetics/analysis
Humans
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
CRISPR-Cas Systems/genetics
Signal Transduction
Endodeoxyribonucleases/genetics/metabolism
Bacterial Proteins
CRISPR-Associated Proteins

@article {pmid39120142,
year = {2024},
author = {Wang, T and Li, A and Zhao, H and Wu, Q and Guo, J and Tian, H and Wang, J and Que, Y and Xu, L},
title = {A novel system with robust compatibility and stability for detecting Sugarcane yellow leaf virus based on CRISPR-Cas12a.},
journal = {Microbiology spectrum},
volume = {12},
number = {9},
pages = {e0114924},
doi = {10.1128/spectrum.01149-24},
pmid = {39120142},
issn = {2165-0497},
mesh = {*Saccharum/virology ; *CRISPR-Cas Systems/genetics ; *Plant Diseases/virology ; *Nucleic Acid Amplification Techniques/methods ; *Luteoviridae/genetics/isolation & purification ; *Plant Leaves/virology ; Molecular Diagnostic Techniques/methods ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Sugarcane yellow leaf virus (SCYLV) can reduce sugarcane productivity. A novel detection system based on reverse transcription-multienzyme isothermal rapid amplification (RT-MIRA) combined with CRISPR-Cas12a, named RT-MIRA-CRISPR-Cas12a, was developed. This innovative approach employs crude leaf extract directly as the reaction template, streamlining the extraction process for simplicity and speed. Combining RT-MIRA and CRISPR-Cas12a in one reaction tube increases the ease of operation while reducing the risk of aerosol contamination. In addition, it exhibits sensitivity equivalent to qPCR, boasting a lower detection limit of 25 copies. Remarkably, the entire process, from sample extraction to reaction completion, requires only 52-57 minutes, just a thermostat water bath. The result can be observed and judged by the naked eye.IMPORTANCESugarcane yellow leaf disease (SCYLD) is an important viral disease that affects sugarcane yield. There is an urgent need for rapid, sensitive, and stable detection methods. The reverse transcription-multienzyme isothermal rapid amplification combined with CRISPR-Cas12a (RT-MIRA-CRISPR-Cas12a) method established in this study has good specificity and high sensitivity. In addition, the system showed good compatibility and stability with the crude leaf extract, as shown by the fact that the crude extract of the positive sample could still be stably detected after 1 week when placed at 4°C. RT-MIRA-CRISPR-Cas12a, reverse transcription polymerase chain reaction (RT-PCR), and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) were used to detect SCYLV on 33 sugarcane leaf samples collected from the field, and it was found that the three methods reached consistent conclusions. This Cas12a-based detection method proves highly suitable for the rapid on-site detection of the SCYLV.},
}

*Saccharum/virology
*CRISPR-Cas Systems/genetics
*Plant Diseases/virology
*Nucleic Acid Amplification Techniques/methods
*Luteoviridae/genetics/isolation & purification
*Plant Leaves/virology
Molecular Diagnostic Techniques/methods
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid39018097,
year = {2024},
author = {Jadlowsky, JK and Chang, JF and Spencer, DH and Warrington, JM and Levine, BL and June, CH and Fraietta, JA and Singh, N},
title = {Regulatory Considerations for Genome-Edited T-cell Therapies.},
journal = {Cancer immunology research},
volume = {12},
number = {9},
pages = {1132-1135},
doi = {10.1158/2326-6066.CIR-24-0482},
pmid = {39018097},
issn = {2326-6074},
support = {//Damon Runyon Cancer Research Foundation (DRCRF)/ ; R37CA285572/BC/NCI NIH HHS/United States ; },
mesh = {Humans ; *Gene Editing ; *T-Lymphocytes/immunology/metabolism ; Genetic Therapy/methods ; Immunotherapy, Adoptive/methods ; Animals ; Receptors, Chimeric Antigen/genetics/immunology ; CRISPR-Cas Systems ; Receptors, Antigen, T-Cell/genetics ; Cell- and Tissue-Based Therapy/methods ; },
abstract = {Methods to engineer the genomes of human cells for therapeutic intervention continue to advance at a remarkable pace. Chimeric antigen receptor-engineered T lymphocytes have pioneered the way for these therapies, initially beginning with insertions of chimeric antigen receptor transgenes into T-cell genomes using classical gene therapy vectors. The broad use of clustered regularly interspaced short palindromic repeats (CRISPR)-based technologies to edit endogenous genes has now opened the door to a new era of precision medicine. To add complexity, many engineered cellular therapies under development integrate gene therapy with genome editing to introduce novel biological functions and enhance therapeutic efficacy. Here, we review the current state of scientific, translational, and regulatory oversight of gene-edited cell products.},
}

Humans
*Gene Editing
*T-Lymphocytes/immunology/metabolism
Genetic Therapy/methods
Immunotherapy, Adoptive/methods
Animals
Receptors, Chimeric Antigen/genetics/immunology
CRISPR-Cas Systems
Receptors, Antigen, T-Cell/genetics
Cell- and Tissue-Based Therapy/methods

@article {pmid38991878,
year = {2024},
author = {Cerdà, P and Aguilera, C and Riera-Mestre, A},
title = {Pioneering the future: CRISPR-Cas9 gene therapy for hereditary hemorrhagic telangiectasia. Author's reply.},
journal = {European journal of internal medicine},
volume = {127},
number = {},
pages = {142-143},
doi = {10.1016/j.ejim.2024.07.003},
pmid = {38991878},
issn = {1879-0828},
mesh = {*Telangiectasia, Hereditary Hemorrhagic/therapy/genetics ; Humans ; *Genetic Therapy/methods ; *CRISPR-Cas Systems ; },
}

*Telangiectasia, Hereditary Hemorrhagic/therapy/genetics
Humans
*Genetic Therapy/methods
*CRISPR-Cas Systems

@article {pmid38977907,
year = {2024},
author = {Giuliano, CJ and Wei, KJ and Harling, FM and Waldman, BS and Farringer, MA and Boydston, EA and Lan, TCT and Thomas, RW and Herneisen, AL and Sanderlin, AG and Coppens, I and Dvorin, JD and Lourido, S},
title = {CRISPR-based functional profiling of the Toxoplasma gondii genome during acute murine infection.},
journal = {Nature microbiology},
volume = {9},
number = {9},
pages = {2323-2343},
pmid = {38977907},
issn = {2058-5276},
support = {R01 AI158501/AI/NIAID NIH HHS/United States ; R01 AI144369/AI/NIAID NIH HHS/United States ; R01 AI145941/AI/NIAID NIH HHS/United States ; R01 AI158501/AI/NIAID NIH HHS/United States ; R01 AI144369/AI/NIAID NIH HHS/United States ; R01 AI145941/AI/NIAID NIH HHS/United States ; },
mesh = {Animals ; *Toxoplasma/genetics ; Mice ; *Genome, Protozoan ; *Host-Parasite Interactions/genetics ; CRISPR-Cas Systems ; Protozoan Proteins/genetics/metabolism ; Toxoplasmosis, Animal/parasitology ; Clustered Regularly Interspaced Short Palindromic Repeats ; Female ; Toxoplasmosis/parasitology ; Antiprotozoal Agents/pharmacology ; Disease Models, Animal ; Pyrimethamine/pharmacology ; },
abstract = {Examining host-pathogen interactions in animals can capture aspects of infection that are obscured in cell culture. Using CRISPR-based screens, we functionally profile the entire genome of the apicomplexan parasite Toxoplasma gondii during murine infection. Barcoded gRNAs enabled bottleneck detection and mapping of population structures within parasite lineages. Over 300 genes with previously unknown roles in infection were found to modulate parasite fitness in mice. Candidates span multiple axes of host-parasite interaction. Rhoptry Apical Surface Protein 1 was characterized as a mediator of host-cell tropism that facilitates repeated invasion attempts. GTP cyclohydrolase I was also required for fitness in mice and druggable through a repurposed compound, 2,4-diamino-6-hydroxypyrimidine. This compound synergized with pyrimethamine against T. gondii and malaria-causing Plasmodium falciparum parasites. This work represents a complete survey of an apicomplexan genome during infection of an animal host and points to novel interfaces of host-parasite interaction.},
}

Animals
*Toxoplasma/genetics
Mice
*Genome, Protozoan
*Host-Parasite Interactions/genetics
CRISPR-Cas Systems
Protozoan Proteins/genetics/metabolism
Toxoplasmosis, Animal/parasitology
Clustered Regularly Interspaced Short Palindromic Repeats
Female
Toxoplasmosis/parasitology
Antiprotozoal Agents/pharmacology
Disease Models, Animal
Pyrimethamine/pharmacology

@article {pmid38962826,
year = {2024},
author = {Xu, J and Ren, J and Xu, K and Fang, M and Ka, M and Xu, F and Wang, X and Wang, J and Han, Z and Feng, G and Zhang, Y and Hai, T and Li, W and Hu, Z},
title = {Elimination of GGTA1, CMAH, β4GalNT2 and CIITA genes in pigs compromises human versus pig xenogeneic immune reactions.},
journal = {Animal models and experimental medicine},
volume = {7},
number = {4},
pages = {584-590},
doi = {10.1002/ame2.12461},
pmid = {38962826},
issn = {2576-2095},
support = {2019YFA0903800//National Key Research and Development Program/ ; 2021YFA0805701//National Key Research and Development Program/ ; 2021YFA0805905//National Key Research and Development Program/ ; 2022YFA1103603//National Key Research and Development Program/ ; YSBR-012//CAS Project for Young Scientists in Basic Research/ ; 2023ZD0407503//STI 2030-Major Project/ ; 32071456//National Natural Science Foundation of China/ ; 82241224//National Natural Science Foundation of China/ ; XDA16030000//Strategic Priority Research Program of the Chinese Academy of Sciences/ ; },
mesh = {Animals ; Humans ; Swine ; *Transplantation, Heterologous ; *N-Acetylgalactosaminyltransferases/genetics ; *Graft Rejection/genetics/immunology ; Galactosyltransferases/genetics ; Nuclear Proteins/genetics ; Mixed Function Oxygenases/genetics ; Animals, Genetically Modified ; Gene Knockout Techniques ; CRISPR-Cas Systems ; Gene Editing ; Trans-Activators ; },
abstract = {BACKGROUND: Pig organ xenotransplantation is a potential solution for the severe organ shortage in clinic, while immunogenic genes need to be eliminated to improve the immune compatibility between humans and pigs. Current knockout strategies are mainly aimed at the genes causing hyperacute immune rejection (HAR) that occurs in the first few hours while adaptive immune reactions orchestrated by CD4 T cell thereafter also cause graft failure, in which process the MHC II molecule plays critical roles.

METHODS: Thus, we generate a 4-gene (GGTA1, CMAH, β4GalNT2, and CIITA) knockout pig by CRISPR/Cas9 and somatic cell nuclear transfer to compromise HAR and CD4 T cell reactions simultaneously.

RESULTS: We successfully obtained 4KO piglets with deficiency in all alleles of genes, and at cellular and tissue levels. Additionally, the safety of our animals after gene editing was verified by using whole-genome sequencing and karyotyping. Piglets have survived for more than one year in the barrier, and also survived for more than 3 months in the conventional environment, suggesting that the piglets without MHC II can be raised in the barrier and then gradually mated in the conventional environment.

CONCLUSIONS: 4KO piglets have lower immunogenicity, are safe in genomic level, and are easier to breed than the model with both MHC I and II deletion.},
}

Animals
Humans
Swine
*Transplantation, Heterologous
*N-Acetylgalactosaminyltransferases/genetics
*Graft Rejection/genetics/immunology
Galactosyltransferases/genetics
Nuclear Proteins/genetics
Mixed Function Oxygenases/genetics
Animals, Genetically Modified
Gene Knockout Techniques
CRISPR-Cas Systems
Gene Editing
Trans-Activators

@article {pmid38879351,
year = {2024},
author = {James, M and Sehgal, VS},
title = {Pioneering the future: CRISPR-Cas9 gene therapy for hereditary hemorrhagic telangiectasia.},
journal = {European journal of internal medicine},
volume = {127},
number = {},
pages = {140-141},
doi = {10.1016/j.ejim.2024.06.012},
pmid = {38879351},
issn = {1879-0828},
mesh = {*Telangiectasia, Hereditary Hemorrhagic/therapy/genetics ; Humans ; *Genetic Therapy/methods ; *CRISPR-Cas Systems ; Gene Editing/methods ; },
}

*Telangiectasia, Hereditary Hemorrhagic/therapy/genetics
Humans
*Genetic Therapy/methods
*CRISPR-Cas Systems
Gene Editing/methods

@article {pmid38874582,
year = {2024},
author = {De Munter, S and Buhl, JL and De Cock, L and Van Parys, A and Daneels, W and Pascal, E and Deseins, L and Ingels, J and Goetgeluk, G and Jansen, H and Billiet, L and Pille, M and Van Duyse, J and Bonte, S and Vandamme, N and Van Dorpe, J and Offner, F and Leclercq, G and Taghon, T and Depla, E and Tavernier, J and Kerre, T and Drost, J and Vandekerckhove, B},
title = {Knocking Out CD70 Rescues CD70-Specific NanoCAR T Cells from Antigen-Induced Exhaustion.},
journal = {Cancer immunology research},
volume = {12},
number = {9},
pages = {1236-1251},
doi = {10.1158/2326-6066.CIR-23-0677},
pmid = {38874582},
issn = {2326-6074},
support = {KW/2196/REG/001/001//Universitair Ziekenhuis Gent (UZ Gent)/ ; 12AP724N//Fonds Wetenschappelijk Onderzoek (FWO)/ ; G028220N//Fonds Wetenschappelijk Onderzoek (FWO)/ ; F2021/IOF-StarTT/107//Ghent University IOF/ ; },
mesh = {Humans ; *CD27 Ligand ; Animals ; Mice ; *Immunotherapy, Adoptive/methods ; *Xenograft Model Antitumor Assays ; *Receptors, Chimeric Antigen/immunology/genetics ; T-Lymphocytes/immunology/metabolism ; Lymphoma, Large B-Cell, Diffuse/immunology/therapy/genetics ; Gene Knockout Techniques ; Cell Line, Tumor ; CRISPR-Cas Systems ; },
abstract = {CD70 is an attractive target for chimeric antigen receptor (CAR) T-cell therapy for the treatment of both solid and liquid malignancies. However, the functionality of CD70-specific CAR T cells is modest. We optimized a CD70-specific VHH-based CAR (nanoCAR). We evaluated the nanoCARs in clinically relevant models in vitro, using co-cultures of CD70-specific nanoCAR T cells with malignant rhabdoid tumor organoids, and in vivo, using a diffuse large B-cell lymphoma patient-derived xenograft (PDX) model. Although the nanoCAR T cells were highly efficient in organoid co-cultures, they showed only modest efficacy in the PDX model. We determined that fratricide was not causing this loss in efficacy but rather CD70 interaction in cis with the nanoCAR-induced exhaustion. Knocking out CD70 in nanoCAR T cells using CRISPR/Cas9 resulted in dramatically enhanced functionality in the diffuse large B-cell lymphoma PDX model. Through single-cell transcriptomics, we obtained evidence that CD70 knockout CD70-specific nanoCAR T cells were protected from antigen-induced exhaustion. In addition, we demonstrated that wild-type CD70-specific nanoCAR T cells already exhibited signs of exhaustion shortly after production. Their gene signature strongly overlapped with gene signatures of exhausted CAR T cells. Conversely, the gene signature of knockout CD70-specific nanoCAR T cells overlapped with the gene signature of CAR T-cell infusion products leading to complete responses in chronic lymphatic leukemia patients. Our data show that CARs targeting endogenous T-cell antigens negatively affect CAR T-cell functionality by inducing an exhausted state, which can be overcome by knocking out the specific target.},
}

Humans
*CD27 Ligand
Animals
Mice
*Immunotherapy, Adoptive/methods
*Xenograft Model Antitumor Assays
*Receptors, Chimeric Antigen/immunology/genetics
T-Lymphocytes/immunology/metabolism
Lymphoma, Large B-Cell, Diffuse/immunology/therapy/genetics
Gene Knockout Techniques
Cell Line, Tumor
CRISPR-Cas Systems

@article {pmid38869428,
year = {2024},
author = {Chang, JF and Wellhausen, N and Engel, NW and Landmann, JH and Hopkins, CR and Salas-McKee, J and Bear, AS and Selli, ME and Agarwal, S and Jadlowsky, JK and Linette, GP and Gill, S and June, CH and Fraietta, JA and Singh, N},
title = {Identification of Core Techniques That Enhance Genome Editing of Human T Cells Expressing Synthetic Antigen Receptors.},
journal = {Cancer immunology research},
volume = {12},
number = {9},
pages = {1136-1146},
doi = {10.1158/2326-6066.CIR-24-0251},
pmid = {38869428},
issn = {2326-6074},
mesh = {Humans ; *Gene Editing/methods ; *T-Lymphocytes/immunology/metabolism ; CRISPR-Cas Systems ; Receptors, Antigen, T-Cell/genetics/immunology ; },
abstract = {Genome editing technologies have seen remarkable progress in recent years, enabling precise regulation of exogenous and endogenous genes. These advances have been extensively applied to the engineering of human T lymphocytes, leading to the development of practice changing therapies for patients with cancer and the promise of synthetic immune cell therapies for a variety of nonmalignant diseases. Many distinct conceptual and technical approaches have been used to edit T-cell genomes, however targeted assessments of which techniques are most effective for manufacturing, gene editing, and transgene expression are rarely reported. Through extensive comparative evaluation, we identified methods that most effectively enhance engineering of research-scale and preclinical T-cell products at critical stages of manufacturing.},
}

Humans
*Gene Editing/methods
*T-Lymphocytes/immunology/metabolism
CRISPR-Cas Systems
Receptors, Antigen, T-Cell/genetics/immunology

@article {pmid37816604,
year = {2024},
author = {Lo, IHY and Matthews, BJ},
title = {Validating Single-Guide RNA for Aedes aegypti Gene Editing.},
journal = {Cold Spring Harbor protocols},
volume = {2024},
number = {9},
pages = {pdb.prot108340},
doi = {10.1101/pdb.prot108340},
pmid = {37816604},
issn = {1559-6095},
mesh = {*Aedes/genetics ; Animals ; *Gene Editing/methods ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *CRISPR-Cas Systems/genetics ; Animals, Genetically Modified/genetics ; },
abstract = {Creating transgenic mosquitoes allows for mechanistic studies of basic mosquito biology and the development of novel vector control strategies. CRISPR-Cas9 gene editing has revolutionized gene editing, including in mosquitoes. This protocol details part of the gene editing process of Aedes aegypti mosquitoes via CRISPR-Cas9, through testing and validating single-guide RNAs (sgRNAs). Gene editing activity varies depending on the sequence of sgRNAs used, so validation of sgRNA activity should be done before large-scale generation of mutants or transgenics. sgRNA is designed using online tools and synthesized in <1 h. Once mutants or transgenics are generated via embryo microinjection, sgRNA activity is validated by quick genotyping polymerase chain reaction (PCR) and DNA sequencing.},
}

*Aedes/genetics
Animals
*Gene Editing/methods
*RNA, Guide, CRISPR-Cas Systems/genetics
*CRISPR-Cas Systems/genetics
Animals, Genetically Modified/genetics

@article {pmid37816601,
year = {2024},
author = {Lo, IHY and Matthews, BJ},
title = {Design and Validation of Guide RNAs for CRISPR-Cas9 Genome Editing in Mosquitoes.},
journal = {Cold Spring Harbor protocols},
volume = {2024},
number = {9},
pages = {pdb.top107688},
doi = {10.1101/pdb.top107688},
pmid = {37816601},
issn = {1559-6095},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Animals ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *Culicidae/genetics ; },
abstract = {CRISPR-Cas9 has revolutionized gene editing for traditional and nontraditional model organisms alike. This tool has opened the door to new mechanistic studies of basic mosquito biology as well as the development of novel vector control strategies based on CRISPR-Cas9, including gene drives that spread genetic elements in the population. Although the promise of the specificity, flexibility, and ease of deployment CRISPR is real, its implementation still requires empirical optimization for each new species of interest, as well as to each genomic target within a given species. Here, we provide an overview of designing and testing single-guide RNAs for the use of CRISPR-based gene editing tools.},
}

*CRISPR-Cas Systems/genetics
*Gene Editing/methods
Animals
*RNA, Guide, CRISPR-Cas Systems/genetics
*Culicidae/genetics

@article {pmid39221932,
year = {2024},
author = {Kongkaew, R and Uttamapinant, C and Patchsung, M},
title = {Point-of-care CRISPR-based Diagnostics with Premixed and Freeze-dried Reagents.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {210},
pages = {},
doi = {10.3791/66703},
pmid = {39221932},
issn = {1940-087X},
mesh = {*Freeze Drying/methods ; *Point-of-Care Systems ; Humans ; *Nucleic Acid Amplification Techniques/methods ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; CRISPR-Cas Systems/genetics ; Indicators and Reagents/chemistry ; Molecular Diagnostic Techniques/methods ; },
abstract = {Molecular diagnostics by Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based detection have high diagnostic accuracy and attributes that are suitable for use at point-of-care settings such as fast turnaround times for results, convenient simple readouts, and no requirement of complicated instruments. However, the reactions can be cumbersome to perform at the point of care due to their many components and manual handling steps. Herein, we provide a step-by-step, optimized protocol for the robust detection of disease pathogens and genetic markers with recombinase-based isothermal amplification and CRISPR-based reagents, which are premixed and then freeze-dried in easily stored and ready-to-use formats. Premixed, freeze-dried reagents can be rehydrated for immediate use and retain high amplification and detection efficiencies. We also provide a troubleshooting guide for commonly found problems upon preparing and using premixed, freeze-dried reagents for CRISPR-based diagnostics, to make the detection platform more accessible to the wider diagnostic/genetic testing communities.},
}

*Freeze Drying/methods
*Point-of-Care Systems
Humans
*Nucleic Acid Amplification Techniques/methods
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
CRISPR-Cas Systems/genetics
Indicators and Reagents/chemistry
Molecular Diagnostic Techniques/methods

@article {pmid39219618,
year = {2024},
author = {Tripathi, L and Ntui, VO and Tripathi, JN},
title = {Application of CRISPR/Cas-based gene-editing for developing better banana.},
journal = {Frontiers in bioengineering and biotechnology},
volume = {12},
number = {},
pages = {1395772},
pmid = {39219618},
issn = {2296-4185},
abstract = {Banana (Musa spp.), including plantain, is one of the major staple food and cash crops grown in over 140 countries in the subtropics and tropics, with around 153 million tons annual global production, feeding about 400 million people. Despite its widespread cultivation and adaptability to diverse environments, banana production faces significant challenges from pathogens and pests that often coexist within agricultural landscapes. Recent advancements in CRISPR/Cas-based gene editing offer transformative solutions to enhance banana resilience and productivity. Researchers at IITA, Kenya, have successfully employed gene editing to confer resistance to diseases such as banana Xanthomonas wilt (BXW) by targeting susceptibility genes and banana streak virus (BSV) by disrupting viral sequences. Other breakthroughs include the development of semi-dwarf plants, and increased β-carotene content. Additionally, non-browning banana have been developed to reduce food waste, with regulatory approval in the Philippines. The future prospects of gene editing in banana looks promising with CRISPR-based gene activation (CRISPRa) and inhibition (CRISPRi) techniques offering potential for improved disease resistance. The Cas-CLOVER system provides a precise alternative to CRISPR/Cas9, demonstrating success in generating gene-edited banana mutants. Integration of precision genetics with traditional breeding, and adopting transgene-free editing strategies, will be pivotal in harnessing the full potential of gene-edited banana. The future of crop gene editing holds exciting prospects for producing banana that thrives across diverse agroecological zones and offers superior nutritional value, ultimately benefiting farmers and consumers. This article highlights the pivotal role of CRISPR/Cas technology in advancing banana resilience, yield and nutritional quality, with significant implications for global food security.},
}

@article {pmid39218584,
year = {2024},
author = {Luo, S and Wu, J and Zhong, M and Sun, J and Ao, H and Cao, X and Liu, J and Ju, H},
title = {An electrochemiluminescent imaging strategy based on CRISPR/Cas12a for ultrasensitive detection of nucleic acid.},
journal = {Analytica chimica acta},
volume = {1324},
number = {},
pages = {343040},
doi = {10.1016/j.aca.2024.343040},
pmid = {39218584},
issn = {1873-4324},
mesh = {*Electrochemical Techniques/methods ; *Luminescent Measurements/methods ; *CRISPR-Cas Systems/genetics ; Humans ; Biosensing Techniques/methods ; DNA, Viral/analysis/genetics ; Human papillomavirus 18/genetics ; Limit of Detection ; Gold/chemistry ; CRISPR-Associated Proteins ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {BACKGROUND: Persistent infection with human papillomavirus (HPV) significantly contributes to the development of cervical cancer. Thus, it is urgent to develop rapid and accurate methods for HPV detection. Herein, we present an ultrasensitive CRISPR/Cas12a-based electrochemiluminescent (ECL) imaging technique for the detection of HPV-18 DNA.

RESULT: The ECL DNA sensor array is constructed by applying black hole quencher (BHQ) and polymer dots (Pdots) co-labeled hairpin DNA (hpDNA) onto a gold-coated indium tin oxide slide (Au-ITO). The ECL imaging method involves an incubation process of target HPV-18 with a mixture of crRNA and Cas12a to activate Cas12a, followed by an incubation of the active Cas12a with the ECL sensor. This interaction causes the indiscriminate cleavage of BHQ from Pdots by digesting hpDNA on the sensor surface, leading to the restoration of the ECL signal of Pdots. The ECL brightness readout demonstrates superior performance of the ECL imaging technique, with a linear detection range of 10 fM-500 pM and a limit-of-detection (LOD) of 5.3 fM.

SIGNIFICANCE: The Cas12a-based ECL imaging approach offers high sensitivity and a broad detection range, making it highly promising for nucleic acid detection applications.},
}

*Electrochemical Techniques/methods
*Luminescent Measurements/methods
*CRISPR-Cas Systems/genetics
Humans
Biosensing Techniques/methods
DNA, Viral/analysis/genetics
Human papillomavirus 18/genetics
Limit of Detection
Gold/chemistry
CRISPR-Associated Proteins
Bacterial Proteins
Endodeoxyribonucleases

@article {pmid39218375,
year = {2024},
author = {Shidan, Z and Song, L and Yumin, Z and Rong, C and Siteng, W and Meirong, L and Guangjin, L},
title = {First Report of Streptococcus agalactiae isolated from a healthy Captive Sichuan golden snub-nosed monkey (Rhinopithecus roxellana) in China.},
journal = {Microbial pathogenesis},
volume = {},
number = {},
pages = {106907},
doi = {10.1016/j.micpath.2024.106907},
pmid = {39218375},
issn = {1096-1208},
abstract = {Streptococcus agalactiae (S. agalactiae) is an opportunistic pathogen, and to date, studies have mainly focused on S. agalactiae strains isolated from humans, dairy cows, and fish. We reported one S. agalactiae strain, named CFFB, which was isolated from a healthy Sichuan golden snub-nosed monkey. Classical bacteriological approaches, as well as, next-generation sequencing, comparative genomics, and mice challenge test were used to characterize this strain. CFFB was identified as serotype III, ST19 combination which is a common type found in human strains. Phylogenetic analysis showed that the genome of CFFB was closely related to human clinical isolates, rather far away from animal strains. In total, CFFB contained fewer virulence-associated genes and antibiotic resistance genes than human isolates that were close to CFFB in evolutionary relationships. In the mice challenge test, CFFB had a relative weak virulence that just caused death in 33% of ICR mice at a dose of 10[8] CFU by intraperitoneal injection, and CFFB was reisolated from the cardiac blood of the dead mice. Meanwhile, two intact prophages (prophage 1 and 2) were identified in the CFFB genome and shared high similarities with phage Javan52 and Javan29 which from human S. agalactiae isolate Gottschalk 1002A and RBH03, respectively. Moreover, the type II-A CRISPR-Cas system was detected in the CFFB genome, and the spacers from CFFB were the same to the streptococci isolates from human. These results suggest that CFFB isolated from healthy Sichuan golden snub-nosed monkeys may have its origin in human S. agalactiae. Our results suggested some genomic similarities between the S. agalactiae colonized in Sichuan golden snub-nosed monkey and those in infected humans.},
}

@article {pmid39218175,
year = {2024},
author = {Hosseini, SA and Elahian, F and Mirzaei, SA},
title = {Innovative genetic scissor strategies and their applications in cancer treatment and prevention: CRISPR modules and challenges.},
journal = {International journal of biological macromolecules},
volume = {},
number = {},
pages = {135239},
doi = {10.1016/j.ijbiomac.2024.135239},
pmid = {39218175},
issn = {1879-0003},
abstract = {There are lots of gene editing tools for targeting genome sequences. Some are almost known, and most are a complete mystery and undiscovered. CRISPR/Cas editing tools have brought about a major revolution in medicine. Researchers have shown that CRISPR can modify DNA much more accurately, economically and easily than previous methods. CRISPR has proven itself effective for the deletion, replacement and insertion of DNA fragments into cell types, tissues and organisms. Recently, combining CRISPR/Cas with factors (transcription factors/repressors, exonucleases, endonucleases, transposons, caspase, fluorescent proteins, oxidoreductive enzymes, DNA/RNA polymerases), and elements (aptamers, barcodes, fluorescent probes, Trigger) have provided genome, transcriptome, proteome and epigenome modification. These modules are being investigated for cancer prevention and therapy and this review focuses on such innovative combinations that hopefully will become a clinical reality in the near future.},
}

@article {pmid39217145,
year = {2024},
author = {Niu, H and Maruoka, M and Noguchi, Y and Kosako, H and Suzuki, J},
title = {Phospholipid scrambling induced by an ion channel/metabolite transporter complex.},
journal = {Nature communications},
volume = {15},
number = {1},
pages = {7566},
pmid = {39217145},
issn = {2041-1723},
support = {21K19261//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; 22H02572//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; 1199566//MEXT | JST | Core Research for Evolutional Science and Technology (CREST)/ ; },
mesh = {Humans ; *Calcium/metabolism ; *Phospholipid Transfer Proteins/metabolism/genetics ; *Phospholipids/metabolism ; HEK293 Cells ; Ion Channels/metabolism/genetics ; Animals ; CRISPR-Cas Systems ; },
abstract = {Cells establish the asymmetrical distribution of phospholipids and alter their distribution by phospholipid scrambling (PLS) to adapt to environmental changes. Here, we demonstrate that a protein complex, consisting of the ion channel Tmem63b and the thiamine transporter Slc19a2, induces PLS upon calcium (Ca[2+]) stimulation. Through revival screening using a CRISPR sgRNA library on high PLS cells, we identify Tmem63b as a PLS-inducing factor. Ca[2+] stimulation-mediated PLS is suppressed by deletion of Tmem63b, while human disease-related Tmem63b mutants induce constitutive PLS. To search for a molecular link between Ca[2+] stimulation and PLS, we perform revival screening on Tmem63b-overexpressing cells, and identify Slc19a2 and the Ca[2+]-activated K[+] channel Kcnn4 as PLS-regulating factors. Deletion of either of these genes decreases PLS activity. Biochemical screening indicates that Tmem63b and Slc19a2 form a heterodimer. These results demonstrate that a Tmem63b/Slc19a2 heterodimer induces PLS upon Ca[2+] stimulation, along with Kcnn4 activation.},
}

Humans
*Calcium/metabolism
*Phospholipid Transfer Proteins/metabolism/genetics
*Phospholipids/metabolism
HEK293 Cells
Ion Channels/metabolism/genetics
Animals
CRISPR-Cas Systems

@article {pmid39213744,
year = {2024},
author = {Araújo, MRB and Prates, FD and Viana, MVC and Santos, LS and Mattos-Guaraldi, AL and Camargo, CH and Sacchi, CT and Campos, KR and Vieira, VV and Santos, MBN and Bokermann, S and Ramos, JN and Azevedo, V},
title = {Genomic analysis of two penicillin- and rifampin-resistant Corynebacterium rouxii strains isolated from cutaneous infections in dogs.},
journal = {Research in veterinary science},
volume = {179},
number = {},
pages = {105396},
doi = {10.1016/j.rvsc.2024.105396},
pmid = {39213744},
issn = {1532-2661},
abstract = {Although diphtheria is a vaccine-preventable disease, numerous cases are still reported around the world, as well as outbreaks in countries, including European ones. Species of the Corynebacterium diphtheriae complex are potentially toxigenic and, therefore, must be considered given the possible consequences, such as the circulation of clones and transmission of antimicrobial resistance and virulence genes. Recently, Corynebacterium rouxii was characterized and included among the valid species of the complex. Therefore, two cases of C. rouxii infection arising from infections in domestic animals are presented here. We provide molecular characterization, phylogenetic analyses, genome sequencing, and CRISPR-Cas analyses to contribute to a better understanding of the molecular bases, pathogenesis, and epidemiological monitoring of this species, which is still little studied. We confirmed its taxonomic position with genome sequencing and in silico analysis and identified the ST-918 for both strains. The clinical isolates were sensitive resistance to benzylpenicillin and rifampin. Antimicrobial resistance genes, including tetB, rpoB2, and rbpA genes, were predicted. The bla and ampC genes were not found. Several virulence factors were also detected, including adhesion, iron uptake systems, gene regulation (dtxR), and post-translational modification (MdbA). Finally, one prophage and the Type I-E CRISPR-Cas system were identified.},
}

@article {pmid39038660,
year = {2024},
author = {Du, Y and Liu, X and Gao, H and Liu, X and Huang, M and Chai, Q and Xing, Z and Zhang, T and Ma, D},
title = {Rapid and one-tube detection of human metapneumovirus using the RT-RPA and CRISPR/Cas12a.},
journal = {Journal of virological methods},
volume = {329},
number = {},
pages = {115001},
doi = {10.1016/j.jviromet.2024.115001},
pmid = {39038660},
issn = {1879-0984},
mesh = {*Metapneumovirus/genetics/isolation & purification ; Humans ; *Paramyxoviridae Infections/diagnosis/virology ; *CRISPR-Cas Systems ; *Sensitivity and Specificity ; RNA, Viral/genetics ; Respiratory Tract Infections/virology/diagnosis ; Nucleic Acid Amplification Techniques/methods ; },
abstract = {Human metapneumovirus (HMPV) is a common pathogen that can cause acute respiratory tract infections and is prevalent worldwide. There is yet no effective vaccine or specific treatment for HMPV. Early, rapid, and accurate detection is essential to treat the disease and control the spread of infection. In this study, we created the One-tube assay by combining Reverse Transcription-Recombinase Polymerase Amplification (RT-RPA) with the CRISPR/Cas12a system. By targeting the nucleoprotein (N) gene of HMPV to design specific primers and CRISPR RNAs (crRNAs), combining RT-RPA and CRISPR/Cas12a, established the One-tube assay. Meanwhile, the reaction conditions of the One-tube assay were optimized to achieve rapid and visual detection of HMPV. This assay could detect HMPV at 1 copy/μL in 30 min, without cross-reactivity with nine other respiratory pathogens. We validated the detection performance using clinical specimens and showed that the coincidence rate was 98.53 %，compared to the quantitative reverse-transcription polymerase chain reaction. The One-tube assay reduced the detection time and simplified the manual operation, while maintaining the detection performance and providing a new platform for HMPV detection.},
}

*Metapneumovirus/genetics/isolation & purification
Humans
*Paramyxoviridae Infections/diagnosis/virology
*CRISPR-Cas Systems
*Sensitivity and Specificity
RNA, Viral/genetics
Respiratory Tract Infections/virology/diagnosis
Nucleic Acid Amplification Techniques/methods

@article {pmid38683016,
year = {2024},
author = {Kachwala, MJ and Hamdard, F and Cicek, D and Dagci, H and Smith, CW and Kalla, N and Yigit, MV},
title = {Universal CRISPR-Cas12a and Toehold RNA Cascade Reaction on Paper Substrate for Visual Salmonella Genome Detection.},
journal = {Advanced healthcare materials},
volume = {13},
number = {22},
pages = {e2400508},
doi = {10.1002/adhm.202400508},
pmid = {38683016},
issn = {2192-2659},
support = {2022-08596//National Institute of Food and Agriculture/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Genome, Bacterial/genetics ; *Salmonella/genetics ; Paper ; Lactuca/microbiology/genetics ; Animals ; Milk/microbiology ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Salmonella, the most prevalent food-borne pathogen, poses significant medical and economic threats. Swift and accurate on-site identification and serotyping of Salmonella is crucial to curb its spread and contamination. Here, a synthetic biology cascade reaction is presented on a paper substrate using CRISPR-Cas12a and recombinase polymerase amplification (RPA), enabling the programming of a standard toehold RNA switch for a genome of choice. This approach employs just one toehold RNA switch design to differentiate between two different Salmonella serotypes, i.e., S. Typhimurium and S. Enteritidis, without the need for reengineering the toehold RNA switch. The sensor exhibits high sensitivity, capable of visually detecting as few as 100 copies of the whole genome from a model Salmonella pathogen on a paper substrate. Furthermore, this robust assay is successfully applied to detect whole genomes in contaminated milk and lettuce samples, demonstrating its potential in real sample analysis. Due to its versatility and practical features, genomes from different organisms can be detected by merely changing a single RNA element in this universal cell-free cascade reaction.},
}

*CRISPR-Cas Systems/genetics
*Genome, Bacterial/genetics
*Salmonella/genetics
Paper
Lactuca/microbiology/genetics
Animals
Milk/microbiology
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid39213392,
year = {2024},
author = {Du, Q and Zhang, Z and Yang, W and Zhou, X and Zhou, N and Wu, C and Bao, J},
title = {CBGDA: a manually curated resource for gene-disease associations based on genome-wide CRISPR.},
journal = {Database : the journal of biological databases and curation},
volume = {2024},
number = {},
pages = {},
doi = {10.1093/database/baae077},
pmid = {39213392},
issn = {1758-0463},
support = {31971162 32071275 U20A20410//National Nature Science Foundation of China/ ; 31971162 32071275 U20A20410//National Nature Science Foundation of China/ ; },
mesh = {Humans ; *Databases, Genetic ; *Data Curation/methods ; Genome-Wide Association Study/methods ; Genetic Predisposition to Disease ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; CRISPR-Cas Systems ; Disease/genetics ; },
abstract = {The field of understanding the association between genes and diseases is rapidly expanding, making it challenging for researchers to keep up with the influx of new publications and genetic datasets. Fortunately, there are now several regularly updated databases available that focus on cataloging gene-disease relationships. The development of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 system has revolutionized the field of gene editing, providing a highly efficient, accurate, and reliable method for exploring gene-disease associations. However, currently, there is no resource specifically dedicated to collecting and integrating the latest experimentally supported gene-disease association data derived from genome-wide CRISPR screening. To address this gap, we have developed the CRISPR-Based Gene-Disease Associations (CBGDA) database, which includes over 200 manually curated gene-disease association data derived from genome-wide CRISPR screening studies. Through CBGDA, users can explore gene-disease association data derived from genome-wide CRISPR screening, gaining insights into the expression patterns of genes in different diseases, associated chemical data, and variant information. This provides a novel perspective on understanding the associations between genes and diseases. What is more, CBGDA integrates data from several other databases and resources, enhancing its comprehensiveness and utility. In summary, CBGDA offers a fresh perspective and comprehensive insights into the research on gene-disease associations. It fills the gap by providing a dedicated resource for accessing up-to-date, experimentally supported gene-disease association data derived from genome-wide CRISPR screening. Database URL: http://cbgda.zhounan.org/main.},
}

Humans
*Databases, Genetic
*Data Curation/methods
Genome-Wide Association Study/methods
Genetic Predisposition to Disease
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
CRISPR-Cas Systems
Disease/genetics

@article {pmid39212859,
year = {2024},
author = {Wu, B and Luo, H and Chen, Z and Amin, B and Yang, M and Li, Z and Wu, S and Salmen, SH and Alharbi, SA and Fang, Z},
title = {Rice Promoter Editing: An Efficient Genetic Improvement Strategy.},
journal = {Rice (New York, N.Y.)},
volume = {17},
number = {1},
pages = {55},
pmid = {39212859},
issn = {1939-8425},
support = {32060064/32260498//National Natural Science Foundation of China/ ; Qiankehepingtairencai-YQK (2023) 002//Guizhou Provincial Excellent Young Talents Project of Science and Technology/ ; Qiankehechengguo (2024) General 116 and Qiankehejichu-ZK (2022) Key 008//Guizhou Provincial Science and Technology Projects/ ; Qiankehezhongyindi (2023) 008//Key Laboratory of Molecular Breeding for Grain and Oil Crops in Guizhou Province/ ; Qianjiaoji (2023) 007//Key Laboratory of Functional Agriculture of Guizhou Provincial Department of Education/ ; Qiandongnan Kehe Support (2023) 06//Qiandongnan Science and Technology Support Project/ ; RSP2024R385//Researchers supporting project, King Saud University, Riyadh, Saudi Arabia/ ; },
abstract = {Gene expression levels in rice (Oryza sativa L.) and other plant species are determined by the promoters, which directly control phenotypic characteristics. As essential components of genes, promoters regulate the intensity, location, and timing of gene expression. They contain numerous regulatory elements and serve as binding sites for proteins that modulate transcription, including transcription factors and RNA polymerases. Genome editing can alter promoter sequences, thereby precisely modifying the expression patterns of specific genes, and ultimately affecting the morphology, quality, and resistance of rice. This paper summarizes research on rice promoter editing conducted in recent years, focusing on improvements in yield, heading date, quality, and disease resistance. It is expected to inform the application of promoter editing and encourage further research and development in crop genetic improvement with promote.},
}

@article {pmid39212458,
year = {2024},
author = {Lorenzo, CD and Blasco-Escámez, D and Beauchet, A and Wytynck, P and Sanches, M and Garcia Del Campo, JR and Inzé, D and Nelissen, H},
title = {Maize mutant screens: from classical methods to new CRISPR-based approaches.},
journal = {The New phytologist},
volume = {},
number = {},
pages = {},
doi = {10.1111/nph.20084},
pmid = {39212458},
issn = {1469-8137},
support = {833866//H2020 European Research Council/ ; },
abstract = {Mutations play a pivotal role in shaping the trajectory and outcomes of a species evolution and domestication. Maize (Zea mays) has been a major staple crop and model for genetic research for more than 100 yr. With the arrival of site-directed mutagenesis and genome editing (GE) driven by the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), maize mutational research is once again in the spotlight. If we combine the powerful physiological and genetic characteristics of maize with the already available and ever increasing toolbox of CRISPR-Cas, prospects for its future trait engineering are very promising. This review aimed to give an overview of the progression and learnings of maize screening studies analyzing forward genetics, natural variation and reverse genetics to focus on recent GE approaches. We will highlight how each strategy and resource has contributed to our understanding of maize natural and induced trait variability and how this information could be used to design the next generation of mutational screenings.},
}

@article {pmid39211481,
year = {2024},
author = {Anbazhagan, P and Parameswari, B and Anitha, K and Chaitra, GV and Bajaru, B and Rajashree, A and Mangrauthia, SK and Yousuf, F and Chalam, VC and Singh, GP},
title = {Advances in plant pathogen detection: integrating recombinase polymerase amplification with CRISPR/Cas systems.},
journal = {3 Biotech},
volume = {14},
number = {9},
pages = {214},
doi = {10.1007/s13205-024-04055-x},
pmid = {39211481},
issn = {2190-572X},
abstract = {Plant pathogens are causing substantial economic losses and thus became a significant threat to global agriculture. Effective and timely detection methods are prerequisite for combating the damages caused by the plant pathogens. In the realm of plant pathogen detection, the isothermal amplification techniques, e.g., recombinase polymerase amplification (RPA) and loop-mediated isothermal amplification (LAMP), have emerged as a fast, precise, and most sensitive alternative to conventional PCR but they often comprise high rates of non-specific amplification and operational complexity. In recent advancements, clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated nuclease Cas systems, particularly Cas12, have emerged as powerful tools for highly sensitive, specific, and rapid pathogen detection. Exploiting the collateral activities of Cas12, which selectively cleaves single-stranded DNA (ssDNA), novel detection platforms have been developed. The mechanism employs the formation of a triple complex molecule comprising guide RNA, Cas12 enzyme, and the substrate target nucleotide sequence. Upon recognition of the target, Cas12 indiscriminately cleaves the DNA strand, leading to the release of fluorescence from the cleaved ssDNA reporter. Integration of isothermal amplification methods with CRISPR/Cas12 enables one-step detection assays, facilitating rapid pathogen identification within 30 min at a single temperature. This integrated RPA-CRISPR/Cas12a approach eliminates the need for RNA extraction and cDNA conversion, allowing direct use of crude plant sap as a template. With an affordable fluorescence visualization system, this portable method achieves 100-fold greater sensitivity than conventional techniques. This review summarizes recent advances in RPA-CRISPR/Cas12a for detecting plant pathogens, covering primer design, field-level portability, and enhanced sensitivity.},
}

@article {pmid39211201,
year = {2024},
author = {Zhang, H and Zhou, C and Mohammad, Z and Zhao, J},
title = {Structural basis of human 20S proteasome biogenesis.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
doi = {10.1101/2024.08.08.607236},
pmid = {39211201},
issn = {2692-8205},
abstract = {New proteasomes are produced to accommodate increases in cellular catabolic demand and prevent the accumulation of cytotoxic proteins. Formation of the proteasomal 20S core complex relies on the function of the five chaperones PAC1-4 and POMP. To understand how these chaperones facilitate proteasome assembly, we tagged the endogenous chaperones using CRISPR/Cas gene editing and examined the chaperone-bound complexes by cryo-EM. We observed an early α-ring intermediate subcomplex that is stabilized by PAC1-4, which transitions to β-ring assembly upon dissociation of PAC3/PAC4 and rearrangement of the PAC1 N-terminal tail. Completion of the β-ring and dimerization of half-proteasomes repositions critical lysine K33 to trigger cleavage of the β pro-peptides, leading to the concerted dissociation of POMP and PAC1/PAC2 to yield mature 20S proteasomes. This study reveals structural insights into critical points along the assembly pathway of the human proteasome and provides a molecular blueprint for 20S biogenesis.},
}

@article {pmid39211128,
year = {2024},
author = {Lin, CP and Li, H and Brogan, DJ and Wang, T and Akbari, OS and Komives, EA},
title = {CRISPR-RNA binding drives structural ordering that primes Cas7-11 for target cleavage.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
doi = {10.1101/2024.08.01.606276},
pmid = {39211128},
issn = {2692-8205},
abstract = {Type III-E CRISPR-Cas effectors, of which Cas7-11 is the first, are single proteins that cleave target RNAs without nonspecific collateral cleavage, opening new possibilities for RNA editing. Biochemical experiments combined with amide hydrogen-deuterium exchange (HDX-MS) experiments provide a first glimpse of the conformational dynamics of apo Cas7-11. HDX-MS revealed the backbone comprised of the four Cas7 zinc-binding RRM folds are well-folded but insertion sequences are highly dynamic and fold upon binding crRNA. The crRNA causes folding of disordered catalytic loops and β-hairpins, stronger interactions at domain-domain interfaces, and folding of the Cas7.1 processing site. Target RNA binding causes only minor ordering around the catalytic loops of Cas7.2 and Cas7.3. We show that Cas7-11 cannot fully process the CRISPR array and that binding of partially processed crRNA induces multiple states in Cas7-11 and reduces target RNA cleavage. The insertion domain shows the most ordering upon binding of mature crRNA. Finally, we show a crRNA-induced conformational change in one of the TPR-CHAT binding sites providing an explanation for why crRNA binding facilitates TPR-CHAT binding. The results provide the first glimpse of the apo state of Cas7-11 and reveal how its structure and function are regulated by crRNA binding.},
}

@article {pmid39210508,
year = {2024},
author = {Raddaoui, A and Chebbi, Y and Frigui, S and Latorre, J and Ammeri, RW and Abdejlil, NB and Torres, C and Mohamed Salah, A and Achour, W},
title = {Genetic characterization of vancomycin-resistant Enterococcus faecium isolates from neutropenic patients in Tunisia: Spread of the pandemic CC17 clone associated with high genetic diversity in Tn1546-like structures.},
journal = {Journal of applied microbiology},
volume = {},
number = {},
pages = {},
doi = {10.1093/jambio/lxae225},
pmid = {39210508},
issn = {1365-2672},
abstract = {AIMS: In Tunisia, limited research has focused on characterizing clinical vancomycin resistant Enterococcus faecium (VREfm). This study aimed to bridge this knowledge gap by molecular characterisation of antimicrobial resistance, determining the genetic elements mediating vancomycin-resistance, and whole-genome sequencing of one representative VREfm isolate.

METHODS AND RESULTS: Over six years (2011-2016), a total of eighty VREfm isolates responsible for infection or colonization were identified from hospitalized patients, with the incidence rate increasing from 2% in 2011 to 27% in 2016. All of these strains harbored the vanA gene. The screening for antimicrobial resistance genes revealed the predominance of ermB, tetM, and aac(6')-Ie-aph(2'')-Ia genes and 81.2% of strains harboured the Tn1545. PFGE identified seven clusters, with two major clusters (belonging to ST117 and ST80) persisting throughout the study period. Seven Tn1546 types were detected, with type VI (truncated transposon) being the most prevalent (57.5%). Whole-genome sequencing revealed a 3,028,373 bp chromosome and five plasmids. Mobile genetic elements and a type I CRISPR-cas locus were identified. Notably, the vanA gene was carried by the classic Tn1546 transposon with ISL3 insertion on a rep17pRUM plasmid.

CONCLUSION: A concerning trend in the prevalence of VREfm essentially attributed to CC17 persistence and to horizontal transfer of multiple genetic variants of truncated vanA-Tn1546.},
}

@article {pmid39210507,
year = {2024},
author = {Fan, K and Gökbağ, B and Tang, S and Li, S and Huang, Y and Wang, L and Cheng, L and Li, L},
title = {Synthetic lethal connectivity and graph transformer improve synthetic lethality prediction.},
journal = {Briefings in bioinformatics},
volume = {25},
number = {5},
pages = {},
doi = {10.1093/bib/bbae425},
pmid = {39210507},
issn = {1477-4054},
mesh = {Humans ; *Synthetic Lethal Mutations ; K562 Cells ; Computational Biology/methods ; CRISPR-Cas Systems ; Algorithms ; Jurkat Cells ; Gene Knockout Techniques ; Neoplasms/genetics ; },
abstract = {Synthetic lethality (SL) has shown great promise for the discovery of novel targets in cancer. CRISPR double-knockout (CDKO) technologies can only screen several hundred genes and their combinations, but not genome-wide. Therefore, good SL prediction models are highly needed for genes and gene pairs selection in CDKO experiments. However, lack of scalable SL properties prevents generalizability of SL interactions to out-of-sample data, thereby hindering modeling efforts. In this paper, we recognize that SL connectivity is a scalable and generalizable SL property. We develop a novel two-step multilayer encoder for individual sample-specific SL prediction model (MLEC-iSL), which predicts SL connectivity first and SL interactions subsequently. MLEC-iSL has three encoders, namely, gene, graph, and transformer encoders. MLEC-iSL achieves high SL prediction performance in K562 (AUPR, 0.73; AUC, 0.72) and Jurkat (AUPR, 0.73; AUC, 0.71) cells, while no existing methods exceed 0.62 AUPR and AUC. The prediction performance of MLEC-iSL is validated in a CDKO experiment in 22Rv1 cells, yielding a 46.8% SL rate among 987 selected gene pairs. The screen also reveals SL dependency between apoptosis and mitosis cell death pathways.},
}

Humans
*Synthetic Lethal Mutations
K562 Cells
Computational Biology/methods
CRISPR-Cas Systems
Algorithms
Jurkat Cells
Gene Knockout Techniques
Neoplasms/genetics

@article {pmid39209994,
year = {2024},
author = {Zhao, Y and Qin, J and Yu, D and Liu, Y and Song, D and Tian, K and Chen, H and Ye, Q and Wang, X and Xu, T and Xuan, H and Sun, N and Ma, W and Zhong, J and Sun, P and Song, Y and Hu, J and Zhao, Y and Hou, X and Meng, X and Jiang, C and Cai, J},
title = {Polymer-locking fusogenic liposomes for glioblastoma-targeted siRNA delivery and CRISPR-Cas gene editing.},
journal = {Nature nanotechnology},
volume = {},
number = {},
pages = {},
pmid = {39209994},
issn = {1748-3395},
support = {82372901//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82073298//National Natural Science Foundation of China (National Science Foundation of China)/ ; 22007051//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82003022//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82272988//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82230086//National Natural Science Foundation of China (National Science Foundation of China)/ ; 2019M660074//China Postdoctoral Science Foundation/ ; LBH-Z19029//Heilongjiang Postdoctoral Science Foundation/ ; LBH-TZ2218//Heilongjiang Postdoctoral Science Foundation/ ; },
abstract = {In patients with glioblastoma (GBM), upregulated midkine (MDK) limits the survival benefits conferred by temozolomide (TMZ). RNA interference (RNAi) and CRISPR-Cas9 gene editing technology are attractive approaches for regulating MDK expression. However, delivering these biologics to GBM tissue is challenging. Here we demonstrate a polymer-locking fusogenic liposome (Plofsome) that can be transported across the blood-brain barrier (BBB) and deliver short interfering RNA or CRISPR-Cas9 ribonucleoprotein complexes into the cytoplasm of GBM cells. Plofsome is designed by integrating a 'lock' into the fusogenic liposome using a traceless reactive oxygen species (ROS)-cleavable linker so that fusion occurs only after crossing the BBB and entering the GBM tissue with high ROS levels. Our results showed that MDK suppression by Plofsomes significantly reduced TMZ resistance and inhibited GBM growth in orthotopic brain tumour models. Importantly, Plofsomes are effective only at tumour sites and not in normal tissues, which improves the safety of combined RNAi and CRISPR-Cas9 therapeutics.},
}

@article {pmid39209602,
year = {2024},
author = {Binan, G and Yalun, W and Xinyan, W and Yongfu, Y and Peng, Z and Yunhaon, C and Xuan, Z and Chenguang, L and Fengwu, B and Ping, X and Qiaoning, H and Shihui, Y},
title = {Efficient genome-editing tools to engineer the recalcitrant non-model industrial microorganism Zymomonas mobilis.},
journal = {Trends in biotechnology},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.tibtech.2024.05.005},
pmid = {39209602},
issn = {1879-3096},
abstract = {Current biotechnology relies on a few well-studied model organisms, such as Escherichia coli and Saccharomyces cerevisiae, for which abundant information and efficient toolkits are available for genetic manipulation, but which lack industrially favorable characteristics. Non-model industrial microorganisms usually do not have effective and/or efficient genome-engineering toolkits, which hampers the development of microbial cell factories to meet the fast-growing bioeconomy. In this study, using the non-model ethanologenic bacterium Zymomonas mobilis as an example, we developed a workflow to mine and temper the elements of restriction-modification (R-M), CRISPR/Cas, toxin-antitoxin (T-A) systems, and native plasmids, which are hidden within industrial microorganisms themselves, as efficient genome-editing toolkits, and established a genome-wide iterative and continuous editing (GW-ICE) system for continuous genome editing with high efficiency. This research not only provides tools and pipelines for engineering the non-model polyploid industrial microorganism Z. mobilis efficiently, but also sets a paradigm to overcome biotechnological limitations in other genetically recalcitrant non-model industrial microorganisms.},
}