@article {pmid38652405,
year = {2024},
author = {Zhao, S and Yin, R and Zhang, M and Zhai, Z and Shen, Z and Mou, Y and Xu, D and Zhou, L and Lai, D},
title = {Efficient gene editing in the slow-growing, non-sporulating, melanized, endophytic fungus Berkleasmium sp. Dzf12 using a CRISPR/Cas9 system.},
journal = {World journal of microbiology & biotechnology},
volume = {40},
number = {6},
pages = {176},
pmid = {38652405},
issn = {1573-0972},
support = {2023YFD1401400//National Key Research and Development Program of China/ ; 2023YFD1401400//National Key Research and Development Program of China/ ; 31872623//National Natural Science Foundation of China/ ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; *Ascomycota/genetics/metabolism ; Endophytes/genetics/metabolism ; Melanins/biosynthesis/metabolism ; Fungal Proteins/genetics/metabolism ; Protoplasts ; },
abstract = {The endophytic fungus Berkleasmium sp. Dzf12 that was isolated from Dioscorea zingiberensis, is a proficient producer of palmarumycins, which are intriguing polyketides of the spirobisnaphthalene class. These compounds displayed a wide range of bioactivities, including antibacterial, antifungal, and cytotoxic activities. However, conventional genetic manipulation of Berkleasmium sp. Dzf12 is difficult and inefficient, partially due to the slow-growing, non-sporulating, and highly pigmented behavior of this fungus. Herein, we developed a CRISPR/Cas9 system suitable for gene editing in Berkleasmium sp. Dzf12. The protoplast preparation was optimized, and the expression of Cas9 in Berkleasmium sp. Dzf12 was validated. To assess the gene disruption efficiency, a putative 1, 3, 6, 8-tetrahydroxynaphthalene synthase encoding gene, bdpks, involved in 1,8-dihydroxynaphthalene (DHN)-melanin biosynthesis, was selected as the target for gene disruption. Various endogenous sgRNA promoters were tested, and different strategies to express sgRNA were compared, resulting in the construction of an optimal system using the U6 snRNA-1 promoter as the sgRNA promoter. Successful disruption of bdpks led to a complete abolishment of the production of spirobisnaphthalenes and melanin. This work establishes a useful gene targeting disruption system for exploration of gene functions in Berkleasmium sp. Dzf12, and also provides an example for developing an efficient CRISPR/Cas9 system to the fungi that are difficult to manipulate using conventional genetic tools.},
}

*Gene Editing/methods
*CRISPR-Cas Systems
*Ascomycota/genetics/metabolism
Endophytes/genetics/metabolism
Melanins/biosynthesis/metabolism
Fungal Proteins/genetics/metabolism
Protoplasts

@article {pmid38652321,
year = {2024},
author = {Wu, H and Sun, Y and Wang, Y and Luo, L and Song, Y},
title = {Advances in miniature CRISPR-Cas proteins and their applications in gene editing.},
journal = {Archives of microbiology},
volume = {206},
number = {5},
pages = {231},
pmid = {38652321},
issn = {1432-072X},
support = {32170173//National Natural Science Foundation of China/ ; ZXL2021422//Gusu Leading Talent Project/ ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; *CRISPR-Associated Proteins/genetics/metabolism ; },
abstract = {The CRISPR-Cas system consists of Cas proteins and single-stranded RNAs that recruit Cas proteins and specifically target the nucleic acid. Some Cas proteins can accurately cleave the target nucleic acid under the guidance of the single-stranded RNAs. Due to its exceptionally high specificity, the CRISPR-Cas system is now widely used in various fields such as gene editing, transcription regulation, and molecular diagnosis. However, the huge size of the most frequently utilized Cas proteins (Cas9, Cas12a, and Cas13, which contain 950-1,400 amino acids) can limit their applicability, especially in eukaryotic gene editing, where larger Cas proteins are difficult to deliver into the target cells. Recently discovered miniature CRISPR-Cas proteins, consisting of only 400 to 800 amino acids, offer the possibility of overcoming this limitation. This article systematically reviews the latest research progress of several miniature CRISPR-Cas proteins (Cas12f, Cas12j, Cas12k, and Cas12m) and their practical applications in the field of gene editing.},
}

*Gene Editing/methods
*CRISPR-Cas Systems
*CRISPR-Associated Proteins/genetics/metabolism

@article {pmid38651269,
year = {2024},
author = {Antony, JS and Herranz, AM and Mohammadian Gol, T and Mailand, S and Monnier, P and Rottenberger, J and Roig-Merino, A and Keller, B and Gowin, C and Milla, M and Beyer, TA and Mezger, M},
title = {Accelerated generation of gene-engineered monoclonal CHO cell lines using FluidFM nanoinjection and CRISPR/Cas9.},
journal = {Biotechnology journal},
volume = {19},
number = {4},
pages = {e2300505},
doi = {10.1002/biot.202300505},
pmid = {38651269},
issn = {1860-7314},
support = {2485-0-0//Fortüne Tübingen/ ; 2412-0-0//Fortüne Tübingen/ ; 440-0-0//UKT-Clinician Scientist Program/ ; //University Children's Hospital of Tübingen/ ; },
mesh = {CHO Cells ; *Cricetulus ; Animals ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Antibodies, Monoclonal/genetics ; Recombinant Proteins/genetics ; Gene Knockout Techniques/methods ; bcl-2-Associated X Protein/genetics/metabolism ; Tetrahydrofolate Dehydrogenase/genetics/metabolism ; Fucosyltransferases/genetics/metabolism ; Cricetinae ; Genetic Engineering/methods ; },
abstract = {Chinese hamster ovary (CHO) cells are the commonly used mammalian host system to manufacture recombinant proteins including monoclonal antibodies. However unfavorable non-human glycoprofile displayed on CHO-produced monoclonal antibodies have negative impacts on product quality, pharmacokinetics, and therapeutic efficiency. Glycoengineering such as genetic elimination of genes involved in glycosylation pathway in CHO cells is a viable solution but constrained due to longer timeline and laborious workflow. Here, in this proof-of-concept (PoC) study, we present a novel approach coined CellEDIT to engineer CHO cells by intranuclear delivery of the CRISPR components to single cells using the FluidFM technology. Co-injection of CRISPR system targeting BAX, DHFR, and FUT8 directly into the nucleus of single cells, enabled us to generate triple knockout CHO-K1 cell lines within a short time frame. The proposed technique assures the origin of monoclonality without the requirement of limiting dilution, cell sorting or positive selection. Furthermore, the approach is compatible to develop both single and multiple knockout clones (FUT8, BAX, and DHFR) in CHO cells. Further analyses on single and multiple knockout clones confirmed the targeted genetic disruption and altered protein expression. The knockout CHO-K1 clones showed the persistence of gene editing during the subsequent passages, compatible with serum free chemically defined media and showed equivalent transgene expression like parental clone.},
}

CHO Cells
*Cricetulus
Animals
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
Antibodies, Monoclonal/genetics
Recombinant Proteins/genetics
Gene Knockout Techniques/methods
bcl-2-Associated X Protein/genetics/metabolism
Tetrahydrofolate Dehydrogenase/genetics/metabolism
Fucosyltransferases/genetics/metabolism
Cricetinae
Genetic Engineering/methods

@article {pmid38650045,
year = {2024},
author = {Fan, XW and Gao, ZF and Ling, DD and Wang, DH and Cui, Y and Du, HQ and Li, CL and Zhou, X},
title = {CRISPR/Cas9 nickase mediated signal amplification integrating with the trans-cleavage activity of Cas12a for highly selective and sensitive detection of single base mutations.},
journal = {Military Medical Research},
volume = {11},
number = {1},
pages = {25},
pmid = {38650045},
issn = {2054-9369},
mesh = {*CRISPR-Cas Systems ; CRISPR-Associated Proteins/genetics ; Endodeoxyribonucleases/genetics ; Humans ; Mutation ; Deoxyribonuclease I ; Bacterial Proteins/genetics ; },
}

*CRISPR-Cas Systems
CRISPR-Associated Proteins/genetics
Endodeoxyribonucleases/genetics
Humans
Mutation
Deoxyribonuclease I
Bacterial Proteins/genetics

@article {pmid38648198,
year = {2024},
author = {Alseth, EO and Custodio, R and Sundius, SA and Kuske, RA and Brown, SP and Westra, ER},
title = {The impact of phage and phage resistance on microbial community dynamics.},
journal = {PLoS biology},
volume = {22},
number = {4},
pages = {e3002346},
pmid = {38648198},
issn = {1545-7885},
abstract = {Where there are bacteria, there will be bacteriophages. These viruses are known to be important players in shaping the wider microbial community in which they are embedded, with potential implications for human health. On the other hand, bacteria possess a range of distinct immune mechanisms that provide protection against bacteriophages, including the mutation or complete loss of the phage receptor, and CRISPR-Cas adaptive immunity. While our previous work showed how a microbial community may impact phage resistance evolution, little is known about the inverse, namely how interactions between phages and these different phage resistance mechanisms affect the wider microbial community in which they are embedded. Here, we conducted a 10-day, fully factorial evolution experiment to examine how phage impact the structure and dynamics of an artificial four-species bacterial community that includes either Pseudomonas aeruginosa wild-type or an isogenic mutant unable to evolve phage resistance through CRISPR-Cas. Additionally, we used mathematical modelling to explore the ecological interactions underlying full community behaviour, as well as to identify general principles governing the impacts of phage on community dynamics. Our results show that the microbial community structure is drastically altered by the addition of phage, with Acinetobacter baumannii becoming the dominant species and P. aeruginosa being driven nearly extinct, whereas P. aeruginosa outcompetes the other species in the absence of phage. Moreover, we find that a P. aeruginosa strain with the ability to evolve CRISPR-based resistance generally does better when in the presence of A. baumannii, but that this benefit is largely lost over time as phage is driven extinct. Finally, we show that pairwise data alone is insufficient when modelling our microbial community, both with and without phage, highlighting the importance of higher order interactions in governing multispecies dynamics in complex communities. Combined, our data clearly illustrate how phage targeting a dominant species allows for the competitive release of the strongest competitor while also contributing to community diversity maintenance and potentially preventing the reinvasion of the target species, and underline the importance of mapping community composition before therapeutically applying phage.},
}

@article {pmid38648173,
year = {2024},
author = {Přibylová, A and Fischer, L},
title = {How to use CRISPR/Cas9 in plants - from target site selection to DNA repair.},
journal = {Journal of experimental botany},
volume = {},
number = {},
pages = {},
doi = {10.1093/jxb/erae147},
pmid = {38648173},
issn = {1460-2431},
abstract = {A tool for precise, target-specific, efficient and affordable genome editing, it is a dream for many researchers, from those who do basic research to those who use it for applied research. Since 2012, we have the tool that almost fulfils such requirements; it is based on CRISPR/Cas systems. However, even CRISPR/Cas has limitations and obstacles that might surprise its users. In this review, we focus on the most frequently used variant, CRISPR/Cas9 from Streptococcus pyogenes, and highlight the key factors affecting its mutagenesis outcomes. Firstly, factors affecting the CRISPR/Cas9 activity, such as the effect of the target sequence, chromatin state or Cas9 variant, and how long it remains in place after cleavage. Secondly, factors affecting the follow-up DNA repair mechanisms include mostly the cell type and cell cycle phase, but also, for example, the type of DNA ends produced by Cas9 cleavage (blunt/staggered). Moreover, we note some differences between using CRISPR/Cas9 in plants, yeasts and animals, as knowledge from individual kingdoms is not fully transferable. Awareness of these factors can increase the likelihood of achieving the expected results of plant genome editing, for which we provide detailed guidelines.},
}

@article {pmid38647391,
year = {2024},
author = {Cho, HY and Yoo, M and Pongkulapa, T and Rabie, H and Muotri, AR and Yin, PT and Choi, JW and Lee, KB},
title = {Magnetic Nanoparticle-Assisted Non-Viral CRISPR-Cas9 for Enhanced Genome Editing to Treat Rett Syndrome.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {},
number = {},
pages = {e2306432},
doi = {10.1002/advs.202306432},
pmid = {38647391},
issn = {2198-3844},
support = {AARG-NTF-21-847862/ALZ/Alzheimer's Association/United States ; },
abstract = {The CRISPR-Cas9 technology has the potential to revolutionize the treatment of various diseases, including Rett syndrome, by enabling the correction of genes or mutations in human patient cells. However, several challenges need to be addressed before its widespread clinical application. These challenges include the low delivery efficiencies to target cells, the actual efficiency of the genome-editing process, and the precision with which the CRISPR-Cas system operates. Herein, the study presents a Magnetic Nanoparticle-Assisted Genome Editing (MAGE) platform, which significantly improves the transfection efficiency, biocompatibility, and genome-editing accuracy of CRISPR-Cas9 technology. To demonstrate the feasibility of the developed technology, MAGE is applied to correct the mutated MeCP2 gene in induced pluripotent stem cell-derived neural progenitor cells (iPSC-NPCs) from a Rett syndrome patient. By combining magnetofection and magnetic-activated cell sorting, MAGE achieves higher multi-plasmid delivery (99.3%) and repairing efficiencies (42.95%) with significantly shorter incubation times than conventional transfection agents without size limitations on plasmids. The repaired iPSC-NPCs showed similar characteristics as wild-type neurons when they differentiated into neurons, further validating MAGE and its potential for future clinical applications. In short, the developed nanobio-combined CRISPR-Cas9 technology offers the potential for various clinical applications, particularly in stem cell therapies targeting different genetic diseases.},
}

@article {pmid38647075,
year = {2024},
author = {Tan, Q and Shi, Y and Duan, C and Li, Q and Gong, T and Li, S and Duan, X and Xie, H and Li, Y and Chen, L},
title = {Simple, sensitive, and visual detection of 12 respiratory pathogens with one-pot-RPA-CRISPR/Cas12a assay.},
journal = {Journal of medical virology},
volume = {96},
number = {4},
pages = {e29624},
doi = {10.1002/jmv.29624},
pmid = {38647075},
issn = {1096-9071},
support = {22YXYJ0120//Medical Research Project of Xi'an Science and Technology Bureau/ ; 2023WGZJ-YB-39//Foreign Expert Service Project of Science and Technology Department of Shaanxi Province/ ; QCYRCXM-2022-56//Qin Chuangyuan Recruited High-level Innovation and Entrepreneurship Talents Project of Science and Technology Department of Shaanxi Province/ ; KY201904011//The Science and Technology Partnership Program, Ministry of Science and Technology of China/ ; 2021056//Sichuan Science and Technology Program/ ; 2021ZYD0085//Central Government Directed Special Funds for Local Science and Technology Development Project/ ; 2021YFH0100//The Sichuan Science and Technology Program/ ; CAMS-2021-I2M-1-060//The CAMS Initiative for Innovative Medicine/ ; },
abstract = {Respiratory infections pose a serious threat to global public health, underscoring the urgent need for rapid, accurate, and large-scale diagnostic tools. In recent years, the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated) system, combined with isothermal amplification methods, has seen widespread application in nucleic acid testing (NAT). However, achieving a single-tube reaction system containing all necessary components is challenging due to the competitive effects between recombinase polymerase amplification (RPA) and CRISPR/Cas reagents. Furthermore, to enable precision medicine, distinguishing between bacterial and viral infections is essential. Here, we have developed a novel NAT method, termed one-pot-RPA-CRISPR/Cas12a, which combines RPA with CRISPR molecular diagnostic technology, enabling simultaneous detection of 12 common respiratory pathogens, including six bacteria and six viruses. RPA and CRISPR/Cas12a reactions are separated by paraffin, providing an independent platform for RPA reactions to generate sufficient target products before being mixed with the CRISPR/Cas12a system. Results can be visually observed under LED blue light. The sensitivity of the one-pot-RPA-CRISPR/Cas12a method is 2.5 × 10[0] copies/μL plasmids, with no cross-reaction with other bacteria or viruses. Additionally, the clinical utility was evaluated by testing clinical isolates of bacteria and virus throat swab samples, demonstrating favorable performance. Thus, our one-pot-RPA-CRISPR/Cas12a method shows immense potential for accurate and large-scale detection of 12 common respiratory pathogens in point-of-care testing.},
}

@article {pmid38591968,
year = {2024},
author = {Xu, J and Yang, H and Sui, Z and Yuan, X and Jia, L and Guo, L},
title = {One-pot isothermal amplification permits recycled activation of CRISPR/Cas12a for sensing terminal deoxynucleotidyl transferase activity.},
journal = {Chemical communications (Cambridge, England)},
volume = {60},
number = {35},
pages = {4683-4686},
doi = {10.1039/d4cc00825a},
pmid = {38591968},
issn = {1364-548X},
mesh = {*Nucleic Acid Amplification Techniques ; *DNA Nucleotidylexotransferase/metabolism ; *CRISPR-Cas Systems/genetics ; Humans ; },
abstract = {This study introduces a one-pot isothermal amplification assay for ultrasensitive analysis of terminal deoxynucleotidyl transferase (TdT) activity. The system realizes recycled activation of CRISPR/Cas12a, enabling exceptional signal amplification. This approach maximizes the simplicity of the detection method, offering a promising avenue for molecular disease diagnosis.},
}

*Nucleic Acid Amplification Techniques
*DNA Nucleotidylexotransferase/metabolism
*CRISPR-Cas Systems/genetics
Humans

@article {pmid38527850,
year = {2024},
author = {Ai, N and Han, CR and Zhao, H and Cheng, SY and Ge, W},
title = {Disruption of Thyroid Hormone Receptor Thrab Leads to Female Infertility in Zebrafish.},
journal = {Endocrinology},
volume = {165},
number = {5},
pages = {},
doi = {10.1210/endocr/bqae037},
pmid = {38527850},
issn = {1945-7170},
support = {//University of Macau/ ; //UMDF/ ; //FDCT/ ; //NSFC/ ; //FDCT Funding Scheme for Postdoctoral Researchers/ ; },
mesh = {Animals ; Female ; *Zebrafish/genetics ; *Infertility, Female/genetics ; *Ovary/metabolism ; Thyroid Hormone Receptors alpha/genetics/metabolism ; Zebrafish Proteins/genetics/metabolism ; Mutation ; CRISPR-Cas Systems ; Reproduction/genetics ; },
abstract = {Thyroid hormones (THs) T4 and T3 are vital for development, growth, and metabolism. Thyroid dysfunction can also cause problems in fertility, suggesting involvement of THs in reproduction. In zebrafish, there exist 2 forms of TH receptor alpha gene (thraa and thrab). Disruption of these genes by CRISPR/Cas9 showed no reproductive irregularities in the thraa mutant; however, inactivation of the thrab gene resulted in female infertility. Although young female mutants (thrabm/m) showed normal ovarian development and folliculogenesis before sexual maturation, they failed to release eggs during oviposition after sexual maturation. This spawning failure was due to oviductal blockage at the genital papilla. The obstruction of the oviduct subsequently caused an accumulation of the eggs in the ovary, resulting in severe ovarian hypertrophy, abdominal distention, and disruption of folliculogenesis. Gene expression analysis showed expression of both TH receptors and estrogen receptors in the genital papilla, suggesting a direct TH action and potential interactions between thyroid and estrogen signaling pathways in controlling genital papilla development and function. In addition to their actions in the reproductive tracts, THs may also have direct effects in the ovary, as suggested by follicle atresia and cessation of folliculogenesis in the heterozygous mutant (thrab+/m), which was normal in all aspects of female reproduction in young and sexually mature fish but exhibited premature ovarian failure in aged females. In summary, this study provides substantial evidence for roles of THs in controlling the development and functions of both reproductive tract and ovary.},
}

Animals
Female
*Zebrafish/genetics
*Infertility, Female/genetics
*Ovary/metabolism
Thyroid Hormone Receptors alpha/genetics/metabolism
Zebrafish Proteins/genetics/metabolism
Mutation
CRISPR-Cas Systems
Reproduction/genetics

@article {pmid38647045,
year = {2024},
author = {Olivi, L and Bagchus, C and Pool, V and Bekkering, E and Speckner, K and Offerhaus, H and Wu, WY and Depken, M and Martens, KJA and Staals, RHJ and Hohlbein, J},
title = {Live-cell imaging reveals the trade-off between target search flexibility and efficiency for Cas9 and Cas12a.},
journal = {Nucleic acids research},
volume = {},
number = {},
pages = {},
doi = {10.1093/nar/gkae283},
pmid = {38647045},
issn = {1362-4962},
support = {024.003.019//The Netherlands Organization of Scientific Research/ ; VI.Vidi.203.074//VIDI/ ; //NWO/ ; },
abstract = {CRISPR-Cas systems have widely been adopted as genome editing tools, with two frequently employed Cas nucleases being SpyCas9 and LbCas12a. Although both nucleases use RNA guides to find and cleave target DNA sites, the two enzymes differ in terms of protospacer-adjacent motif (PAM) requirements, guide architecture and cleavage mechanism. In the last years, rational engineering led to the creation of PAM-relaxed variants SpRYCas9 and impLbCas12a to broaden the targetable DNA space. By employing their catalytically inactive variants (dCas9/dCas12a), we quantified how the protein-specific characteristics impact the target search process. To allow quantification, we fused these nucleases to the photoactivatable fluorescent protein PAmCherry2.1 and performed single-particle tracking in cells of Escherichia coli. From our tracking analysis, we derived kinetic parameters for each nuclease with a non-targeting RNA guide, strongly suggesting that interrogation of DNA by LbdCas12a variants proceeds faster than that of SpydCas9. In the presence of a targeting RNA guide, both simulations and imaging of cells confirmed that LbdCas12a variants are faster and more efficient in finding a specific target site. Our work demonstrates the trade-off of relaxing PAM requirements in SpydCas9 and LbdCas12a using a powerful framework, which can be applied to other nucleases to quantify their DNA target search.},
}

@article {pmid38646626,
year = {2024},
author = {Wang, Y and Pang, F},
title = {Diagnosis of bovine viral diarrhea virus: an overview of currently available methods.},
journal = {Frontiers in microbiology},
volume = {15},
number = {},
pages = {1370050},
pmid = {38646626},
issn = {1664-302X},
abstract = {Bovine viral diarrhea virus (BVDV) is the causative agent of bovine viral diarrhea (BVD), which results in significant economic losses in the global cattle industry. Fortunately, various diagnostic methods available for BVDV have been established. They include etiological methods, such as virus isolation (VI); serological methods, such as enzyme-linked immunosorbent assay (ELISA), immunofluorescence assay (IFA), and immunohistochemistry (IHC); molecular methods, such as reverse transcription-polymerase chain reaction (RT-PCR), real-time PCR, digital droplet PCR (ddPCR), loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA), and CRISPR-Cas system; and biosensors. This review summarizes the current diagnostic methods for BVDV, discussing their advantages and disadvantages, and proposes future perspectives for the diagnosis of BVDV, with the intention of providing valuable guidance for effective diagnosis and control of BVD disease.},
}

@article {pmid38644403,
year = {2024},
author = {Zhang, C and Xu, J and Wu, Y and Xu, C and Xu, P},
title = {Base Editors-Mediated Gene Therapy in Hematopoietic Stem Cells for Hematologic Diseases.},
journal = {Stem cell reviews and reports},
volume = {},
number = {},
pages = {},
pmid = {38644403},
issn = {2629-3277},
support = {82170119//the National Natural Science Foundation of China/ ; 202310285172Y//College Student Innovation and Entrepreneurship Training Program of Jiangsu Province/ ; 2022YFC2502700，2022YFC2502701，2022YFC2502702//the National Key Research and Development Program of China/ ; },
abstract = {Base editors, developed from the CRISPR/Cas system, consist of components such as deaminase and Cas variants. Since their emergence in 2016, the precision, efficiency, and safety of base editors have been gradually optimized. The feasibility of using base editors in gene therapy has been demonstrated in several disease models. Compared with the CRISPR/Cas system, base editors have shown great potential in hematopoietic stem cells (HSCs) and HSC-based gene therapy, because they do not generate double-stranded breaks (DSBs) while achieving the precise realization of single-base substitutions. This precise editing mechanism allows for the permanent correction of genetic defects directly at their source within HSCs, thus promising a lasting therapeutic effect. Recent advances in base editors are expected to significantly increase the number of clinical trials for HSC-based gene therapies. In this review, we summarize the development and recent progress of DNA base editors, discuss their applications in HSC gene therapy, and highlight the prospects and challenges of future clinical stem cell therapies.},
}

@article {pmid38643972,
year = {2024},
author = {Kogay, R and Wolf, YI and Koonin, EV},
title = {Defence systems and horizontal gene transfer in bacteria.},
journal = {Environmental microbiology},
volume = {26},
number = {4},
pages = {e16630},
pmid = {38643972},
issn = {1462-2920},
support = {Z01 LM000073/ImNIH/Intramural NIH HHS/United States ; },
mesh = {*Gene Transfer, Horizontal ; *Bacteria/genetics/virology ; *CRISPR-Cas Systems ; Genome, Bacterial ; Interspersed Repetitive Sequences ; Bacteriophages/genetics ; Evolution, Molecular ; Prophages/genetics ; },
abstract = {Horizontal gene transfer (HGT) is a fundamental process in prokaryotic evolution, contributing significantly to diversification and adaptation. HGT is typically facilitated by mobile genetic elements (MGEs), such as conjugative plasmids and phages, which often impose fitness costs on their hosts. However, a considerable number of bacterial genes are involved in defence mechanisms that limit the propagation of MGEs, suggesting they may actively restrict HGT. In our study, we investigated whether defence systems limit HGT by examining the relationship between the HGT rate and the presence of 73 defence systems across 12 bacterial species. We discovered that only six defence systems, three of which were different CRISPR-Cas subtypes, were associated with a reduced gene gain rate at the species evolution scale. Hosts of these defence systems tend to have a smaller pangenome size and fewer phage-related genes compared to genomes without these systems. This suggests that these defence mechanisms inhibit HGT by limiting prophage integration. We hypothesize that the restriction of HGT by defence systems is species-specific and depends on various ecological and genetic factors, including the burden of MGEs and the fitness effect of HGT in bacterial populations.},
}

*Gene Transfer, Horizontal
*Bacteria/genetics/virology
*CRISPR-Cas Systems
Genome, Bacterial
Interspersed Repetitive Sequences
Bacteriophages/genetics
Evolution, Molecular
Prophages/genetics

@article {pmid38641067,
year = {2024},
author = {Ganguly, C and Rostami, S and Long, K and Aribam, SD and Rajan, R},
title = {Unity among the diverse RNA-guided CRISPR-Cas interference mechanisms.},
journal = {The Journal of biological chemistry},
volume = {},
number = {},
pages = {107295},
doi = {10.1016/j.jbc.2024.107295},
pmid = {38641067},
issn = {1083-351X},
abstract = {CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated) systems are adaptive immune systems that protect bacteria and archaea from invading mobile genetic elements (MGEs). The Cas protein-CRISPR RNA (crRNA) complex uses complementarity of the crRNA "guide" region to specifically recognize the invader genome. CRISPR effectors that perform targeted destruction of the foreign genome have emerged independently as multi-subunit protein complexes (Class 1 systems) and as single multi-domain proteins (Class 2). These different CRISPR-Cas systems can cleave RNA, DNA, and protein in an RNA-guided manner to eliminate the invader, and in some cases, they initiate programmed cell death/dormancy. The versatile mechanisms of the different CRISPR-Cas systems to target and destroy nucleic acids have been adapted to develop various programmable-RNA-guided tools and have revolutionized the development of fast, accurate, and accessible genomic applications. In this review, we present the structure and interference mechanisms of different CRISPR-Cas systems and an analysis of their unified features. The three types of Class 1 systems (I, III, and IV) have a conserved right-handed helical filamentous structure that provides a backbone for sequence-specific targeting while using unique proteins with distinct mechanisms to destroy the invader. Similarly, all three Class 2 types (II, V, and VI) have a bilobed architecture that binds the RNA-DNA/RNA hybrid and uses different nuclease domains to cleave invading MGEs. Additionally, we highlight the mechanistic similarities of CRISPR-Cas enzymes with other RNA cleaving enzymes and briefly present the evolutionary routes of the different CRISPR-Cas systems.},
}

@article {pmid38640680,
year = {2024},
author = {Qiu, M and Yuan, Z and Li, N and Yang, X and Zhang, X and Jiang, Y and Zhao, Q and Man, C},
title = {Self-assembled bifunctional nanoflower-enabled CRISPR/Cas biosensing platform for dual-readout detection of Salmonella enterica.},
journal = {Journal of hazardous materials},
volume = {471},
number = {},
pages = {134323},
doi = {10.1016/j.jhazmat.2024.134323},
pmid = {38640680},
issn = {1873-3336},
abstract = {Sensitive detection and point-of-care test of bacterial pathogens is of great significance in safeguarding the public health worldwide. Inspired by the characteristics of horseradish peroxidase (HRP), we synthesized a hybrid nanoflower with peroxidase-like activity via a three-component self-assembled strategy. Interestingly, the prepared nanozyme not only could act as an alternative to HRP for colorimetric biosensing, but also function as a unique signal probe that could be recognized by a pregnancy test strip. By combining the bifunctional properties of hybrid nanoflower, isothermal amplification of LAMP, and the specific recognition and non-specific cleavage properties of CRISPR/Cas12a system, the dual-readout CRISPR/Cas12a biosensor was developed for sensitive and rapid detection of Salmonella enterica. Moreover, this platform in the detection of Salmonella enterica had limits of detection of 1 cfu/mL (colorimetric assay) in the linear range of 10[1]-10[8] cfu/mL and 10[2] cfu/mL (lateral flow assay) in the linear range of 10[2]-10[8] cfu/mL, respectively. Furthermore, the developed biosensor exhibited good recoveries in the spiked samples (lake water and milk) with varying concentrations of Salmonella enterica. This work provides new insights for the design of multifunctional nanozyme and the development of innovative dual-readout CRISPR/Cas system-based biosensing platform for the detection of pathogens.},
}

@article {pmid38639864,
year = {2024},
author = {Rahimi, A and Sameei, P and Mousavi, S and Ghaderi, K and Hassani, A and Hassani, S and Alipour, S},
title = {Application of CRISPR/Cas9 System in the Treatment of Alzheimer's Disease and Neurodegenerative Diseases.},
journal = {Molecular neurobiology},
volume = {},
number = {},
pages = {},
pmid = {38639864},
issn = {1559-1182},
abstract = {Alzheimer's, Parkinson's, and Huntington's are some of the most common neurological disorders, which affect millions of people worldwide. Although there have been many treatments for these diseases, there are still no effective treatments to treat or completely stop these disorders. Perhaps the lack of proper treatment for these diseases can be related to various reasons, but the poor results related to recent clinical research also prompted doctors to look for new treatment approaches. In this regard, various researchers from all over the world have provided many new treatments, one of which is CRISPR/Cas9. Today, the CRISPR/Cas9 system is mostly used for genetic modifications in various species. In addition, by using the abilities available in the CRISPR/Cas9 system, researchers can either remove or modify DNA sequences, which in this way can establish a suitable and useful treatment method for the treatment of genetic diseases that have undergone mutations. We conducted a non-systematic review of articles and study results from various databases, including PubMed, Medline, Web of Science, and Scopus, in recent years. and have investigated new treatment methods in neurodegenerative diseases with a focus on Alzheimer's disease. Then, in the following sections, the treatment methods were classified into three groups: anti-tau, anti-amyloid, and anti-APOE regimens. Finally, we discussed various applications of the CRISPR/Cas-9 system in Alzheimer's disease. Today, using CRISPR/Cas-9 technology, scientists create Alzheimer's disease models that have a more realistic phenotype and reveal the processes of pathogenesis; following the screening of defective genes, they establish treatments for this disease.},
}

@article {pmid38637512,
year = {2024},
author = {Schwartz, EA and Bravo, JPK and Ahsan, M and Macias, LA and McCafferty, CL and Dangerfield, TL and Walker, JN and Brodbelt, JS and Palermo, G and Fineran, PC and Fagerlund, RD and Taylor, DW},
title = {RNA targeting and cleavage by the type III-Dv CRISPR effector complex.},
journal = {Nature communications},
volume = {15},
number = {1},
pages = {3324},
pmid = {38637512},
issn = {2041-1723},
support = {R01 GM141329/GM/NIGMS NIH HHS/United States ; R35 GM138348/GM/NIGMS NIH HHS/United States ; R35 GM139658/GM/NIGMS NIH HHS/United States ; F31 CA257404/CA/NCI NIH HHS/United States ; },
mesh = {RNA/metabolism ; *RNA, Catalytic/metabolism ; CRISPR-Cas Systems/genetics ; DNA/metabolism ; Catalytic Domain ; *CRISPR-Associated Proteins/genetics/metabolism ; RNA Cleavage ; },
abstract = {CRISPR-Cas are adaptive immune systems in bacteria and archaea that utilize CRISPR RNA-guided surveillance complexes to target complementary RNA or DNA for destruction[1-5]. Target RNA cleavage at regular intervals is characteristic of type III effector complexes[6-8]. Here, we determine the structures of the Synechocystis type III-Dv complex, an apparent evolutionary intermediate from multi-protein to single-protein type III effectors[9,10], in pre- and post-cleavage states. The structures show how multi-subunit fusion proteins in the effector are tethered together in an unusual arrangement to assemble into an active and programmable RNA endonuclease and how the effector utilizes a distinct mechanism for target RNA seeding from other type III effectors. Using structural, biochemical, and quantum/classical molecular dynamics simulation, we study the structure and dynamics of the three catalytic sites, where a 2'-OH of the ribose on the target RNA acts as a nucleophile for in line self-cleavage of the upstream scissile phosphate. Strikingly, the arrangement at the catalytic residues of most type III complexes resembles the active site of ribozymes, including the hammerhead, pistol, and Varkud satellite ribozymes. Our work provides detailed molecular insight into the mechanisms of RNA targeting and cleavage by an important intermediate in the evolution of type III effector complexes.},
}

RNA/metabolism
*RNA, Catalytic/metabolism
CRISPR-Cas Systems/genetics
DNA/metabolism
Catalytic Domain
*CRISPR-Associated Proteins/genetics/metabolism
RNA Cleavage

@article {pmid38637031,
year = {2024},
author = {Zhang, X and Zhu, L and Yang, L and Liu, G and Qiu, S and Xiong, X and Huang, K and Xiao, T and Zhu, L},
title = {A sensitive and versatile electrochemical sensor based on hybridization chain reaction and CRISPR/Cas12a system for antibiotic detection.},
journal = {Analytica chimica acta},
volume = {1304},
number = {},
pages = {342562},
doi = {10.1016/j.aca.2024.342562},
pmid = {38637031},
issn = {1873-4324},
mesh = {*Anti-Bacterial Agents ; Electrochemical Techniques/methods ; CRISPR-Cas Systems ; *Biosensing Techniques/methods ; Nucleic Acid Hybridization ; },
abstract = {A sensitive electrochemical platform was constructed with NH2-Cu-MOF as electrochemical probe to detect antibiotics using CRISPR/Cas12a system triggered by hybridization chain reaction (HCR). The sensing system consists of two HCR systems. HCR1 occurred on the electrode surface independent of the target, generating long dsDNA to connect signal probes and producing a strong electrochemical signal. HCR2 was triggered by target, and the resulting dsDNA products activated the CRISPR/Cas12a, thereby resulting in effective and rapid cleavage of the trigger of HCR1, hindering the occurrence of HCR1, and reducing the number of NH2-Cu-MOF on the electrode surface. Eventually, significant signal change depended on the target was obtained. On this basis and with the help of the programmability of DNA, kanamycin and ampicillin were sensitively detected with detection limits of 60 fM and 10 fM (S/N = 3), respectively. Furthermore, the sensing platform showed good detection performance in milk and livestock wastewater samples, demonstrating its great application prospects in the detection of antibiotics in food and environmental water samples.},
}

*Anti-Bacterial Agents
Electrochemical Techniques/methods
CRISPR-Cas Systems
*Biosensing Techniques/methods
Nucleic Acid Hybridization

@article {pmid38636316,
year = {2024},
author = {Wang, X and Deng, X and Zhang, Y and Dong, W and Rao, Q and Huang, Q and Tang, F and Shen, R and Xu, H and Jin, Z and Tang, Y and Du, D},
title = {A rapid and sensitive one-pot platform integrating fluorogenic RNA aptamers and CRISPR-Cas13a for visual detection of monkeypox virus.},
journal = {Biosensors & bioelectronics},
volume = {257},
number = {},
pages = {116268},
doi = {10.1016/j.bios.2024.116268},
pmid = {38636316},
issn = {1873-4235},
abstract = {The recent global upsurge in Monkeypox virus (MPXV) outbreaks underscores the critical need for rapid and precise diagnostic solutions, particularly in resource-constrained settings. The gold standard diagnostic method, qRT-PCR, is hindered by its time-consuming nature, requirement for nucleic acid purification, expensive equipment, and the need for highly trained personnel. Traditional CRISPR/Cas fluorescence assays, relying on trans-cleavage of ssDNA/RNA reporters labeled with costly fluorophores and quenchers, pose challenges that limit their widespread application, especially for point-of-care testing (POCT). In this study, we utilized a cost-effective and stable fluorogenic RNA aptamer (Mango III), specifically binding and illuminating the fluorophore TO3-3 PEG-Biotin Fluorophore (TO3), as a reporter for Cas13a trans-cleavage activity. We propose a comprehensive strategy integrating RNA aptamer, recombinase-aided amplification (RAA), and CRISPR-Cas13a systems for the molecular detection of MPXV target. Leveraging the inherent collateral cleavage properties of the Cas13a system, we established high-sensitivity and specificity assays to distinguish MPXV from other Orthopoxviruses (OPVs). A streamlined one-pot protocol was developed to mitigate aerosol contamination risks. Our aptamer-coupled RAA-Cas13a one-pot detection method achieved a Limit of Detection (LoD) of 4 copies of target MPXV DNA in just 40 min. Validation using clinical MPX specimens confirmed the rapid and reliable application of our RAA-Cas13a-Apt assays without nucleic acid purification procedure, highlighting its potential as a point-of-care testing solution. These results underscore the user-friendliness and effectiveness of our one-pot RAA-Cas13a-Apt diagnostic platform, poised to revolutionize disease detection and management.},
}

@article {pmid38635331,
year = {2024},
author = {},
title = {Correction to: Generation of a Commercial-Scale Founder Population of Porcine Reproductive and Respiratory Syndrome Virus Resistant Pigs Using CRISPR-Cas by Burger et al. The CRISPR Journal, 2023;7(1):12-28; DOI: 10.1089/crispr.2023.0061.},
journal = {The CRISPR journal},
volume = {7},
number = {2},
pages = {131},
doi = {10.1089/crispr.2023.0061.correx},
pmid = {38635331},
issn = {2573-1602},
}

@article {pmid38635329,
year = {2024},
author = {Bouchareb, A and Biggs, D and Alghadban, S and Preece, C and Davies, B},
title = {Increasing Knockin Efficiency in Mouse Zygotes by Transient Hypothermia.},
journal = {The CRISPR journal},
volume = {7},
number = {2},
pages = {111-119},
doi = {10.1089/crispr.2023.0077},
pmid = {38635329},
issn = {2573-1602},
mesh = {Animals ; Humans ; Mice ; *Gene Editing ; CRISPR-Cas Systems/genetics ; Zygote/metabolism ; *Hypothermia/metabolism ; Recombinational DNA Repair/genetics ; },
abstract = {Integration of a point mutation to correct or edit a gene requires the repair of the CRISPR-Cas9-induced double-strand break by homology-directed repair (HDR). This repair pathway is more active in late S and G2 phases of the cell cycle, whereas the competing pathway of nonhomologous end-joining (NHEJ) operates throughout the cell cycle. Accordingly, modulation of the cell cycle by chemical perturbation or simply by the timing of gene editing to shift the editing toward the S/G2 phase has been shown to increase HDR rates. Using a traffic light reporter in mouse embryonic stem cells and a fluorescence conversion reporter in human-induced pluripotent stem cells, we confirm that a transient cold shock leads to an increase in the rate of HDR, with a corresponding decrease in the rate of NHEJ repair. We then investigated whether a similar cold shock could lead to an increase in the rate of HDR in the mouse embryo. By analyzing the efficiency of gene editing using single nucleotide polymorphism changes and loxP insertion at three different genetic loci, we found that a transient reduction in temperature after zygote electroporation of CRISPR-Cas9 ribonucleoprotein with a single-stranded oligodeoxynucleotide repair template did indeed increase knockin efficiency, without affecting embryonic development. The efficiency of gene editing with and without the cold shock was first assessed by genotyping blastocysts. As a proof of concept, we then confirmed that the modified embryo culture conditions were compatible with live births by targeting the coat color gene tyrosinase and observing the repair of the albino mutation. Taken together, our data suggest that a transient cold shock could offer a simple and robust way to improve knockin outcomes in both stem cells and zygotes.},
}

Animals
Humans
Mice
*Gene Editing
CRISPR-Cas Systems/genetics
Zygote/metabolism
*Hypothermia/metabolism
Recombinational DNA Repair/genetics

@article {pmid38635328,
year = {2024},
author = {Mohamad Zamberi, NN and Abuhamad, AY and Low, TY and Mohtar, MA and Syafruddin, SE},
title = {dCas9 Tells Tales: Probing Gene Function and Transcription Regulation in Cancer.},
journal = {The CRISPR journal},
volume = {7},
number = {2},
pages = {73-87},
doi = {10.1089/crispr.2023.0078},
pmid = {38635328},
issn = {2573-1602},
mesh = {*Gene Editing ; CRISPR-Cas Systems/genetics ; CRISPR-Associated Protein 9/genetics ; Epigenesis, Genetic ; *Neoplasms/genetics ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-based genome editing is evolving into an essential tool in the field of biological and medical research. Notably, the development of catalytically deactivated Cas9 (dCas9) enzyme has substantially broadened its traditional boundaries in gene editing or perturbation. The conjugation of dCas9 with various molecular effectors allows precise control over transcriptional processes, epigenetic modifications, visualization of chromosomal dynamics, and several other applications. This expanded repertoire of CRISPR-Cas9 applications has emerged as an invaluable molecular tool kit that empowers researchers to comprehensively interrogate and gain insights into health and diseases. This review delves into the advancements in Cas9 protein engineering, specifically on the generation of various dCas9 tools that have significantly enhanced the CRISPR-based technology capability and versatility. We subsequently discuss the multifaceted applications of dCas9, especially in interrogating the regulation and function of genes that involve in supporting cancer pathogenesis. In addition, we also delineate the designing and utilization of dCas9-based tools as well as highlighting its current constraints and transformative potentials in cancer research.},
}

*Gene Editing
CRISPR-Cas Systems/genetics
CRISPR-Associated Protein 9/genetics
Epigenesis, Genetic
*Neoplasms/genetics

@article {pmid38635327,
year = {2024},
author = {Barrangou, R},
title = {CRISPR Pigs Portend a New Era of Xenotransplantation.},
journal = {The CRISPR journal},
volume = {7},
number = {2},
pages = {71},
doi = {10.1089/crispr.2024.29173.editorial},
pmid = {38635327},
issn = {2573-1602},
mesh = {Animals ; Swine/genetics ; Transplantation, Heterologous ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *CRISPR-Cas Systems/genetics ; Gene Editing ; Animals, Genetically Modified/genetics ; },
}

Animals
Swine/genetics
Transplantation, Heterologous
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
*CRISPR-Cas Systems/genetics
Gene Editing
Animals, Genetically Modified/genetics

@article {pmid38635326,
year = {2024},
author = {Wang, M and Rieber, L and van Baaren, J and Morgan, M and Merrett, S and McDowell, I and Bowen, T},
title = {Diverse Class 2 CRISPR Effectors as Active Nucleases with Expanded Targeting Capabilities.},
journal = {The CRISPR journal},
volume = {7},
number = {2},
pages = {120-130},
doi = {10.1089/crispr.2023.0058},
pmid = {38635326},
issn = {2573-1602},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing ; Endonucleases/genetics ; RNA ; },
abstract = {CRISPR-Cas systems have proven effective in a variety of applications due to their ease of use and relatively high editing efficiency. Yet, any individual CRISPR-Cas system has inherent limitations, necessitating a diversity of RNA-guided nucleases to suit applications with distinct needs. We searched through metagenomic sequences to identify RNA-guided nucleases and found enzymes from diverse CRISPR-Cas types and subtypes, the most promising of which we developed into gene-editing platforms. Based on prior annotations of the metagenomic sequences, we establish the likely taxa and sampling locations where Class 2 CRISPR-Cas systems active in eukaryotes may be found. The newly discovered systems show robust capabilities as gene editors and base editors.},
}

*CRISPR-Cas Systems/genetics
*Gene Editing
Endonucleases/genetics
RNA

@article {pmid38633802,
year = {2024},
author = {Wang, Q and Kline, EC and Gilligan-Steinberg, SD and Lai, JJ and Hull, IT and Olanrewaju, AO and Panpradist, N and Lutz, BR},
title = {Sensitive Pathogen Detection and Drug Resistance Characterization Using Pathogen-Derived Enzyme Activity Amplified by LAMP or CRISPR-Cas.},
journal = {medRxiv : the preprint server for health sciences},
volume = {},
number = {},
pages = {},
pmid = {38633802},
support = {P30 AI027757/AI/NIAID NIH HHS/United States ; R33 AI140460/AI/NIAID NIH HHS/United States ; },
abstract = {Pathogens encapsulate or encode their own suite of enzymes to facilitate replication in the host. The pathogen-derived enzymes possess specialized activities that are essential for pathogen replication and have naturally been candidates for drug targets. Phenotypic assays detecting the activities of pathogen-derived enzymes and characterizing their inhibition under drugs offer an opportunity for pathogen detection, drug resistance testing for individual patients, and as a research tool for new drug development. Here, we used HIV as an example to develop assays targeting the reverse transcriptase (RT) enzyme encapsulated in HIV for sensitive detection and phenotypic characterization, with the potential for point-of-care (POC) applications. Specifically, we targeted the complementary (cDNA) generation activity of the HIV RT enzyme by adding engineered RNA as substrates for HIV RT enzyme to generate cDNA products, followed by cDNA amplification and detection facilitated by loop-mediated isothermal amplification (LAMP) or CRISPR-Cas systems. To guide the assay design, we first used qPCR to characterize the cDNA generation activity of HIV RT enzyme. In the LAMP-mediated Product-Amplified RT activity assay (LamPART), the cDNA generation and LAMP amplification were combined into one pot with novel assay designs. When coupled with direct immunocapture of HIV RT enzyme for sample preparation and endpoint lateral flow assays for detection, LamPART detected as few as 20 copies of HIV RT enzyme spiked into 25μL plasma (fingerstick volume), equivalent to a single virion. In the Cas-mediated Product-Amplified RT activity assay (CasPART), we tailored the substrate design to achieve a LoD of 2e4 copies (1.67fM) of HIV RT enzyme. Furthermore, with its phenotypic characterization capability, CasPART was used to characterize the inhibition of HIV RT enzyme under antiretroviral drugs and differentiate between wild-type and mutant HIV RT enzyme for potential phenotypic drug resistance testing. Moreover, the CasPART assay can be readily adapted to target the activity of other pathogen-derived enzymes. As a proof-of-concept, we successfully adapted CasPART to detect HIV integrase with a sensitivity of 83nM. We anticipate the developed approach of detecting enzyme activity with product amplification has the potential for a wide range of pathogen detection and phenotypic characterization.},
}

@article {pmid38632224,
year = {2024},
author = {De Carluccio, G and Fusco, V and di Bernardo, D},
title = {Engineering a synthetic gene circuit for high-performance inducible expression in mammalian systems.},
journal = {Nature communications},
volume = {15},
number = {1},
pages = {3311},
pmid = {38632224},
issn = {2041-1723},
support = {STAR PLUS//Compagnia di San Paolo (Fondazione Compagnia di San Paolo)/ ; TIGEM//Fondazione Telethon (Telethon Foundation)/ ; 826121//EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Societal Challenges | H2020 Health (H2020 Societal Challenges - Health, Demographic Change and Well-being)/ ; DICMAPI//Università degli Studi di Napoli Federico II (University of Naples Federico II)/ ; },
mesh = {Animals ; *Genes, Synthetic ; *Gene Expression Regulation ; Transcription Factors/genetics ; Gene Regulatory Networks ; Promoter Regions, Genetic ; Mammals/genetics ; CRISPR-Cas Systems ; },
abstract = {Inducible gene expression systems can be used to control the expression of a gene of interest by means of a small-molecule. One of the most common designs involves engineering a small-molecule responsive transcription factor (TF) and its cognate promoter, which often results in a compromise between minimal uninduced background expression (leakiness) and maximal induced expression. Here, we focus on an alternative strategy using quantitative synthetic biology to mitigate leakiness while maintaining high expression, without modifying neither the TF nor the promoter. Through mathematical modelling and experimental validations, we design the CASwitch, a mammalian synthetic gene circuit based on combining two well-known network motifs: the Coherent Feed-Forward Loop (CFFL) and the Mutual Inhibition (MI). The CASwitch combines the CRISPR-Cas endoribonuclease CasRx with the state-of-the-art Tet-On3G inducible gene system to achieve high performances. To demonstrate the potentialities of the CASwitch, we apply it to three different scenarios: enhancing a whole-cell biosensor, controlling expression of a toxic gene and inducible production of Adeno-Associated Virus (AAV) vectors.},
}

Animals
*Genes, Synthetic
*Gene Expression Regulation
Transcription Factors/genetics
Gene Regulatory Networks
Promoter Regions, Genetic
Mammals/genetics
CRISPR-Cas Systems

@article {pmid38632100,
year = {2024},
author = {Yan-Chun, B and Ling-Li, D and Zai-Xia, L and Feng-Ying, M and Yu, W and Yong-Bin, L and Ming-Juan, G and Ri-Su, N and Wen-Guang, Z},
title = {Progress on CRISPR/Cas9 system in the genetic improvement of livestock and poultry.},
journal = {Yi chuan = Hereditas},
volume = {46},
number = {3},
pages = {219-231},
doi = {10.16288/j.yczz.24-021},
pmid = {38632100},
issn = {0253-9772},
mesh = {Animals ; *CRISPR-Cas Systems ; *Livestock/genetics ; Poultry/genetics ; Gene Editing/methods ; Gene Knock-In Techniques ; },
abstract = {CRISPR/Cas9 gene editing technology, as a highly efficient genome editing method, has been extensively employed in the realm of animal husbandry for genetic improvement. With its remarkable efficiency and precision, this technology has revolutionized the field of animal husbandry. Currently, CRISPR/Cas9-based gene knockout, gene knock-in and gene modification techniques are widely employed to achieve precise enhancements in crucial production traits of livestock and poultry species. In this review, we summarize the operational principle and development history of CRISPR/Cas9 technology. Additionally, we highlight the research advancements utilizing this technology in muscle growth and development, fiber growth, milk quality composition, disease resistance breeding, and animal welfare within the livestock and poultry sectors. Our aim is to provide a more comprehensive understanding of the application of CRISPR/Cas9 technology in gene editing for livestock and poultry.},
}

Animals
*CRISPR-Cas Systems
*Livestock/genetics
Poultry/genetics
Gene Editing/methods
Gene Knock-In Techniques

@article {pmid38631624,
year = {2024},
author = {Hu, H and Liu, L and Wei, XY and Duan, JJ and Deng, JY and Pei, DS},
title = {Revolutionizing aquatic eco-environmental monitoring: Utilizing the RPA-Cas-FQ detection platform for zooplankton.},
journal = {The Science of the total environment},
volume = {},
number = {},
pages = {172414},
doi = {10.1016/j.scitotenv.2024.172414},
pmid = {38631624},
issn = {1879-1026},
abstract = {The integration of recombinase polymerase amplification (RPA) with CRISPR/Cas technology has revolutionized molecular diagnostics and pathogen detection due to its unparalleled sensitivity and trans-cleavage ability. However, its potential in the ecological and environmental monitoring scenarios for aquatic ecosystems remains largely unexplored, particularly in accurate qualitative/quantitative detection, and its actual performance in handling complex real environmental samples. Using zooplankton as a model, we have successfully optimized the RPA-CRISPR/Cas12a fluorescence detection platform (RPA-Cas-FQ), providing several crucial "technical tips". Our findings indicate the sensitivity of CRISPR/Cas12a alone is 5 × 10[9] copies/reaction, which can be dramatically increased to 5 copies/reaction when combined with RPA. The optimized RPA-Cas-FQ enables reliable qualitative and semi-quantitative detection within 50 min, and exhibits a good linear relationship between fluorescence intensity and DNA concentration (R[2] = 0.956-0.974***). Additionally, we developed a rapid and straightforward identification procedure for single zooplankton by incorporating heat-lysis and DNA-barcode techniques. We evaluated the platform's effectiveness using real environmental DNA (eDNA) samples from the Three Gorges Reservoir, confirming its practicality. The eDNA-RPA-Cas-FQ demonstrated strong consistency (Kappa = 0.43***) with eDNA-Metabarcoding in detecting species presence/absence in the reservoir. Furthermore, the two semi-quantitative eDNA quantification technologies showed a strong positive correlation (R[2] = 0.58-0.87***). This platform also has the potential to monitor environmental pollutants by selecting appropriate indicator species. The novel insights and methodologies presented in this study represent a significant advancement in meeting the complex needs of aquatic ecosystem protection and monitoring.},
}

@article {pmid38630829,
year = {2024},
author = {Tan, E and Wan, T and Pan, Q and Duan, J and Zhang, S and Wang, R and Gao, P and Lv, J and Wang, H and Li, D and Ping, Y and Cheng, Y},
title = {Dual-responsive nanocarriers for efficient cytosolic protein delivery and CRISPR-Cas9 gene therapy of inflammatory skin disorders.},
journal = {Science advances},
volume = {10},
number = {16},
pages = {eadl4336},
pmid = {38630829},
issn = {2375-2548},
mesh = {Animals ; Mice ; *CRISPR-Cas Systems ; *Gene Editing ; NLR Family, Pyrin Domain-Containing 3 Protein/genetics ; Gene Transfer Techniques ; Genetic Therapy ; Polymers/chemistry ; Ribonucleoproteins/genetics ; },
abstract = {Developing protein drugs that can target intracellular sites remains a challenge due to their inadequate membrane permeability. Efficient carriers for cytosolic protein delivery are required for protein-based drugs, cancer vaccines, and CRISPR-Cas9 gene therapies. Here, we report a screening process to identify highly efficient materials for cytosolic protein delivery from a library of dual-functionalized polymers bearing both boronate and lipoic acid moieties. Both ligands were found to be crucial for protein binding, endosomal escape, and intracellular protein release. Polymers with higher grafting ratios exhibit remarkable efficacies in cytosolic protein delivery including enzymes, monoclonal antibodies, and Cas9 ribonucleoprotein while preserving their activity. Optimal polymer successfully delivered Cas9 ribonucleoprotein targeting NLRP3 to disrupt NLRP3 inflammasomes in vivo and ameliorate inflammation in a mouse model of psoriasis. Our study presents a promising option for the discovery of highly efficient materials tailored for cytosolic delivery of specific proteins and complexes such as Cas9 ribonucleoprotein.},
}

Animals
Mice
*CRISPR-Cas Systems
*Gene Editing
NLR Family, Pyrin Domain-Containing 3 Protein/genetics
Gene Transfer Techniques
Genetic Therapy
Polymers/chemistry
Ribonucleoproteins/genetics

@article {pmid38629716,
year = {2024},
author = {Johnson, GA and Gould, SI and Sánchez-Rivera, FJ},
title = {Deconstructing cancer with precision genome editing.},
journal = {Biochemical Society transactions},
volume = {52},
number = {2},
pages = {803-819},
doi = {10.1042/BST20230984},
pmid = {38629716},
issn = {1470-8752},
mesh = {Humans ; *Gene Editing/methods ; *Neoplasms/genetics/therapy ; *CRISPR-Cas Systems ; Mutation ; Animals ; Precision Medicine ; Genome, Human ; },
abstract = {Recent advances in genome editing technologies are allowing investigators to engineer and study cancer-associated mutations in their endogenous genetic contexts with high precision and efficiency. Of these, base editing and prime editing are quickly becoming gold-standards in the field due to their versatility and scalability. Here, we review the merits and limitations of these precision genome editing technologies, their application to modern cancer research, and speculate how these could be integrated to address future directions in the field.},
}

Humans
*Gene Editing/methods
*Neoplasms/genetics/therapy
*CRISPR-Cas Systems
Mutation
Animals
Precision Medicine
Genome, Human

@article {pmid38627399,
year = {2024},
author = {Bi, M and Su, W and Li, J and Mo, X},
title = {Insights into the inhibition of protospacer integration via direct interaction between Cas2 and AcrVA5.},
journal = {Nature communications},
volume = {15},
number = {1},
pages = {3256},
pmid = {38627399},
issn = {2041-1723},
support = {32071476//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {*CRISPR-Associated Proteins/metabolism ; CRISPR-Cas Systems ; },
abstract = {Spacer acquisition step in CRISPR-Cas system involves the recognition and subsequent integration of protospacer by the Cas1-Cas2 complex in CRISPR-Cas systems. Here we report an anti-CRISPR protein, AcrVA5, and reveal the mechanisms by which it strongly inhibits protospacer integration. Our biochemical data shows that the integration by Cas1-Cas2 was abrogated in the presence of AcrVA5. AcrVA5 exhibits low binding affinity towards Cas2 and acetylates Cas2 at Lys[55] on the binding interface of the Cas2 and AcrVA5 N-terminal peptide complex to inhibit the Cas2-mediated endonuclease activity. Moreover, a detailed structural comparison between our crystal structure and homolog structure shows that binding of AcrVA5 to Cas2 causes steric hindrance to the neighboring protospacer resulting in the partial disassembly of the Cas1-Cas2 and protospacer complex, as demonstrated by electrophoretic mobility shift assay. Our study focuses on this mechanism of spacer acquisition inhibition and provides insights into the biology of CRISPR-Cas systems.},
}

*CRISPR-Associated Proteins/metabolism
CRISPR-Cas Systems

@article {pmid38613479,
year = {2024},
author = {Cao, H and Mao, K and Yang, J and Wu, Q and Hu, J and Zhang, H},
title = {High-Throughput μPAD with Cascade Signal Amplification through Dual Enzymes for arsM in Paddy Soil.},
journal = {Analytical chemistry},
volume = {96},
number = {16},
pages = {6337-6346},
doi = {10.1021/acs.analchem.3c05958},
pmid = {38613479},
issn = {1520-6882},
mesh = {*Soil/chemistry ; Horseradish Peroxidase/metabolism/chemistry ; CRISPR-Cas Systems ; Oryza/chemistry ; Soil Pollutants/analysis ; Lab-On-A-Chip Devices ; Endodeoxyribonucleases/metabolism/chemistry ; High-Throughput Screening Assays/methods ; Bacterial Proteins/genetics/metabolism ; CRISPR-Associated Proteins/metabolism ; Limit of Detection ; Nucleic Acid Amplification Techniques/methods ; },
abstract = {The arsM gene is a critical biomarker for the potential risk of arsenic exposure in paddy soil. However, on-site screening of arsM is limited by the lack of high-throughput point-of-use (POU) methods. Here, a multiplex CRISPR/Cas12a microfluidic paper-based analytical device (μPAD) was constructed for the high-throughput POU analysis of arsM, with cascade amplification driven by coupling crRNA-enhanced Cas12a and horseradish peroxidase (HRP)-modified probes. First, seven crRNAs were designed to recognize arsM, and their LODs and background signal intensities were evaluated. Next, a step-by-step iterative approach was utilized to develop and optimize coupling systems, which improved the sensitivity 32 times and eliminated background signal interference. Then, ssDNA reporters modified with HRP were introduced to further lower the LOD to 16 fM, and the assay results were visible to the naked eye. A multiplex channel microfluidic paper-based chip was developed for the reaction integration and simultaneous detection of 32 samples and generated a recovery rate between 87.70 and 114.05%, simplifying the pretreatment procedures and achieving high-throughput POU analysis. Finally, arsM in Wanshan paddy soil was screened on site, and the arsM abundance ranged from 1.05 × 10[6] to 6.49 × 10[7] copies/g; this result was not affected by the environmental indicators detected in the study. Thus, a coupling crRNA-based cascade amplification method for analyzing arsM was constructed, and a microfluidic device was developed that contains many more channels than previous paper chips, greatly improving the analytical performance in paddy soil samples and providing a promising tool for the on-site screening of arsM at large scales.},
}

*Soil/chemistry
Horseradish Peroxidase/metabolism/chemistry
CRISPR-Cas Systems
Oryza/chemistry
Soil Pollutants/analysis
Lab-On-A-Chip Devices
Endodeoxyribonucleases/metabolism/chemistry
High-Throughput Screening Assays/methods
Bacterial Proteins/genetics/metabolism
CRISPR-Associated Proteins/metabolism
Limit of Detection
Nucleic Acid Amplification Techniques/methods

@article {pmid38608689,
year = {2024},
author = {Vos, PD and Gandadireja, AP and Rossetti, G and Siira, SJ and Mantegna, JL and Filipovska, A and Rackham, O},
title = {Mutational rescue of the activity of high-fidelity Cas9 enzymes.},
journal = {Cell reports methods},
volume = {4},
number = {4},
pages = {100756},
doi = {10.1016/j.crmeth.2024.100756},
pmid = {38608689},
issn = {2667-2375},
mesh = {Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *CRISPR-Associated Protein 9/genetics/metabolism ; HEK293 Cells ; Mutation ; Endonucleases/genetics/metabolism ; },
abstract = {Programmable DNA endonucleases derived from bacterial genetic defense systems, exemplified by CRISPR-Cas9, have made it significantly easier to perform genomic modifications in living cells. However, unprogrammed, off-target modifications can have serious consequences, as they often disrupt the function or regulation of non-targeted genes and compromise the safety of therapeutic gene editing applications. High-fidelity mutants of Cas9 have been established to enable more accurate gene editing, but these are typically less efficient. Here, we merge the strengths of high-fidelity Cas9 and hyperactive Cas9 variants to provide an enzyme, which we dub HyperDriveCas9, that yields the desirable properties of both parents. HyperDriveCas9 functions efficiently in mammalian cells and introduces insertion and deletion mutations into targeted genomic regions while maintaining a favorable off-target profile. HyperDriveCas9 is a precise and efficient tool for gene editing applications in science and medicine.},
}

Humans
*Gene Editing/methods
*CRISPR-Cas Systems/genetics
*CRISPR-Associated Protein 9/genetics/metabolism
HEK293 Cells
Mutation
Endonucleases/genetics/metabolism

@article {pmid38604773,
year = {2024},
author = {Li, QN and Ma, AX and Wang, DX and Dai, ZQ and Wu, SL and Lu, S and Zhu, LN and Jiang, HX and Pang, DW and Kong, DM},
title = {Allosteric Activator-Regulated CRISPR/Cas12a System Enables Biosensing and Imaging of Intracellular Endogenous and Exogenous Targets.},
journal = {Analytical chemistry},
volume = {96},
number = {16},
pages = {6426-6435},
doi = {10.1021/acs.analchem.4c00555},
pmid = {38604773},
issn = {1520-6882},
mesh = {*CRISPR-Cas Systems/genetics ; *Biosensing Techniques/methods ; Humans ; *MicroRNAs/analysis/metabolism ; Allosteric Regulation ; CRISPR-Associated Proteins/metabolism ; Endodeoxyribonucleases/metabolism/chemistry ; Bacterial Proteins/metabolism/chemistry/genetics ; HEK293 Cells ; },
abstract = {Sensors designed based on the trans-cleavage activity of CRISPR/Cas12a systems have opened up a new era in the field of biosensing. The current design of CRISPR/Cas12-based sensors in the "on-off-on" mode mainly focuses on programming the activator strand (AS) to indirectly switch the trans-cleavage activity of Cas12a in response to target information. However, this design usually requires the help of additional auxiliary probes to keep the activator strand in an initially "blocked" state. The length design and dosage of the auxiliary probe need to be strictly optimized to ensure the lowest background and the best signal-to-noise ratio. This will inevitably increase the experiment complexity. To solve this problem, we propose using AS after the "RESET" effect to directly regulate the Cas12a enzymatic activity. Initially, the activator strand was rationally designed to be embedded in a hairpin structure to deprive its ability to activate the CRISPR/Cas12a system. When the target is present, target-mediated strand displacement causes the conformation change in the AS, the hairpin structure is opened, and the CRISPR/Cas12a system is reactivated; the switchable structure of AS can be used to regulate the degree of activation of Cas12a according to the target concentration. Due to the advantages of low background and stability, the CRISPR/Cas12a-based strategy can not only image endogenous biomarkers (miR-21) in living cells but also enable long-term and accurate imaging analysis of the process of exogenous virus invasion of cells. Release and replication of virus genome in host cells are indispensable hallmark events of cell infection by virus; sensitive monitoring of them is of great significance to revealing virus infection mechanism and defending against viral diseases.},
}

*CRISPR-Cas Systems/genetics
*Biosensing Techniques/methods
Humans
*MicroRNAs/analysis/metabolism
Allosteric Regulation
CRISPR-Associated Proteins/metabolism
Endodeoxyribonucleases/metabolism/chemistry
Bacterial Proteins/metabolism/chemistry/genetics
HEK293 Cells

@article {pmid38599073,
year = {2024},
author = {Li, H and Qiao, S and Zhang, H and Qiao, Y and Liu, J and Li, Y},
title = {Highly sensitive and selective demethylase FTO detection using a DNAzyme-mediated CRISPR/Cas12a signal cascade amplification electrochemiluminescence biosensor with C-CN/PCNV heterojunction as emitter.},
journal = {Biosensors & bioelectronics},
volume = {256},
number = {},
pages = {116276},
doi = {10.1016/j.bios.2024.116276},
pmid = {38599073},
issn = {1873-4235},
mesh = {*Biosensing Techniques ; *Alpha-Ketoglutarate-Dependent Dioxygenase FTO/chemistry/metabolism ; Humans ; *DNA, Catalytic/chemistry ; *Luminescent Measurements ; *Electrochemical Techniques/methods ; *Limit of Detection ; *CRISPR-Cas Systems ; Nitriles/chemistry ; Endodeoxyribonucleases/chemistry/metabolism ; CRISPR-Associated Proteins/chemistry ; Adenosine/analogs & derivatives/analysis/chemistry ; Nanostructures/chemistry ; Ferrous Compounds/chemistry ; Bacterial Proteins/chemistry/genetics ; *Metallocenes ; },
abstract = {Fat mass and obesity-associated protein (FTO) has gained attention as the first RNA N6-methyladenosine (m[6]A) modification eraser due to its overexpression being associated with various cancers. In this study, an electrochemiluminescence (ECL) biosensor for the detection of demethylase FTO was developed based on DNAzyme-mediated CRISPR/Cas12a signal cascade amplification system and carboxylated carbon nitride nanosheets/phosphorus-doped nitrogen-vacancy modified carbon nitride nanosheets (C-CN/PCNV) heterojunction as the emitter. The biosensor was constructed by modifying the C-CN/PCNV heterojunction and a ferrocene-tagged probe (ssDNA-Fc) on a glassy carbon electrode. The presence of FTO removes the m[6]A modification on the catalytic core of DNAzyme, restoring its cleavage activity and generating activator DNA. This activator DNA further activates the trans-cleavage ability of Cas12a, leading to the cleavage of the ssDNA-Fc and the recovery of the ECL signal. The C-CN/PCNV heterojunction prevents electrode passivation and improves the electron-hole recombination, resulting in significantly enhanced ECL signal. The biosensor demonstrates high sensitivity with a low detection limit of 0.63 pM in the range from 1.0 pM to 100 nM. Furthermore, the biosensor was successfully applied to detect FTO in cancer cell lysate and screen FTO inhibitors, showing great potential in early clinical diagnosis and drug discovery.},
}

*Biosensing Techniques
*Alpha-Ketoglutarate-Dependent Dioxygenase FTO/chemistry/metabolism
Humans
*DNA, Catalytic/chemistry
*Luminescent Measurements
*Electrochemical Techniques/methods
*Limit of Detection
*CRISPR-Cas Systems
Nitriles/chemistry
Endodeoxyribonucleases/chemistry/metabolism
CRISPR-Associated Proteins/chemistry
Adenosine/analogs & derivatives/analysis/chemistry
Nanostructures/chemistry
Ferrous Compounds/chemistry
Bacterial Proteins/chemistry/genetics
*Metallocenes

@article {pmid38574662,
year = {2024},
author = {Zhang, H and Zhang, M and Zhou, Y and Qiao, Z and Gao, L and Cao, L and Yin, H and Wang, M},
title = {Organic photoelectrochemical transistor aptasensor for dual-mode detection of DEHP with CRISPR-Cas13a assisted signal amplification.},
journal = {Journal of hazardous materials},
volume = {470},
number = {},
pages = {134175},
doi = {10.1016/j.jhazmat.2024.134175},
pmid = {38574662},
issn = {1873-3336},
mesh = {*Aptamers, Nucleotide/chemistry ; *Diethylhexyl Phthalate/chemistry/analysis ; *Biosensing Techniques/methods ; *Electrochemical Techniques/instrumentation/methods ; *CRISPR-Cas Systems ; *Transistors, Electronic ; Water Pollutants, Chemical/analysis ; Polystyrenes/chemistry ; Limit of Detection ; Nucleic Acid Amplification Techniques ; *Thiophenes ; },
abstract = {Emerging organic photoelectrochemical transistors (OPECTs) with inherent amplification capabilities, good biocompatibility and even self-powered operation have emerged as a promising detection tool, however, they are still not widely studied for pollutant detection. In this paper, a novel OPECT dual-mode aptasensor was constructed for the ultrasensitive detection of di(2-ethylhexyl) phthalate (DEHP). MXene/In2S3/In2O3 Z-scheme heterojunction was used as a light fuel for ion modulation in sensitive gated OPECT biosensing. A transistor system based on poly(3,4-ethylenedioxythiophene):poly(styrene sulfonate) (PEDOT:PSS) converted biological events associated with photosensitive gate achieving nearly a thousand-fold higher current gain at zero bias voltage. This work quantified the target DEHP by aptamer-specific induction of CRISPR-Cas13a trans-cutting activity with target-dependent rolling circle amplification as the signal amplification unit, and incorporated the signal changes strategy of biocatalytic precipitation and TMB color development. Combining OPECT with the auxiliary validation of colorimetry (CM), high sensitivity and accurate detection of DEHP were achieved with a linear range of 0.1 pM to 200 pM and a minimum detection limit of 0.02 pM. This study not only provides a new method for the detection of DEHP, but also offers a promising prospect for the gating and application of the unique OPECT.},
}

*Aptamers, Nucleotide/chemistry
*Diethylhexyl Phthalate/chemistry/analysis
*Biosensing Techniques/methods
*Electrochemical Techniques/instrumentation/methods
*CRISPR-Cas Systems
*Transistors, Electronic
Water Pollutants, Chemical/analysis
Polystyrenes/chemistry
Limit of Detection
Nucleic Acid Amplification Techniques
*Thiophenes

@article {pmid38570687,
year = {2024},
author = {Schevenels, G and Cabochette, P and America, M and Vandenborne, A and De Grande, L and Guenther, S and He, L and Dieu, M and Christou, B and Vermeersch, M and Germano, RFV and Perez-Morga, D and Renard, P and Martin, M and Vanlandewijck, M and Betsholtz, C and Vanhollebeke, B},
title = {A brain-specific angiogenic mechanism enabled by tip cell specialization.},
journal = {Nature},
volume = {628},
number = {8009},
pages = {863-871},
pmid = {38570687},
issn = {1476-4687},
mesh = {*Zebrafish ; Animals ; *Brain/cytology/blood supply/metabolism ; *Blood-Brain Barrier/metabolism/cytology ; *Neovascularization, Physiologic ; *Endothelial Cells/metabolism/cytology ; *Basement Membrane/metabolism ; *Collagen Type IV/metabolism ; Wnt Proteins/metabolism ; CRISPR-Cas Systems/genetics ; Humans ; Organ Specificity ; Wnt Signaling Pathway ; Zebrafish Proteins/metabolism/genetics ; Cell Movement ; Meninges/cytology/blood supply/metabolism ; },
abstract = {Vertebrate organs require locally adapted blood vessels[1,2]. The gain of such organotypic vessel specializations is often deemed to be molecularly unrelated to the process of organ vascularization. Here, opposing this model, we reveal a molecular mechanism for brain-specific angiogenesis that operates under the control of Wnt7a/b ligands-well-known blood-brain barrier maturation signals[3-5]. The control mechanism relies on Wnt7a/b-dependent expression of Mmp25, which we find is enriched in brain endothelial cells. CRISPR-Cas9 mutagenesis in zebrafish reveals that this poorly characterized glycosylphosphatidylinositol-anchored matrix metalloproteinase is selectively required in endothelial tip cells to enable their initial migration across the pial basement membrane lining the brain surface. Mechanistically, Mmp25 confers brain invasive competence by cleaving meningeal fibroblast-derived collagen IV α5/6 chains within a short non-collagenous region of the central helical part of the heterotrimer. After genetic interference with the pial basement membrane composition, the Wnt-β-catenin-dependent organotypic control of brain angiogenesis is lost, resulting in properly patterned, yet blood-brain-barrier-defective cerebrovasculatures. We reveal an organ-specific angiogenesis mechanism, shed light on tip cell mechanistic angiodiversity and thereby illustrate how organs, by imposing local constraints on angiogenic tip cells, can select vessels matching their distinctive physiological requirements.},
}

*Zebrafish
Animals
*Brain/cytology/blood supply/metabolism
*Blood-Brain Barrier/metabolism/cytology
*Neovascularization, Physiologic
*Endothelial Cells/metabolism/cytology
*Basement Membrane/metabolism
*Collagen Type IV/metabolism
Wnt Proteins/metabolism
CRISPR-Cas Systems/genetics
Humans
Organ Specificity
Wnt Signaling Pathway
Zebrafish Proteins/metabolism/genetics
Cell Movement
Meninges/cytology/blood supply/metabolism

@article {pmid38554702,
year = {2024},
author = {Köhler, AR and Haußer, J and Harsch, A and Bernhardt, S and Häußermann, L and Brenner, LM and Lungu, C and Olayioye, MA and Bashtrykov, P and Jeltsch, A},
title = {Modular dual-color BiAD sensors for locus-specific readout of epigenome modifications in single cells.},
journal = {Cell reports methods},
volume = {4},
number = {4},
pages = {100739},
doi = {10.1016/j.crmeth.2024.100739},
pmid = {38554702},
issn = {2667-2375},
mesh = {Humans ; *Epigenome ; *DNA Methylation ; *Single-Cell Analysis/methods ; *Histones/metabolism/genetics ; Epigenesis, Genetic ; Genetic Loci ; Chromatin/metabolism/genetics ; Biosensing Techniques/methods ; Color ; HEK293 Cells ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; },
abstract = {Dynamic changes in the epigenome at defined genomic loci play crucial roles during cellular differentiation and disease development. Here, we developed dual-color bimolecular anchor detector (BiAD) sensors for high-sensitivity readout of locus-specific epigenome modifications by fluorescence microscopy. Our BiAD sensors comprise an sgRNA/dCas9 complex as anchor and double chromatin reader domains as detector modules, both fused to complementary parts of a split IFP2.0 fluorophore, enabling its reconstitution upon binding of both parts in close proximity. In addition, a YPet fluorophore is recruited to the sgRNA to mark the genomic locus of interest. With these dual-color BiAD sensors, we detected H3K9me2/3 and DNA methylation and their dynamic changes upon RNAi or inhibitor treatment with high sensitivity at endogenous genomic regions. Furthermore, we showcased locus-specific H3K36me2/3 readout as well as H3K27me3 and H3K9me2/3 enrichment on the inactive X chromosome, highlighting the broad applicability of our dual-color BiAD sensors for single-cell epigenome studies.},
}

Humans
*Epigenome
*DNA Methylation
*Single-Cell Analysis/methods
*Histones/metabolism/genetics
Epigenesis, Genetic
Genetic Loci
Chromatin/metabolism/genetics
Biosensing Techniques/methods
Color
HEK293 Cells
RNA, Guide, CRISPR-Cas Systems/genetics/metabolism

@article {pmid38538798,
year = {2024},
author = {Radko-Juettner, S and Yue, H and Myers, JA and Carter, RD and Robertson, AN and Mittal, P and Zhu, Z and Hansen, BS and Donovan, KA and Hunkeler, M and Rosikiewicz, W and Wu, Z and McReynolds, MG and Roy Burman, SS and Schmoker, AM and Mageed, N and Brown, SA and Mobley, RJ and Partridge, JF and Stewart, EA and Pruett-Miller, SM and Nabet, B and Peng, J and Gray, NS and Fischer, ES and Roberts, CWM},
title = {Targeting DCAF5 suppresses SMARCB1-mutant cancer by stabilizing SWI/SNF.},
journal = {Nature},
volume = {628},
number = {8007},
pages = {442-449},
pmid = {38538798},
issn = {1476-4687},
mesh = {Animals ; Female ; Humans ; Male ; Mice ; Cell Line, Tumor ; CRISPR-Cas Systems ; Gene Editing ; *Mutation ; *Neoplasms/genetics/metabolism ; *SMARCB1 Protein/deficiency/genetics/metabolism ; Tumor Suppressor Proteins/deficiency/genetics/metabolism ; *Multiprotein Complexes/chemistry/metabolism ; Proteolysis ; Ubiquitin/metabolism ; },
abstract = {Whereas oncogenes can potentially be inhibited with small molecules, the loss of tumour suppressors is more common and is problematic because the tumour-suppressor proteins are no longer present to be targeted. Notable examples include SMARCB1-mutant cancers, which are highly lethal malignancies driven by the inactivation of a subunit of SWI/SNF (also known as BAF) chromatin-remodelling complexes. Here, to generate mechanistic insights into the consequences of SMARCB1 mutation and to identify vulnerabilities, we contributed 14 SMARCB1-mutant cell lines to a near genome-wide CRISPR screen as part of the Cancer Dependency Map Project[1-3]. We report that the little-studied gene DDB1-CUL4-associated factor 5 (DCAF5) is required for the survival of SMARCB1-mutant cancers. We show that DCAF5 has a quality-control function for SWI/SNF complexes and promotes the degradation of incompletely assembled SWI/SNF complexes in the absence of SMARCB1. After depletion of DCAF5, SMARCB1-deficient SWI/SNF complexes reaccumulate, bind to target loci and restore SWI/SNF-mediated gene expression to levels that are sufficient to reverse the cancer state, including in vivo. Consequently, cancer results not from the loss of SMARCB1 function per se, but rather from DCAF5-mediated degradation of SWI/SNF complexes. These data indicate that therapeutic targeting of ubiquitin-mediated quality-control factors may effectively reverse the malignant state of some cancers driven by disruption of tumour suppressor complexes.},
}

Animals
Female
Humans
Male
Mice
Cell Line, Tumor
CRISPR-Cas Systems
Gene Editing
*Mutation
*Neoplasms/genetics/metabolism
*SMARCB1 Protein/deficiency/genetics/metabolism
Tumor Suppressor Proteins/deficiency/genetics/metabolism
*Multiprotein Complexes/chemistry/metabolism
Proteolysis
Ubiquitin/metabolism

@article {pmid38532709,
year = {2024},
author = {Sale, JE and Stoddard, BL},
title = {CRISPR in Nucleic Acids Research: the sequel.},
journal = {Nucleic acids research},
volume = {52},
number = {7},
pages = {3489-3492},
doi = {10.1093/nar/gkae159},
pmid = {38532709},
issn = {1362-4962},
mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; Humans ; Clustered Regularly Interspaced Short Palindromic Repeats ; Nucleic Acids/genetics ; },
}

*CRISPR-Cas Systems
*Gene Editing/methods
Humans
Clustered Regularly Interspaced Short Palindromic Repeats
Nucleic Acids/genetics

@article {pmid38526223,
year = {2024},
author = {Franks, SNJ and Heon-Roberts, R and Ryan, BJ},
title = {CRISPRi: a way to integrate iPSC-derived neuronal models.},
journal = {Biochemical Society transactions},
volume = {52},
number = {2},
pages = {539-551},
doi = {10.1042/BST20230190},
pmid = {38526223},
issn = {1470-8752},
mesh = {*Induced Pluripotent Stem Cells/cytology/metabolism ; Humans ; *Neurodegenerative Diseases/therapy/genetics/metabolism ; *Neurons/metabolism/cytology ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Animals ; Gene Editing ; },
abstract = {The genetic landscape of neurodegenerative diseases encompasses genes affecting multiple cellular pathways which exert effects in an array of neuronal and glial cell-types. Deconvolution of the roles of genes implicated in disease and the effects of disease-associated variants remains a vital step in the understanding of neurodegeneration and the development of therapeutics. Disease modelling using patient induced pluripotent stem cells (iPSCs) has enabled the generation of key cell-types associated with disease whilst maintaining the genomic variants that predispose to neurodegeneration. The use of CRISPR interference (CRISPRi), alongside other CRISPR-perturbations, allows the modelling of the effects of these disease-associated variants or identifying genes which modify disease phenotypes. This review summarises the current applications of CRISPRi in iPSC-derived neuronal models, such as fluorescence-activated cell sorting (FACS)-based screens, and discusses the future opportunities for disease modelling, identification of disease risk modifiers and target/drug discovery in neurodegeneration.},
}

*Induced Pluripotent Stem Cells/cytology/metabolism
Humans
*Neurodegenerative Diseases/therapy/genetics/metabolism
*Neurons/metabolism/cytology
*CRISPR-Cas Systems
Clustered Regularly Interspaced Short Palindromic Repeats
Animals
Gene Editing

@article {pmid38499498,
year = {2024},
author = {Gawlitt, S and Collins, SP and Yu, Y and Blackman, SA and Barquist, L and Beisel, CL},
title = {Expanding the flexibility of base editing for high-throughput genetic screens in bacteria.},
journal = {Nucleic acids research},
volume = {52},
number = {7},
pages = {4079-4097},
doi = {10.1093/nar/gkae174},
pmid = {38499498},
issn = {1362-4962},
support = {1R35GM119561/NH/NIH HHS/United States ; 865973/ERC_/European Research Council/International ; 1R35GM119561/NH/NIH HHS/United States ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; *Escherichia coli/genetics ; High-Throughput Screening Assays/methods ; Genome, Bacterial/genetics ; CRISPR-Associated Protein 9/genetics/metabolism ; Streptococcus/genetics ; Streptococcus pyogenes/genetics/enzymology ; Machine Learning ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Genome-wide screens have become powerful tools for elucidating genotype-to-phenotype relationships in bacteria. Of the varying techniques to achieve knockout and knockdown, CRISPR base editors are emerging as promising options. However, the limited number of available, efficient target sites hampers their use for high-throughput screening. Here, we make multiple advances to enable flexible base editing as part of high-throughput genetic screening in bacteria. We first co-opt the Streptococcus canis Cas9 that exhibits more flexible protospacer-adjacent motif recognition than the traditional Streptococcus pyogenes Cas9. We then expand beyond introducing premature stop codons by mutating start codons. Next, we derive guide design rules by applying machine learning to an essentiality screen conducted in Escherichia coli. Finally, we rescue poorly edited sites by combining base editing with Cas9-induced cleavage of unedited cells, thereby enriching for intended edits. The efficiency of this dual system was validated through a conditional essentiality screen based on growth in minimal media. Overall, expanding the scope of genome-wide knockout screens with base editors could further facilitate the investigation of new gene functions and interactions in bacteria.},
}

*Gene Editing/methods
*CRISPR-Cas Systems
*Escherichia coli/genetics
High-Throughput Screening Assays/methods
Genome, Bacterial/genetics
CRISPR-Associated Protein 9/genetics/metabolism
Streptococcus/genetics
Streptococcus pyogenes/genetics/enzymology
Machine Learning
RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid38351238,
year = {2024},
author = {Asanomi, Y and Kimura, T and Shimoda, N and Shigemizu, D and Niida, S and Ozaki, K},
title = {CRISPR/Cas9-mediated knock-in cells of the late-onset Alzheimer's disease-risk variant, SHARPIN G186R, reveal reduced NF-κB pathway and accelerated Aβ secretion.},
journal = {Journal of human genetics},
volume = {69},
number = {5},
pages = {171-176},
pmid = {38351238},
issn = {1435-232X},
support = {JP21dk0207045//Japan Agency for Medical Research and Development (AMED)/ ; JP22dk0207060//Japan Agency for Medical Research and Development (AMED)/ ; JP21de0107002//Japan Agency for Medical Research and Development (AMED)/ ; JP21dk0207052//Japan Agency for Medical Research and Development (AMED)/ ; JP18kk0205009//Japan Agency for Medical Research and Development (AMED)/ ; JP18kk0205012//Japan Agency for Medical Research and Development (AMED)/ ; 21-23//National Center for Geriatrics and Gerontology (NCGG)/ ; 21-24//National Center for Geriatrics and Gerontology (NCGG)/ ; 23-7//National Center for Geriatrics and Gerontology (NCGG)/ ; },
mesh = {Humans ; *Alzheimer Disease/genetics/metabolism/pathology ; *CRISPR-Cas Systems ; *NF-kappa B/metabolism/genetics ; *Amyloid beta-Peptides/metabolism/genetics ; Signal Transduction/genetics ; Gene Knock-In Techniques ; },
}

Humans
*Alzheimer Disease/genetics/metabolism/pathology
*CRISPR-Cas Systems
*NF-kappa B/metabolism/genetics
*Amyloid beta-Peptides/metabolism/genetics
Signal Transduction/genetics
Gene Knock-In Techniques

@article {pmid38627378,
year = {2024},
author = {Wang, Y and Chen, H and Lin, K and Han, Y and Gu, Z and Wei, H and Mu, K and Wang, D and Liu, L and Jin, R and Song, R and Rong, Z and Wang, S},
title = {Ultrasensitive single-step CRISPR detection of monkeypox virus in minutes with a vest-pocket diagnostic device.},
journal = {Nature communications},
volume = {15},
number = {1},
pages = {3279},
pmid = {38627378},
issn = {2041-1723},
mesh = {Humans ; Male ; *Monkeypox virus ; Biological Assay ; Cell Death ; *Exanthema ; Nucleotidyltransferases ; Nucleic Acid Amplification Techniques ; Sensitivity and Specificity ; CRISPR-Cas Systems ; Recombinases ; },
abstract = {The emerging monkeypox virus (MPXV) has raised global health concern, thereby highlighting the need for rapid, sensitive, and easy-to-use diagnostics. Here, we develop a single-step CRISPR-based diagnostic platform, termed SCOPE (Streamlined CRISPR On Pod Evaluation platform), for field-deployable ultrasensitive detection of MPXV in resource-limited settings. The viral nucleic acids are rapidly released from the rash fluid swab, oral swab, saliva, and urine samples in 2 min via a streamlined viral lysis protocol, followed by a 10-min single-step recombinase polymerase amplification (RPA)-CRISPR/Cas13a reaction. A pod-shaped vest-pocket analysis device achieves the whole process for reaction execution, signal acquisition, and result interpretation. SCOPE can detect as low as 0.5 copies/µL (2.5 copies/reaction) of MPXV within 15 min from the sample input to the answer. We validate the developed assay on 102 clinical samples from male patients / volunteers, and the testing results are 100% concordant with the real-time PCR. SCOPE achieves a single-molecular level sensitivity in minutes with a simplified procedure performed on a miniaturized wireless device, which is expected to spur substantial progress to enable the practice application of CRISPR-based diagnostics techniques in a point-of-care setting.},
}

Humans
Male
*Monkeypox virus
Biological Assay
Cell Death
*Exanthema
Nucleotidyltransferases
Nucleic Acid Amplification Techniques
Sensitivity and Specificity
CRISPR-Cas Systems
Recombinases

@article {pmid38623982,
year = {2024},
author = {Volodina, OV and Fabrichnikova, AR and Anuchina, AA and Mishina, OS and Lavrov, AV and Smirnikhina, SA},
title = {Evolution of Prime Editing Systems: Move Forward to the Treatment of Hereditary Diseases.},
journal = {Current gene therapy},
volume = {},
number = {},
pages = {},
doi = {10.2174/0115665232295117240405070809},
pmid = {38623982},
issn = {1875-5631},
abstract = {The development of gene therapy using genome editing tools recently became relevant. With the invention of programmable nucleases, it became possible to treat hereditary diseases due to introducing targeted double strand break in the genome followed by homology directed repair (HDR) or non-homologous end-joining (NHEJ) reparation. CRISPR-Cas9 is more efficient and easier to use in comparison with other programmable nucleases. To improve the efficiency and safety of this gene editing tool, various modifications CRISPR-Cas9 basis were created in recent years, such as prime editing - in this system, Cas9 nickase is fused with reverse transcriptase and guide RNA, which contains a desired correction. Prime editing demonstrates equal or higher correction efficiency as HDR-mediated editing and much less off-target effect due to inducing nick. There are several studies in which prime editing is used to correct mutations in which researchers reported little or no evidence of off-target effects. The system can also be used to functionally characterize disease variants. However, prime editing still has several limitations that could be further improved. The effectiveness of the method is not yet high enough to apply it in clinical trials. Delivery of prime editors is also a big challenge due to their size. In the present article, we observe the development of the platform, and discuss the candidate proteins for efficiency enhancing, main delivery methods and current applications of prime editing.},
}

@article {pmid38514901,
year = {2024},
author = {Zhai, D and Li, L and Chen, C and Wang, X and Liu, R and Shan, Y},
title = {INPP5E Regulates the Distribution of Phospholipids on Cilia in RPE1 Cells.},
journal = {Journal of clinical laboratory analysis},
volume = {38},
number = {7},
pages = {e25031},
pmid = {38514901},
issn = {1098-2825},
mesh = {*Cilia/metabolism ; *Phosphoric Monoester Hydrolases/metabolism/genetics ; Humans ; Cell Line ; Phosphatidylinositol 4,5-Diphosphate/metabolism ; Retinal Pigment Epithelium/metabolism/cytology ; Phosphatidylinositol Phosphates/metabolism ; CRISPR-Cas Systems ; Phospholipids/metabolism ; },
abstract = {BACKGROUND: Primary cilia are static microtubule-based structures protruding from the cell surface and present on most vertebrate cells. The appropriate localization of phospholipids is essential for cilia formation and stability. INPP5E is a cilia-localized inositol 5-phosphatase; its deletion alters the phosphoinositide composition in the ciliary membrane, disrupting ciliary function.

METHODS: The EGFP-2xP4M[SidM], PH[PLCδ1]-EGFP, and SMO-tRFP plasmids were constructed by the Gateway system to establish a stable RPE1 cell line. The INPP5E KO RPE1 cell line was constructed with the CRISPR/Cas9 system. The localization of INPP5E and the distribution of PI(4,5)P2 and PI4P were examined by immunofluorescence microscopy. The fluorescence intensity co-localized with cilia was quantified by ImageJ.

RESULTS: In RPE1 cells, PI4P is localized at the ciliary membrane, whereas PI(4,5)P2 is localized at the base of cilia. Knocking down or knocking out INPP5E alters this distribution, resulting in the distribution of PI(4,5)P2 along the ciliary membrane and the disappearance of PI4P from the cilia. Meanwhile, PI(4,5)P2 is located in the ciliary membrane labeled by SMO-tRFP.

CONCLUSIONS: INPP5E regulates the distribution of phosphoinositide on cilia. PI(4,5)P2 localizes at the ciliary membrane labeled with SMO-tRFP, indicating that ciliary pocket membrane contains PI(4,5)P2, and phosphoinositide composition in early membrane structures may differ from that in mature ciliary membrane.},
}

*Cilia/metabolism
*Phosphoric Monoester Hydrolases/metabolism/genetics
Humans
Cell Line
Phosphatidylinositol 4,5-Diphosphate/metabolism
Retinal Pigment Epithelium/metabolism/cytology
Phosphatidylinositol Phosphates/metabolism
CRISPR-Cas Systems
Phospholipids/metabolism

@article {pmid38355914,
year = {2024},
author = {McLaurin, KA and Li, H and Khalili, K and Mactutus, CF and Booze, RM},
title = {HIV-1 mRNA knockdown with CRISPR/CAS9 enhances neurocognitive function.},
journal = {Journal of neurovirology},
volume = {30},
number = {1},
pages = {71-85},
pmid = {38355914},
issn = {1538-2443},
support = {DA059310/DA/NIDA NIH HHS/United States ; DA056288/DA/NIDA NIH HHS/United States ; MH106392/MH/NIMH NIH HHS/United States ; NS100624/NS/NINDS NIH HHS/United States ; DA059310/DA/NIDA NIH HHS/United States ; DA056288/DA/NIDA NIH HHS/United States ; MH106392/MH/NIMH NIH HHS/United States ; NS100624/NS/NINDS NIH HHS/United States ; },
mesh = {Animals ; *HIV-1/genetics/physiology ; Rats ; *CRISPR-Cas Systems ; *Rats, Transgenic ; *RNA, Messenger/genetics/metabolism ; *Gene Editing/methods ; Neuroglia/virology/metabolism ; Dependovirus/genetics ; HIV Infections/virology/genetics ; Gene Knockdown Techniques ; RNA, Viral/genetics ; Cognition/physiology ; Humans ; },
abstract = {Mixed glia are infiltrated with HIV-1 virus early in the course of infection leading to the development of a persistent viral reservoir in the central nervous system. Modification of the HIV-1 genome using gene editing techniques, including CRISPR/Cas9, has shown great promise towards eliminating HIV-1 viral reservoirs; whether these techniques are capable of removing HIV-1 viral proteins from mixed glia, however, has not been systematically evaluated. Herein, the efficacy of adeno-associated virus 9 (AAV9)-CRISPR/Cas9 gene editing for eliminating HIV-1 messenger RNA (mRNA) from cortical mixed glia was evaluated in vitro and in vivo. In vitro, a within-subjects experimental design was utilized to treat mixed glia isolated from neonatal HIV-1 transgenic (Tg) rats with varying doses (0, 0.9, 1.8, 2.7, 3.6, 4.5, or 5.4 µL corresponding to a physical titer of 0, 4.23 × 10[9], 8.46 × 10[9], 1.269 × 10[10], 1.692 × 10[10], 2.115 × 10[10], and 2.538 × 10[10] gc/µL) of CRISPR/Cas9 for 72 h. Dose-dependent decreases in the number of HIV-1 mRNA, quantified using an innovative in situ hybridization technique, were observed in a subset (i.e., n = 5 out of 8) of primary mixed glia. In vivo, HIV-1 Tg rats were retro-orbitally inoculated with CRISPR/Cas9 for two weeks, whereby treatment resulted in profound excision (i.e., approximately 53.2%) of HIV-1 mRNA from the medial prefrontal cortex. Given incomplete excision of the HIV-1 viral genome, the clinical relevance of HIV-1 mRNA knockdown for eliminating neurocognitive impairments was evaluated via examination of temporal processing, a putative neurobehavioral mechanism underlying HIV-1-associated neurocognitive disorders (HAND). Indeed, treatment with CRISPR/Cas9 protractedly, albeit not permanently, restored the developmental trajectory of temporal processing. Proof-of-concept studies, therefore, support the susceptibility of mixed glia to gene editing and the potential of CRISPR/Cas9 to serve as a novel therapeutic strategy for HAND, even in the absence of full viral eradication.},
}

Animals
*HIV-1/genetics/physiology
Rats
*CRISPR-Cas Systems
*Rats, Transgenic
*RNA, Messenger/genetics/metabolism
*Gene Editing/methods
Neuroglia/virology/metabolism
Dependovirus/genetics
HIV Infections/virology/genetics
Gene Knockdown Techniques
RNA, Viral/genetics
Cognition/physiology
Humans

@article {pmid38624901,
year = {2022},
author = {Mondal, S and Halder, SK and Mondal, KC},
title = {Tailoring in fungi for next generation cellulase production with special reference to CRISPR/CAS system.},
journal = {Systems microbiology and biomanufacturing},
volume = {2},
number = {1},
pages = {113-129},
pmid = {38624901},
issn = {2662-7663},
abstract = {Cellulose is the utmost plenteous source of biopolymer in our earth, and fungi are the most efficient and ubiquitous organism in degrading the cellulosic biomass by synthesizing cellulases. Tailoring through genetic manipulation has played a substantial role in constructing novel fungal strains towards improved cellulase production of desired traits. However, the traditional methods of genetic manipulation of fungi are time-consuming and tedious. With the availability of the full-genome sequences of several industrially relevant filamentous fungi, CRISPR-CAS (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein) technology has come into the focus for the proficient development of manipulated strains of filamentous fungi. This review summarizes the mode of action of cellulases, transcription level regulation for cellulase expression, various traditional strategies of genetic manipulation with CRISPR-CAS technology to develop modified fungal strains for a preferred level of cellulase production, and the futuristic trend in this arena of research.},
}

@article {pmid38622798,
year = {2024},
author = {Peng, W and Gao, M and Zhu, X and Liu, X and Yang, G and Li, S and Liu, Y and Bai, L and Yang, J and Bao, J},
title = {Visual screening of CRISPR/Cas9 editing efficiency based on micropattern arrays for editing porcine cells.},
journal = {Biotechnology journal},
volume = {19},
number = {4},
pages = {e2300691},
doi = {10.1002/biot.202300691},
pmid = {38622798},
issn = {1860-7314},
support = {82270662//National Natural Science Foundation of China/ ; 82070640//National Natural Science Foundation of China/ ; 2022-YF05-01255-SN//Technology Innovation R&D Project of Chengdu Science and Technology Bureau/ ; 22ZDYF1406//Key R&D Project of Sichuan Science and Technology Department/ ; ZYJC21014//1.3.5 project for disciplines of excellence, West China Hospital Sichuan University/ ; },
mesh = {Animals ; Swine ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Gene Knockout Techniques ; Genome ; RNA, Messenger/genetics ; },
abstract = {CRISPR/Cas9 technology, combined with somatic cell nuclear transplantation (SCNT), represents the primary approach to generating gene-edited pigs. The inefficiency in acquiring gene-edited nuclear donors is attributed to low editing and delivery efficiency, both closely linked to the selection of CRISPR/Cas9 forms. However, there is currently no direct method to evaluate the efficiency of CRISPR/Cas9 editing in porcine genomes. A platform based on fluorescence reporting signals and micropattern arrays was developed in this study, to visually assess the efficiency of gene editing. The optimal specifications for culturing porcine cells, determined by the quantity and state of cells grown on micropattern arrays, were a diameter of 200 µm and a spacing of 150 µm. By visualizing the area of fluorescence loss and measuring the gray value of the micropattern arrays, it was quickly determined that the mRNA form targeting porcine cells exhibited the highest editing efficiency compared to DNA and Ribonucleoprotein (RNP) forms of CRISPR/Cas9. Subsequently, four homozygotes of the β4GalNT2 gene knockout were successfully obtained through the mRNA form, laying the groundwork for the subsequent generation of gene-edited pigs. This platform facilitates a quick, simple, and effective evaluation of gene knockout efficiency. Additionally, it holds significant potential for swiftly testing novel gene editing tools, assessing delivery methods, and tailoring evaluation platforms for various cell types.},
}

Animals
Swine
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
Gene Knockout Techniques
Genome
RNA, Messenger/genetics

@article {pmid38622404,
year = {2024},
author = {Freen-van Heeren, JJ},
title = {Employing CRISPR-Cas9 to Enhance T Cell Effector Function.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2782},
number = {},
pages = {195-208},
pmid = {38622404},
issn = {1940-6029},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *T-Lymphocytes ; Gene Editing/methods ; Genetic Engineering/methods ; Cytokines/genetics ; },
abstract = {As part of the adaptive immune system, T cells are critical to maintain immune homeostasis. T cells provide protective immunity by killing infected cells and combatting cancerous cells. To do so, T cells produce and secrete effector molecules, such as granzymes, perforin, and cytokines such as tumor necrosis factor α and interferon γ. However, in immune suppressive environments, such as tumors, T cells gradually lose the capacity to perform their effector function. One way T cell effector function can be enhanced is through genetic engineering with tools such as clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9). This protocol explains in a step-by-step fashion how to perform a controlled electroporation-based CRISPR experiment to enhance human T cell effector function. Of note, these steps are suitable for CRISPR-mediated genome editing in T cells in general and can thus also be used to study proteins of interest that do not influence T cell effector function.},
}

Humans
*CRISPR-Cas Systems/genetics
*T-Lymphocytes
Gene Editing/methods
Genetic Engineering/methods
Cytokines/genetics

@article {pmid38622403,
year = {2024},
author = {Liao, X and Li, L},
title = {CRISPR-Cas9-Induced Gene Editing in Primary Human Monocytes.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2782},
number = {},
pages = {189-193},
pmid = {38622403},
issn = {1940-6029},
mesh = {Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems ; Monocytes/metabolism ; CRISPR-Associated Protein 9/genetics ; },
abstract = {Monocytes play important and diverse roles in both homeostatic and inflammatory immune responses. The CRISPR-Cas9 system in lentiviral vectors has been widely used to manipulate specific genes of immortal monocyte cell lines to study monocyte functions. However, human primary monocytes are refractory to this method with low gene knockout (KO) efficiency. In this chapter, we developed an in vitro gene-editing procedure for primary human monocytes with a consistent and high-gene KO efficiency via a ribonucleoprotein (RNP) complex consisting of Cas9 protein and single-guide RNA (sgRNA). This method can be adapted to study the functions of targeted signaling molecules involved in modulating monocyte polarization in primary human monocytes.},
}

Humans
*Gene Editing/methods
*CRISPR-Cas Systems/genetics
RNA, Guide, CRISPR-Cas Systems
Monocytes/metabolism
CRISPR-Associated Protein 9/genetics

@article {pmid38622379,
year = {2024},
author = {Prostova, M and Kanevskaya, A and Panteleev, V and Lisitskaya, L and Perfilova Tugaeva, KV and Sluchanko, NN and Esyunina, D and Kulbachinskiy, A},
title = {DNA-targeting short Argonautes complex with effector proteins for collateral nuclease activity and bacterial population immunity.},
journal = {Nature microbiology},
volume = {},
number = {},
pages = {},
pmid = {38622379},
issn = {2058-5276},
support = {22-14-00182//Russian Science Foundation (RSF)/ ; 22-14-00182//Russian Science Foundation (RSF)/ ; 22-14-00182//Russian Science Foundation (RSF)/ ; 22-14-00182//Russian Science Foundation (RSF)/ ; 22-14-00182//Russian Science Foundation (RSF)/ ; 22-14-00182//Russian Science Foundation (RSF)/ ; },
abstract = {Two prokaryotic defence systems, prokaryotic Argonautes (pAgos) and CRISPR-Cas, detect and cleave invader nucleic acids using complementary guides and the nuclease activities of pAgo or Cas proteins. However, not all pAgos are active nucleases. A large clade of short pAgos bind nucleic acid guides but lack nuclease activity, suggesting a different mechanism of action. Here we investigate short pAgos associated with a putative effector nuclease, NbaAgo from Novosphingopyxis baekryungensis and CmeAgo from Cupriavidus metallidurans. We show that these pAgos form a heterodimeric complex with co-encoded effector nucleases (short prokaryotic Argonaute, DNase and RNase associated (SPARDA)). RNA-guided target DNA recognition unleashes the nuclease activity of SPARDA leading to indiscriminate collateral cleavage of DNA and RNA. Activation of SPARDA by plasmids or phages results in degradation of cellular DNA and cell death or dormancy, conferring target-specific population protection and expanding the range of known prokaryotic immune systems.},
}

@article {pmid38622229,
year = {2024},
author = {Xiang, X and Yang, H and Yuan, X and Dong, X and Mai, S and Zhang, Q and Chen, L and Cao, D and Chen, H and Guo, W and Li, L},
title = {CRISPR/Cas9-mediated editing of GmDWF1 brassinosteroid biosynthetic gene induces dwarfism in soybean.},
journal = {Plant cell reports},
volume = {43},
number = {5},
pages = {116},
pmid = {38622229},
issn = {1432-203X},
support = {32071926//The National Natural Science Foundation of China/ ; 32072087//The National Natural Science Foundation of China/ ; CAAS-OCRI-XKPY-202103//Agricultural Science and Technology Innovation Program of Chinese Academy of Agricultural Sciences/ ; },
mesh = {*Brassinosteroids/metabolism ; Glycine max/genetics ; CRISPR-Cas Systems/genetics ; Mutation/genetics ; *Arabidopsis/metabolism ; Gene Editing ; Gene Expression Regulation, Plant/genetics ; },
abstract = {The study on the GmDWF1-deficient mutant dwf1 showed that GmDWF1 plays a crucial role in determining soybean plant height and yield by influencing the biosynthesis of brassinosteroids. Soybean has not adopted the Green Revolution, such as reduced height for increased planting density, which have proven beneficial for cereal crops. Our research identified the soybean genes GmDWF1a and GmDWF1b, homologous to Arabidopsis AtDWF1, and found that they are widely expressed, especially in leaves, and linked to the cellular transport system, predominantly within the endoplasmic reticulum and intracellular vesicles. These genes are essential for the synthesis of brassinosteroids (BR). Single mutants of GmDWF1a and GmDWF1b, as well as double mutants of both genes generated through CRISPR/Cas9 genome editing, exhibit a dwarf phenotype. The single-gene mutant exhibits moderate dwarfism, while the double mutant shows more pronounced dwarfism. Despite the reduced stature, all types of mutants preserve their node count. Notably, field tests have shown that the single GmDWF1a mutant produced significantly more pods than wild-type plants. Spraying exogenous brassinolide (BL) can compensate for the loss in plant height induced by the decrease in endogenous BRs. Comparing transcriptome analyses of the GmDWF1a mutant and wild-type plants revealed a significant impact on the expression of many genes that influence soybean growth. Identifying the GmDWF1a and GmDWF1b genes could aid in the development of compact, densely planted soybean varieties, potentially boosting productivity.},
}

*Brassinosteroids/metabolism
Glycine max/genetics
CRISPR-Cas Systems/genetics
Mutation/genetics
*Arabidopsis/metabolism
Gene Editing
Gene Expression Regulation, Plant/genetics

@article {pmid38622170,
year = {2024},
author = {Lee, SH and Wang, CY and Li, IJ and Abe, G and Ota, KG},
title = {Exploring the origin of a unique mutant allele in twin-tail goldfish using CRISPR/Cas9 mutants.},
journal = {Scientific reports},
volume = {14},
number = {1},
pages = {8716},
pmid = {38622170},
issn = {2045-2322},
support = {112-2311-B-001-033//National Science and Technology Council/ ; },
mesh = {Animals ; *Goldfish/genetics ; Alleles ; *CRISPR-Cas Systems ; Biological Evolution ; Mutation ; Phenotype ; Animals, Domestic/genetics ; },
abstract = {Artificial selection has been widely applied to genetically fix rare phenotypic features in ornamental domesticated animals. For many of these animals, the mutated loci and alleles underlying rare phenotypes are known. However, few studies have explored whether these rare genetic mutations might have been fixed due to competition among related mutated alleles or if the fixation occurred due to contingent stochastic events. Here, we performed genetic crossing with twin-tail ornamental goldfish and CRISPR/Cas9-mutated goldfish to investigate why only a single mutated allele-chdS with a E127X stop codon (also called chdA[E127X])-gives rise to the twin-tail phenotype in the modern domesticated goldfish population. Two closely related chdS mutants were generated with CRISPR/Cas9 and compared with the E127X allele in F2 and F3 generations. Both of the CRISPR/Cas9-generated alleles were equivalent to the E127X allele in terms of penetrance/expressivity of the twin-tail phenotype and viability of carriers. These findings indicate that multiple truncating mutations could have produced viable twin-tail goldfish. Therefore, the absence of polymorphic alleles for the twin-tail phenotype in modern goldfish likely stems from stochastic elimination or a lack of competing alleles in the common ancestor. Our study is the first experimental comparison of a singular domestication-derived allele with CRISPR/Cas9-generated alleles to understand how genetic fixation of a unique genotype and phenotype may have occurred. Thus, our work may provide a conceptual framework for future investigations of rare evolutionary events in domesticated animals.},
}

Animals
*Goldfish/genetics
Alleles
*CRISPR-Cas Systems
Biological Evolution
Mutation
Phenotype
Animals, Domestic/genetics

@article {pmid38621214,
year = {2024},
author = {Srivastava, R and Davison, CW and Krull, AG and Entriken, SM and Zumbrock, A and Cortes Hidalgo, MD and Adair, KJ and Escherich, AM and Lara, JN and Neverman, EC and Hodnefield, M and McElligtot, E and Sandquist, EJ and Ogilvie, C and Lafontant, P and Essner, JJ},
title = {An Undergraduate Course in CRISPR/Cas9-Mediated Gene Editing in Zebrafish.},
journal = {Zebrafish},
volume = {21},
number = {2},
pages = {162-170},
doi = {10.1089/zeb.2023.0091},
pmid = {38621214},
issn = {1557-8542},
mesh = {Humans ; Animals ; *Gene Editing/methods ; *CRISPR-Cas Systems ; Zebrafish/genetics ; RNA, Guide, CRISPR-Cas Systems ; Students ; },
abstract = {We have developed a one-credit semester-long research experience for undergraduate students that involves the use of CRISPR/Cas9 to edit genes in zebrafish. The course is available to students at all stages of their undergraduate training and can be taken up to four times. Students select a gene of interest to edit as the basis of their semester-long project. To select a gene, exploration of developmental processes and human disease is encouraged. As part of the course, students use basic bioinformatic tools, design guide RNAs, inject zebrafish embryos, and analyze both the molecular consequences of gene editing and phenotypic outcomes. Over the 10 years we have offered the course, enrollment has grown from less than 10 students to more than 60 students per semester. Each year, we choose a different gene editing strategy to explore based on recent publications of gene editing methodologies. These have included making CRISPants, targeted integrations, and large gene deletions. In this study, we present how we structure the course and our assessment of the course over the past 3 years.},
}

Humans
Animals
*Gene Editing/methods
*CRISPR-Cas Systems
Zebrafish/genetics
RNA, Guide, CRISPR-Cas Systems
Students

@article {pmid38621205,
year = {2024},
author = {Davison, C and Harzman, H and Nicholson, J and Entriken, S and Mobley, K and Krull, A and Singhal, M and Skow, C and Matthews, N and Kopp, L and Gillette, B and Weide, TJ and Hukvari, JR and Stumpf, SCP and Feldmann, OM and McGrail, M and Srivastava, R and Essner, JJ},
title = {Tagging the tjp1a Gene in Zebrafish with Monomeric Red Fluorescent Protein Using Biotin Homology Arms.},
journal = {Zebrafish},
volume = {21},
number = {2},
pages = {191-197},
doi = {10.1089/zeb.2023.0096},
pmid = {38621205},
issn = {1557-8542},
mesh = {Animals ; *Zebrafish/genetics ; *CRISPR-Cas Systems ; Red Fluorescent Protein ; Biotin/genetics ; Endothelial Cells ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Tjp1a and other tight junction and adherens proteins play important roles in cell-cell adhesion, scaffolding, and forming seals between cells in epithelial and endothelial tissues. In this study, we labeled Tjp1a of zebrafish with the monomeric red fluorescent protein (mRFP) using CRISPR/Cas9-mediated targeted integration of biotin-labeled polymerase chain reaction (PCR) generated templates. Labeling Tjp1a with RFP allowed us to follow membrane and junctional dynamics of epithelial and endothelial cells throughout zebrafish embryo development. For targeted integration, we used short 35 bp homology arms on each side of the Cas9 genomic target site at the C-terminal of the coding sequence in tjp1a. Through PCR using 5' biotinylated primers containing the homology arms, we generated a double-stranded template for homology directed repair containing a flexible linker followed by RFP. Cas9 protein was complexed with the tjp1a gRNA before mixing with the repair template and microinjected into one-cell zebrafish embryos. We confirmed and recovered a precise integration allele at the desired site at the tjp1a C-terminus. Examination of fluorescence reveals RFP cell-cell junctional labeling using confocal imaging. We are currently using this stable tjp1a-mRFP[is86] line to examine the behavior and interactions between cells during vascular formation in zebrafish.},
}

Animals
*Zebrafish/genetics
*CRISPR-Cas Systems
Red Fluorescent Protein
Biotin/genetics
Endothelial Cells
RNA, Guide, CRISPR-Cas Systems

@article {pmid38616491,
year = {2024},
author = {Tang, X and Ren, Q and Yan, X and Zhang, R and Liu, L and Han, Q and Zheng, X and Qi, Y and Song, H and Zhang, Y},
title = {Boosting genome editing in plants with single transcript unit surrogate reporter systems.},
journal = {Plant communications},
volume = {},
number = {},
pages = {100921},
doi = {10.1016/j.xplc.2024.100921},
pmid = {38616491},
issn = {2590-3462},
abstract = {CRISPR-Cas-based genome editing holds immense promise for advancing plant genomics and crop enhancement. However, the challenge of low editing activity complicates the identification of edited events. In this study, we introduce multiple Single Transcript Unit Surrogate Reporter (STU-SR) systems to enhance the selection of genome-edited plants. These systems utilize the same sgRNAs designed for endogenous genes to edit reporter genes, establishing a direct link between reporter gene editing activity and that of endogenous genes. Various strategies are employed to restore functional reporter genes post-genome editing, including efficient single strand annealing (SSA) for homologous recombination in STU-SR-SSA systems. STU-SR-BE systems leverage base editing to reinstate the start codon, enriching C-to-T and A-to-G base editing events. Our results showcase the effectiveness of these STU-SR systems in enhancing genome editing events in monocot rice, encompassing Cas9 nuclease-based targeted mutagenesis, cytosine base editing, and adenine base editing. The systems exhibit compatibility with Cas9 variants, such as the PAM-less SpRY, and are demonstrated to boost genome editing in Brassica oleracea, a dicot vegetable crop. In summary, we have developed highly efficient and versatile STU-SR systems for enrichment of genome-edited plants.},
}

@article {pmid38583236,
year = {2024},
author = {Wong, BL and Mendoza, HG and Jacobsen, CS and Beal, PA},
title = {RNA sequences that direct selective ADAR editing from a SELEX library bearing 8-azanebularine.},
journal = {Bioorganic & medicinal chemistry},
volume = {104},
number = {},
pages = {117700},
doi = {10.1016/j.bmc.2024.117700},
pmid = {38583236},
issn = {1464-3391},
mesh = {Humans ; Adenosine ; *Adenosine Deaminase/metabolism ; Base Sequence ; *Purine Nucleosides ; *Ribonucleosides ; RNA, Double-Stranded ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Adenosine Deaminases Acting on RNA (ADARs) catalyze the deamination of adenosine to inosine in double-stranded RNA (dsRNA). ADARs' ability to recognize and edit dsRNA is dependent on local sequence context surrounding the edited adenosine and the length of the duplex. A deeper understanding of how editing efficiency is affected by mismatches, loops, and bulges around the editing site would aid in the development of therapeutic gRNAs for ADAR-mediated site-directed RNA editing (SDRE). Here, a SELEX (systematic evolution of ligands by exponential enrichment) approach was employed to identify dsRNA substrates that bind to the deaminase domain of human ADAR2 (hADAR2d) with high affinity. A library of single-stranded RNAs was hybridized with a fixed-sequence target strand containing the nucleoside analog 8-azanebularine that mimics the adenosine deamination transition state. The presence of this nucleoside analog in the library biased the screen to identify hit sequences compatible with adenosine deamination at the site of 8-azanebularine modification. SELEX also identified non-duplex structural elements that supported editing at the target site while inhibiting editing at bystander sites.},
}

Humans
Adenosine
*Adenosine Deaminase/metabolism
Base Sequence
*Purine Nucleosides
*Ribonucleosides
RNA, Double-Stranded
RNA, Guide, CRISPR-Cas Systems

@article {pmid38527624,
year = {2024},
author = {Piñón Hofbauer, J and Guttmann-Gruber, C and Wally, V and Sharma, A and Gratz, IK and Koller, U},
title = {Challenges and progress related to gene editing in rare skin diseases.},
journal = {Advanced drug delivery reviews},
volume = {208},
number = {},
pages = {115294},
doi = {10.1016/j.addr.2024.115294},
pmid = {38527624},
issn = {1872-8294},
mesh = {Humans ; *Gene Editing ; CRISPR-Cas Systems/genetics ; Quality of Life ; Skin ; *Skin Diseases/genetics/therapy ; },
abstract = {Genodermatoses represent a large group of inherited skin disorders encompassing clinically-heterogeneous conditions that manifest in the skin and other organs. Depending on disease variant, associated clinical manifestations and secondary complications can severely impact patients' quality of life and currently available treatments are transient and not curative. Multiple emerging approaches using CRISPR-based technologies offer promising prospects for therapy. Here, we explore current advances and challenges related to gene editing in rare skin diseases, including different strategies tailored to mutation type and structural organization of the affected gene, considerations for in vivo and ex vivo applications, the critical issue of delivery into the skin, and immune aspects of therapy. Against the backdrop of a landmark FDA approval for the first re-dosable gene replacement therapy for a rare genetic skin disorder, gene editing approaches are inching closer to the clinics and the possibility of a local permanent cure for patients affected by these disorders.},
}

Humans
*Gene Editing
CRISPR-Cas Systems/genetics
Quality of Life
Skin
*Skin Diseases/genetics/therapy

@article {pmid38517011,
year = {2024},
author = {Lau, MSH and Madika, A and Zhang, Y and Minton, NP},
title = {Parageobacillus thermoglucosidasius Strain Engineering Using a Theophylline Responsive RiboCas for Controlled Gene Expression.},
journal = {ACS synthetic biology},
volume = {13},
number = {4},
pages = {1237-1245},
doi = {10.1021/acssynbio.3c00735},
pmid = {38517011},
issn = {2161-5063},
mesh = {*CRISPR-Cas Systems/genetics ; *Theophylline ; Gene Editing ; Gene Expression ; *Bacillaceae ; },
abstract = {The relentless increase in atmospheric greenhouse gas concentrations as a consequence of the exploitation of fossil resources compels the adoption of sustainable routes to chemical and fuel manufacture based on biological fermentation processes. The use of thermophilic chassis in such processes is an attractive proposition; however, their effective exploitation will require improved genome editing tools. In the case of the industrially relevant chassis Parageobacillus thermoglucosidasius, CRISPR/Cas9-based gene editing has been demonstrated. The constitutive promoter used, however, accentuates the deleterious nature of Cas9, causing decreased transformation and low editing efficiencies, together with an increased likelihood of off-target effects or alternative mutations. Here, we rectify this issue by controlling the expression of Cas9 through the use of a synthetic riboswitch that is dependent on the nonmetabolized, nontoxic, and cheap inducer, theophylline. We demonstrate that the riboswitches are dose-dependent, allowing for controlled expression of the target gene. Through their use, we were then able to address the deleterious nature of Cas9 and produce an inducible system, RiboCas93. The benefits of RiboCas93 were demonstrated by increased transformation efficiency of the editing vectors, improved efficiency in mutant generation (100%), and a reduction of Cas9 toxicity, as indicated by a reduction in the number of single nucleotide polymorphisms (SNPs) observed. This new system provides a quick and efficient way to produce mutants in P. thermoglucosidasius.},
}

*CRISPR-Cas Systems/genetics
*Theophylline
Gene Editing
Gene Expression
*Bacillaceae

@article {pmid38508753,
year = {2024},
author = {Carvajal-Moreno, J and Wang, X and Hernandez, VA and Mondal, M and Zhao, X and Yalowich, JC and Elton, TS},
title = {Use of CRISPR/Cas9 with Homology-Directed Repair to Gene-Edit Topoisomerase IIβ in Human Leukemia K562 Cells: Generation of a Resistance Phenotype.},
journal = {The Journal of pharmacology and experimental therapeutics},
volume = {389},
number = {2},
pages = {186-196},
doi = {10.1124/jpet.123.002038},
pmid = {38508753},
issn = {1521-0103},
mesh = {Humans ; Etoposide/pharmacology ; K562 Cells ; DNA Topoisomerases, Type II/genetics/metabolism ; Mitoxantrone ; CRISPR-Cas Systems/genetics ; Codon, Nonsense ; *Antineoplastic Agents/pharmacology ; *Leukemia ; DNA ; Phenotype ; },
abstract = {DNA topoisomerase IIβ (TOP2β/180; 180 kDa) is a nuclear enzyme that regulates DNA topology by generation of short-lived DNA double-strand breaks, primarily during transcription. TOP2β/180 can be a target for DNA damage-stabilizing anticancer drugs, whose efficacy is often limited by chemoresistance. Our laboratory previously demonstrated reduced levels of TOP2β/180 (and the paralog TOP2α/170) in an acquired etoposide-resistant human leukemia (K562) clonal cell line, K/VP.5, in part due to overexpression of microRNA-9-3p/5p impacting post-transcriptional events. To evaluate the effect on drug sensitivity upon reduction/elimination of TOP2β/180, a premature stop codon was generated at the TOP2β/180 gene exon 19/intron 19 boundary (AGAA//GTAA→ATAG//GTAA) in parental K562 cells (which contain four TOP2β/180 alleles) by CRISPR/Cas9 editing with homology-directed repair to disrupt production of full-length TOP2β/180. Gene-edited clones were identified and verified by quantitative polymerase chain reaction and Sanger sequencing, respectively. Characterization of TOP2β/180 gene-edited clones, with one or all four TOP2β/180 alleles mutated, revealed partial or complete loss of TOP2β mRNA/protein, respectively. The loss of TOP2β/180 protein correlated with decreased (2-{4-[(7-chloro-2-quinoxalinyl)oxy]phenoxy}p
ropionic acid)-induced DNA damage and partial resistance in growth inhibition assays. Partial resistance to mitoxantrone was also noted in the gene-edited clone with all four TOP2β/180 alleles modified. No cross-resistance to etoposide or mAMSA was noted in the gene-edited clones. Results demonstrated the role of TOP2β/180 in drug sensitivity/resistance in K562 cells and revealed differential paralog activity of TOP2-targeted agents. SIGNIFICANCE STATEMENT: Data indicated that CRISPR/Cas9 editing of the exon 19/intron 19 boundary in the TOP2β/180 gene to introduce a premature stop codon resulted in partial to complete disruption of TOP2β/180 expression in human leukemia (K562) cells depending on the number of edited alleles. Edited clones were partially resistant to mitoxantrone and XK469, while lacking resistance to etoposide and mAMSA. Results demonstrated the import of TOP2β/180 in drug sensitivity/resistance in K562 cells and revealed differential paralog activity of TOP2-targeted agents.},
}

Humans
Etoposide/pharmacology
K562 Cells
DNA Topoisomerases, Type II/genetics/metabolism
Mitoxantrone
CRISPR-Cas Systems/genetics
Codon, Nonsense
*Antineoplastic Agents/pharmacology
*Leukemia
DNA
Phenotype

@article {pmid38493007,
year = {2024},
author = {Dimitrievska, M and Bansal, D and Vitale, M and Strouboulis, J and Miccio, A and Nicolaides, KH and El Hoss, S and Shangaris, P and Jacków-Malinowska, J},
title = {Revolutionising healing: Gene Editing's breakthrough against sickle cell disease.},
journal = {Blood reviews},
volume = {65},
number = {},
pages = {101185},
doi = {10.1016/j.blre.2024.101185},
pmid = {38493007},
issn = {1532-1681},
mesh = {Humans ; Gene Editing/methods ; CRISPR-Cas Systems ; *Anemia, Sickle Cell/genetics/therapy ; *Hemoglobinopathies/genetics ; Fetal Hemoglobin/genetics ; },
abstract = {Recent advancements in gene editing illuminate new potential therapeutic approaches for Sickle Cell Disease (SCD), a debilitating monogenic disorder caused by a point mutation in the β-globin gene. Despite the availability of several FDA-approved medications for symptomatic relief, allogeneic hematopoietic stem cell transplantation (HSCT) remains the sole curative option, underscoring a persistent need for novel treatments. This review delves into the growing field of gene editing, particularly the extensive research focused on curing haemoglobinopathies like SCD. We examine the use of techniques such as CRISPR-Cas9 and homology-directed repair, base editing, and prime editing to either correct the pathogenic variant into a non-pathogenic or wild-type one or augment fetal haemoglobin (HbF) production. The article elucidates ways to optimize these tools for efficacious gene editing with minimal off-target effects and offers insights into their effective delivery into cells. Furthermore, we explore clinical trials involving alternative SCD treatment strategies, such as LentiGlobin therapy and autologous HSCT, distilling the current findings. This review consolidates vital information for the clinical translation of gene editing for SCD, providing strategic insights for investigators eager to further the development of gene editing for SCD.},
}

Humans
Gene Editing/methods
CRISPR-Cas Systems
*Anemia, Sickle Cell/genetics/therapy
*Hemoglobinopathies/genetics
Fetal Hemoglobin/genetics

@article {pmid38479385,
year = {2024},
author = {Ma, X and Yin, J and Qiao, L and Wan, H and Liu, X and Zhou, Y and Wu, J and Niu, L and Wu, M and Wang, X and Ye, H},
title = {A programmable targeted protein-degradation platform for versatile applications in mammalian cells and mice.},
journal = {Molecular cell},
volume = {84},
number = {8},
pages = {1585-1600.e7},
doi = {10.1016/j.molcel.2024.02.019},
pmid = {38479385},
issn = {1097-4164},
mesh = {Mice ; Animals ; *CRISPR-Cas Systems ; *Gene Editing ; CRISPR-Associated Protein 9/genetics/metabolism ; Proteins/metabolism ; Proteolysis ; Mammals/metabolism ; },
abstract = {Myriad physiological and pathogenic processes are governed by protein levels and modifications. Controlled protein activity perturbation is essential to studying protein function in cells and animals. Based on Trim-Away technology, we screened for truncation variants of E3 ubiquitinase Trim21 with elevated efficiency (ΔTrim21) and developed multiple ΔTrim21-based targeted protein-degradation systems (ΔTrim-TPD) that can be transfected into host cells. Three ΔTrim-TPD variants are developed to enable chemical and light-triggered programmable activation of TPD in cells and animals. Specifically, we used ΔTrim-TPD for (1) red-light-triggered inhibition of HSV-1 virus proliferation by degrading the packaging protein gD, (2) for chemical-triggered control of the activity of Cas9/dCas9 protein for gene editing, and (3) for blue-light-triggered degradation of two tumor-associated proteins for spatiotemporal inhibition of melanoma tumor growth in mice. Our study demonstrates that multiple ΔTrim21-based controllable TPD systems provide powerful tools for basic biology research and highlight their potential biomedical applications.},
}

Mice
Animals
*CRISPR-Cas Systems
*Gene Editing
CRISPR-Associated Protein 9/genetics/metabolism
Proteins/metabolism
Proteolysis
Mammals/metabolism

@article {pmid38460553,
year = {2024},
author = {Ren, QW and Liu, TY and Lan, HJ and Li, ZC and Huang, MJ and Zhao, YT and Chen, Y and Liao, LN and Ma, XH and Liu, JZ},
title = {Partially knocking out NtPDK1a/1b/1c/1d simultaneously in Nicotiana tabacum using CRISPR/CAS9 technology results in auxin-related developmental defects.},
journal = {Plant science : an international journal of experimental plant biology},
volume = {343},
number = {},
pages = {112057},
doi = {10.1016/j.plantsci.2024.112057},
pmid = {38460553},
issn = {1873-2259},
mesh = {*Nicotiana/genetics ; *Arabidopsis/genetics/metabolism ; Indoleacetic Acids/metabolism ; CRISPR-Cas Systems ; Protein Kinases/genetics ; Plants/metabolism ; Gene Expression Regulation, Plant ; },
abstract = {The eukaryotic AGC protein kinase subfamily (protein kinase A/ protein kinase G/ protein kinase C-family) is involved in regulating numerous biological processes across kingdoms, including growth and development, and apoptosis. PDK1(3-phosphoinositide-dependent protein kinase 1) is a conserved serine/threonine kinase in eukaryotes, which is both a member of AGC kinase and a major regulator of many other downstream AGC protein kinase family members. Although extensively investigated in model plant Arabidopsis, detailed reports for tobacco PDK1s have been limited. To better understand the functions of PDK1s in tobacco, CRISPR/CAS9 transgenic lines were generated in tetraploid N. tabacum, cv. Samsun (NN) with 5-7 of the 8 copies of 4 homologous PDK1 genes in tobacco genome (NtPDK1a/1b/1c/1d homologs) simultaneously knocked out. Numerous developmental defects were observed in these NtPDK1a/1b/1c/1d CRISPR/CAS9 lines, including cotyledon fusion leaf shrinkage, uneven distribution of leaf veins, convex veins, root growth retardation, and reduced fertility, all of which reminiscence of impaired polar auxin transport. The severity of these defects was correlated with the number of knocked out alleles of NtPDK1a/1b/1c/1d. Consistent with the observation in Arabidopsis, it was found that the polar auxin transport, and not auxin biosynthesis, was significantly compromised in these knockout lines compared with the wild type tobacco plants. The fact that no homozygous plant with all 8 NtPDK1a/1b/1c/1d alleles being knocked out suggested that knocking out 8 alleles of NtPDK1a/1b/1c/1d could be lethal. In conclusion, our results indicated that NtPDK1s are versatile AGC kinases that participate in regulation of tobacco growth and development via modulating polar auxin transport. Our results also indicated that CRISPR/CAS9 technology is a powerful tool in resolving gene redundancy in polyploidy plants.},
}

*Nicotiana/genetics
*Arabidopsis/genetics/metabolism
Indoleacetic Acids/metabolism
CRISPR-Cas Systems
Protein Kinases/genetics
Plants/metabolism
Gene Expression Regulation, Plant

@article {pmid38613390,
year = {2024},
author = {Cardiff, RAL and Faulkner, ID and Beall, JG and Carothers, JM and Zalatan, JG},
title = {CRISPR-Cas tools for simultaneous transcription & translation control in bacteria.},
journal = {Nucleic acids research},
volume = {},
number = {},
pages = {},
doi = {10.1093/nar/gkae275},
pmid = {38613390},
issn = {1362-4962},
support = {MCB 2225632//National Science Foundation/ ; },
abstract = {Robust control over gene translation at arbitrary mRNA targets is an outstanding challenge in microbial synthetic biology. The development of tools that can regulate translation will greatly expand our ability to precisely control genes across the genome. In Escherichia coli, most genes are contained in multi-gene operons, which are subject to polar effects where targeting one gene for repression leads to silencing of other genes in the same operon. These effects pose a challenge for independently regulating individual genes in multi-gene operons. Here, we use CRISPR-dCas13 to address this challenge. We find dCas13-mediated repression exhibits up to 6-fold lower polar effects compared to dCas9. We then show that we can selectively activate single genes in a synthetic multi-gene operon by coupling dCas9 transcriptional activation of an operon with dCas13 translational repression of individual genes within the operon. We also show that dCas13 and dCas9 can be multiplexed for improved biosynthesis of a medically-relevant human milk oligosaccharide. Taken together, our findings suggest that combining transcriptional and translational control can access effects that are difficult to achieve with either mode independently. These combined tools for gene regulation will expand our abilities to precisely engineer bacteria for biotechnology and perform systematic genetic screens.},
}

@article {pmid38612794,
year = {2024},
author = {Stahl, F and Evert, BO and Han, X and Breuer, P and Wüllner, U},
title = {Spinocerebellar Ataxia Type 3 Pathophysiology-Implications for Translational Research and Clinical Studies.},
journal = {International journal of molecular sciences},
volume = {25},
number = {7},
pages = {},
pmid = {38612794},
issn = {1422-0067},
mesh = {Humans ; Animals ; *Machado-Joseph Disease/genetics ; Translational Research, Biomedical ; *Spinocerebellar Ataxias/genetics ; Translational Science, Biomedical ; Animals, Genetically Modified ; },
abstract = {The spinocerebellar ataxias (SCA) comprise a group of inherited neurodegenerative diseases. Machado-Joseph Disease (MJD) or spinocerebellar ataxia 3 (SCA3) is the most common autosomal dominant form, caused by the expansion of CAG repeats within the ataxin-3 (ATXN3) gene. This mutation results in the expression of an abnormal protein containing long polyglutamine (polyQ) stretches that confers a toxic gain of function and leads to misfolding and aggregation of ATXN3 in neurons. As a result of the neurodegenerative process, SCA3 patients are severely disabled and die prematurely. Several screening approaches, e.g., druggable genome-wide and drug library screenings have been performed, focussing on the reduction in stably overexpressed ATXN3(polyQ) protein and improvement in the resultant toxicity. Transgenic overexpression models of toxic ATXN3, however, missed potential modulators of endogenous ATXN3 regulation. In another approach to identify modifiers of endogenous ATXN3 expression using a CRISPR/Cas9-modified SK-N-SH wild-type cell line with a GFP-T2A-luciferase (LUC) cassette under the control of the endogenous ATXN3 promotor, four statins were identified as potential activators of expression. We here provide an overview of the high throughput screening approaches yet performed to find compounds or genomic modifiers of ATXN3(polyQ) toxicity in different SCA3 model organisms and cell lines to ameliorate and halt SCA3 progression in patients. Furthermore, the putative role of cholesterol in neurodegenerative diseases (NDDs) in general and SCA3 in particular is discussed.},
}

Humans
Animals
*Machado-Joseph Disease/genetics
Translational Research, Biomedical
*Spinocerebellar Ataxias/genetics
Translational Science, Biomedical
Animals, Genetically Modified

@article {pmid38609947,
year = {2024},
author = {Im, SH and Jang, M and Park, JH and Chung, HJ},
title = {Finely tuned ionizable lipid nanoparticles for CRISPR/Cas9 ribonucleoprotein delivery and gene editing.},
journal = {Journal of nanobiotechnology},
volume = {22},
number = {1},
pages = {175},
pmid = {38609947},
issn = {1477-3155},
support = {2021R1A2C2011763//National Research Foundation of Korea/ ; 2021R1A2C2011763//National Research Foundation of Korea/ ; HI22C2010//Ministry of Health and Welfare/ ; },
mesh = {*Gene Editing ; *CRISPR-Cas Systems ; Cell Line ; Ribonucleoproteins/genetics ; *Liposomes ; *Nanoparticles ; },
abstract = {Nonviral delivery of the CRISPR/Cas9 system provides great benefits for in vivo gene therapy due to the low risk of side effects. However, in vivo gene editing by delivering the Cas9 ribonucleoprotein (RNP) is challenging due to the poor delivery into target tissues and cells. Here, we introduce an effective delivery method for the CRISPR/Cas9 RNPs by finely tuning the formulation of ionizable lipid nanoparticles. The LNPs delivering CRISPR/Cas9 RNPs (CrLNPs) are demonstrated to induce gene editing with high efficiencies in various cancer cell lines in vitro. Furthermore, we show that CrLNPs can be delivered into tumor tissues with high efficiency, as well as induce significant gene editing in vivo. The current study presents an effective platform for nonviral delivery of the CRISPR/Cas9 system that can be applied as an in vivo gene editing therapeutic for treating various diseases such as cancer and genetic disorders.},
}

*Gene Editing
*CRISPR-Cas Systems
Cell Line
Ribonucleoproteins/genetics
*Liposomes
*Nanoparticles

@article {pmid38609863,
year = {2024},
author = {Chen, X and Zhong, S and Zhan, Y and Zhang, X},
title = {CRISPR-Cas9 applications in T cells and adoptive T cell therapies.},
journal = {Cellular & molecular biology letters},
volume = {29},
number = {1},
pages = {52},
pmid = {38609863},
issn = {1689-1392},
support = {82203862//National Natural Science Foundation of China/ ; 222300420344//Natural Science Foundation of Henan Province/ ; SBGJ202102140//Medical Science Technology Program of Henan/ ; LHGJ20200273//Medical Science Technology Program of Henan/ ; },
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *T-Lymphocytes ; Cell Differentiation ; Inflammation ; Lymphocyte Activation ; },
abstract = {T cell immunity is central to contemporary cancer and autoimmune therapies, encompassing immune checkpoint blockade and adoptive T cell therapies. Their diverse characteristics can be reprogrammed by different immune challenges dependent on antigen stimulation levels, metabolic conditions, and the degree of inflammation. T cell-based therapeutic strategies are gaining widespread adoption in oncology and treating inflammatory conditions. Emerging researches reveal that clustered regularly interspaced palindromic repeats-associated protein 9 (CRISPR-Cas9) genome editing has enabled T cells to be more adaptable to specific microenvironments, opening the door to advanced T cell therapies in preclinical and clinical trials. CRISPR-Cas9 can edit both primary T cells and engineered T cells, including CAR-T and TCR-T, in vivo and in vitro to regulate T cell differentiation and activation states. This review first provides a comprehensive summary of the role of CRISPR-Cas9 in T cells and its applications in preclinical and clinical studies for T cell-based therapies. We also explore the application of CRISPR screen high-throughput technology in editing T cells and anticipate the current limitations of CRISPR-Cas9, including off-target effects and delivery challenges, and envisioned improvements in related technologies for disease screening, diagnosis, and treatment.},
}

Humans
*CRISPR-Cas Systems/genetics
*T-Lymphocytes
Cell Differentiation
Inflammation
Lymphocyte Activation

@article {pmid38609574,
year = {2024},
author = {De Castro, V and Galaine, J and Loyon, R and Godet, Y},
title = {CRISPR-Cas gene knockouts to optimize engineered T cells for cancer immunotherapy.},
journal = {Cancer gene therapy},
volume = {},
number = {},
pages = {},
pmid = {38609574},
issn = {1476-5500},
support = {2022-0047//Ligue Contre le Cancer/ ; },
abstract = {While CAR-T and tgTCR-T therapies have exhibited noteworthy and promising outcomes in hematologic and solid tumors respectively, a set of distinct challenges remains. Consequently, the quest for novel strategies has become imperative to safeguard and more effectively release the full functions of engineered T cells. These factors are intricately linked to the success of adoptive cell therapy. Recently, CRISPR-based technologies have emerged as a major breakthrough for maintaining T cell functions. These technologies have allowed the discovery of T cells' negative regulators such as specific cell-surface receptors, cell-signaling proteins, and transcription factors that are involved in the development or maintenance of T cell dysfunction. By employing a CRISPR-genic invalidation approach to target these negative regulators, it has become possible to prevent the emergence of hypofunctional T cells. This review revisits the establishment of the dysfunctional profile of T cells before delving into a comprehensive summary of recent CRISPR-gene invalidations, with each invalidation contributing to the enhancement of engineered T cells' antitumor capacities. The narrative unfolds as we explore how these advancements were discovered and identified, marking a significant advancement in the pursuit of superior adoptive cell therapy.},
}

@article {pmid38609487,
year = {2024},
author = {Naveed, M and Tahir, F and Aziz, T and Waseem, M and Makhdoom, SI and Ali, N and Alharbi, M and Albekairi, TH and Alasmari, AF},
title = {Molecular identification of Proteus mirabilis, Vibrio species leading to CRISPR-Cas9 modification of tcpA and UreC genes causing cholera and UTI.},
journal = {Scientific reports},
volume = {14},
number = {1},
pages = {8563},
pmid = {38609487},
issn = {2045-2322},
mesh = {Humans ; *Cholera ; Proteus mirabilis/genetics ; Cadmium/toxicity ; CRISPR-Cas Systems/genetics ; RNA, Ribosomal, 16S ; Wastewater ; RNA, Guide, CRISPR-Cas Systems ; *Vibrio/genetics ; *Urinary Tract Infections ; },
abstract = {Heavy metal accumulation increases rapidly in the environment due to anthropogenic activities and industrialization. The leather and surgical industry produces many contaminants containing heavy metals. Cadmium, a prominent contaminant, is linked to severe health risks, notably kidney and liver damage, especially among individuals exposed to contaminated wastewater. This study aims to leverage the natural cadmium resistance mechanisms in bacteria for bioaccumulation purposes. The industrial wastewater samples, characterized by an alarming cadmium concentration of 29.6 ppm, 52 ppm, and 76.4 ppm-far exceeding the recommended limit of 0.003 ppm-were subjected to screening for cadmium-resistant bacteria using cadmium-supplemented media with CdCl2. 16S rRNA characterization identified Vibrio cholerae and Proteus mirabilis as cadmium-resistant bacteria in the collected samples. Subsequently, the cadmium resistance-associated cadA gene was successfully amplified in Vibrio species and Proteus mirabilis, revealing a product size of 623 bp. Further analysis of the identified bacteria included the examination of virulent genes, specifically the tcpA gene (472 bp) associated with cholera and the UreC gene (317 bp) linked to urinary tract infections. To enhance the bioaccumulation of cadmium, the study proposes the potential suppression of virulent gene expression through in-silico gene-editing tools such as CRISPR-Cas9. A total of 27 gRNAs were generated for UreC, with five selected for expression. Similarly, 42 gRNA sequences were generated for tcpA, with eight chosen for expression analysis. The selected gRNAs were integrated into the lentiCRISPR v2 expression vector. This strategic approach aims to facilitate precise gene editing of disease-causing genes (tcpA and UreC) within the bacterial genome. In conclusion, this study underscores the potential utility of Vibrio species and Proteus mirabilis as effective candidates for the removal of cadmium from industrial wastewater, offering insights for future environmental remediation strategies.},
}

Humans
*Cholera
Proteus mirabilis/genetics
Cadmium/toxicity
CRISPR-Cas Systems/genetics
RNA, Ribosomal, 16S
Wastewater
RNA, Guide, CRISPR-Cas Systems
*Vibrio/genetics
*Urinary Tract Infections

@article {pmid38609352,
year = {2024},
author = {McLean, ZL and Gao, D and Correia, K and Roy, JCL and Shibata, S and Farnum, IN and Valdepenas-Mellor, Z and Kovalenko, M and Rapuru, M and Morini, E and Ruliera, J and Gillis, T and Lucente, D and Kleinstiver, BP and Lee, JM and MacDonald, ME and Wheeler, VC and Mouro Pinto, R and Gusella, JF},
title = {Splice modulators target PMS1 to reduce somatic expansion of the Huntington's disease-associated CAG repeat.},
journal = {Nature communications},
volume = {15},
number = {1},
pages = {3182},
pmid = {38609352},
issn = {2041-1723},
support = {NS091161//U.S. Department of Health & Human Services | NIH | National Institute of Neurological Disorders and Stroke (NINDS)/ ; NS105709//U.S. Department of Health & Human Services | NIH | National Institute of Neurological Disorders and Stroke (NINDS)/ ; NS119471//U.S. Department of Health & Human Services | NIH | National Institute of Neurological Disorders and Stroke (NINDS)/ ; NS049206//U.S. Department of Health & Human Services | NIH | National Institute of Neurological Disorders and Stroke (NINDS)/ ; NS126420//U.S. Department of Health & Human Services | NIH | National Institute of Neurological Disorders and Stroke (NINDS)/ ; DP2-CA281401//U.S. Department of Health & Human Services | NIH | National Cancer Institute (NCI)/ ; },
mesh = {Humans ; *Huntington Disease/genetics ; Exons/genetics ; Gene Expression Profiling ; Heterozygote ; Homozygote ; MutL Proteins ; Neoplasm Proteins ; },
abstract = {Huntington's disease (HD) is a dominant neurological disorder caused by an expanded HTT exon 1 CAG repeat that lengthens huntingtin's polyglutamine tract. Lowering mutant huntingtin has been proposed for treating HD, but genetic modifiers implicate somatic CAG repeat expansion as the driver of onset. We find that branaplam and risdiplam, small molecule splice modulators that lower huntingtin by promoting HTT pseudoexon inclusion, also decrease expansion of an unstable HTT exon 1 CAG repeat in an engineered cell model. Targeted CRISPR-Cas9 editing shows this effect is not due to huntingtin lowering, pointing instead to pseudoexon inclusion in PMS1. Homozygous but not heterozygous inactivation of PMS1 also reduces CAG repeat expansion, supporting PMS1 as a genetic modifier of HD and a potential target for therapeutic intervention. Although splice modulation provides one strategy, genome-wide transcriptomics also emphasize consideration of cell-type specific effects and polymorphic variation at both target and off-target sites.},
}

Humans
*Huntington Disease/genetics
Exons/genetics
Gene Expression Profiling
Heterozygote
Homozygote
MutL Proteins
Neoplasm Proteins

@article {pmid38579141,
year = {2024},
author = {Moon, SY and Zhang, D and Chen, SC and Lamey, TM and Thompson, JA and McLaren, TL and Chen, FK and McLenachan, S},
title = {Rapid Variant Pathogenicity Analysis by CRISPR Activation of CRB1 Gene Expression in Patient-Derived Fibroblasts.},
journal = {The CRISPR journal},
volume = {7},
number = {2},
pages = {100-110},
doi = {10.1089/crispr.2023.0065},
pmid = {38579141},
issn = {2573-1602},
mesh = {Humans ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Reactive Oxygen Species/metabolism ; Virulence ; *CRISPR-Cas Systems ; Gene Editing ; Gene Expression ; Eye Proteins/genetics/metabolism ; Membrane Proteins/genetics ; Nerve Tissue Proteins/genetics/metabolism ; },
abstract = {Inherited retinal diseases (IRDs) are a heterogeneous group of blinding genetic disorders caused by pathogenic variants in genes expressed in the retina. In this study, we sought to develop a method for rapid evaluation of IRD gene variant pathogenicity by inducing expression of retinal genes in patient-derived fibroblasts using CRISPR-activation (CRISPRa). We demonstrate CRISPRa of CRB1 expression in fibroblasts derived from patients with retinitis pigmentosa, enabling investigation of pathogenic mechanisms associated with specific variants. We show the CRB1 c.4005 + 1G>A variant caused exon 11 skipping in CRISPR-activated fibroblasts and retinal organoids (ROs) derived from the same RP12 patient. The c.652 + 5G>C variant was shown to enhance exon 2 skipping in CRISPR-activated fibroblasts and differentially affected CRB1 isoform expression in fibroblasts and ROs. Our study demonstrates an accessible platform for transcript screening of IRD gene variants in patient-derived fibroblasts, which can potentially be applied for rapid pathogenicity assessments of any gene variant.},
}

Humans
*Clustered Regularly Interspaced Short Palindromic Repeats
Reactive Oxygen Species/metabolism
Virulence
*CRISPR-Cas Systems
Gene Editing
Gene Expression
Eye Proteins/genetics/metabolism
Membrane Proteins/genetics
Nerve Tissue Proteins/genetics/metabolism

@article {pmid38578788,
year = {2024},
author = {Ma, S and Ni, X and Chen, S and Qiao, X and Xu, X and Chen, W and Champer, J and Huang, J},
title = {A small-molecule approach to restore female sterility phenotype targeted by a homing suppression gene drive in the fruit pest Drosophila suzukii.},
journal = {PLoS genetics},
volume = {20},
number = {4},
pages = {e1011226},
pmid = {38578788},
issn = {1553-7404},
mesh = {Female ; Animals ; Humans ; Drosophila/genetics ; Drosophila melanogaster/genetics ; *Gene Drive Technology ; *Infertility, Female/genetics ; CRISPR-Cas Systems ; Fruit ; RNA, Guide, CRISPR-Cas Systems ; Phenotype ; },
abstract = {CRISPR-based gene drives offer promising prospects for controlling disease-transmitting vectors and agricultural pests. A significant challenge for successful suppression-type drive is the rapid evolution of resistance alleles. One approach to mitigate the development of resistance involves targeting functionally constrained regions using multiple gRNAs. In this study, we constructed a 3-gRNA homing gene drive system targeting the recessive female fertility gene Tyrosine decarboxylase 2 (Tdc2) in Drosophila suzukii, a notorious fruit pest. Our investigation revealed only a low level of homing in the germline, but feeding octopamine restored the egg-laying defects in Tdc2 mutant females, allowing easier line maintenance than for other suppression drive targets. We tested the effectiveness of a similar system in Drosophila melanogaster and constructed additional split drive systems by introducing promoter-Cas9 transgenes to improve homing efficiency. Our findings show that genetic polymorphisms in wild populations may limit the spread of gene drive alleles, and the position effect profoundly influences Cas9 activity. Furthermore, this study highlights the potential of conditionally rescuing the female infertility caused by the gene drive, offering a valuable tool for the industrial-scale production of gene drive transgenic insects.},
}

Female
Animals
Humans
Drosophila/genetics
Drosophila melanogaster/genetics
*Gene Drive Technology
*Infertility, Female/genetics
CRISPR-Cas Systems
Fruit
RNA, Guide, CRISPR-Cas Systems
Phenotype

@article {pmid38564198,
year = {2024},
author = {Levrier, G},
title = {Making the Search for Genome Editing Tortured Phrases a Collective Effort.},
journal = {The CRISPR journal},
volume = {7},
number = {2},
pages = {72},
doi = {10.1089/crispr.2024.0015},
pmid = {38564198},
issn = {2573-1602},
mesh = {*Gene Editing ; *CRISPR-Cas Systems ; },
}

*Gene Editing
*CRISPR-Cas Systems

@article {pmid38564197,
year = {2024},
author = {Lima, L and Berni, M and Mota, J and Bressan, D and Julio, A and Cavalcante, R and Macias, V and Li, Z and Rasgon, JL and Bier, E and Araujo, H},
title = {Gene Editing in the Chagas Disease Vector Rhodnius prolixus by Cas9-Mediated ReMOT Control.},
journal = {The CRISPR journal},
volume = {7},
number = {2},
pages = {88-99},
doi = {10.1089/crispr.2023.0076},
pmid = {38564197},
issn = {2573-1602},
mesh = {Animals ; Female ; Gene Editing/methods ; *Rhodnius/genetics/parasitology ; CRISPR-Cas Systems ; Insect Vectors/parasitology ; *Chagas Disease/genetics/parasitology ; },
abstract = {Rhodnius prolixus is currently the model vector of choice for studying Chagas disease transmission, a debilitating disease caused by Trypanosoma cruzi parasites. However, transgenesis and gene editing protocols to advance the field are still lacking. Here, we tested protocols for the maternal delivery of CRISPR-Cas9 (clustered regularly spaced palindromic repeats/Cas-9 associated) elements to developing R. prolixus oocytes and strategies for the identification of insertions and deletions (indels) in target loci of resulting gene-edited generation zero (G0) nymphs. We demonstrate successful gene editing of the eye color markers Rp-scarlet and Rp-white, and the cuticle color marker Rp-yellow, with highest effectiveness obtained using Receptor-Mediated Ovary Transduction of Cargo (ReMOT Control) with the ovary-targeting BtKV ligand. These results provide proof of concepts for generating somatic mutations in R. prolixus and potentially for generating germ line-edited lines in triatomines, laying the foundation for gene editing protocols that could lead to the development of novel control strategies for vectors of Chagas disease.},
}

Animals
Female
Gene Editing/methods
*Rhodnius/genetics/parasitology
CRISPR-Cas Systems
Insect Vectors/parasitology
*Chagas Disease/genetics/parasitology

@article {pmid38447647,
year = {2024},
author = {Wang, W and Wang, S and Meng, X and Zhao, Y and Li, N and Wang, T and Feng, N and Yan, F and Xia, X},
title = {A virus-like particle candidate vaccine based on CRISPR/Cas9 gene editing technology elicits broad-spectrum protection against SARS-CoV-2.},
journal = {Antiviral research},
volume = {225},
number = {},
pages = {105854},
doi = {10.1016/j.antiviral.2024.105854},
pmid = {38447647},
issn = {1872-9096},
mesh = {Mice ; Animals ; Humans ; SARS-CoV-2 ; *Vaccines, Virus-Like Particle ; COVID-19 Vaccines ; CRISPR-Cas Systems ; Gene Editing ; *COVID-19 ; Antibodies, Viral ; Antibodies, Neutralizing ; },
abstract = {The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with frequent mutations has seriously damaged the effectiveness of the 2019 coronavirus disease (COVID-19) vaccine. There is an urgent need to develop a broad-spectrum vaccine while elucidating the underlying immune mechanisms. Here, we developed a SARS-CoV-2 virus-like particles (VLPs) vaccine based on the Canarypox-virus vector (ALVAC-VLPs) using CRISPR/Cas9. Immunization with ALVAC-VLPs showed the effectively induce SARS-CoV-2 specific T and B cell responses to resist the lethal challenge of mouse adaptive strains. Notably, ALVAC-VLPs conferred protection in golden hamsters against SARS-CoV-2 Wuhan-Hu-1 (wild-type, WT) and variants (Beta, Delta, Omicron BA.1, and BA.2), as evidenced by the prevention of weight loss, reduction in lung and turbinate tissue damage, and decreased viral load. Further investigation into the mechanism of immune response induced by ALVAC-VLPs revealed that toll-like receptor 4 (TLR4) mediates the recruitment of dendritic cells (DCs) to secondary lymphoid organs, thereby initiating follicle assisted T (Tfh) cell differentiation, the proliferation of germinal center (GC) B cells and plasma cell production. These findings demonstrate the immunogenicity and efficacy of the safe ALVAC-VLPs vaccine against SARS-CoV-2 and provide valuable insight into the development of COVID-19 vaccine strategies.},
}

Mice
Animals
Humans
SARS-CoV-2
*Vaccines, Virus-Like Particle
COVID-19 Vaccines
CRISPR-Cas Systems
Gene Editing
*COVID-19
Antibodies, Viral
Antibodies, Neutralizing

@article {pmid38366553,
year = {2024},
author = {Zhang, T and Zhao, F and Li, J and Sun, X and Zhang, X and Wang, H and Fan, P and Lai, L and Li, Z and Sui, T},
title = {Programmable RNA 5-methylcytosine (m5C) modification of cellular RNAs by dCasRx conjugated methyltransferase and demethylase.},
journal = {Nucleic acids research},
volume = {52},
number = {6},
pages = {2776-2791},
pmid = {38366553},
issn = {1362-4962},
support = {32101226//National Natural Science Foundation of China/ ; YESS20210189//China Association for Science and Technology/ ; },
mesh = {Animals ; Mice ; *5-Methylcytosine/metabolism ; *Methylation ; *Methyltransferases/genetics/metabolism ; RNA, Transfer/metabolism ; *Genetic Techniques ; CRISPR-Cas Systems ; Humans ; *RNA Editing ; },
abstract = {5-Methylcytosine (m5C), an abundant RNA modification, plays a crucial role in regulating RNA fate and gene expression. While recent progress has been made in understanding the biological roles of m5C, the inability to introduce m5C at specific sites within transcripts has hindered efforts to elucidate direct links between specific m5C and phenotypic outcomes. Here, we developed a CRISPR-Cas13d-based tool, named reengineered m5C modification system (termed 'RCMS'), for targeted m5C methylation and demethylation in specific transcripts. The RCMS editors consist of a nuclear-localized dCasRx conjugated to either a methyltransferase, NSUN2/NSUN6, or a demethylase, the catalytic domain of mouse Tet2 (ten-eleven translocation 2), enabling the manipulation of methylation events at precise m5C sites. We demonstrate that the RCMS editors can direct site-specific m5C incorporation and demethylation. Furthermore, we confirm their effectiveness in modulating m5C levels within transfer RNAs and their ability to induce changes in transcript abundance and cell proliferation through m5C-mediated mechanisms. These findings collectively establish RCMS editors as a focused epitranscriptome engineering tool, facilitating the identification of individual m5C alterations and their consequential effects.},
}

Animals
Mice
*5-Methylcytosine/metabolism
*Methylation
*Methyltransferases/genetics/metabolism
RNA, Transfer/metabolism
*Genetic Techniques
CRISPR-Cas Systems
Humans
*RNA Editing

@article {pmid38149939,
year = {2024},
author = {Guri-Lamce, I and AlRokh, Y and Kim, Y and Maeshima, R and Graham, C and Hart, SL and McGrath, JA and Jacków-Malinowska, J},
title = {Topical gene editing therapeutics using lipid nanoparticles: 'gene creams' for genetic skin diseases?.},
journal = {The British journal of dermatology},
volume = {190},
number = {5},
pages = {617-627},
doi = {10.1093/bjd/ljad528},
pmid = {38149939},
issn = {1365-2133},
mesh = {Humans ; Gene Editing ; CRISPR-Cas Systems ; Gene Transfer Techniques ; *Nanoparticles ; *Skin Diseases, Genetic/genetics ; *Liposomes ; },
abstract = {Patients living with inherited skin diseases have benefited from recent advances in DNA sequencing technologies that provide new or improved diagnostics. However, developing and delivering new treatments for the 'genodermatoses' remains challenging. The goal of creating topical preparations that can recover the inherent gene pathology remains largely aspirational. However, recent progress in two fields - the chemistry of topical delivery formulations (lipid nanoparticles) and the molecular biology of gene repair (CRISPR-Cas9, base and prime editing) - presents new opportunities to address this unmet need. In this review, we discuss how lipid nanoparticle delivery vehicles could be used to deliver gene-editing tools to formulate topical 'gene creams' suitable for the treatment of genodermatoses. We summarize the historical landscape of topical therapeutics and advances in gene editing that may herald an era of new therapies for patients with inherited skin disorders.},
}

Humans
Gene Editing
CRISPR-Cas Systems
Gene Transfer Techniques
*Nanoparticles
*Skin Diseases, Genetic/genetics
*Liposomes

@article {pmid38609262,
year = {2024},
author = {Su, Z and Wang, X and Chen, X and Ding, L and Zeng, X and Xu, J and Peng, C},
title = {Novel CRISPR/SpRY system for rapid detection of CRISPR/Cas-mediated gene editing in rice.},
journal = {Analytica chimica acta},
volume = {1303},
number = {},
pages = {342519},
doi = {10.1016/j.aca.2024.342519},
pmid = {38609262},
issn = {1873-4324},
mesh = {*Oryza/genetics ; CRISPR-Cas Systems/genetics ; Gene Editing ; Biological Assay ; Biotechnology ; RNA ; },
abstract = {The gene editing technology represented by clustered rule-interspersed short palindromic repeats (CRISPR)/Cas9 has developed as a common tool in the field of biotechnology. Many gene-edited products in plant varieties have recently been commercialized. However, the rapid on-site visual detection of gene-edited products without instrumentation remains challenging. This study aimed to develop a novel and efficient method, termed the CRISPR/SpRY detection platform, for the rapid screening of CRISPR/Cas9-induced mutants based on CRISPR/SpRY-mediated in vitro cleavage using rice (Oryza sativa L.) samples genetically edited at the TGW locus as an example. We designed the workflow of the CRISPR/SpRY detection platform and conducted a feasibility assessment. Subsequently, we optimized the reaction system of CRISPR/SpRY, and developed a one-pot CRISPR/SpRY assay by integrating recombinase polymerase amplification (RPA). The sensitivity of the method was further verified using recombinant plasmids. The proposed method successfully identified various types of mutations, including insertions, deletions (indels), and nucleotide substitutions, with excellent sensitivity. Finally, the applicability of this method was validated using different rice samples. The entire process was completed in less than an hour, with a limit of detection as low as 1%. Compared with previous methods, our approach is simple to operate, instrumentation-free, cost-effective, and time-efficient. The primary significance lies in the liberation of our developed system from the limitations imposed using protospacer adjacent motif sequences. This expands the scope and versatility of the CRISPR-based detection platform, making it a promising and groundbreaking platform for detecting mutations induced by gene editing.},
}

*Oryza/genetics
CRISPR-Cas Systems/genetics
Gene Editing
Biological Assay
Biotechnology
RNA

@article {pmid38609257,
year = {2024},
author = {He, W and Li, X and Li, X and Guo, M and Zhang, M and Hu, R and Li, M and Ding, S and Yan, Y},
title = {Split activator of CRISPR/Cas12a for direct and sensitive detection of microRNA.},
journal = {Analytica chimica acta},
volume = {1303},
number = {},
pages = {342477},
doi = {10.1016/j.aca.2024.342477},
pmid = {38609257},
issn = {1873-4324},
mesh = {*MicroRNAs/genetics ; CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems ; *Nucleic Acids ; DNA, Single-Stranded/genetics ; },
abstract = {CRISPR/Cas12a-based nucleic acid assays have been increasingly used for molecular diagnostics. However, most current CRISPR/Cas12a-based RNA assays require the conversion of RNA into DNA by preamplification strategies, which increases the complexity of detection. Here, we found certain chimeric DNA-RNA hybrid single strands could activate the trans-cleavage activity of Cas12a, and then discovered the activating effect of split ssDNA and RNA when they are present simultaneously. As proof of concept, split nucleic acid-activated Cas12a (SNA-Cas12a) strategy was developed for direct detection of miR-155. By adding a short ssDNA to the proximal end of the crRNA spacer sequence, we realized the direct detection of RNA targets using Cas12a. With the assistance of ssDNA, we extended the limitation that CRISPR/Cas12a cannot be activated by RNA targets. In addition, by taking advantage of the programmability of crRNA, the length of its binding to DNA and RNA was optimized to achieve the optimal efficiency in activating Cas12a. The SNA-Cas12a method enabled sensitive miR-155 detection at pM level. This method was simple, rapid, and specific. Thus, we proposed a new Cas12a-based RNA detection strategy that expanded the application of CRISPR/Cas12a.},
}

*MicroRNAs/genetics
CRISPR-Cas Systems
RNA, Guide, CRISPR-Cas Systems
*Nucleic Acids
DNA, Single-Stranded/genetics

@article {pmid38609223,
year = {2024},
author = {Ma, J and Qian, C and Hu, Q and Zhang, J and Gu, G and Liang, X and Zhang, L},
title = {The bacteriome-coupled phage communities continuously contract and shift to orchestrate the traditional rice vinegar fermentation.},
journal = {Food research international (Ottawa, Ont.)},
volume = {184},
number = {},
pages = {114244},
doi = {10.1016/j.foodres.2024.114244},
pmid = {38609223},
issn = {1873-7145},
mesh = {Humans ; *Oryza ; Acetic Acid ; Fermentation ; *Bacteriophages/genetics ; *Microbiota/genetics ; },
abstract = {Amounts of microbiome studies have uncovered the microbial communities of traditional food fermentations, while in which the phageome development with time is poorly understood. Here, we conducted a study to decipher both phageome and bacteriome of the traditional rice vinegar fermentation. The vinegar phageomes showed significant differences in the alpha diversity, network density and clustering coefficient over time. Peduoviridae had the highest relative abundance. Moreover, the phageome negatively correlated to the cognate bacteriome in alpha diversity, and undergone constantly contracting and shifting across the temporal scale. Nevertheless, 257 core virial clusters (VCs) persistently occurred with time whatever the significant impacts imposed by the varied physiochemical properties. Glycoside hydrolase (GH) and glycosyltransferase (GT) families genes displayed the higher abundances across all samples. Intriguingly, diversely structuring of toxin-antitoxin systems (TAs) and CRISPR-Cas arrays were frequently harbored by phage genomes. Their divergent organization and encoding attributes underlie the multiple biological roles in modulation of network and/or contest of phage community as well as bacterial host community. This phageome-wide mapping will fuel the current insights of phage community ecology in other traditional fermented ecosystems that are challenging to decipher.},
}

Humans
*Oryza
Acetic Acid
Fermentation
*Bacteriophages/genetics
*Microbiota/genetics

@article {pmid38607027,
year = {2024},
author = {Chua, R and Wang, L and Singaraja, R and Ghosh, S},
title = {Functional and Multi-Omics Effects of an Optimized CRISPR-Mediated FURIN Depletion in U937 Monocytes.},
journal = {Cells},
volume = {13},
number = {7},
pages = {},
doi = {10.3390/cells13070588},
pmid = {38607027},
issn = {2073-4409},
support = {2U54GM104940, R01HL146462-01/NH/NIH HHS/United States ; },
mesh = {Humans ; Animals ; Mice ; *Monocytes/metabolism ; *Furin/genetics/metabolism ; U937 Cells ; Clustered Regularly Interspaced Short Palindromic Repeats ; Multiomics ; RNA, Guide, CRISPR-Cas Systems ; Cytokines/genetics ; },
abstract = {The pro-protein convertase FURIN (PCSK3) is implicated in a wide range of normal and pathological biological processes such as infectious diseases, cancer and cardiovascular diseases. Previously, we performed a systemic inhibition of FURIN in a mouse model of atherosclerosis and demonstrated significant plaque reduction and alterations in macrophage function. To understand the cellular mechanisms affected by FURIN inhibition in myeloid cells, we optimized a CRISPR-mediated gene deletion protocol for successfully deriving hemizygous (HZ) and nullizygous (NZ) FURIN knockout clones in U937 monocytic cells using lipotransfection-based procedures and a dual guide RNA delivery strategy. We observed differences in monocyte and macrophage functions involving phagocytosis, lipid accumulation, cell migration, inflammatory gene expression, cytokine release patterns, secreted proteomics (cytokines) and whole-genome transcriptomics between wild-type, HZ and NZ FURIN clones. These studies provide a mechanistic basis on the possible roles of myeloid cell FURIN in cardiovascular disorders.},
}

Humans
Animals
Mice
*Monocytes/metabolism
*Furin/genetics/metabolism
U937 Cells
Clustered Regularly Interspaced Short Palindromic Repeats
Multiomics
RNA, Guide, CRISPR-Cas Systems
Cytokines/genetics

@article {pmid38607007,
year = {2024},
author = {Wang, S and Zhu, Y and Du, S and Zheng, Y},
title = {Preclinical Advances in LNP-CRISPR Therapeutics for Solid Tumor Treatment.},
journal = {Cells},
volume = {13},
number = {7},
pages = {},
pmid = {38607007},
issn = {2073-4409},
mesh = {Humans ; CRISPR-Cas Systems/genetics ; Gene Editing ; Genetic Therapy ; *Neoplasms/genetics/therapy ; *Nanostructures ; },
abstract = {Solid tumors, with their intricate cellular architecture and genetic heterogeneity, have long posed therapeutic challenges. The advent of the CRISPR genome editing system offers a promising, precise genetic intervention. However, the journey from bench to bedside is fraught with hurdles, chief among them being the efficient delivery of CRISPR components to tumor cells. Lipid nanoparticles (LNPs) have emerged as a potential solution. This biocompatible nanomaterial can encapsulate the CRISPR/Cas9 system, ensuring targeted delivery while mitigating off-target effects. Pre-clinical investigations underscore the efficacy of LNP-mediated CRISPR delivery, with marked disruption of oncogenic pathways and subsequent tumor regression. Overall, CRISPR/Cas9 technology, when combined with LNPs, presents a groundbreaking approach to cancer therapy, offering precision, efficacy, and potential solutions to current limitations. While further research and clinical testing are required, the future of personalized cancer treatment based on CRISPR/Cas9 holds immense promise.},
}

Humans
CRISPR-Cas Systems/genetics
Gene Editing
Genetic Therapy
*Neoplasms/genetics/therapy
*Nanostructures

@article {pmid38605715,
year = {2024},
author = {Shen, Q and Ruan, H and Zhang, H and Wu, T and Zhu, K and Han, W and Dong, R and Ming, T and Qi, H and Zhang, Y},
title = {Utilization of CRISPR-Cas genome editing technology in filamentous fungi: function and advancement potentiality.},
journal = {Frontiers in microbiology},
volume = {15},
number = {},
pages = {1375120},
pmid = {38605715},
issn = {1664-302X},
abstract = {Filamentous fungi play a crucial role in environmental pollution control, protein secretion, and the production of active secondary metabolites. The evolution of gene editing technology has significantly improved the study of filamentous fungi, which in the past was laborious and time-consuming. But recently, CRISPR-Cas systems, which utilize small guide RNA (sgRNA) to mediate clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas), have demonstrated considerable promise in research and application for filamentous fungi. The principle, function, and classification of CRISPR-Cas, along with its application strategies and research progress in filamentous fungi, will all be covered in the review. Additionally, we will go over general matters to take into account when editing a genome with the CRISPR-Cas system, including the creation of vectors, different transformation methodologies, multiple editing approaches, CRISPR-mediated transcriptional activation (CRISPRa) or interference (CRISPRi), base editors (BEs), and Prime editors (PEs).},
}

@article {pmid38578074,
year = {2024},
author = {Meng, JN and Xu, ZK and Li, PR and Zeng, X and Liu, Y and Xu, ZL and Wang, J and Ding, Y and Shen, X},
title = {Universal and Naked-Eye Diagnostic Platform for Emetic Bacillus cereus Based on RPA-Assisted CRISPR/Cas12a.},
journal = {Journal of agricultural and food chemistry},
volume = {72},
number = {15},
pages = {8823-8830},
doi = {10.1021/acs.jafc.3c06744},
pmid = {38578074},
issn = {1520-5118},
mesh = {*Food Microbiology ; *Emetics ; Recombinases/genetics ; Bacillus cereus/genetics ; CRISPR-Cas Systems ; Sensitivity and Specificity ; Nucleotidyltransferases/genetics ; },
abstract = {Emetic Bacillus cereus (B. cereus), which can cause emetic food poisoning and in some cases even fulminant liver failure and death, has aroused widespread concern. Herein, a universal and naked-eye diagnostic platform for emetic B. cereus based on recombinase polymerase amplification (RPA)-assisted CRISPR/Cas12a was developed by targeting the cereulide synthetase biosynthetic gene (cesB). The diagnostic platform enabled one-pot detection by adding components at the bottom and cap of the tube separately. The visual limit of detection of RPA-CRISPR/Cas12a for gDNA and cells of emetic B. cereus was 10[-2] ng μL[-1] and 10[2] CFU mL[-1], respectively. Meanwhile, it maintained the same sensitivity in the rice, milk, and cooked meat samples even if the gDNA was extracted by simple boiling. The whole detection process can be finished within 40 min, and the single cell of emetic B. cereus was able to be recognized through enrichment for 2-5 h. The good specificity, high sensitivity, rapidity, and simplicity of the RPA-assisted CRISPR/Cas12a diagnostic platform made it serve as a potential tool for the on-site detection of emetic B. cereus in food matrices. In addition, the RPA-assisted CRISPR/Cas12a assay is the first application in emetic B. cereus detection.},
}

*Food Microbiology
*Emetics
Recombinases/genetics
Bacillus cereus/genetics
CRISPR-Cas Systems
Sensitivity and Specificity
Nucleotidyltransferases/genetics

@article {pmid38575365,
year = {2024},
author = {Wang, Y and Du, P and Shao, Y and Wang, W and Liu, Y and Ma, Y and Hu, P and Cao, J and Wang, X and Abd El-Aty, AM},
title = {An Innovative and Efficient Fluorescent Detection Technique for Salmonella in Animal-Derived Foods Using the CRISPR/Cas12a-HCR System Combined with PCR/RAA.},
journal = {Journal of agricultural and food chemistry},
volume = {72},
number = {15},
pages = {8831-8839},
doi = {10.1021/acs.jafc.3c08829},
pmid = {38575365},
issn = {1520-5118},
mesh = {Animals ; Cattle ; *CRISPR-Cas Systems ; Coloring Agents ; DNA, Single-Stranded ; Recombinases ; Salmonella/genetics ; Polymerase Chain Reaction ; *Biosensing Techniques ; },
abstract = {Here, we present a method for Salmonella detection using clustered regularly interspaced short palindromic repeats associated with the CRISPR-associated protein 12a-hybridization chain reaction (CRISPR/Cas12a-HCR) system combined with polymerase chain reaction/recombinase-assisted amplification (PCR/RAA) technology. The approach relies on the Salmonella invA gene as a biorecognition element and its amplification through PCR and RAA. In the presence of the target gene, Cas12a, guided by crRNA, recognizes and cleaves the amplification product, initiating the HCR. Fluorescently labeled single-stranded DNA (ssDNA) H1 and H2 were introduced, and the Salmonella concentration was determined based on the fluorescence intensity from the triggered HCR. Both assays demonstrate high specificity, sensitivity, simplicity, and rapidity. The detection range was 2 × 10[1]-2 × 10[9] CFU/mL, with an LOD of 20 CFU/mL, and the entire process enabled specific and rapid Salmonella detection within 85-105 min. Field-incurred spiked recovery tests were conducted in mutton and beef samples using both assays, demonstrating satisfactory recovery and accuracy in animal-derived foods. By combining CRISPR/Cas12a with hybridization chain reaction technology, this study presents a rapid and sensitive Salmonella detection method that is crucial for identifying pathogenic bacteria and monitoring food safety.},
}

Animals
Cattle
*CRISPR-Cas Systems
Coloring Agents
DNA, Single-Stranded
Recombinases
Salmonella/genetics
Polymerase Chain Reaction
*Biosensing Techniques

@article {pmid38564120,
year = {2024},
author = {Sarkar, P and Santiago Vazquez, J and Zhou, M and Levy, A and Mou, Z and Orbović, V},
title = {Multiplexed gene editing in citrus by using a multi-intron containing Cas9 gene.},
journal = {Transgenic research},
volume = {33},
number = {1-2},
pages = {59-66},
pmid = {38564120},
issn = {1573-9368},
support = {2018-70016-27392//USDA/NIFA Emergency Citrus Disease Research and Extension Program/ ; },
mesh = {*Gene Editing ; CRISPR-Cas Systems/genetics ; Introns ; *Citrus/genetics ; RNA, Guide, CRISPR-Cas Systems ; RNA, Transfer/genetics ; },
abstract = {Several expression systems have been developed in clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (CRISPR/Cas9) framework allowing for gene editing of disease-associated genes across diverse citrus varieties. In this study, we present a new approach employing a multi-intron containing Cas9 gene plus multiple gRNAs separated with tRNA sequences to target the phytoene desaturase gene in both 'Carrizo' citrange and 'Duncan' grapefruit. Notably, using this unified vector significantly boosted editing efficiency in both citrus varieties, showcasing mutations in all three designated targets. The implementation of this multiplex gene editing system with a multi-intron-containing Cas9 plus a gRNA-tRNA array demonstrates a promising avenue for efficient citrus genome editing, equipping us with potent tools in the ongoing battle against several diseases such as canker and huanglongbing.},
}

*Gene Editing
CRISPR-Cas Systems/genetics
Introns
*Citrus/genetics
RNA, Guide, CRISPR-Cas Systems
RNA, Transfer/genetics

@article {pmid38504013,
year = {2024},
author = {Abkallo, HM and Arbuthnot, P and Auer, TO and Berger, DK and Burger, J and Chakauya, E and Concordet, JP and Diabate, A and Di Donato, V and Groenewald, JH and Guindo, A and Koekemoer, LL and Nazare, F and Nolan, T and Okumu, F and Orefuwa, E and Paemka, L and Prieto-Godino, L and Runo, S and Sadler, M and Tesfaye, K and Tripathi, L and Wondji, C},
title = {Making genome editing a success story in Africa.},
journal = {Nature biotechnology},
volume = {42},
number = {4},
pages = {551-554},
pmid = {38504013},
issn = {1546-1696},
mesh = {*Gene Editing ; *CRISPR-Cas Systems/genetics ; Africa ; Genome ; },
}

*Gene Editing
*CRISPR-Cas Systems/genetics
Africa
Genome

@article {pmid38497640,
year = {2024},
author = {Wang, P and Du, X and Zhao, Y and Wang, W and Cai, T and Tang, K and Wang, X},
title = {Combining CRISPR/Cas9 and natural excision for the precise and complete removal of mobile genetic elements in bacteria.},
journal = {Applied and environmental microbiology},
volume = {90},
number = {4},
pages = {e0009524},
pmid = {38497640},
issn = {1098-5336},
support = {32070175//MOST | National Natural Science Foundation of China (NSFC)/ ; 42188102//MOST | National Natural Science Foundation of China (NSFC)/ ; 91951203//MOST | National Natural Science Foundation of China (NSFC)/ ; 2022YFC3103600//MOST | National Key Research and Development Program of China (NKPs)/ ; 2019BT02Y262//Local Innovative and Research Teams Project of Guangdong Pearl River Talents Program/ ; 2021345//Youth Innovation Promotion Association of the Chinese Academy of Sciences (CAS YIPA)/ ; },
mesh = {*CRISPR-Cas Systems ; *RNA, Guide, CRISPR-Cas Systems ; Bacteria/genetics ; Genomic Islands ; Gene Transfer, Horizontal ; Plasmids/genetics ; Interspersed Repetitive Sequences ; },
abstract = {Horizontal gene transfer, facilitated by mobile genetic elements (MGEs), is an adaptive evolutionary process that contributes to the evolution of bacterial populations and infectious diseases. A variety of MGEs not only can integrate into the bacterial genome but also can survive or even replicate like plasmids in the cytoplasm, thus requiring precise and complete removal for studying their strategies in benefiting host cells. Existing methods for MGE removal, such as homologous recombination-based deletion and excisionase-based methods, have limitations in effectively eliminating certain MGEs. To overcome these limitations, we developed the Cas9-NE method, which combines the CRISPR/Cas9 system with the natural excision of MGEs. In this approach, a specialized single guide RNA (sgRNA) element is designed with a 20-nucleotide region that pairs with the MGE sequence. This sgRNA is expressed from a plasmid that also carries the Cas9 gene. By utilizing the Cas9-NE method, both the integrative and circular forms of MGEs can be precisely and completely eliminated through Cas9 cleavage, generating MGE-removed cells. We have successfully applied the Cas9-NE method to remove four representative MGEs, including plasmids, prophages, and genomic islands, from Vibrio strains. This new approach not only enables various investigations on MGEs but also has significant implications for the rapid generation of strains for commercial purposes.IMPORTANCEMobile genetic elements (MGEs) are of utmost importance for bacterial adaptation and pathogenicity, existing in various forms and multiple copies within bacterial cells. Integrated MGEs play dual roles in bacterial hosts, enhancing the fitness of the host by delivering cargo genes and potentially modifying the bacterial genome through the integration/excision process. This process can lead to alterations in promoters or coding sequences or even gene disruptions at integration sites, influencing the physiological functions of host bacteria. Here, we developed a new approach called Cas9-NE, allowing them to maintain the natural sequence changes associated with MGE excision. Cas9-NE allows the one-step removal of integrated and circular MGEs, addressing the challenge of eliminating various MGE forms efficiently. This approach simplifies MGE elimination in bacteria, expediting research on MGEs.},
}

*CRISPR-Cas Systems
*RNA, Guide, CRISPR-Cas Systems
Bacteria/genetics
Genomic Islands
Gene Transfer, Horizontal
Plasmids/genetics
Interspersed Repetitive Sequences

@article {pmid38323580,
year = {2024},
author = {Romano, A and Mortellaro, A},
title = {The New Frontiers of Gene Therapy and Gene Editing in Inflammatory Diseases.},
journal = {Human gene therapy},
volume = {35},
number = {7-8},
pages = {219-231},
doi = {10.1089/hum.2023.210},
pmid = {38323580},
issn = {1557-7422},
mesh = {Animals ; Humans ; *Gene Editing/methods ; Quality of Life ; Genetic Therapy/methods ; Genetic Engineering ; *Severe Combined Immunodeficiency ; CRISPR-Cas Systems ; },
abstract = {Inflammatory diseases are conditions characterized by abnormal and often excessive immune responses, leading to tissue and organ inflammation. The complexity of these disorders arises from the intricate interplay of genetic factors and immune responses, which challenges conventional therapeutic approaches. However, the field of genetic manipulation has sparked unprecedented optimism in addressing these complex disorders. This review aims to comprehensively explore the application of gene therapy and gene editing in the context of inflammatory diseases, offering solutions that range from correcting genetic defects to precise immune modulation. These therapies have exhibited remarkable potential in ameliorating symptoms, improving quality of life, and even achieving disease remission. As we delve into recent breakthroughs and therapeutic applications, we illustrate how these advancements offer novel and transformative solutions for conditions that have traditionally eluded conventional treatments. By examining successful case studies and preclinical research, we emphasize the favorable results and substantial transformative impacts that gene-based interventions have demonstrated in patients and animal models of inflammatory diseases such as chronic granulomatous disease, cryopyrin-associated syndromes, and adenosine deaminase 2 deficiency, as well as those of multifactorial origins such as arthropathies (osteoarthritis, rheumatoid arthritis) and inflammatory bowel disease. In conclusion, gene therapy and gene editing offer transformative opportunities to address the underlying causes of inflammatory diseases, ushering in a new era of precision medicine and providing hope for personalized, targeted treatments.},
}

Animals
Humans
*Gene Editing/methods
Quality of Life
Genetic Therapy/methods
Genetic Engineering
*Severe Combined Immunodeficiency
CRISPR-Cas Systems

@article {pmid38251667,
year = {2024},
author = {Rai, R and Steinberg, Z and Romito, M and Zinghirino, F and Hu, YT and White, N and Naseem, A and Thrasher, AJ and Turchiano, G and Cavazza, A},
title = {CRISPR/Cas9-Based Disease Modeling and Functional Correction of Interleukin 7 Receptor Alpha Severe Combined Immunodeficiency in T-Lymphocytes and Hematopoietic Stem Cells.},
journal = {Human gene therapy},
volume = {35},
number = {7-8},
pages = {269-283},
doi = {10.1089/hum.2023.100},
pmid = {38251667},
issn = {1557-7422},
mesh = {Infant, Newborn ; Humans ; *Severe Combined Immunodeficiency/genetics/therapy ; T-Lymphocytes/metabolism ; CRISPR-Cas Systems ; Hematopoietic Stem Cells/metabolism ; Gene Editing/methods ; Receptors, Interleukin-7/genetics/metabolism ; },
abstract = {Interleukin 7 Receptor alpha Severe Combined Immunodeficiency (IL7R-SCID) is a life-threatening disorder caused by homozygous mutations in the IL7RA gene. Defective IL7R expression in humans hampers T cell precursors' proliferation and differentiation during lymphopoiesis resulting in the absence of T cells in newborns, who succumb to severe infections and death early after birth. Previous attempts to tackle IL7R-SCID by viral gene therapy have shown that unregulated IL7R expression predisposes to leukemia, suggesting the application of targeted gene editing to insert a correct copy of the IL7RA gene in its genomic locus and mediate its physiological expression as a more feasible therapeutic approach. To this aim, we have first developed a CRISPR/Cas9-based IL7R-SCID disease modeling system that recapitulates the disease phenotype in primary human T cells and hematopoietic stem and progenitor cells (HSPCs). Then, we have designed a knockin strategy that targets IL7RA exon 1 and introduces through homology-directed repair a corrective, promoterless IL7RA cDNA followed by a reporter cassette through AAV6 transduction. Targeted integration of the corrective cassette in primary T cells restored IL7R expression and rescued functional downstream IL7R signaling. When applied to HSPCs further induced to differentiate into T cells in an Artificial Thymic Organoid system, our gene editing strategy overcame the T cell developmental block observed in IL7R-SCID patients, while promoting full maturation of T cells with physiological and developmentally regulated IL7R expression. Finally, genotoxicity assessment of the CRISPR/Cas9 platform in HSPCs using biased and unbiased technologies confirmed the safety of the strategy, paving the way for a new, efficient, and safe therapeutic option for IL7R-SCID patients.},
}

Infant, Newborn
Humans
*Severe Combined Immunodeficiency/genetics/therapy
T-Lymphocytes/metabolism
CRISPR-Cas Systems
Hematopoietic Stem Cells/metabolism
Gene Editing/methods
Receptors, Interleukin-7/genetics/metabolism

@article {pmid38159103,
year = {2024},
author = {Wang, R and Li, Y and Xu, S and Huang, Q and Tu, M and Zhu, Y and Cen, H and Dong, J and Jiang, L and Yao, X},
title = {Genome-wide association study reveals the genetic basis for petal-size formation in rapeseed (Brassica napus) and CRISPR-Cas9-mediated mutagenesis of BnFHY3 for petal-size reduction.},
journal = {The Plant journal : for cell and molecular biology},
volume = {118},
number = {2},
pages = {373-387},
doi = {10.1111/tpj.16609},
pmid = {38159103},
issn = {1365-313X},
support = {2022YFD1200404//National Key Research and Development Program of China/ ; },
mesh = {*Brassica napus/genetics ; Genome-Wide Association Study ; CRISPR-Cas Systems ; Plant Breeding ; *Brassica rapa/genetics ; Mutagenesis ; },
abstract = {Petals in rapeseed (Brassica napus) serve multiple functions, including protection of reproductive organs, nutrient acquisition, and attraction of pollinators. However, they also cluster densely at the top, forming a thick layer that absorbs and reflects a considerable amount of photosynthetically active radiation. Breeding genotypes with large, small, or even petal-less varieties, requires knowledge of primary genes for allelic selection and manipulation. However, our current understanding of petal-size regulation is limited, and the lack of markers and pre-breeding materials hinders targeted petal-size breeding. Here, we conducted a genome-wide association study on petal size using 295 diverse accessions. We identified 20 significant single nucleotide polymorphisms and 236 genes associated with petal-size variation. Through a cross-analysis of genomic and transcriptomic data, we focused on 14 specific genes, from which molecular markers for diverging petal-size features can be developed. Leveraging CRISPR-Cas9 technology, we successfully generated a quadruple mutant of Far-Red Elongated Hypocotyl 3 (q-bnfhy3), which exhibited smaller petals compared to the wild type. Our study provides insights into the genetic basis of petal-size regulation in rapeseed and offers abundant potential molecular markers for breeding. The q-bnfhy3 mutant unveiled a novel role of FHY3 orthologues in regulating petal size in addition to previously reported functions.},
}

*Brassica napus/genetics
Genome-Wide Association Study
CRISPR-Cas Systems
Plant Breeding
*Brassica rapa/genetics
Mutagenesis

@article {pmid38069547,
year = {2024},
author = {Zhang, M and Feng, J and Li, Y and Qin, PZ and Chai, Y},
title = {Generation of tamoxifen-inducible Tfap2b-CreER[T2] mice using CRISPR-Cas9.},
journal = {Genesis (New York, N.Y. : 2000)},
volume = {62},
number = {1},
pages = {e23582},
pmid = {38069547},
issn = {1526-968X},
support = {R01 DE012711/NH/NIH HHS/United States ; R01 DE022503/NH/NIH HHS/United States ; R01 DE012711/DE/NIDCR NIH HHS/United States ; T90 DE021982/DE/NIDCR NIH HHS/United States ; R01 DE022503/DE/NIDCR NIH HHS/United States ; },
mesh = {Mice ; Animals ; *CRISPR-Cas Systems ; *Tamoxifen/pharmacology ; Mice, Transgenic ; Red Fluorescent Protein ; Integrases/genetics/metabolism ; },
abstract = {Tfap2b, a pivotal transcription factor, plays critical roles within neural crest cells and their derived lineage. To unravel the intricate lineage dynamics and contribution of these Tfap2b+ cells during craniofacial development, we established a Tfap2b-CreER[T2] knock-in transgenic mouse line using the CRISPR-Cas9-mediated homologous direct repair. By breeding with tdTomato reporter mice and initiating Cre activity through tamoxifen induction at distinct developmental time points, we show the Tfap2b lineage within the key neural crest-derived domains, such as the facial mesenchyme, midbrain, cerebellum, spinal cord, and limbs. Notably, the migratory neurons stemming from the dorsal root ganglia are visible subsequent to Cre activity initiated at E8.5. Intriguingly, Tfap2b+ cells, serving as the progenitors for limb development, show activity predominantly commencing at E10.5. Across the mouse craniofacial landscape, Tfap2b exhibits a widespread presence throughout the facial organs. Here we validate its role as a marker of progenitors in tooth development and have confirmed that this process initiates from E12.5. Our study not only validates the Tfap2b-CreER[T2] transgenic line, but also provides a powerful tool for lineage tracing and genetic targeting of Tfap2b-expressing cells and their progenitor in a temporally and spatially regulated manner during the intricate process of development and organogenesis.},
}

Mice
Animals
*CRISPR-Cas Systems
*Tamoxifen/pharmacology
Mice, Transgenic
Red Fluorescent Protein
Integrases/genetics/metabolism

@article {pmid38062734,
year = {2024},
author = {Whittaker, TE and Moula, SE and Bahal, S and Bakri, FG and Hayajneh, WA and Daoud, AK and Naseem, A and Cavazza, A and Thrasher, AJ and Santilli, G},
title = {Multidimensional Response Surface Methodology for the Development of a Gene Editing Protocol for p67[phox]-Deficient Chronic Granulomatous Disease.},
journal = {Human gene therapy},
volume = {35},
number = {7-8},
pages = {298-312},
pmid = {38062734},
issn = {1557-7422},
support = {/WT_/Wellcome Trust/United Kingdom ; 104807/WT_/Wellcome Trust/United Kingdom ; MR/S021930/1/MRC_/Medical Research Council/United Kingdom ; },
mesh = {Humans ; *Granulomatous Disease, Chronic/genetics/therapy ; Gene Editing ; Genetic Therapy/methods ; Antigens, CD34/genetics ; Hematopoietic Stem Cells/metabolism ; CRISPR-Cas Systems ; },
abstract = {Replacing a faulty gene with a correct copy has become a viable therapeutic option as a result of recent progress in gene editing protocols. Targeted integration of therapeutic genes in hematopoietic stem cells has been achieved for multiple genes using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system and Adeno-Associated Virus (AAV) to carry a donor template. Although this is a promising strategy to correct genetic blood disorders, it is associated with toxicity and loss of function in CD34[+] hematopoietic stem and progenitor cells, which has hampered clinical application. Balancing the maximum achievable correction against deleterious effects on the cells is critical. However, multiple factors are known to contribute, and the optimization process is laborious and not always clearly defined. We have developed a flexible multidimensional Response Surface Methodology approach for optimization of gene correction. Using this approach, we could rapidly investigate and select editing conditions for CD34[+] cells with the best possible balance between correction and cell/colony-forming unit (CFU) loss in a parsimonious one-shot experiment. This method revealed that using relatively low doses of AAV2/6 and CRISPR/Cas9 ribonucleoprotein complex, we can preserve the fitness of CD34[+] cells and, at the same time, achieve high levels of targeted gene insertion. We then used these optimized editing conditions for the correction of p67[phox]-deficient chronic granulomatous disease (CGD), an autosomal recessive disorder of blood phagocytic cells resulting in severe recurrent bacterial and fungal infections and achieved rescue of p67[phox] expression and functional correction of CD34[+]-derived neutrophils from a CGD patient.},
}

Humans
*Granulomatous Disease, Chronic/genetics/therapy
Gene Editing
Genetic Therapy/methods
Antigens, CD34/genetics
Hematopoietic Stem Cells/metabolism
CRISPR-Cas Systems

@article {pmid37997697,
year = {2024},
author = {Zhang, D and Boch, J},
title = {Development of TALE-adenine base editors in plants.},
journal = {Plant biotechnology journal},
volume = {22},
number = {5},
pages = {1067-1077},
pmid = {37997697},
issn = {1467-7652},
mesh = {Humans ; *Cytosine Deaminase/genetics ; *Adenine ; Gene Editing/methods ; DNA/genetics ; CRISPR-Cas Systems ; *Cytidine Deaminase ; *Proteins ; },
abstract = {Base editors enable precise nucleotide changes at targeted genomic loci without requiring double-stranded DNA breaks or repair templates. TALE-adenine base editors (TALE-ABEs) are genome editing tools, composed of a DNA-binding domain from transcription activator-like effectors (TALEs), an engineered adenosine deaminase (TadA8e), and a cytosine deaminase domain (DddA), that allow A•T-to-G•C editing in human mitochondrial DNA. However, the editing ability of TALE-ABEs in plants apart from chloroplast DNA has not been described, so far, and the functional role how DddA enhances TadA8e is still unclear. We tested a series of TALE-ABEs with different deaminase fusion architectures in Nicotiana benthamiana and rice. The results indicate that the double-stranded DNA-specific cytosine deaminase DddA can boost the activities of single-stranded DNA-specific deaminases (TadA8e or APOBEC3A) on double-stranded DNA. We analysed A•T-to-G•C editing efficiencies in a β-glucuronidase reporter system and showed precise adenine editing in genomic regions with high product purity in rice protoplasts. Furthermore, we have successfully regenerated rice plants with A•T-to-G•C mutations in the chloroplast genome using TALE-ABE. Consequently, TALE-adenine base editors provide alternatives for crop improvement and gene therapy by editing nuclear or organellar genomes.},
}

Humans
*Cytosine Deaminase/genetics
*Adenine
Gene Editing/methods
DNA/genetics
CRISPR-Cas Systems
*Cytidine Deaminase
*Proteins

@article {pmid37400521,
year = {2024},
author = {Wessels, HH and Stirn, A and Méndez-Mancilla, A and Kim, EJ and Hart, SK and Knowles, DA and Sanjana, NE},
title = {Prediction of on-target and off-target activity of CRISPR-Cas13d guide RNAs using deep learning.},
journal = {Nature biotechnology},
volume = {42},
number = {4},
pages = {628-637},
pmid = {37400521},
issn = {1546-1696},
support = {DP2HG010099//U.S. Department of Health & Human Services | NIH | National Human Genome Research Institute (NHGRI)/ ; },
mesh = {Humans ; *RNA, Guide, CRISPR-Cas Systems ; CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Deep Learning ; RNA ; Gene Editing ; },
abstract = {Transcriptome engineering applications in living cells with RNA-targeting CRISPR effectors depend on accurate prediction of on-target activity and off-target avoidance. Here we design and test ~200,000 RfxCas13d guide RNAs targeting essential genes in human cells with systematically designed mismatches and insertions and deletions (indels). We find that mismatches and indels have a position- and context-dependent impact on Cas13d activity, and mismatches that result in G-U wobble pairings are better tolerated than other single-base mismatches. Using this large-scale dataset, we train a convolutional neural network that we term targeted inhibition of gene expression via gRNA design (TIGER) to predict efficacy from guide sequence and context. TIGER outperforms the existing models at predicting on-target and off-target activity on our dataset and published datasets. We show that TIGER scoring combined with specific mismatches yields the first general framework to modulate transcript expression, enabling the use of RNA-targeting CRISPRs to precisely control gene dosage.},
}

Humans
*RNA, Guide, CRISPR-Cas Systems
CRISPR-Cas Systems/genetics
Clustered Regularly Interspaced Short Palindromic Repeats
*Deep Learning
RNA
Gene Editing

@article {pmid37365261,
year = {2024},
author = {},
title = {Efficient A-to-C base editing with high specificity.},
journal = {Nature biotechnology},
volume = {42},
number = {4},
pages = {578-579},
pmid = {37365261},
issn = {1546-1696},
support = {32025023//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {*Gene Editing ; *CRISPR-Cas Systems/genetics ; },
}

*Gene Editing
*CRISPR-Cas Systems/genetics

@article {pmid37322276,
year = {2024},
author = {Chen, L and Hong, M and Luan, C and Gao, H and Ru, G and Guo, X and Zhang, D and Zhang, S and Li, C and Wu, J and Randolph, PB and Sousa, AA and Qu, C and Zhu, Y and Guan, Y and Wang, L and Liu, M and Feng, B and Song, G and Liu, DR and Li, D},
title = {Adenine transversion editors enable precise, efficient A•T-to-C•G base editing in mammalian cells and embryos.},
journal = {Nature biotechnology},
volume = {42},
number = {4},
pages = {638-650},
pmid = {37322276},
issn = {1546-1696},
support = {82100773//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32025023//National Natural Science Foundation of China (National Science Foundation of China)/ ; U01AI142756//Foundation for the National Institutes of Health (Foundation for the National Institutes of Health, Inc.)/ ; },
mesh = {Animals ; Mice ; Humans ; *Gene Editing ; *Adenine/metabolism ; Mutation ; Cytosine/metabolism ; Adenosine ; CRISPR-Cas Systems/genetics ; Mammals/genetics ; },
abstract = {Base editors have substantial promise in basic research and as therapeutic agents for the correction of pathogenic mutations. The development of adenine transversion editors has posed a particular challenge. Here we report a class of base editors that enable efficient adenine transversion, including precise A•T-to-C•G editing. We found that a fusion of mouse alkyladenine DNA glycosylase (mAAG) with nickase Cas9 and deaminase TadA-8e catalyzed adenosine transversion in specific sequence contexts. Laboratory evolution of mAAG significantly increased A-to-C/T conversion efficiency up to 73% and expanded the targeting scope. Further engineering yielded adenine-to-cytosine base editors (ACBEs), including a high-accuracy ACBE-Q variant, that precisely install A-to-C transversions with minimal Cas9-independent off-targeting effects. ACBEs mediated high-efficiency installation or correction of five pathogenic mutations in mouse embryos and human cell lines. Founder mice showed 44-56% average A-to-C edits and allelic frequencies of up to 100%. Adenosine transversion editors substantially expand the capabilities and possible applications of base editing technology.},
}

Animals
Mice
Humans
*Gene Editing
*Adenine/metabolism
Mutation
Cytosine/metabolism
Adenosine
CRISPR-Cas Systems/genetics
Mammals/genetics

@article {pmid38605260,
year = {2024},
author = {Rafiq, MS and Shabbir, MA and Raza, A and Irshad, S and Asghar, A and Maan, MK and Gondal, MA and Hao, H},
title = {CRISPR-Cas System: A New Dawn to Combat Antibiotic Resistance.},
journal = {BioDrugs : clinical immunotherapeutics, biopharmaceuticals and gene therapy},
volume = {},
number = {},
pages = {},
pmid = {38605260},
issn = {1179-190X},
support = {32172914//the National Natural Science Foundation of China/ ; 2021YFD1800600//Key Technology Research and Development Program of Shandong Province/ ; . 2662022DKYJC005//the Fundamental Research Funds for the Central Universities/ ; },
abstract = {Antimicrobial resistance (AMR) can potentially harm global public health. Horizontal gene transfer (HGT), which speeds up the emergence of AMR and increases the burden of drug resistance in mobile genetic elements (MGEs), is the primary method by which AMR genes are transferred across bacterial pathogens. New approaches are urgently needed to halt the spread of bacterial diseases and antibiotic resistance. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), an RNA-guided adaptive immune system, protects prokaryotes from foreign DNA like plasmids and phages. This approach may be essential in limiting horizontal gene transfer and halting the spread of antibiotic resistance. The CRISPR-Cas system has been crucial in identifying and understanding resistance mechanisms and developing novel therapeutic approaches. This review article investigates the CRISPR-Cas system's potential as a tool to combat bacterial AMR. Antibiotic-resistant bacteria can be targeted and eliminated by the CRISPR-Cas system. It has been proven to be an efficient method for removing carbapenem-resistant plasmids and regaining antibiotic susceptibility. The CRISPR-Cas system has enormous potential as a weapon against bacterial AMR. It precisely targets and eliminates antibiotic-resistant bacteria, facilitates resistance mechanism identification, and offers new possibilities in diagnostics and therapeutics.},
}