@article {pmid39716183,
year = {2024},
author = {Cohen, S and Bergman, S and Lynn, N and Tuller, T},
title = {A tool for CRISPR-Cas9 sgRNA evaluation based on computational models of gene expression.},
journal = {Genome medicine},
volume = {16},
number = {1},
pages = {152},
pmid = {39716183},
issn = {1756-994X},
mesh = {*CRISPR-Cas Systems ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Humans ; Software ; Computational Biology/methods ; Gene Editing/methods ; Gene Knockout Techniques ; Computer Simulation ; Models, Genetic ; Gene Expression ; Gene Expression Regulation ; },
abstract = {BACKGROUND: CRISPR is widely used to silence genes by inducing mutations expected to nullify their expression. While numerous computational tools have been developed to design single-guide RNAs (sgRNAs) with high cutting efficiency and minimal off-target effects, only a few tools focus specifically on predicting gene knockouts following CRISPR. These tools consider factors like conservation, amino acid composition, and frameshift likelihood. However, they neglect the impact of CRISPR on gene expression, which can dramatically affect the success of CRISPR-induced gene silencing attempts. Furthermore, information regarding gene expression can be useful even when the objective is not to silence a gene. Therefore, a tool that considers gene expression when predicting CRISPR outcomes is lacking.

RESULTS: We developed EXPosition, the first computational tool that combines models predicting gene knockouts after CRISPR with models that forecast gene expression, offering more accurate predictions of gene knockout outcomes. EXPosition leverages deep-learning models to predict key steps in gene expression: transcription, splicing, and translation initiation. We showed our tool performs better at predicting gene knockout than existing tools across 6 datasets, 4 cell types and ~207k sgRNAs. We also validated our gene expression models using the ClinVar dataset by showing enrichment of pathogenic mutations in high-scoring mutations according to our models.

CONCLUSIONS: We believe EXPosition will enhance both the efficiency and accuracy of genome editing projects, by directly predicting CRISPR's effect on various aspects of gene expression. EXPosition is available at http://www.cs.tau.ac.il/~tamirtul/EXPosition . The source code is available at https://github.com/shaicoh3n/EXPosition .},
}

*CRISPR-Cas Systems
*RNA, Guide, CRISPR-Cas Systems/genetics
Humans
Software
Computational Biology/methods
Gene Editing/methods
Gene Knockout Techniques
Computer Simulation
Models, Genetic
Gene Expression
Gene Expression Regulation

@article {pmid39715238,
year = {2024},
author = {Qiu, M and Tian, Y and Wang, H and Yang, J and Qu, B and Jiang, Y and Zhao, Q and Zhang, X and Man, C},
title = {CRISPR/Cas System Meets CLICK-17 DNAzyme: A Click Chemistry-Based Fluorescence Biosensing Platform Designed for Highly Sensitive Detection of Salmonella.},
journal = {Analytical chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.analchem.4c05316},
pmid = {39715238},
issn = {1520-6882},
abstract = {Salmonella is one of the most dangerous and contagious foodborne pathogens, posing a significant threat to public health and food safety. In this study, we developed a click chemistry-based fluorescence biosensing platform for highly sensitive detection of Salmonella enterica (S. enterica) by integrating the trans-cleavage activity of CRISPR/Cas12a with the CLICK17-mediated copper(II)-dependent azide-alkyne cycloaddition (Cu(II)AAC) click reaction. Herein, CLICK-17 can provide binding sites for Cu ions and high redox stability for one or much catalytically vital Cu[+] within its active sites, which facilitate the click reaction. With the existence of only Cu[2+], CLICK17 still can catalyze the click reaction between 3-butyn-1-ol and 3-azido-7-hydroxycoumarin to produce a fluorescence signal. By integrating the recombinase polymerase amplification (RPA), specific recognition, and trans-cleavage ability of the CRISPR/Cas12a system and the CLICK17-catalyzed Cu(II)AAC click reaction, the established biosensor obtained high detection sensitivity. This CLICK17-assisted CRISPR/Cas12a fluorescence biosensor was used for the detection of S. enterica with a limit of detection (LOD) as low as 1 cfu/mL in a wide linear detection range of 6 × 10[1]-6 × 10[7] cfu/mL. Moreover, the developed biosensor exhibited high specificity and anti-interference capability and had a recovery of 93%-104% in detection of S. enterica in spiked milk, infant formula, orange juice, and meat samples. This study provides a promising CRISPR/Cas12a-based fluorescence biosensor for the detection of foodborne pathogens.},
}

@article {pmid39714464,
year = {2024},
author = {Böck, D and Wilhelm, M and Mumenthaler, J and Carpanese, DF and Kulcsár, PI and d'Aquin, S and Cremonesi, A and Rassi, A and Häberle, J and Patriarchi, T and Schwank, G},
title = {Base editing of Ptbp1 in neurons alleviates symptoms in a mouse model of Parkinson's disease.},
journal = {eLife},
volume = {13},
number = {},
pages = {},
pmid = {39714464},
issn = {2050-084X},
support = {310030_185293/SNSF_/Swiss National Science Foundation/Switzerland ; 310030_196455/SNSF_/Swiss National Science Foundation/Switzerland ; FN20-0000000203//Novartis Foundation for Medical-Biological Research/ ; 196287/SNSF_/Swiss National Science Foundation/Switzerland ; 891959/ERC_/European Research Council/International ; },
mesh = {Animals ; *Polypyrimidine Tract-Binding Protein/metabolism/genetics ; Mice ; *Disease Models, Animal ; *Heterogeneous-Nuclear Ribonucleoproteins/metabolism/genetics ; *Parkinson Disease/genetics/metabolism ; *Astrocytes/metabolism ; Dopaminergic Neurons/metabolism ; Gene Editing ; Male ; Corpus Striatum/metabolism ; Tyrosine 3-Monooxygenase/metabolism/genetics ; Mice, Inbred C57BL ; },
abstract = {Parkinson's disease (PD) is a multifactorial disease caused by irreversible progressive loss of dopaminergic neurons (DANs). Recent studies have reported the successful conversion of astrocytes into DANs by repressing polypyrimidine tract binding protein 1 (PTBP1), which led to the rescue of motor symptoms in a chemically-induced mouse model of PD. However, follow-up studies have questioned the validity of this astrocyte-to-DAN conversion model. Here, we devised an adenine base editing strategy to downregulate PTBP1 in astrocytes and neurons in a chemically-induced PD mouse model. While PTBP1 downregulation in astrocytes had no effect, PTBP1 downregulation in neurons of the striatum resulted in the expression of the DAN marker tyrosine hydroxylase (TH) in non-dividing neurons, which was associated with an increase in striatal dopamine concentrations and a rescue of forelimb akinesia and spontaneous rotations. Phenotypic analysis using multiplexed iterative immunofluorescence imaging further revealed that most of these TH-positive cells co-expressed the dopaminergic marker DAT and the pan-neuronal marker NEUN, with the majority of these triple-positive cells being classified as mature GABAergic neurons. Additional research is needed to fully elucidate the molecular mechanisms underlying the expression of the observed markers and understand how the formation of these cells contributes to the rescue of spontaneous motor behaviors. Nevertheless, our findings support a model where downregulation of neuronal, but not astrocytic, PTBP1 can mitigate symptoms in PD mice.},
}

Animals
*Polypyrimidine Tract-Binding Protein/metabolism/genetics
Mice
*Disease Models, Animal
*Heterogeneous-Nuclear Ribonucleoproteins/metabolism/genetics
*Parkinson Disease/genetics/metabolism
*Astrocytes/metabolism
Dopaminergic Neurons/metabolism
Gene Editing
Male
Corpus Striatum/metabolism
Tyrosine 3-Monooxygenase/metabolism/genetics
Mice, Inbred C57BL

@article {pmid39661519,
year = {2024},
author = {Pan, Q and Zhang, Z and Xiong, Y and Bao, Y and Chen, T and Xu, P and Liu, Z and Ma, H and Yu, Y and Zhou, Z and Wei, W},
title = {Mapping functional elements of the DNA damage response through base editor screens.},
journal = {Cell reports},
volume = {43},
number = {12},
pages = {115047},
doi = {10.1016/j.celrep.2024.115047},
pmid = {39661519},
issn = {2211-1247},
mesh = {*DNA Damage ; Humans ; *DNA Repair ; *Gene Editing/methods ; CRISPR-Cas Systems/genetics ; Protein Serine-Threonine Kinases/metabolism/genetics ; Cisplatin/pharmacology ; Mutation/genetics ; Replication Protein A/metabolism/genetics ; HEK293 Cells ; },
abstract = {Maintaining genomic stability is vital for cellular equilibrium. In this study, we combined CRISPR-mediated base editing with pooled screening technologies to identify numerous mutations in lysine residues and protein-coding genes. The loss of these lysine residues and genes resulted in either sensitivity or resistance to DNA-damaging agents. Among the identified variants, we characterized both loss-of-function and gain-of-function mutations in response to DNA damage. Notably, we discovered that the K494 mutation of C17orf53 disrupts its interaction with RPA proteins, leading to increased sensitivity to cisplatin. Additionally, our analysis identified STK35 as a previously unrecognized gene involved in DNA damage response (DDR) pathways, suggesting that it may play a critical role in DNA repair. We believe that this resource will offer valuable insights into the broader functions of DNA damage response genes and accelerate research on variants relevant to cancer therapy.},
}

*DNA Damage
Humans
*DNA Repair
*Gene Editing/methods
CRISPR-Cas Systems/genetics
Protein Serine-Threonine Kinases/metabolism/genetics
Cisplatin/pharmacology
Mutation/genetics
Replication Protein A/metabolism/genetics
HEK293 Cells

@article {pmid39660762,
year = {2024},
author = {Zeng, T and Wu, Q and Liu, Y and Qi, Q and Shen, W and Gu, W and Zhang, Y and Xiong, W and Xie, Z and Qi, X and Tian, T and Zhou, X},
title = {Unraveling the Cleavage Reaction of Hydroxylamines with Cyclopropenones Considering Biocompatibility.},
journal = {Journal of the American Chemical Society},
volume = {146},
number = {51},
pages = {35077-35089},
doi = {10.1021/jacs.4c09757},
pmid = {39660762},
issn = {1520-5126},
mesh = {*Cyclopropanes/chemistry/pharmacology ; Humans ; HeLa Cells ; *Hydroxylamines/chemistry ; Biocompatible Materials/chemistry/pharmacology/chemical synthesis ; Density Functional Theory ; Molecular Structure ; CRISPR-Cas Systems ; Prodrugs/chemistry/pharmacology/chemical synthesis ; },
abstract = {We develop a latent biocompatible cleavage reaction involving the hitherto unexplored interaction between hydroxylamines and cyclopropenones. Our study addresses the regioselectivity challenges commonly observed in asymmetric cyclopropenone transformations, substantiated by variations in substrate, Density Functional Theory calculations, and in situ NMR analysis. This reaction is characterized by high efficiency, broad substrate scope, stability, latent biocompatibility, and mild reaction conditions. Significantly, it facilitates fluorescence activation and functions as a controlled release mechanism for prodrugs, showing great promise in biological assays. Our success in achieving the controlled release of nitrogen mustard in HeLa cells underscores its potential application in cellular contexts. Additionally, we introduce a simple and highly efficient method for synthesizing α, β-substituted pentenolides, applicable to a variety of substrates. Moreover, we extend this cleavage reaction to the CRISPR-Cas9 system, achieving precise, on-demand regulation of guide RNA activity. The introduction of this cleavage reaction offers a promising tool for biochemical research and biotechnological applications.},
}

*Cyclopropanes/chemistry/pharmacology
Humans
HeLa Cells
*Hydroxylamines/chemistry
Biocompatible Materials/chemistry/pharmacology/chemical synthesis
Density Functional Theory
Molecular Structure
CRISPR-Cas Systems
Prodrugs/chemistry/pharmacology/chemical synthesis

@article {pmid39658506,
year = {2024},
author = {Shen, H and Weng, Z and Zhao, H and Song, H and Wang, F and Fan, C and Song, P},
title = {Random Sanitization in DNA Information Storage Using CRISPR-Cas12a.},
journal = {Journal of the American Chemical Society},
volume = {146},
number = {51},
pages = {35155-35164},
doi = {10.1021/jacs.4c11380},
pmid = {39658506},
issn = {1520-5126},
mesh = {*CRISPR-Cas Systems/genetics ; DNA/chemistry ; Information Storage and Retrieval ; DNA, Single-Stranded/chemistry ; Thermodynamics ; },
abstract = {DNA information storage provides an excellent solution for metadata storage due to its high density, programmability, and long-term stability. However, current research primarily focuses on the processes of storing and reading data, lacking comprehensive solutions for secure metadata wiping. Herein, we present a method of random sanitization in DNA information storage using CRISPR-Cas12a (RSDISC) based on precise control of the thermodynamic energy of primer-template hybridization. We utilize the collateral cleavage (trans-activity) of single-stranded DNA (ssDNA) by CRISPR-Cas12a to achieve selective sanitization of files in metadata. This method enables ssDNA degradation with different GC contents, lengths, and secondary structures to achieve a sanitization efficiency up to 99.9% for 28,258 oligonucleotides in DNA storage within one round. We demonstrate that the number of erasable files could reach 10[12] based on a model of primer-template hybridization efficiency. Overall, RSDISC provides a random sanitization approach to set the foundation of information encryption, file classification, memory deallocation, and accurate reading in DNA storage.},
}

*CRISPR-Cas Systems/genetics
DNA/chemistry
Information Storage and Retrieval
DNA, Single-Stranded/chemistry
Thermodynamics

@article {pmid36200515,
year = {2025},
author = {Abdallah, NA and Elsharawy, H and Abulela, HA and Thilmony, R and Abdelhadi, AA and Elarabi, NI},
title = {Multiplex CRISPR/Cas9-mediated genome editing to address drought tolerance in wheat.},
journal = {GM crops & food},
volume = {16},
number = {1},
pages = {1-17},
doi = {10.1080/21645698.2022.2120313},
pmid = {36200515},
issn = {2164-5701},
mesh = {*Triticum/genetics/growth & development ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Plants, Genetically Modified/genetics ; *Droughts ; Genome, Plant/genetics ; Plant Proteins/genetics ; Mutation/genetics ; Plant Leaves/genetics ; Drought Resistance ; },
abstract = {Genome editing tools have rapidly been adopted by plant scientists for crop improvement. Genome editing using a multiplex sgRNA-CRISPR/Cas9 genome editing system is a useful technique for crop improvement in monocot species. In this study, we utilized precise gene editing techniques to generate wheat 3'(2'), 5'-bisphosphate nucleotidase (TaSal1) mutants using a multiplex sgRNA-CRISPR/Cas9 genome editing system. Five active TaSal1 homologous genes were found in the genome of Giza168 in addition to another apparently inactive gene on chromosome 4A. Three gRNAs were designed and used to target exons 4, 5 and 7 of the five wheat TaSal1 genes. Among the 120 Giza168 transgenic plants, 41 lines exhibited mutations and produced heritable TaSal1 mutations in the M1 progeny and 5 lines were full 5 gene knock-outs. These mutant plants exhibit a rolled-leaf phenotype in young leaves and bended stems, but there were no significant changes in the internode length and width, leaf morphology, and stem shape. Anatomical and scanning electron microscope studies of the young leaves of mutated TaSal1 lines showed closed stomata, increased stomata width and increase in the size of the bulliform cells. Sal1 mutant seedlings germinated and grew better on media containing polyethylene glycol than wildtype seedlings. Our results indicate that the application of the multiplex sgRNA-CRISPR/Cas9 genome editing is efficient tool for mutating more multiple TaSal1 loci in hexaploid wheat.},
}

*Triticum/genetics/growth & development
*Gene Editing/methods
*CRISPR-Cas Systems/genetics
*Plants, Genetically Modified/genetics
*Droughts
Genome, Plant/genetics
Plant Proteins/genetics
Mutation/genetics
Plant Leaves/genetics
Drought Resistance

@article {pmid39714446,
year = {2024},
author = {Petazzi, P and Ventura, T and Luongo, FP and McClafferty, H and May, A and Taylor, HA and Shipston, MJ and Romanò, N and Forrester, LM and Menendez, P and Fidanza, A},
title = {A novel human pluripotent stem cell gene activation system identifies IGFBP2 as a mediator in the production of haematopoietic progenitors in vitro.},
journal = {eLife},
volume = {13},
number = {},
pages = {},
pmid = {39714446},
issn = {2050-084X},
support = {S002219/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; EHA RAG 2021//European Hematology Association/ ; Research Global Award//American Society for Hematology/ ; Precision Medicine PhD scholarship/MRC_/Medical Research Council/United Kingdom ; Tissue Repair PhD studentship//College of Medicine and Veterinary Medicine, University of Edinburgh/ ; Traineeship Program 2016/2017//Erasmus +/ ; PERIS program//Catalan Government/ ; RTC-2018-4603-1//MINECO/ ; },
mesh = {*Insulin-Like Growth Factor Binding Protein 2/metabolism/genetics ; Humans ; *Cell Differentiation ; *Hematopoietic Stem Cells/metabolism ; Induced Pluripotent Stem Cells/metabolism ; Transcriptional Activation ; CRISPR-Cas Systems ; Transcription Factors/metabolism/genetics ; Pluripotent Stem Cells/metabolism ; },
abstract = {A major challenge in the stem cell biology field is the ability to produce fully functional cells from induced pluripotent stem cells (iPSCs) that are a valuable resource for cell therapy, drug screening, and disease modelling. Here, we developed a novel inducible CRISPR-mediated activation strategy (iCRISPRa) to drive the expression of multiple endogenous transcription factors (TFs) important for in vitro cell fate and differentiation of iPSCs to haematopoietic progenitor cells. This work has identified a key role for IGFBP2 in developing haematopoietic progenitors. We first identified nine candidate TFs that we predicted to be involved in blood cell emergence during development, then generated tagged gRNAs directed to the transcriptional start site of these TFs that could also be detected during single-cell RNA sequencing (scRNAseq). iCRISPRa activation of these endogenous TFs resulted in a significant expansion of arterial-fated endothelial cells expressing high levels of IGFBP2, and our analysis indicated that IGFBP2 is involved in the remodelling of metabolic activity during in vitro endothelial to haematopoietic transition. As well as providing fundamental new insights into the mechanisms of haematopoietic differentiation, the broader applicability of iCRISPRa provides a valuable tool for studying dynamic processes in development and for recapitulating abnormal phenotypes characterised by ectopic activation of specific endogenous gene expression in a wide range of systems.},
}

*Insulin-Like Growth Factor Binding Protein 2/metabolism/genetics
Humans
*Cell Differentiation
*Hematopoietic Stem Cells/metabolism
Induced Pluripotent Stem Cells/metabolism
Transcriptional Activation
CRISPR-Cas Systems
Transcription Factors/metabolism/genetics
Pluripotent Stem Cells/metabolism

@article {pmid39714100,
year = {2024},
author = {Cui, Q and Zhang, Z and Qin, L and Teng, Z and Wang, Z and Wu, W and Fan, L and Su, J and Hao, Y and Qin, J and Zhang, L and Wang, Q and Zhuang, Y and Zheng, H and Zhang, S and Geng, X and Zhu, L and Chen, Y and Lu, B and Li, Y and Zhu, X},
title = {Interleukin-37 promotes wound healing in diabetic mice by inhibiting the MAPK/NLRP3 pathway.},
journal = {Journal of diabetes investigation},
volume = {},
number = {},
pages = {},
doi = {10.1111/jdi.14389},
pmid = {39714100},
issn = {2040-1124},
support = {3030294002//Multidisciplinary Diagnosis and Treatment (MDT) Construction Project of Diabetic Foot/ ; 82400978//National Natural Science Foundation of China/ ; 82000790//National Natural Science Foundation of China/ ; 82200949//National Natural Science Foundation of China/ ; DGF501069/015//Multidisciplinary Diagnosis and Treatment (MDT) demonstration project in research hospital, Shanghai Medical College, Fudan University/ ; },
abstract = {AIMS/INTRODUCTION: Diabetic foot ulcer (DFU) is a prevalent complication of diabetes characterized by heightened inflammation and impaired wound-healing processes. Interleukin-37 (IL-37) is a natural suppressor of innate inflammation. Here, we aim to investigate the potential of IL-37 in enhancing the healing process of diabetic wounds.

MATERIALS AND METHODS: The skin samples of DFU and non-diabetic patients during foot and ankle orthopedic surgery were collected. The IL-37 transgenic mice (IL-37Tg) were created using CRISPR/Cas-mediated genome engineering. Mice were administered streptozotocin (STZ, 150 mg/kg) to induce a diabetic model. After 4 weeks, an equidistant full-thickness excisional wound measuring 8 mm was created on the central back of each mouse and allowed to heal naturally. Body weight and blood glucose levels were measured weekly. The wound area was measured, and skin samples were collected on Day 10 for further Quantitative polymerase chain reaction (qPCR) and WB detection and RNA sequencing analysis.

RESULTS: The proinflammation cytokines such as TNF-α and IL-1β and the MAPK signaling pathway were significantly increased in the wound margin of DFU patients. Compared with diabetic mice, diabetic IL-37Tg mice showed a significantly accelerated healing process. The enriched signaling pathways in RNA sequencing included cytokine-cytokine receptor interaction, TNF signaling pathway, and NOD-like receptor signaling pathway. Through QPCR and WB detection, we found that IL-37 could inhibit the activated MAPK and NOD-like signaling pathway, reducing TNF-α, IL-1β, and NLRP3 expression in the diabetic wound.

CONCLUSIONS: IL-37 promotes skin wound healing in diabetic mice, providing a new possible target for treating diabetic wounds.},
}

@article {pmid39713824,
year = {2024},
author = {Karesh, WB},
title = {Shifting from wildlife disease threats to wildlife health.},
journal = {Revue scientifique et technique (International Office of Epizootics)},
volume = {Special Edition},
number = {},
pages = {141-144},
doi = {10.20506/rst.SE.3568},
pmid = {39713824},
issn = {0253-1933},
mesh = {Animals ; *Animals, Wild ; Humans ; Zoonoses/prevention & control ; Conservation of Natural Resources ; Animal Diseases/prevention & control/epidemiology ; },
abstract = {The evolution of wildlife disease management and surveillance, as documented in the World Organisation for Animal Health's Scientific and Technical Review, reflects a deepening understanding of the links between wildlife health, ecosystem integrity and human well-being. Early work, beginning with the World Assembly of Delegates in 1954, primarily focused on diseases like rabies. This focus expanded over time to include broader concerns such as the impacts of climate change, habitat loss and increased human-wildlife interactions on wildlife health. By the late 20th century, the emphasis had shifted towards improved practices for wildlife disease control and the development of advanced diagnostic methods and vaccines. Articles in the Review highlight the growing complexity of wildlife diseases and the need for holistic management strategies. The adoption in recent years of cutting-edge technologies like CRISPR-Cas systems and metagenomics points to a future of more proactive and integrated approaches to wildlife disease management. There is still a need to address not just the consequences of wildlife diseases but also their anthropogenic drivers. The latest perspectives advocate for nature-based solutions, expanded partnerships and systems-level thinking to effectively tackle 21st-century challenges in wildlife and biodiversity conservation.},
}

Animals
*Animals, Wild
Humans
Zoonoses/prevention & control
Conservation of Natural Resources
Animal Diseases/prevention & control/epidemiology

@article {pmid39712894,
year = {2024},
author = {Wang, X and Yang, R and Tang, T and Zhou, Y and Chen, H and Jiang, Y and Zhang, S and Qin, S and Wang, M and Wang, C},
title = {One-pot MCDA-CRISPR-Cas-based detection platform for point-of-care testing of severe acute respiratory syndrome coronavirus 2.},
journal = {Frontiers in microbiology},
volume = {15},
number = {},
pages = {1503356},
pmid = {39712894},
issn = {1664-302X},
abstract = {Compared to quantitative real-time PCR (q-PCR), CRISPR-Cas-mediated technology is more suitable for point-of-care testing (POCT) and has potential for wider application in the future. Generally, the operational procedure of CRISPR-Cas-mediated diagnostic method consists of two independent steps, the reaction of signal amplification and the CRISPR-Cas-mediated signal detection. Complex multi-step procedures can easily lead to cross-contamination. To develop a convenient and rapid method for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection, we propose a MCTOP method (Multiple cross displacement amplification-CRISPR-Cas12b-based testing in one-pot), which targets the open reading frame 1ab (ORF1ab) and nucleocapsid protein (N) gene of SARS-CoV-2. This method combines MCDA isothermal amplification and CRISPR-Cas-mediated sequence-specific detection into a one-pot reaction. The optimal reaction was achieved with isothermal amplification of 40 min and CRISPR-Cas-based detection of 15 min, both at 64°C. Then, the results can be visualized by the real-time fluorescence instrument and also lateral flow biosensor. The lowest detection limit of the proposed method is 10 copies of each of target sequences, and it has no cross-reactivity with non-SARS-CoV-2 templates. In a clinical test of 70 pharyngeal swab samples, MCTOP assay showed a specificity of 100% and sensitivities of 98 and 96% for the real-time fluorescence instrument and lateral flow biosensor, respectively. The MCTOP developed in this study is a rapid, convenient, highly sensitive, and specific method for SARS-CoV-2 nucleic acid detection. It can be used as an effective point-of-care testing (POCT) tool for clinical diagnosis and epidemiologic surveillance of SARS-CoV-2 infections, especially suitable for the basic, field and clinical laboratory.},
}

@article {pmid39710679,
year = {2024},
author = {Jia, X and Yuan, B and Wang, W and Wang, K and Ling, D and Wei, M and Hu, Y and Guo, W and Chen, Z and Du, L and Jin, Y},
title = {Gene editing tool-loaded biomimetic cationic vesicles with highly efficient bacterial internalization for in vivo eradication of pathogens.},
journal = {Journal of nanobiotechnology},
volume = {22},
number = {1},
pages = {787},
pmid = {39710679},
issn = {1477-3155},
support = {7232261//Beijing Natural Science Foundation of China/ ; },
mesh = {*Gene Editing/methods ; *Pseudomonas aeruginosa/drug effects/genetics ; *CRISPR-Cas Systems ; *Plasmids/genetics ; *Acinetobacter baumannii/drug effects/genetics ; Animals ; *Cations/chemistry ; Mice ; Humans ; Pseudomonas Infections/drug therapy ; Anti-Bacterial Agents/pharmacology/chemistry ; Drug Resistance, Multiple, Bacterial/drug effects/genetics ; Acinetobacter Infections/drug therapy ; COVID-19 ; Female ; Mice, Inbred BALB C ; Biomimetics/methods ; },
abstract = {In the post-COVID-19 era, drug-resistant bacterial infections emerge as one of major death causes, where multidrug-resistant Acinetobacter baumannii (MRAB) and drug-resistant Pseudomonas aeruginosa (DRPA) represent primary pathogens. However, the classical antibiotic strategy currently faces the bottleneck of drug resistance. We develop an antimicrobial strategy that applies the selective delivery of CRISPR/Cas9 plasmids to pathogens with biomimetic cationic hybrid vesicles (BCVs), irrelevant to bacterial drug resistance. CRISPR/Cas9 plasmids were constructed, replicating in MRAB or DRPA and expressing ribonucleic proteins, leading to irreparable chromosomal lesions; however, delivering the negatively charged plasmids with extremely large molecular weight to the pathogens at the infection site became a huge challenge. We found that the BCVs integrating the bacterial out membrane vesicles and cationic lipids efficiently delivered the plasmids in vitro/in vivo to the pathogens followed by effective internalization. The BCVs were used by intratracheal or topical hydrogel application against MRAB pulmonary infection or DRPA wound infection, and both of the two pathogens were eradicated from the lung or the wound. CRISPR/Cas9 plasmid-loaded BCVs become a promising medication for drug-resistant bacteria infections.},
}

*Gene Editing/methods
*Pseudomonas aeruginosa/drug effects/genetics
*CRISPR-Cas Systems
*Plasmids/genetics
*Acinetobacter baumannii/drug effects/genetics
Animals
*Cations/chemistry
Mice
Humans
Pseudomonas Infections/drug therapy
Anti-Bacterial Agents/pharmacology/chemistry
Drug Resistance, Multiple, Bacterial/drug effects/genetics
Acinetobacter Infections/drug therapy
COVID-19
Female
Mice, Inbred BALB C
Biomimetics/methods

@article {pmid39666461,
year = {2024},
author = {Yan, B and Liu, Y and Cai, Y and Liu, Y and Chen, Y},
title = {Protocol for establishing CRISPR-Cas12a for efficient genome editing of Pseudomonas aeruginosa phages.},
journal = {STAR protocols},
volume = {5},
number = {4},
pages = {103488},
doi = {10.1016/j.xpro.2024.103488},
pmid = {39666461},
issn = {2666-1667},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Pseudomonas aeruginosa/genetics/virology ; Genome, Viral/genetics ; Pseudomonas Phages/genetics ; Plasmids/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {We developed an efficient type V CRISPR-Cas12a system tailored specifically for Pseudomonas aeruginosa phages, showcasing its remarkable cleavage activity and the ability to precisely introduce genetic modifications, including point mutations, deletions, and insertions, into phage genomes. Here, we present a protocol for establishing CRISPR-Cas12a for genome editing of Pseudomonas aeruginosa phages. We describe steps for the construction of pCRISPR-12a plasmid and guide RNA and the utilization of the type V CRISPR-Cas12a system for precise genetic editing of phages. For complete details on the use and execution of this protocol, please refer to Chen et al.[1].},
}

*CRISPR-Cas Systems/genetics
*Gene Editing/methods
*Pseudomonas aeruginosa/genetics/virology
Genome, Viral/genetics
Pseudomonas Phages/genetics
Plasmids/genetics
RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid39661507,
year = {2024},
author = {Ordóñez, A and Ron, D and Harding, HP},
title = {Protocol for iterative enrichment of integrated sgRNAs via derivative CRISPR-Cas9 libraries from genomic DNA of sorted fixed cells.},
journal = {STAR protocols},
volume = {5},
number = {4},
pages = {103493},
doi = {10.1016/j.xpro.2024.103493},
pmid = {39661507},
issn = {2666-1667},
mesh = {*CRISPR-Cas Systems/genetics ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Humans ; DNA/genetics ; Gene Library ; Lentivirus/genetics ; Gene Editing/methods ; },
abstract = {Here, we present a protocol for iterative enrichment of integrated single guide RNA (sgRNA) via derivative CRISPR-Cas9 from genomic DNA (gDNA) of phenotypically sorted fixed cells. We describe steps for high-scale lentiviral production, genome-wide screening, extracting gDNA from fixed cells, cloning of integrated sgRNAs, and high-scale transformation. This protocol introduces three key advantages: (1) applicability to fixed cells, (2) bypassing epigenetic drift, and (3) pause points lowering the contamination risk. We believe this approach will benefit researchers applying somatic cell genetics in cell biology. For complete details on the use and execution of this protocol, please refer to Ordoñez et al.[1].},
}

*CRISPR-Cas Systems/genetics
*RNA, Guide, CRISPR-Cas Systems/genetics
Humans
DNA/genetics
Gene Library
Lentivirus/genetics
Gene Editing/methods

@article {pmid39487981,
year = {2024},
author = {Sonawala, U and Derevnina, L and Eves-van den Akker, S},
title = {Protocol for Cas9-targeted long-read sequencing in Globodera pallida and Globodera rostochiensis.},
journal = {STAR protocols},
volume = {5},
number = {4},
pages = {103427},
pmid = {39487981},
issn = {2666-1667},
mesh = {Animals ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Tylenchoidea/genetics ; Solanum tuberosum/parasitology/genetics ; CRISPR-Cas Systems/genetics ; High-Throughput Nucleotide Sequencing/methods ; CRISPR-Associated Protein 9/genetics/metabolism ; Sequence Analysis, DNA/methods ; },
abstract = {We present a protocol to achieve a higher depth of long-read sequencing of region(s) of interest in potato cyst nematodes without amplification using a Cas9-based Nanopore enrichment approach. We describe steps for designing high-fidelity guide RNAs to be used with Cas9 nuclease, extracting high-molecular-weight DNA from the nematodes, and dephosphorylating genomic DNA ends. We then detail procedures for using Cas9-guide RNA complex to make targeted cleavage of the region of interest followed by a Nanopore library preparation. For complete details on the use and execution of this protocol, please refer to Sonawala et al.[1].},
}

Animals
*RNA, Guide, CRISPR-Cas Systems/genetics
Tylenchoidea/genetics
Solanum tuberosum/parasitology/genetics
CRISPR-Cas Systems/genetics
High-Throughput Nucleotide Sequencing/methods
CRISPR-Associated Protein 9/genetics/metabolism
Sequence Analysis, DNA/methods

@article {pmid39471176,
year = {2024},
author = {Bao, Y and Wei, W},
title = {Protocol for high-throughput screening of functional lysine residues in cell fitness.},
journal = {STAR protocols},
volume = {5},
number = {4},
pages = {103418},
pmid = {39471176},
issn = {2666-1667},
mesh = {Humans ; *Lysine/metabolism/chemistry ; *High-Throughput Screening Assays/methods ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; Gene Editing/methods ; CRISPR-Cas Systems/genetics ; Genome, Human ; HEK293 Cells ; },
abstract = {Amino acid residues are crucial to protein structure and function and have links to various human diseases. Here, we present a protocol for screening functional lysine residues across the human genome. We describe steps for designing lysine codon-targeting single-guide RNAs (sgRNAs), constructing an sgRNA library, conducting cell fitness screenings, and acquiring screening results. This approach leverages base editing and high-throughput screening techniques to systematically examine functional amino acid residues. For complete details on the use and execution of this protocol, please refer to Bao et al.[1].},
}

Humans
*Lysine/metabolism/chemistry
*High-Throughput Screening Assays/methods
RNA, Guide, CRISPR-Cas Systems/genetics/metabolism
Gene Editing/methods
CRISPR-Cas Systems/genetics
Genome, Human
HEK293 Cells

@article {pmid39427312,
year = {2024},
author = {Mao, Q and Jiang, J and Ye, Q and Wang, H and Lin, CP},
title = {Protocol for the derivation and culture of murine trophoblast organoids for CRISPR-Cas9 screening.},
journal = {STAR protocols},
volume = {5},
number = {4},
pages = {103405},
pmid = {39427312},
issn = {2666-1667},
mesh = {Animals ; *Trophoblasts/cytology/metabolism ; Mice ; *CRISPR-Cas Systems/genetics ; *Organoids/cytology/metabolism ; Female ; RNA, Guide, CRISPR-Cas Systems/genetics ; Pregnancy ; Cell Culture Techniques/methods ; Cell Differentiation/genetics ; Placenta/cytology/metabolism ; },
abstract = {Murine trophoblast organoids present a more balanced array of trophoblast subtypes, rendering them a suitable platform for CRISPR-Cas9-based screening. Here, we present a protocol for the derivation and culture of murine trophoblast organoids from trophoblast stem cells or placentae. We describe steps for establishing and differentiating murine trophoblast organoids, the characterization of trophoblast organoids in both conditions, the generation of focused single guide RNA (sgRNA) libraries, and the subsequent screening using those libraries in murine trophoblast organoids. For complete details on the use and execution of this protocol, please refer to Mao et al.[1].},
}

Animals
*Trophoblasts/cytology/metabolism
Mice
*CRISPR-Cas Systems/genetics
*Organoids/cytology/metabolism
Female
RNA, Guide, CRISPR-Cas Systems/genetics
Pregnancy
Cell Culture Techniques/methods
Cell Differentiation/genetics
Placenta/cytology/metabolism

@article {pmid39427309,
year = {2024},
author = {Xiao, YX and Wei, J and Moffat, J},
title = {Protocol for CRISPR-based endogenous protein tagging in mammalian cells.},
journal = {STAR protocols},
volume = {5},
number = {4},
pages = {103404},
pmid = {39427309},
issn = {2666-1667},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; HEK293 Cells ; Transfection/methods ; Animals ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Electroporation/methods ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; },
abstract = {Tracking the localization and proximal interaction partners of endogenous proteins provides valuable functional insight. Here, we present a protocol for CRISPR-based endogenous protein tagging in mammalian cells. We describe steps for endogenously tagging human TSC22D2 and MAP4, including designing Cas9 and Cas12a guides for knockin, modularized repair template design and cloning, and procedures for lipid transfection and electroporation. This protocol accommodates Cas nucleases in plasmid expression or ribonucleoprotein complex (RNP) formats. This "endo-tagging" approach offers flexibility and broad applicability. For complete details on the use and execution of this protocol, please refer to Xiao et al.[1].},
}

Humans
*CRISPR-Cas Systems/genetics
HEK293 Cells
Transfection/methods
Animals
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Electroporation/methods
RNA, Guide, CRISPR-Cas Systems/genetics/metabolism

@article {pmid39392744,
year = {2024},
author = {Kiesler, P and Lee, SS and Norris, AL and Miller, MF and Mercado, CJ and Moyer, AL and Maragh, S},
title = {Protocol for CRISPR-Cas9 genome editing of a swine cell line via electroporation.},
journal = {STAR protocols},
volume = {5},
number = {4},
pages = {103385},
doi = {10.1016/j.xpro.2024.103385},
pmid = {39392744},
issn = {2666-1667},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Electroporation/methods ; Swine ; Cell Line ; Male ; Testis/cytology/metabolism ; Transfection/methods ; },
abstract = {Genome editing technology is being used in animals for a variety of purposes, including improvement of animal and public health outcomes. Characterization of genome editing reagents and anticipated genomic alterations is an essential step toward the development of an edited animal. Here, we present a protocol for genome editing in the swine testicular (ST) cell line. We describe steps for evaluating CRISPR-Cas9 complex functionality in vitro, delivering editing molecules into cells by transfection, and assessing target editing via Sanger sequencing.},
}

Animals
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
*Electroporation/methods
Swine
Cell Line
Male
Testis/cytology/metabolism
Transfection/methods

@article {pmid39342621,
year = {2024},
author = {Dhaliwal, NK and Weng, OY and Li, Y},
title = {Protocol for the efficient and inducible generation of CRISPR-Cas9-edited human cortical neurons from the iCas9-iNgn2 hPSCs.},
journal = {STAR protocols},
volume = {5},
number = {4},
pages = {103352},
pmid = {39342621},
issn = {2666-1667},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Neurons/cytology/metabolism ; *Gene Editing/methods ; Pluripotent Stem Cells/cytology/metabolism ; Cerebral Cortex/cytology/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; Nerve Tissue Proteins/genetics/metabolism ; Lentivirus/genetics ; Basic Helix-Loop-Helix Transcription Factors ; },
abstract = {Generation of CRISPR-Cas9-edited cortical neurons from human pluripotent stem cells (hPSCs) enables the study of gene functions and neural disease mechanisms. Here, we present a protocol for developing iCas9-iNgn2 hPSC, an inducible cell line that allows the simultaneous induction of the neuralizing transcription factor Ngn2 and the Cas9 nuclease to rapidly generate edited human cortical neurons. We describe the steps of the protocol from transducing iCas9-iNgn2 with guide RNA-containing lentivirus to producing edited cortical neurons in about 2 weeks. For complete details on the use and execution of this protocol, please refer to Dhaliwal et al.[1].},
}

Humans
*CRISPR-Cas Systems/genetics
*Neurons/cytology/metabolism
*Gene Editing/methods
Pluripotent Stem Cells/cytology/metabolism
Cerebral Cortex/cytology/metabolism
RNA, Guide, CRISPR-Cas Systems/genetics
Nerve Tissue Proteins/genetics/metabolism
Lentivirus/genetics
Basic Helix-Loop-Helix Transcription Factors

@article {pmid39277866,
year = {2024},
author = {Hu, X and Wang, Y},
title = {Protocol to identify receptors of secreted proteins through CRISPR-Cas9 whole-genome screening technology.},
journal = {STAR protocols},
volume = {5},
number = {4},
pages = {103315},
pmid = {39277866},
issn = {2666-1667},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; RNA, Guide, CRISPR-Cas Systems/genetics ; MCF-7 Cells ; Receptors, Cell Surface/genetics/metabolism ; Flow Cytometry/methods ; Lentivirus/genetics ; },
abstract = {The interaction between cell surface receptors and their ligands is crucial for intercellular communication. However, current techniques for identifying direct receptor-ligand interactions remain limited. Here, we present a protocol to identify receptors of secreted proteins using a genome-scale CRISPR-Cas9 knockout genetic screening approach. We describe steps for creating a single-guide RNA (sgRNA) lentivirus library, infecting stable Cas9-MCF7 cells, staining with tagged Cholesin, and sorting non-binding cells via flow cytometry. We then detail procedures for extracting DNA, amplifying sgRNAs, and sequencing. For complete details on the use and execution of this protocol, please refer to Hu et al.[1].},
}

*CRISPR-Cas Systems/genetics
Humans
RNA, Guide, CRISPR-Cas Systems/genetics
MCF-7 Cells
Receptors, Cell Surface/genetics/metabolism
Flow Cytometry/methods
Lentivirus/genetics

@article {pmid38886504,
year = {2024},
author = {Luna, SE and Camarena, J and Hampton, JP and Majeti, KR and Charlesworth, CT and Soupene, E and Selvaraj, S and Jia, K and Sheehan, VA and Cromer, MK and Porteus, MH},
title = {Enhancement of erythropoietic output by Cas9-mediated insertion of a natural variant in haematopoietic stem and progenitor cells.},
journal = {Nature biomedical engineering},
volume = {8},
number = {12},
pages = {1540-1552},
pmid = {38886504},
issn = {2157-846X},
support = {Junior Faculty Scholar Award//American Society of Hematology (ASH)/ ; Minority Medical Student Award//American Society of Hematology (ASH)/ ; Medical Scientist Training Program//SU | School of Medicine, Stanford University (Stanford School of Medicine, Stanford University)/ ; Stanford Medical Scholars Research Program//SU | School of Medicine, Stanford University (Stanford School of Medicine, Stanford University)/ ; },
mesh = {Humans ; *Hematopoietic Stem Cells/metabolism/cytology ; *Gene Editing/methods ; *Receptors, Erythropoietin/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *Erythropoiesis/genetics ; CRISPR-Associated Protein 9/metabolism/genetics ; Antigens, CD34/metabolism ; beta-Thalassemia/genetics ; },
abstract = {Some gene polymorphisms can lead to monogenic diseases, whereas other polymorphisms may confer beneficial traits. A well-characterized example is congenital erythrocytosis-the non-pathogenic hyper-production of red blood cells-that is caused by a truncated erythropoietin receptor. Here we show that Cas9-mediated genome editing in CD34[+] human haematopoietic stem and progenitor cells (HSPCs) can recreate the truncated form of the erythropoietin receptor, leading to substantial increases in erythropoietic output. We also show that combining the expression of the cDNA of a truncated erythropoietin receptor with a previously reported genome-editing strategy to fully replace the HBA1 gene with an HBB transgene in HSPCs (to restore normal haemoglobin production in cells with a β-thalassaemia phenotype) gives the edited HSPCs and the healthy red blood cell phenotype a proliferative advantage. Combining knowledge of human genetics with precise genome editing to insert natural human variants into therapeutic cells may facilitate safer and more effective genome-editing therapies for patients with genetic diseases.},
}

Humans
*Hematopoietic Stem Cells/metabolism/cytology
*Gene Editing/methods
*Receptors, Erythropoietin/genetics/metabolism
*CRISPR-Cas Systems/genetics
*Erythropoiesis/genetics
CRISPR-Associated Protein 9/metabolism/genetics
Antigens, CD34/metabolism
beta-Thalassemia/genetics

@article {pmid38092857,
year = {2024},
author = {Webber, BR and Johnson, MJ and Skeate, JG and Slipek, NJ and Lahr, WS and DeFeo, AP and Mills, LJ and Qiu, X and Rathmann, B and Diers, MD and Wick, B and Henley, T and Choudhry, M and Starr, TK and McIvor, RS and Moriarity, BS},
title = {Cas9-induced targeted integration of large DNA payloads in primary human T cells via homology-mediated end-joining DNA repair.},
journal = {Nature biomedical engineering},
volume = {8},
number = {12},
pages = {1553-1570},
pmid = {38092857},
issn = {2157-846X},
support = {R01 AI146009/AI/NIAID NIH HHS/United States ; R01 AI161017/AI/NIAID NIH HHS/United States ; R37 CA276345/CA/NCI NIH HHS/United States ; T32HL007062-46//Foundation for the National Institutes of Health (Foundation for the National Institutes of Health, Inc.)/ ; },
mesh = {Humans ; Animals ; *T-Lymphocytes/immunology ; Mice ; DNA End-Joining Repair ; CRISPR-Cas Systems/genetics ; DNA ; Receptors, Antigen, T-Cell/genetics/immunology/metabolism ; CRISPR-Associated Protein 9/metabolism/genetics ; },
abstract = {The reliance on viral vectors for the production of genetically engineered immune cells for adoptive cellular therapies remains a translational bottleneck. Here we report a method leveraging the DNA repair pathway homology-mediated end joining, as well as optimized reagent composition and delivery, for the Cas9-induced targeted integration of large DNA payloads into primary human T cells with low toxicity and at efficiencies nearing those of viral vectors (targeted knock-in of 1-6.7 kb payloads at rates of up to 70% at multiple targeted genomic loci and with cell viabilities of over 80%). We used the method to produce T cells with an engineered T-cell receptor or a chimaeric antigen receptor and show that the cells maintained low levels of exhaustion markers and excellent capacities for proliferation and cytokine production and that they elicited potent antitumour cytotoxicity in vitro and in mice. The method is readily adaptable to current good manufacturing practices and scale-up processes, and hence may be used as an alternative to viral vectors for the production of genetically engineered T cells for cancer immunotherapies.},
}

Humans
Animals
*T-Lymphocytes/immunology
Mice
DNA End-Joining Repair
CRISPR-Cas Systems/genetics
DNA
Receptors, Antigen, T-Cell/genetics/immunology/metabolism
CRISPR-Associated Protein 9/metabolism/genetics

@article {pmid39709563,
year = {2024},
author = {Shepelev, MV and Komkov, DS and Golubev, DS and Borovikova, SE and Mazurov, DV and Kruglova, NA},
title = {[Donor DNA Modification with Cas9 Targeting Sites Improves the Efficiency of MTC34 Knock-in into the CXCR4 Locus].},
journal = {Molekuliarnaia biologiia},
volume = {58},
number = {4},
pages = {590-600},
pmid = {39709563},
issn = {0026-8984},
mesh = {Humans ; *CRISPR-Cas Systems ; *Receptors, CXCR4/genetics/metabolism ; *Gene Knock-In Techniques/methods ; Gene Editing/methods ; Plasmids/genetics/metabolism ; CRISPR-Associated Protein 9/genetics/metabolism ; DNA/genetics/metabolism ; Cell Line ; DNA Breaks, Double-Stranded ; HIV-1/genetics/metabolism ; },
abstract = {To successfully apply the genome editing technology using the CRISPR/Cas9 system in the clinic, it is necessary to achieve a high efficiency of knock-in, which is insertion of a genetic construct into a given locus of the target cell genome. One of the approaches to increase the efficiency of knock-in is to modify donor DNA with the same Cas9 targeting sites (CTS) that are used to induce double-strand breaks (DSBs) in the cell genome (the double-cut donor method). Another approach is based on introducing truncated CTS (tCTS), including a PAM site and 16 proximal nucleotides, into the donor DNA. Presumably, tCTS sites do not induce cleavage of the donor plasmid, but can support its transport into the nucleus by Cas9. However, the exact mechanisms whereby these two donor DNA modifications increase the knock-in level are unknown. In this study, the modifications were tested for effect on the knock-in efficiency of the MTC34 genetic construct encoding the HIV-1 fusion inhibitory peptide MT-C34 into the CXCR4 locus of the CEM/R5 T-cell line. When full-length CTSs were introduced into the donor plasmid DNA, the knock-in level was doubled regardless of the CTS number or position relative to the donor sequence. Modifications with tCTSs did not affect the knock-in levels. In vitro, both CTS and tCTS were efficiently cleaved by Cas9. To understand the mechanism of action of these modifications in detail, it is necessary to evaluate their cleavage both in vitro and in vivo.},
}

Humans
*CRISPR-Cas Systems
*Receptors, CXCR4/genetics/metabolism
*Gene Knock-In Techniques/methods
Gene Editing/methods
Plasmids/genetics/metabolism
CRISPR-Associated Protein 9/genetics/metabolism
DNA/genetics/metabolism
Cell Line
DNA Breaks, Double-Stranded
HIV-1/genetics/metabolism

@article {pmid39709562,
year = {2024},
author = {Golubev, DS and Komkov, DS and Shepelev, MV and Mazurov, DV and Kruglova, NA},
title = {[Methods to Increase the Efficiency of Knock-in of a Construct Encoding the HIV-1 Fusion Inhibitor, MT-C34 Peptide, into the CXCR4 Locus in the CEM/R5 T Cell Line].},
journal = {Molekuliarnaia biologiia},
volume = {58},
number = {4},
pages = {575-589},
pmid = {39709562},
issn = {0026-8984},
mesh = {*Receptors, CXCR4/genetics/metabolism ; Humans ; *Gene Knock-In Techniques ; *HIV-1/genetics/drug effects ; HIV Fusion Inhibitors/pharmacology ; CRISPR-Cas Systems ; Cell Line ; DNA End-Joining Repair/drug effects/genetics ; Gene Editing/methods ; Nuclear Localization Signals/genetics ; Plasmids/genetics/metabolism ; T-Lymphocytes/metabolism/drug effects ; NF-kappa B/metabolism/genetics ; CRISPR-Associated Protein 9/genetics/metabolism ; HIV Infections/genetics/virology/drug therapy ; },
abstract = {The low knock-in efficiency, especially in primary human cells, limits the use of the genome editing technology for therapeutic purposes, rendering it important to develop approaches for increasing the knock-in levels. In this work, the efficiencies of several approaches were studied using a model of knock-in of a construct coding for the peptide HIV fusion inhibitor MT-C34 into the human CXCR4 locus in the CEM/R5 T cell line. First, donor DNA modification was evaluated as a means to improve the efficiency of plasmid transport into the nucleus. The donor plasmid was modified to include the simian virus 40 (SV40) DNA nuclear targeting sequence (DTS) or binding sites for the transcription factor NF-κB, whose effects on the knock-in levels have not been described. The modification was ineffective in the model of MT-C34 knock-in into the CXCR4 locus. A second approach consisted in modification of Cas9 nuclease by introducing two additional nuclear localization signals (NLSs) and increased the knock-in level by 30%. Finally, blocking DNA repair via the nonhomologous end joining (NHEJ) pathway with DNA-dependent protein kinase inhibitors caused a 1.8-fold increase in knock-in. A combination of the last two approaches caused an additive effect. Thus, increasing the number of NLSs in the Cas9 protein and inhibiting DNA repair via the NHEJ pathway significantly increased the level of knock-in of the HIV-1 fusion inhibitory peptide into the clinically relevant locus CXCR4. The finding can be used to develop effective gene therapy approaches for treating HIV infection.},
}

*Receptors, CXCR4/genetics/metabolism
Humans
*Gene Knock-In Techniques
*HIV-1/genetics/drug effects
HIV Fusion Inhibitors/pharmacology
CRISPR-Cas Systems
Cell Line
DNA End-Joining Repair/drug effects/genetics
Gene Editing/methods
Nuclear Localization Signals/genetics
Plasmids/genetics/metabolism
T-Lymphocytes/metabolism/drug effects
NF-kappa B/metabolism/genetics
CRISPR-Associated Protein 9/genetics/metabolism
HIV Infections/genetics/virology/drug therapy

@article {pmid39709560,
year = {2024},
author = {Averina, OA and Kuznetsova, SA and Permyakov, OA and Sergiev, PV},
title = {[How to Shift the Equilibrium of DNA Break Repair in Favor of Homologous Recombination].},
journal = {Molekuliarnaia biologiia},
volume = {58},
number = {4},
pages = {525-548},
pmid = {39709560},
issn = {0026-8984},
mesh = {Humans ; Animals ; *DNA Breaks, Double-Stranded ; *DNA End-Joining Repair ; *Recombinational DNA Repair ; CRISPR-Cas Systems ; Gene Editing/methods ; Cell Cycle/genetics ; Homologous Recombination ; },
abstract = {The CRISPR/Cas technology of targeted genome editing made it possible to carry out genetic engineering manipulations with eukaryotic genomes with a high efficiency. Targeted induction of site-specific DNA breaks is one of the key stages of the technology. The cell repairs the breaks via one of the two pathways, nonhomologous end joining (NHEJ) and homology-driven repair (HDR). The choice of the DNA repair pathway is determined by the architecture of the DNA break region formed as a result of terminal resection and depends on the cell cycle phase. NHEJ is the main pathway of double-strand break (DSB) repair in mammalian cells and involves a nonspecific ligation reaction. The reaction accuracy depends on the structure of break ends, and various insertions or deletions may arise as a result in the target genome region. Integration of a necessary sequence into the genome occurs via HDR, which requires a template with homology regions flanking a DSB. Introducing a genetic construct into a particular genomic locus is an important task, but is currently intricate and laborious to perform. However, the choice of the repair pathway can be of principal importance for basic research of gene functions and construction of animal models of human diseases to develop therapies. The review summarizes and systematizes the available information on strategies designed to increase the HDR efficiency. The strategies that most efficiently shift the balance towards HDR include use of NHEJ inhibitors, regulation of the key factors of homologous recombination, control of the cell cycle and chromatin status, and construction of HDR templates.},
}

Humans
Animals
*DNA Breaks, Double-Stranded
*DNA End-Joining Repair
*Recombinational DNA Repair
CRISPR-Cas Systems
Gene Editing/methods
Cell Cycle/genetics
Homologous Recombination

@article {pmid39709559,
year = {2024},
author = {Averina, OA and Kuznetsova, SA and Permyakov, OA and Sergiev, PV},
title = {[Current Knowledge of Base Editing and Prime Editing].},
journal = {Molekuliarnaia biologiia},
volume = {58},
number = {4},
pages = {508-524},
pmid = {39709559},
issn = {0026-8984},
mesh = {*Gene Editing/methods ; Humans ; *CRISPR-Cas Systems ; DNA Breaks, Double-Stranded ; Animals ; Genetic Engineering/methods ; DNA/genetics ; },
abstract = {Modern genetic engineering technologies, such as base editing and prime editing (PE), have proven to provide the efficient and reliable genome editing tools that obviate the need for donor templates and double-strand breaks (DSBs) introduced in DNA. Relatively new, they quickly gained recognition for their accuracy, simplicity, and multiplexing capabilities. The review summarizes the new literature on the technologies and considers their architecture, methods to create editors, specificity, efficiency, and versatility. Advantages and disadvantages of the editors are discussed along with their prospective use in basic and applied research. The review may be useful for planning genome editing studies and analyzing their results to solve various problems of fundamental biology, biotechnology, medicine, and agriculture.},
}

*Gene Editing/methods
Humans
*CRISPR-Cas Systems
DNA Breaks, Double-Stranded
Animals
Genetic Engineering/methods
DNA/genetics

@article {pmid39708170,
year = {2024},
author = {Feng, R and Mao, K and Zhang, H and Zhu, H and Du, W and Yang, Z and Wang, S},
title = {Portable microfluidic devices for monitoring antibiotic resistance genes in wastewater.},
journal = {Mikrochimica acta},
volume = {192},
number = {1},
pages = {19},
pmid = {39708170},
issn = {1436-5073},
support = {42377456//the National Natural Science Foundation of China/ ; 2023415//the Youth Innovation Promotion Association CAS/ ; CAS-ANSO-FS-2024-34//CAS-ANSO Fellowship/ ; Qiankehe Jichu-ZK [2022] Yiban 565, Qiankehe Platform Talents-GCC [2023] 046//Guizhou Provincial Science and Technology Projects/ ; },
mesh = {*Wastewater/microbiology ; *Lab-On-A-Chip Devices ; Nucleic Acid Amplification Techniques/methods ; Drug Resistance, Microbial/genetics ; Genes, Bacterial ; CRISPR-Cas Systems/genetics ; Anti-Bacterial Agents ; Microfluidic Analytical Techniques/instrumentation/methods ; Drug Resistance, Bacterial/genetics ; },
abstract = {Antibiotic resistance genes (ARGs) pose serious threats to environmental and public health, and monitoring ARGs in wastewater is a growing need because wastewater is an important source. Microfluidic devices can integrate basic functional units involved in sample assays on a small chip, through the precise control and manipulation of micro/nanofluids in micro/nanoscale spaces, demonstrating the great potential of ARGs detection in wastewater. Here, we (1) summarize the state of the art in microfluidics for recognizing ARGs, (2) determine the strengths and weaknesses of portable microfluidic chips, and (3) assess the potential of portable microfluidic chips to detect ARGs in wastewater. Isothermal nucleic acid amplification and CRISPR/Cas are two commonly used identification elements for the microfluidic detection of ARGs. The former has better sensitivity due to amplification, but false positives due to inappropriate primer design and contamination; the latter has better specificity. The combination of the two can achieve complementarity to a certain extent. Compared with traditional microfluidic chips, low-cost and biocompatible paper-based microfluidics is a very attractive test for ARGs, whose fluid flow in paper does not require external force, but it is weaker in terms of repeatability and high-throughput detection. Due to that only a handful of portable microfluidics detect ARGs in wastewater, fabricating high-throughput microfluidic chips, developing and optimizing recognition techniques for the highly selective and sensitive identification and quantification of a wide range of ARGs in complex wastewater matrices are needed.},
}

*Wastewater/microbiology
*Lab-On-A-Chip Devices
Nucleic Acid Amplification Techniques/methods
Drug Resistance, Microbial/genetics
Genes, Bacterial
CRISPR-Cas Systems/genetics
Anti-Bacterial Agents
Microfluidic Analytical Techniques/instrumentation/methods
Drug Resistance, Bacterial/genetics

@article {pmid39707688,
year = {2024},
author = {Garay-Novillo, JN and Ruiz-Masó, JÁ and Del Solar, G and Barra, JL},
title = {Easy-Curing and pH-Regulated CRISPR-Cas9 Plasmids for Gene Editing and Plasmid Curing in Lactococcus cremoris.},
journal = {Microbial biotechnology},
volume = {17},
number = {12},
pages = {e70060},
pmid = {39707688},
issn = {1751-7915},
support = {CSIC 2022AEP028//Consejo Superior de Investigaciones Científicas/ ; 2023/ResMi-00000018//Secretaría de Ciencia y Tecnología - Universidad Nacional de Córdoba and Ministerio de Ciencia y Tecnología - Córdoba/ ; PICT Start-Up 2018-00811//Agencia Nacional de Promoción Científica y Tecnológica/ ; CONSOLIDAR RESOL-2023-258-E-UNC-SECYT#ACTIP//Secretaría de Ciencia y Tecnología - Universidad Nacional de Córdoba/ ; MHE200027-EMHE-CSIC-2016-program//Consejo Superior de Investigaciones Científicas/ ; },
mesh = {*Plasmids/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *Genetic Vectors/genetics ; *Lactococcus/genetics ; Hydrogen-Ion Concentration ; },
abstract = {In this work, we developed a plasmid-based CRISPR-Cas9 strategy for editing Lactococcus cremoris, which allows easy generation of plasmid-free strains with the desired modification. We constructed versatile shuttle vectors based on the theta-type pAMβ1 promiscuous replicon and p15A ori, expressing both the Cas9 nuclease gene (under pH-regulated promoters derived from P170) and a single-guide RNA for specific targeting (under a strong constitutive promoter). The vectors designed for plasmid targeting were very effective for low- and high-copy-number plasmid curing in L. cremoris, and their targeting efficiency was shown to be tunable by regulating cas9 expression. For chromosome editing, we implemented a host-independent method that enhances double-homologous recombination events using plasmids expressing the genes encoding λRed-phage Redβ recombinase and Escherichia coli single-stranded DNA binding protein (EcSSB). By coupling either the endogenous recombination machinery or the Redβ-EcSSB-assisted recombination system with our novel chromosome-targeting CRISPR-Cas9 plasmids, we efficiently generated and selected thousands of gene-edited cells. Examination of the impact of the constructed CRISPR-Cas9 vectors on host fitness revealed no Cas9-associated toxicity, and, remarkably, these vectors exhibited a very high loss rate when growing the bacterial host cells in the absence of selective pressure.},
}

*Plasmids/genetics
*Gene Editing/methods
*CRISPR-Cas Systems
*Genetic Vectors/genetics
*Lactococcus/genetics
Hydrogen-Ion Concentration

@article {pmid39626969,
year = {2024},
author = {Haussmann, IU and Dix, TC and McQuarrie, DWJ and Dezi, V and Hans, AI and Arnold, R and Soller, M},
title = {Structure-optimized sgRNA selection with PlatinumCRISPr for efficient Cas9 generation of knockouts.},
journal = {Genome research},
volume = {34},
number = {12},
pages = {2279-2292},
doi = {10.1101/gr.279479.124},
pmid = {39626969},
issn = {1549-5469},
mesh = {*RNA, Guide, CRISPR-Cas Systems/genetics ; Animals ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Humans ; CRISPR-Associated Protein 9/metabolism/genetics ; Gene Knockout Techniques/methods ; Drosophila/genetics ; Software ; Drosophila Proteins/genetics/metabolism ; Drosophila melanogaster/genetics ; },
abstract = {A single guide RNA (sgRNA) directs Cas9 nuclease for gene-specific scission of double-stranded DNA. High Cas9 activity is essential for efficient gene editing to generate gene deletions and gene replacements by homologous recombination. However, cleavage efficiency is below 50% for more than half of randomly selected sgRNA sequences in human cell culture screens or model organisms. We used in vitro assays to determine intrinsic molecular parameters for maximal sgRNA activity including correct folding of sgRNAs and Cas9 structural information. From the comparison of over 10 data sets, we find major constraints in sgRNA design originating from defective secondary structure of the sgRNA, sequence context of the seed region, GC context, and detrimental motifs, but we also find considerable variation among different prediction tools when applied to different data sets. To aid selection of efficient sgRNAs, we developed web-based PlatinumCRISPr, an sgRNA design tool to evaluate base-pairing and sequence composition parameters for optimal design of highly efficient sgRNAs for Cas9 genome editing. We applied this tool to select sgRNAs to efficiently generate gene deletions in Drosophila Ythdc1 and Ythdf, that bind to N [6] methylated adenosines (m[6]A) in mRNA. However, we discovered that generating small deletions with sgRNAs and Cas9 leads to ectopic reinsertion of the deleted DNA fragment elsewhere in the genome. These insertions can be removed by standard genetic recombination and chromosome exchange. These new insights into sgRNA design and the mechanisms of CRISPR-Cas9 genome editing advance the efficient use of this technique for safer applications in humans.},
}

*RNA, Guide, CRISPR-Cas Systems/genetics
Animals
*CRISPR-Cas Systems
*Gene Editing/methods
Humans
CRISPR-Associated Protein 9/metabolism/genetics
Gene Knockout Techniques/methods
Drosophila/genetics
Software
Drosophila Proteins/genetics/metabolism
Drosophila melanogaster/genetics

@article {pmid39533823,
year = {2024},
author = {Yamagishi, K and Ike, M and Tokuyasu, K},
title = {Construction of a genome-editing system for the thermophilic actinomycete Streptomyces thermodiastaticus K5 strain.},
journal = {Bioscience, biotechnology, and biochemistry},
volume = {89},
number = {1},
pages = {80-87},
doi = {10.1093/bbb/zbae157},
pmid = {39533823},
issn = {1347-6947},
support = {2020-2022//National Agriculture and Food Research Organization/ ; },
mesh = {*Gene Editing/methods ; *Streptomyces/genetics ; *Plasmids/genetics ; Uracil-DNA Glycosidase/genetics/metabolism ; beta-Glucosidase/genetics/metabolism ; CRISPR-Cas Systems ; Genome, Bacterial ; Mutation ; Lignin/metabolism ; },
abstract = {Thermophilic actinomycetes significantly contribute to the terrestrial carbon cycle via the rapid degradation of lignocellulosic polysaccharides in composts. In this study, a genome-editing system was constructed for the thermophilic actinomycete Streptomyces thermodiastaticus K5 strain, which was isolated from compost. The genome-editing plasmid (pGEK5) harboring nickase Cas9 was derived from the high-copy plasmid pL99 and used for the K5 strain. It was found that pGEK5 could easily be lost from the transformed clone through cultivation on apramycin-free medium and spore formation, enabling its reuse for subsequent genome-editing cycles. With the aid of this plasmid, mutations were sequentially introduced to 2 uracil-DNA glycosylase genes (Udg1 and Udg2) and 1 β-glucosidase gene (Bgl1). Thus, the genome-editing system using pGEK5 enables us to start the functional modification of this thermophilic actinomycete, especially for improved conversion of lignocellulosic biomass.},
}

*Gene Editing/methods
*Streptomyces/genetics
*Plasmids/genetics
Uracil-DNA Glycosidase/genetics/metabolism
beta-Glucosidase/genetics/metabolism
CRISPR-Cas Systems
Genome, Bacterial
Mutation
Lignin/metabolism

@article {pmid39317648,
year = {2024},
author = {Wang, S and Wang, J and Li, B and Zhang, J},
title = {Photoactivable CRISPR for Biosensing and Cancer Therapy.},
journal = {Chembiochem : a European journal of chemical biology},
volume = {25},
number = {24},
pages = {e202400685},
doi = {10.1002/cbic.202400685},
pmid = {39317648},
issn = {1439-7633},
support = {22274072//National Natural Science Foundation of China/ ; 2024300408//Fundamental Research Funds for the Central Universities/ ; },
mesh = {Humans ; *Neoplasms/therapy/diagnosis/genetics ; *Biosensing Techniques ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Genetic Therapy ; Light ; },
abstract = {Photoactivable CRISPR technology represents a transformative approach in the field of genome editing, offering unprecedented control over gene editing with high spatial and temporal precision. By harnessing the power of light to modulate the activity of CRISPR components, this innovative strategy enables precise regulation of Cas proteins, guide RNAs, and ribonucleoprotein complexes. Recent advancements in optical control methodologies, including the development of photoactivable nanocarriers, have significantly expanded the potential applications of CRISPR in biomedical fields. This Concept highlights the latest developments in designing photoactivable CRISPR systems and their promising applications in biosensing and cancer therapy. Additionally, the remaining challenges and future trends are also discussed. It is expected that the photoactivable CRISPR would facilitate translating more precise gene therapies into clinical use.},
}

Humans
*Neoplasms/therapy/diagnosis/genetics
*Biosensing Techniques
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
Genetic Therapy
Light

@article {pmid39298089,
year = {2024},
author = {Wu, K and Wu, Z and Li, X},
title = {Clinical diagnostic value of CRISPR-Cas13a-based molecular technology for tuberculosis.},
journal = {Irish journal of medical science},
volume = {193},
number = {6},
pages = {2889-2895},
pmid = {39298089},
issn = {1863-4362},
mesh = {Humans ; *CRISPR-Cas Systems ; Male ; Female ; Adult ; Middle Aged ; Mycobacterium tuberculosis/genetics/isolation & purification ; Tuberculosis/diagnosis ; Bronchoalveolar Lavage Fluid/microbiology ; Sensitivity and Specificity ; Tuberculosis, Pulmonary/diagnosis ; Aged ; },
abstract = {OBJECTIVE: To address the clinical diagnostic value of CRISPR-Cas13a-based molecular technology for tuberculosis (TB).

METHODS: The 189 suspected TB patients were simultaneously sent for acid-fast staining smear of bronchoalveolar lavage fluid, MGIT 960 cultures, Xpert MTB/RIF assay, and CRISPR-Cas13a assay. Using the final clinical diagnosis as the gold standard, the TB and non-TB groups were determined, and the diagnostic values of the four assays and the combined test in TB were compared. Using MGIT 960 culture as the gold standard, the diagnostic value of CRISPR-Cas13a assay was explored in TB, and the concordance between the CRISPR-Cas13a assay and MGIT 960 culture was compared.

RESULTS: The 189 preliminary diagnosed patients with suspected TB were diagnosed, with 147 in the TB group and 42 in the non-TB group. Taking the final clinical diagnosis as the gold standard, the sensitivity, negative predictive value, and accuracy of CRISPR-Cas13a assay, MGIT 960 culture, and XpertMTB/RIF assay were higher than those of acid-fast staining smear; by comparing the area under the ROC curve, the diagnostic value of the CRISPR-Cas13a assay, MGIT 960 culture, and XpertMTB/RIF assay was superior to that of acid-fast staining smear (all P < 0.05). Using the MGIT 960 culture results as the gold standard, there was a moderate concordance between the CRISPR-Cas13a assay and the MGIT 960 culture (kappa = 0.666).

CONCLUSION: Bronchoalveolar lavage fluid CRISPR-Cas13a assay has high application value in the clinical diagnosis of TB and can be recommended for the initial screening of patients with suspected TB.},
}

Humans
*CRISPR-Cas Systems
Male
Female
Adult
Middle Aged
Mycobacterium tuberculosis/genetics/isolation & purification
Tuberculosis/diagnosis
Bronchoalveolar Lavage Fluid/microbiology
Sensitivity and Specificity
Tuberculosis, Pulmonary/diagnosis
Aged

@article {pmid39147927,
year = {2025},
author = {Xiao, Q and Li, G and Han, D and Wang, H and Yao, M and Ma, T and Zhou, J and Zhang, Y and Zhang, X and He, B and Yuan, Y and Shi, L and Li, T and Yang, H and Huang, J and Zhang, H},
title = {Engineered IscB-ωRNA system with expanded target range for base editing.},
journal = {Nature chemical biology},
volume = {21},
number = {1},
pages = {100-108},
pmid = {39147927},
issn = {1552-4469},
support = {31925016//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82021001//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82271048//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {Animals ; *Gene Editing/methods ; Mice ; Humans ; *Muscular Dystrophy, Duchenne/genetics/metabolism ; HEK293 Cells ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; RNA/metabolism/chemistry/genetics ; CRISPR-Cas Systems ; Bacterial Proteins/genetics/metabolism/chemistry ; Dependovirus/genetics ; },
abstract = {As the evolutionary ancestor of Cas9 nuclease, IscB proteins serve as compact RNA-guided DNA endonucleases and nickases, making them strong candidates for base editing. Nevertheless, the narrow targeting scope limits the application of IscB systems; thus, it is necessary to find more IscBs that recognize different target-adjacent motifs (TAMs). Here, we identified 10 of 19 uncharacterized IscB proteins from uncultured microbes with activity in mammalian cells. Through protein and ωRNA engineering, we further enhanced the activity of IscB ortholog IscB.m16 and expanded its TAM scope from MRNRAA to NNNGNA, resulting in a variant named IscB.m16*. By fusing the deaminase domains with IscB.m16* nickase, we generated IscB.m16*-derived base editors that exhibited robust base-editing efficiency in mammalian cells and effectively restored Duchenne muscular dystrophy proteins in diseased mice through single adeno-associated virus delivery. Thus, this study establishes a set of compact base-editing tools for basic research and therapeutic applications.},
}

Animals
*Gene Editing/methods
Mice
Humans
*Muscular Dystrophy, Duchenne/genetics/metabolism
HEK293 Cells
RNA, Guide, CRISPR-Cas Systems/genetics/metabolism
RNA/metabolism/chemistry/genetics
CRISPR-Cas Systems
Bacterial Proteins/genetics/metabolism/chemistry
Dependovirus/genetics

@article {pmid39707395,
year = {2024},
author = {Xu, W and Zhang, S and Qin, H and Yao, K},
title = {From bench to bedside: cutting-edge applications of base editing and prime editing in precision medicine.},
journal = {Journal of translational medicine},
volume = {22},
number = {1},
pages = {1133},
pmid = {39707395},
issn = {1479-5876},
support = {82471107//National Natural Science Foundation of China/ ; 31970930//National Natural Science Foundation of China/ ; 2020CFA069//Natural Science Foundation of Hubei Province/ ; 2018CFB434//Natural Science Foundation of Hubei Province/ ; },
mesh = {Humans ; *Precision Medicine/methods ; *Gene Editing/methods ; CRISPR-Cas Systems/genetics ; Translational Research, Biomedical ; Animals ; Genetic Therapy/methods ; },
abstract = {CRISPR-based gene editing technology theoretically allows for precise manipulation of any genetic target within living cells, achieving the desired sequence modifications. This revolutionary advancement has fundamentally transformed the field of biomedicine, offering immense clinical potential for treating and correcting genetic disorders. In the treatment of most genetic diseases, precise genome editing that avoids the generation of mixed editing byproducts is considered the ideal approach. This article reviews the current progress of base editors and prime editors, elaborating on specific examples of their applications in the therapeutic field, and highlights opportunities for improvement. Furthermore, we discuss the specific performance of these technologies in terms of safety and efficacy in clinical applications, and analyze the latest advancements and potential directions that could influence the future development of genome editing technologies. Our goal is to outline the clinical relevance of this rapidly evolving scientific field and preview a roadmap for successful DNA base editing therapies for the treatment of hereditary or idiopathic diseases.},
}

Humans
*Precision Medicine/methods
*Gene Editing/methods
CRISPR-Cas Systems/genetics
Translational Research, Biomedical
Animals
Genetic Therapy/methods

@article {pmid39706269,
year = {2024},
author = {Li, X and Wei, Y and Wang, SY and Wang, SG and Xia, PF},
title = {One-for-all gene inactivation via PAM-independent base editing in bacteria.},
journal = {The Journal of biological chemistry},
volume = {},
number = {},
pages = {108113},
doi = {10.1016/j.jbc.2024.108113},
pmid = {39706269},
issn = {1083-351X},
abstract = {Base editing is preferable for bacterial gene inactivation without generating double strand breaks, requiring homology recombination or highly efficient DNA delivery capability. However, the potential of base editing is limited by the adjoined dependence on the editing window and protospacer adjacent motif (PAM). Herein, we report an unconstrained base editing system to enable the inactivation of any genes of interest (GOIs) in bacteria. We employed a dCas9 derivative, dSpRY, and activation-induced cytidine deaminase to build a PAM-independent base editor. Then, we programmed the base editor to exclude the START codon of a GOI instead of introducing premature STOP codons to obtain a universal approach for gene inactivation, namely XSTART, with an overall efficiency approaching 100%. By using XSTART, we successfully manipulated the amino acid metabolisms in Escherichia coli, generating glutamine, arginine, and aspartate auxotrophic strains. While we observed a high frequency of off-target events as a trade-off for increased efficiency, refining the regulatory system of XSTART to limit expression levels reduced off-target events by over 60% without sacrificing efficiency, aligning our results with previously reported levels. Finally, the effectiveness of XSTART was also demonstrated in probiotic E. coli Nissle 1917 and photoautotrophic cyanobacterium Synechococcus elongatus, illustrating its potential in reprogramming diverse bacteria.},
}

@article {pmid39706164,
year = {2024},
author = {Cenik, BK and Aoi, Y and Iwanaszko, M and Howard, BC and Morgan, MA and Andersen, GD and Bartom, ET and Shilatifard, A},
title = {TurboCas: A method for locus-specific labeling of genomic regions and isolating their associated protein interactome.},
journal = {Molecular cell},
volume = {84},
number = {24},
pages = {4929-4944.e8},
doi = {10.1016/j.molcel.2024.11.007},
pmid = {39706164},
issn = {1097-4164},
mesh = {Humans ; *Promoter Regions, Genetic ; *Transcription Factors/metabolism/genetics ; *Chromatin/metabolism/genetics ; HEK293 Cells ; RNA Polymerase II/metabolism/genetics ; Protein Binding ; CRISPR-Cas Systems ; Cell Cycle Proteins/metabolism/genetics ; Positive Transcriptional Elongation Factor B/metabolism/genetics ; Proto-Oncogene Proteins c-myc/genetics/metabolism ; Proto-Oncogene Proteins c-fos/genetics/metabolism ; Gene Expression Regulation ; Heat-Shock Response/genetics ; Protein Interaction Maps ; Bromodomain Containing Proteins ; },
abstract = {Regulation of gene expression during development and stress response requires the concerted action of transcription factors and chromatin-binding proteins. Because this process is cell-type specific and varies with cellular conditions, mapping of chromatin factors at individual regulatory loci is crucial for understanding cis-regulatory control. Previous methods only characterize static protein binding. We present "TurboCas," a method combining a proximity-labeling (PL) enzyme, miniTurbo, with CRISPR-dCas9 that allows for efficient and site-specific labeling of chromatin factors in mammalian cells. Validating TurboCas at the FOS promoter, we identify proteins recruited upon heat shock, cross-validated via RNA polymerase II and P-TEFb immunoprecipitation. These methodologies reveal canonical and uncharacterized factors that function to activate expression of heat-shock-responsive genes. Applying TurboCas to the MYC promoter, we identify two P-TEFb coactivators, the super elongation complex (SEC) and BRD4, as MYC co-regulators. TurboCas provides a genome-specific targeting PL, with the potential to deepen our molecular understanding of transcriptional regulatory pathways in development and stress response.},
}

Humans
*Promoter Regions, Genetic
*Transcription Factors/metabolism/genetics
*Chromatin/metabolism/genetics
HEK293 Cells
RNA Polymerase II/metabolism/genetics
Protein Binding
CRISPR-Cas Systems
Cell Cycle Proteins/metabolism/genetics
Positive Transcriptional Elongation Factor B/metabolism/genetics
Proto-Oncogene Proteins c-myc/genetics/metabolism
Proto-Oncogene Proteins c-fos/genetics/metabolism
Gene Expression Regulation
Heat-Shock Response/genetics
Protein Interaction Maps
Bromodomain Containing Proteins

@article {pmid39705367,
year = {2024},
author = {Su, Z and Zhang, W and Shi, Y and Cui, T and Xu, Y and Yang, R and Huang, M and Zhou, C and Zhang, H and Lu, T and Qu, J and He, ZG and Gan, J and Feng, Y},
title = {A bacterial methyltransferase that initiates biotin synthesis, an attractive anti-ESKAPE druggable pathway.},
journal = {Science advances},
volume = {10},
number = {51},
pages = {eadp3954},
pmid = {39705367},
issn = {2375-2548},
mesh = {*Biotin/biosynthesis ; *Methyltransferases/metabolism/chemistry ; *Acinetobacter baumannii/enzymology ; *Klebsiella pneumoniae/enzymology ; Bacterial Proteins/metabolism/genetics/chemistry ; Adenosine/analogs & derivatives/metabolism ; Anti-Bacterial Agents/pharmacology ; Models, Molecular ; Humans ; CRISPR-Cas Systems ; },
abstract = {The covalently attached cofactor biotin plays pivotal roles in central metabolism. The top-priority ESKAPE-type pathogens, Acinetobacter baumannii and Klebsiella pneumoniae, constitute a public health challenge of global concern. Despite the fact that the late step of biotin synthesis is a validated anti-ESKAPE drug target, the primary stage remains fragmentarily understood. We report the functional definition of two BioC isoenzymes (AbBioC for A. baumannii and KpBioC for K. pneumoniae) that act as malonyl-ACP methyltransferase and initiate biotin synthesis. The physiological requirement of biotin is diverse within ESKAPE pathogens. CRISPR-Cas9-based inactivation of bioC rendered A. baumannii and K. pneumoniae biotin auxotrophic. The availability of soluble AbBioC enabled the in vitro reconstitution of DTB/biotin synthesis. We solved two crystal structures of AbBioC bound to SAM cofactor (2.54 angstroms) and sinefungin (SIN) inhibitor (1.72 angstroms). Structural and functional study provided molecular basis for SIN inhibition of BioC. We demonstrated that BioC methyltransferase plays dual roles in K. pneumoniae infection and A. baumannii colistin resistance.},
}

*Biotin/biosynthesis
*Methyltransferases/metabolism/chemistry
*Acinetobacter baumannii/enzymology
*Klebsiella pneumoniae/enzymology
Bacterial Proteins/metabolism/genetics/chemistry
Adenosine/analogs & derivatives/metabolism
Anti-Bacterial Agents/pharmacology
Models, Molecular
Humans
CRISPR-Cas Systems

@article {pmid39705306,
year = {2024},
author = {Hou, J and Guo, P and Wang, J and Han, D and Tan, W},
title = {Artificial dynamic structure ensemble-guided rational design of a universal RNA aptamer-based sensing tag.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {121},
number = {52},
pages = {e2414793121},
doi = {10.1073/pnas.2414793121},
pmid = {39705306},
issn = {1091-6490},
mesh = {*Aptamers, Nucleotide/chemistry ; Humans ; *Biosensing Techniques/methods ; *RNA, Guide, CRISPR-Cas Systems/genetics ; CRISPR-Cas Systems ; Gene Editing/methods ; Nucleic Acid Conformation ; RNA/chemistry ; Fluorescent Dyes/chemistry ; },
abstract = {Artificially functional RNAs, such as fluorogenic RNA aptamer (FRApt)-based biosensing tag, represent significant advancements in various biological applications but are limited by the lack of insight into dynamic structure ensembles and universal design concepts. Through the development of an artificial RNA structure ensemble, we rationally established an RNA reconstitution model, "SSPepper-Apt," to generate a universal fluorogenic RNA biosensing tag. By utilizing various target-recognizing RNA motifs, SSPepper-Apt enables the modular generation of sensing tags for low-background, highly selective imaging of metabolites, peptides, and proteins in living cells. Additionally, by employing single guide RNA (sgRNA) as the recognition RNA motif, SSPepper-Apt generates fluorescence in both CRISPR-mediated imaging and gene editing only when the Cas9-sgRNA complex is successfully assembled; therefore, it can be an effective sgRNA screening tool for gene editing. Our fluorogenic RNA-sensing tag provides a universal approach for constructing functional RNA systems, avoiding the laborious and time-consuming process of sequence combination, and expanding the application of synthetic biological tools.},
}

*Aptamers, Nucleotide/chemistry
Humans
*Biosensing Techniques/methods
*RNA, Guide, CRISPR-Cas Systems/genetics
CRISPR-Cas Systems
Gene Editing/methods
Nucleic Acid Conformation
RNA/chemistry
Fluorescent Dyes/chemistry

@article {pmid39704190,
year = {2024},
author = {Fan, Z and Xu, L and Cao, Y and Liu, T and Tian, Y and Pan, Z and Mo, Y and Wang, X and Zhu, X and Gao, Y and Zhang, X and Pan, CQ and Wang, L and Ren, F},
title = {One-Pot Assay Based on CRISPR/Cas13a Technology for HEV RNA Point-of-Care Testing.},
journal = {Journal of medical virology},
volume = {96},
number = {12},
pages = {e70115},
pmid = {39704190},
issn = {1096-9071},
support = {//This study was supported by the National Natural Science Foundation of China (82002243, 82100653), Key Projects of the Beijing Municipal Education Commission's Science and Technology Plan (KZ202010025035), Chinese Institutes for Medical Research, Beijing (Grant No. CX24PY23), Beijing Hospitals Authority Youth Programme (QML20201702), Talent Cultivation Plan of Climbing the Peak of Beijing Municipal Hospital Administration (DFL20221503), Beijing Natural Science Foundation-Changping Innovation Joint Fund (L234046), Training Fund for Open Projects at Clinical Institutes and Departments of Capital Medical University (CCMU2023ZKYXZ003), High-Level Public Health Technical Talents Project of Beijing (Subject Leaders-02-13, xuekegugan-03-48)./ ; },
mesh = {*Hepatitis E virus/genetics/isolation & purification ; *Point-of-Care Testing ; *Hepatitis E/diagnosis/virology ; *RNA, Viral/genetics ; Humans ; *CRISPR-Cas Systems ; *Sensitivity and Specificity ; Animals ; Limit of Detection ; Reverse Transcriptase Polymerase Chain Reaction/methods ; Genotype ; Nucleic Acid Amplification Techniques/methods ; },
abstract = {Hepatitis E virus (HEV) poses a serious threat to both public health and animal food safety, thereby highlighting the demands for rapid, sensitive, and easy-to-use detection. This study aimed to develop a One-Pot assay using CRISPR/Cas13a for detecting HEV RNA, suitable for point-of-care testing (POCT) in resource-limited settings. CRISPR/Cas13a combined with reverse transcription polymerase chain reaction (RT-PCR) and reverse transcription recombinase-aided amplification (RT-RAA) was applied to a One-Pot assay device. Additionally, a large cohort of HEV-infected patient (154) and animal (104) specimens was utilized for validation. The RT-PCR/RT-RAA + CRISPR/Cas13a assays for HEV RNA detection (genotypes: HEV-1, HEV-3, and HEV-4) were established, optimized, and validated, achieving a limit of detection (LoD) of 1 copy/μL and 100% specificity. In the application validation for HEV infection, the positive rates of the RT-PCR + CRISPR and RT-RAA + CRISPR assays were 98.6% and 89.6% for patients, and 96.6% and 88.8% for animals, respectively, which were superior to those of RT-qPCR. Furthermore, sample rapid lysis, reagent lyophilization, and the One-Pot device were integrated to construct a One-Pot assay with an LoD of 10[2] copies/μL. Despite slight decreases in sensitivity, the One-Pot assay significantly reduces the assay time to 35 min, making it easy to perform, minimizing contamination, and meeting the requirements for screening. We developed a One-Pot assay of HEV RNA using the CRISPR/Cas13a which effectively realizes a POCT test and maximizes the impetus for POCT implementation and shows potential as a valuable tool for detecting and monitoring HEV infection.},
}

*Hepatitis E virus/genetics/isolation & purification
*Point-of-Care Testing
*Hepatitis E/diagnosis/virology
*RNA, Viral/genetics
Humans
*CRISPR-Cas Systems
*Sensitivity and Specificity
Animals
Limit of Detection
Reverse Transcriptase Polymerase Chain Reaction/methods
Genotype
Nucleic Acid Amplification Techniques/methods

@article {pmid39703747,
year = {2024},
author = {Fallah, T and Shafiei, M},
title = {Comprehensive Analysis of CRISPR-Cas Systems and Their Influence on Antibiotic Resistance in Salmonella enterica Strains.},
journal = {Bioinformatics and biology insights},
volume = {18},
number = {},
pages = {11779322241307984},
pmid = {39703747},
issn = {1177-9322},
abstract = {Salmonella enterica is a gram-negative bacterium that demonstrates a remarkable ability to acquire antibiotic resistance genes (ARGs). The role of the CRISPR-Cas system in influencing antibiotic resistance in S. enterica is still under investigation. This study explores the distribution and impact of CRISPR-Cas systems on antibiotic resistance by analyzing 316 S. enterica genomes. We conducted sequence alignments, phylogenetic analyses, and conservation studies on Cas genes, direct repeats (DRs), and leader sequences. Promoter predictions and RNA secondary structure analyses were also performed. ARGs were identified, and their correlation with Cas gene clusters was evaluated. Our findings revealed that 82.33% of strains possess complete CRISPR-Cas systems, while 17.66% have orphan CRISPRs. We identified 290 distinct DRs, most of which formed stable stem-loop structures, although no promoter regions were detected within the leader sequences. Most spacers were chromosome-targeting, with a smaller proportion homologous to phages and plasmids. Importantly, strains with complete CRISPR-Cas systems showed a higher incidence of ARGs compared with those with orphan or no CRISPR systems. Specifically, the incidence of ARGs was 54.3% higher in strains with complete CRISPR-Cas systems than in strains without CRISPR-Cas systems, and 15.1% higher than in strains with orphan CRISPRs. Spearman's correlation analysis confirmed a statistically significant but weak correlation between the presence of Cas genes and the frequency of ARGs (P-value = 3.892e-06). These results suggest that CRISPR-Cas systems may play a role in the acquisition of ARGs, potentially through mutations under antibiotic pressure. Future studies should investigate mutations, particularly in Cas3-the signature protein of type I CRISPR-Cas systems. In addition, experimental validation, such as culturing S. enterica strains with complete CRISPR-Cas systems under different antibiotic conditions, followed by sequencing to assess the uptake or absence of newly acquired ARGs, would help clarify the potential role of CRISPR-Cas systems in bacterial adaptation to antimicrobial pressures.},
}

@article {pmid39702810,
year = {2024},
author = {Bhagat, M and Kamal, R and Sharma, J and Kaur, K and Sharma, A and Thakur, GS and Bhatia, R and Awasthi, A},
title = {Gene Therapy: Towards a New Era of Medicine.},
journal = {AAPS PharmSciTech},
volume = {26},
number = {1},
pages = {17},
pmid = {39702810},
issn = {1530-9932},
mesh = {Humans ; *Genetic Therapy/methods/trends ; Animals ; *Genetic Vectors ; CRISPR-Cas Systems ; Gene Editing/methods ; Gene Transfer Techniques ; },
abstract = {Over the past years, many significant advances have been made in the field of gene therapy and shown promising results in clinical trials conducted. Gene therapy aims at modifying or replacing a defective, inefficient, or nonfunctional gene with a healthy, functional gene by administration of genome material into the cell to cure genetic diseases. Various methods have been devised to do this by using several viral and non-viral vectors which are either administered by in vivo or ex vivo technique. Viral vectors are best suitable for this therapy due to their potential to invade cells and deliver their genetic material whereas non-viral vectors are less efficient than viral vectors but possess some advantages such as less immunogenic response and large gene carrying capacity. Recent advances in biotechnology such as CRISPR-Cas9 mediated genome engineering and Cancer treatment with Chimeric antigen receptor (CAR) T-cell therapy are addressed in this review. This review article also delves into some recent research studies, gene therapy trials, and its applications, laying out future hopes for gene therapy in the treatment of various diseases namely haemophilia, Muscular dystrophy, SCID, Sickle cell disease, Familial Hypercholesterolemia, Cystic Fibrosis. Additionally, it also includes various nanoformulations and clinical trial data related to gene therapy.},
}

Humans
*Genetic Therapy/methods/trends
Animals
*Genetic Vectors
CRISPR-Cas Systems
Gene Editing/methods
Gene Transfer Techniques

@article {pmid39702692,
year = {2024},
author = {Du, W and Meister, LL and van Grinsven, T and Branco Dos Santos, F},
title = {Efficient Multiplex Genome Editing of the Cyanobacterium Synechocystis sp. PCC6803 via CRISPR-Cas12a.},
journal = {Biotechnology and bioengineering},
volume = {},
number = {},
pages = {},
doi = {10.1002/bit.28910},
pmid = {39702692},
issn = {1097-0290},
abstract = {Cyanobacteria have been genetically modified to convert CO2 into biochemical products, but efficient genetic engineering tools, including CRISPR-Cas systems, remain limited. This is primarily due to the polyploid nature of cyanobacteria, which hinders their effectiveness. Here, we address the latter by specifically (i) modifying the RSF1010-based replicative plasmid to simplify cloning efforts while maintaining high conjugation efficiency; (ii) improving the design of the guide RNA (gRNA) to facilitate chromosomal cleavage; (iii) introducing template DNA fragments as pure plasmids via natural transformation; and (iv) using sacB to facilitate replicative plasmid curing. With this system, the replicative plasmid containing both Cas12a and gRNA is introduced to Synechocystis sp. PCC6803 cells via conjugation to cleave the circular chromosomes. Template DNA plasmid that has meanwhile been assimilated will then repair it achieving the desired genetic modifications. This system was validated by successfully deleting various "neutral" chromosomal loci, both individually and collectively, as well as targeting an essential gene, sll1797. With the sacB-sucrose counter-selection, all deletions were simultaneously made markerless in < 4 weeks. Moreover, we also integrate YFP with various protein degradation tags into the chromosome, allowing for their characterization at the chromosomal level. We foresee this system will greatly facilitate future genome engineering in cyanobacteria.},
}

@article {pmid39702666,
year = {2024},
author = {Kou, Z and Wang, S and Luo, X and Xu, J and Tomberlin, JK and Huang, Y},
title = {Wingless strain created using binary transgenic CRISPR/Cas9 alleviates concerns about mass rearing of Hermetia illucens.},
journal = {Communications biology},
volume = {7},
number = {1},
pages = {1652},
pmid = {39702666},
issn = {2399-3642},
support = {32021001//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32100381//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {Animals ; *CRISPR-Cas Systems ; *Animals, Genetically Modified ; *Diptera/genetics ; Female ; Male ; },
abstract = {Larvae of the black soldier fly Hermetia illucens have potential as a natural waste recycler and subsequent use as protein-rich feed for livestock. A common question about the insect-farming processes is, what about the concerns of mass escape of insects from large populations? Here, we present a binary transgenic CRISPR/Cas9 system to generate wingless strain with the potential to address this issue. We identified gonad-specific promoters in vivo and evaluated use of the two strongest promoters, nanos and exuperantia, to drive Cas9 expression. We found that crossing the Hiexu-Cas9 with transgenic sgRNA-expressing insects resulted in higher knockout efficiency of the marker gene white. The Hiexu-Cas9 strain exhibited a maternal deposition of Cas9 that caused more effective knockout in the progeny of female Cas9-expressing individuals. Using this system, we generated wingless mutants lacking mating ability, which can be maintained in colony through a genetic cross of two single strain. These insects are less likely to escape and would be unable to successfully mate if they did escape. Taken together, this study validates effective genetic tools that can be used for gene function studies and industrial applications in black soldier fly and provides an approach to alleviate the concern about massive rearing.},
}

Animals
*CRISPR-Cas Systems
*Animals, Genetically Modified
*Diptera/genetics
Female
Male

@article {pmid39607706,
year = {2024},
author = {Deng, C and Xin, R and Li, X and Zhang, J and Fan, L and Qiu, Y and Zhao, L},
title = {Optogenetic control of Corynebacterium glutamicum gene expression.},
journal = {Nucleic acids research},
volume = {52},
number = {22},
pages = {14260-14276},
doi = {10.1093/nar/gkae1149},
pmid = {39607706},
issn = {1362-4962},
support = {32301214//National Natural Science Foundation of China/ ; //Shanghai Municipal Education Commission/ ; },
mesh = {*Corynebacterium glutamicum/genetics/metabolism ; *Gene Expression Regulation, Bacterial ; *Optogenetics/methods ; Light ; CRISPR-Cas Systems ; Transcription Factors/metabolism/genetics ; Bioreactors ; Bacterial Proteins/metabolism/genetics ; Oligosaccharides/metabolism/biosynthesis ; Metabolic Engineering/methods ; },
abstract = {Corynebacterium glutamicum is a key industrial workhorse for producing amino acids and high-value chemicals. Balancing metabolic flow between cell growth and product synthesis is crucial for enhancing production efficiency. Developing dynamic, broadly applicable, and minimally toxic gene regulation tools for C. glutamicum remains challenging, as optogenetic tools ideal for dynamic regulatory strategies have not yet been developed. This study introduces an advanced light-controlled gene expression system using light-controlled RNA-binding proteins (RBP), a first for Corynebacterium glutamicum. We established a gene expression regulation system, 'LightOnC.glu', utilizing the light-controlled RBP to construct light-controlled transcription factors in C. glutamicum. Simultaneously, we developed a high-performance light-controlled gene interference system using CRISPR/Cpf1 tools. The metabolic flow in the synthesis network was designed to enable the production of chitin oligosaccharides (CHOSs) and chondroitin sulphate oligosaccharides A (CSA) for the first time in C. glutamicum. Additionally, a light-controlled bioreactor was constructed, achieving a CHOSs production concentration of 6.2 g/L, the highest titer recorded for CHOSs biosynthesis to date. Herein, we have established a programmable light-responsive genetic circuit in C. glutamicum, advancing the theory of dynamic regulation based on light signaling. This breakthrough has potential applications in optimizing metabolic modules in other chassis cells and synthesizing other compounds.},
}

*Corynebacterium glutamicum/genetics/metabolism
*Gene Expression Regulation, Bacterial
*Optogenetics/methods
Light
CRISPR-Cas Systems
Transcription Factors/metabolism/genetics
Bioreactors
Bacterial Proteins/metabolism/genetics
Oligosaccharides/metabolism/biosynthesis
Metabolic Engineering/methods

@article {pmid39607705,
year = {2024},
author = {Kadam, A and Shilo, S and Naor, H and Wainstein, A and Brilon, Y and Feldman, T and Minden, M and Kaushansky, N and Chapal-Ilani, N and Shlush, L},
title = {Utilizing insights of DNA repair machinery to discover MMEJ deletions and novel mechanisms.},
journal = {Nucleic acids research},
volume = {52},
number = {22},
pages = {e106},
doi = {10.1093/nar/gkae1132},
pmid = {39607705},
issn = {1362-4962},
support = {isf-nsfc 2427/18//Rising Tide Foundation/ ; //Steven B. Rubenstein research fund/ ; //Applebaum Foundation/ ; //Ernest and Bonnie Beutler Research Program/ ; //Sagol Institute for Longevity Research/ ; //Barry and Eleanore Reznik Family Cancer Research fund/ ; 1123/21//ISF-IPMP-Israel Precision Medicine Program/ ; },
mesh = {Humans ; *Sequence Deletion ; *DNA End-Joining Repair ; Algorithms ; DNA Repair/genetics ; Exome/genetics ; CRISPR-Cas Systems ; Leukemia, Myeloid, Acute/genetics ; Female ; G-Quadruplexes ; Breast Neoplasms/genetics ; },
abstract = {We developed Del-read, an algorithm targeting medium-sized deletions (6-100 bp) in short-reads, which are challenging for current variant callers relying on alignment. Our focus was on Micro-Homolog mediated End Joining deletions (MMEJ-dels), prevalent in myeloid malignancies. MMEJ-dels follow a distinct pattern, occurring between two homologies, allowing us to generate a comprehensive list of MMEJ-dels in the exome. Using Del-read, we identified numerous novel germline and somatic MMEJ-dels in BEAT-AML and TCGA-breast datasets. Validation in 672 healthy individuals confirmed their presence. These novel MMEJ-dels were linked to genomic features associated with replication stress, like G-quadruplexes and minisatellite. Additionally, we observed a new category of MMEJ-dels with an imperfect-match at the flanking sequences of the homologies, suggesting a mechanism involving mispairing in homology alignment. We demonstrated robustness of the repair system despite CRISPR/Cas9-induced mismatches in the homologies. Further analysis of the canonical ASXL1 deletion revealed a diverse array of these imperfect-matches. This suggests a potentially more flexible and error-prone MMEJ repair system than previously understood. Our findings highlight Del-read's potential in uncovering previously undetected deletions and deepen our understanding of repair mechanisms.},
}

Humans
*Sequence Deletion
*DNA End-Joining Repair
Algorithms
DNA Repair/genetics
Exome/genetics
CRISPR-Cas Systems
Leukemia, Myeloid, Acute/genetics
Female
G-Quadruplexes
Breast Neoplasms/genetics

@article {pmid39569586,
year = {2024},
author = {Kanke, KL and Rayner, RE and Bozik, J and Abel, E and Venugopalan, A and Suu, M and Nouri, R and Stack, JT and Guo, G and Vetter, TA and Cormet-Boyaka, E and Hester, ME and Vaidyanathan, S},
title = {Single-stranded DNA with internal base modifications mediates highly efficient knock-in in primary cells using CRISPR-Cas9.},
journal = {Nucleic acids research},
volume = {52},
number = {22},
pages = {13561-13576},
doi = {10.1093/nar/gkae1069},
pmid = {39569586},
issn = {1362-4962},
support = {HL151900-03/HL/NHLBI NIH HHS/United States ; VAIDYA22A0-KB//Cystic Fibrosis Foundation/ ; UL1TR002733//Center for Clinical and Translational Science/ ; //Ohio State University/ ; MCCOY19Ro//Nationwide Children's Hospital/ ; //Comprehensive Transplant Center Human Tissue Biorepository, OSU Wexner Medical Center/ ; DK106829//National Institute of Diabetes, Digestive and Kidney Diseases/ ; HL151900-03/HL/NHLBI NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; Humans ; *DNA, Single-Stranded/genetics/metabolism ; *Gene Knock-In Techniques ; *Induced Pluripotent Stem Cells/metabolism ; Receptors, CCR5/genetics/metabolism ; Gene Editing/methods ; Cells, Cultured ; Hematopoietic Stem Cells/metabolism ; },
abstract = {Single-stranded DNA (ssDNA) templates along with Cas9 have been used for knocking-in exogenous sequences in the genome but suffer from low efficiency. Here, we show that ssDNA with chemical modifications in 12-19% of internal bases, which we denote as enhanced ssDNA (esDNA), improve knock-in (KI) by 2-3-fold compared to end-modified ssDNA in airway basal stem cells (ABCs), CD34 + hematopoietic cells (CD34 + cells), T-cells and endothelial cells. Over 50% of alleles showed KI in three clinically relevant loci (CFTR, HBB and CCR5) in ABCs using esDNA and up to 70% of alleles showed KI in the HBB locus in CD34 + cells in the presence of a DNA-PKcs inhibitor. This level of correction is therapeutically relevant and is comparable to adeno-associated virus-based templates. The esDNA templates did not improve KI in induced pluripotent stem cells (iPSCs). This may be due to the absence of the nuclease TREX1 in iPSCs. Indeed, knocking out TREX1 in other cells improved KI using unmodified ssDNA. esDNA can be used to modify 20-30 bp regions in primary cells for therapeutic applications and biological modeling. The use of this approach for gene length insertions will require new methods to produce long chemically modified ssDNA in scalable quantities.},
}

*CRISPR-Cas Systems
Humans
*DNA, Single-Stranded/genetics/metabolism
*Gene Knock-In Techniques
*Induced Pluripotent Stem Cells/metabolism
Receptors, CCR5/genetics/metabolism
Gene Editing/methods
Cells, Cultured
Hematopoietic Stem Cells/metabolism

@article {pmid39569582,
year = {2024},
author = {Arantes, PR and Chen, X and Sinha, S and Saha, A and Patel, AC and Sample, M and Nierzwicki, Ł and Lapinaite, A and Palermo, G},
title = {Dimerization of the deaminase domain and locking interactions with Cas9 boost base editing efficiency in ABE8e.},
journal = {Nucleic acids research},
volume = {52},
number = {22},
pages = {13931-13944},
doi = {10.1093/nar/gkae1066},
pmid = {39569582},
issn = {1362-4962},
support = {//Advanced Cyberinfrastructure Coordination Ecosystem: Services & Support (ACCESS) program/ ; CHE-2144823//National Science Foundation/ ; R01GM141329/NH/NIH HHS/United States ; BIO230007//Pittsburgh Supercomputer Center/ ; DP2 GM149550/GM/NIGMS NIH HHS/United States ; TC-24-063//Camille and Henry Dreyfus Foundation/ ; MCB160059//San Diego Supercomputing Center/ ; FG-2023-20431//Sloan Foundation/ ; 2138259//National Science Foundation/ ; R01GM141329/NH/NIH HHS/United States ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; *CRISPR-Associated Protein 9/genetics/metabolism/chemistry ; *DNA/metabolism/genetics/chemistry ; Protein Multimerization ; Humans ; Adenosine Deaminase/metabolism/genetics/chemistry ; Protein Domains ; Deamination ; Molecular Dynamics Simulation ; Adenine/metabolism/chemistry ; },
abstract = {CRISPR-based DNA adenine base editors (ABEs) hold remarkable promises to address human genetic diseases caused by point mutations. ABEs were developed by combining CRISPR-Cas9 with a transfer RNA (tRNA) adenosine deaminase enzyme and through directed evolution, conferring the ability to deaminate DNA. However, the molecular mechanisms driving the efficient DNA deamination in the evolved ABEs remain unresolved. Here, extensive molecular simulations and biochemical experiments reveal the biophysical basis behind the astonishing base editing efficiency of ABE8e, the most efficient ABE to date. We demonstrate that the ABE8e's DNA deaminase domain, TadA8e, forms remarkably stable dimers compared to its tRNA-deaminating progenitor and that the strength of TadA dimerization is crucial for DNA deamination. The TadA8e dimer forms robust interactions involving its R98 and R129 residues, the RuvC domain of Cas9 and the DNA. These locking interactions are exclusive to ABE8e, distinguishing it from its predecessor, ABE7.10, and are indispensable to boost DNA deamination. Additionally, we identify three critical residues that drive the evolution of ABE8e toward improved base editing by balancing the enzyme's activity and stability, reinforcing the TadA8e dimer and improving the ABE8e's functionality. These insights offer new directions to engineer superior ABEs, advancing the design of safer precision genome editing tools.},
}

*Gene Editing/methods
*CRISPR-Cas Systems
*CRISPR-Associated Protein 9/genetics/metabolism/chemistry
*DNA/metabolism/genetics/chemistry
Protein Multimerization
Humans
Adenosine Deaminase/metabolism/genetics/chemistry
Protein Domains
Deamination
Molecular Dynamics Simulation
Adenine/metabolism/chemistry

@article {pmid39530229,
year = {2024},
author = {Song, X and Chen, Z and Sun, W and Yang, H and Guo, L and Zhao, Y and Li, Y and Ren, Z and Shi, J and Liu, C and Ma, P and Huang, X and Ji, Q and Sun, B},
title = {CRISPR-AsCas12f1 couples out-of-protospacer DNA unwinding with exonuclease activity in the sequential target cleavage.},
journal = {Nucleic acids research},
volume = {52},
number = {22},
pages = {14030-14042},
doi = {10.1093/nar/gkae989},
pmid = {39530229},
issn = {1362-4962},
support = {32271505//National Natural Science Foundation of China/ ; 22ZR1441900//Natural Science Foundation of Shanghai/ ; SLST-YS2024004//ShanghaiTech University School of Life Science and Technology Development Fund/ ; },
mesh = {*CRISPR-Cas Systems ; *DNA/metabolism/chemistry/genetics ; *DNA Cleavage ; CRISPR-Associated Proteins/metabolism/genetics ; Gene Editing/methods ; Bacterial Proteins/metabolism/genetics/chemistry ; RNA, Guide, CRISPR-Cas Systems/metabolism/genetics ; Exonucleases/metabolism/genetics ; },
abstract = {Type V-F CRISPR-Cas12f is a group of hypercompact RNA-guided nucleases that present a versatile in vivo delivery platform for gene therapy. Upon target recognition, Acidibacillus sulfuroxidans Cas12f (AsCas12f1) distinctively engenders three DNA break sites, two of which are located outside the protospacer. Combining ensemble and single-molecule approaches, we elucidate the molecular details underlying AsCas12f1-mediated DNA cleavages. We find that following the protospacer DNA unwinding and non-target strand (NTS) DNA nicking, AsCas12f1 surprisingly carries out bidirectional exonucleolytic cleavage from the nick. Subsequently, DNA unwinding is extended to the out-of-protospacer region, and AsCas12f1 gradually digests the unwound DNA beyond the protospacer. Eventually, the single endonucleolytic target-strand DNA cleavage at 3 nt downstream of the protospacer readily dissociates the ternary AsCas12f1-sgRNA-DNA complex from the protospacer adjacent motif-distal end, leaving a staggered double-strand DNA break. The coupling between the unwinding and cleavage of both protospacer and out-of-protospacer DNA is promoted by Mg2+. Kinetic analysis on the engineered AsCas12f1-v5.1 variant identifies the only accelerated step of the protospacer NTS DNA trimming within the sequential DNA cleavage. Our findings provide a dynamic view of AsCas12f1 catalyzing DNA unwinding-coupled nucleolytic cleavage and help with practical improvements of Cas12f-based genome editing tools.},
}

*CRISPR-Cas Systems
*DNA/metabolism/chemistry/genetics
*DNA Cleavage
CRISPR-Associated Proteins/metabolism/genetics
Gene Editing/methods
Bacterial Proteins/metabolism/genetics/chemistry
RNA, Guide, CRISPR-Cas Systems/metabolism/genetics
Exonucleases/metabolism/genetics

@article {pmid39530221,
year = {2024},
author = {Pale, LM and Khatib, JB and Nusawardhana, A and Straka, J and Nicolae, CM and Moldovan, GL},
title = {CRISPR knockout genome-wide screens identify the HELQ-RAD52 axis in regulating the repair of cisplatin-induced single-stranded DNA gaps.},
journal = {Nucleic acids research},
volume = {52},
number = {22},
pages = {13832-13848},
doi = {10.1093/nar/gkae998},
pmid = {39530221},
issn = {1362-4962},
support = {R01 GM134681/GM/NIGMS NIH HHS/United States ; R01 ES026184/ES/NIEHS NIH HHS/United States ; R01ES026184/NH/NIH HHS/United States ; F31 CA275340/CA/NCI NIH HHS/United States ; R01 CA244417/CA/NCI NIH HHS/United States ; 4D01_2024_1002//Four Diamonds Transformative Patient-Oriented Cancer Research Project/ ; R01ES026184/NH/NIH HHS/United States ; },
mesh = {*Cisplatin/pharmacology ; *DNA, Single-Stranded/genetics/metabolism ; Humans ; *Rad52 DNA Repair and Recombination Protein/genetics/metabolism ; *DNA Helicases/genetics/metabolism ; *DNA Repair/drug effects/genetics ; CRISPR-Cas Systems ; Antineoplastic Agents/pharmacology ; DNA Damage ; Gene Knockout Techniques ; DNA Primase/genetics/metabolism ; Cell Line, Tumor ; BRCA2 Protein/genetics/metabolism ; },
abstract = {Treatment with genotoxic agents, such as platinum compounds, is still the mainstay therapeutical approach for the majority of cancers. Our understanding of the mechanisms of action of these drugs is, however, imperfect and continuously evolving. Recent advances highlighted single-stranded DNA (ssDNA) gap accumulation as a potential determinant underlying cisplatin chemosensitivity, at least in some genetic backgrounds, such as BRCA mutations. Cisplatin-induced ssDNA gaps form upon restart of DNA synthesis downstream of cisplatin-induced lesions through repriming catalyzed by the PRIMPOL enzyme. Here, we show that PRIMPOL overexpression in otherwise wild-type cells results in accumulation of cisplatin-induced ssDNA gaps without sensitizing cells to cisplatin, suggesting that ssDNA gap accumulation does not confer cisplatin sensitivity in BRCA-proficient cells. To understand how ssDNA gaps may cause cellular sensitivity, we employed CRISPR-mediated genome-wide genetic screening to identify factors which enable the cytotoxicity of cisplatin-induced ssDNA gaps. We found that the helicase HELQ specifically suppresses cisplatin sensitivity in PRIMPOL-overexpressing cells, and this is associated with reduced ssDNA accumulation. We moreover identify RAD52 as a mediator of this pathway. RAD52 promotes ssDNA gap accumulation through a BRCA-mediated mechanism. Our work identified the HELQ-RAD52-BRCA axis as a regulator of ssDNA gap processing and cisplatin sensitization.},
}

*Cisplatin/pharmacology
*DNA, Single-Stranded/genetics/metabolism
Humans
*Rad52 DNA Repair and Recombination Protein/genetics/metabolism
*DNA Helicases/genetics/metabolism
*DNA Repair/drug effects/genetics
CRISPR-Cas Systems
Antineoplastic Agents/pharmacology
DNA Damage
Gene Knockout Techniques
DNA Primase/genetics/metabolism
Cell Line, Tumor
BRCA2 Protein/genetics/metabolism

@article {pmid39491497,
year = {2025},
author = {Pillai, A and Verma, V and Galande, S},
title = {CHARM and EvoETR: Precision epigenetic tools for gene silencing.},
journal = {BioEssays : news and reviews in molecular, cellular and developmental biology},
volume = {47},
number = {1},
pages = {e2400186},
doi = {10.1002/bies.202400186},
pmid = {39491497},
issn = {1521-1878},
support = {JCB/2019/000013//Science and Engineering Research Board/ ; },
mesh = {Animals ; *Gene Silencing ; *Gene Editing/methods ; *Epigenesis, Genetic ; Mice ; Humans ; CRISPR-Cas Systems ; DNA Methylation ; Proprotein Convertase 9/genetics/metabolism ; Prions/genetics/metabolism ; },
abstract = {With the advent of gene editing technologies like CRISPR/Cas9, it has become possible to edit genomic regions of interest for research and therapeutic purposes. These technologies have also been adapted to alter gene expression without changing their DNA sequence, allowing epigenetic edits. While genetic editors make edits by cutting the genome at specified regions, epigenetic editors leverage the same targeting mechanism but act based on the epigenetic modifier fused to them, such as a methyltransferase. Here, we discuss two recently employed epigenetic editors (epi-editors) that silenced target genes involved in disease to mitigate their effects. Neumann et al. reported the construction of an epigenetic editor called CHARM that could methylate and silence the prion gene in mouse brains and subsequently switch itself off. Additionally, Capelluti et al. developed an epi-editor called EvoETR that knocked down Pcsk9 in the murine liver to reduce LDL levels. We aim to highlight the design principles underlying the design of these epi-editors to inform future editor designs.},
}

Animals
*Gene Silencing
*Gene Editing/methods
*Epigenesis, Genetic
Mice
Humans
CRISPR-Cas Systems
DNA Methylation
Proprotein Convertase 9/genetics/metabolism
Prions/genetics/metabolism

@article {pmid39441374,
year = {2025},
author = {Guccio, N and Alcaino, C and Miedzybrodzka, EL and Santos-Hernández, M and Smith, CA and Davison, A and Bany Bakar, R and Kay, RG and Reimann, F and Gribble, FM},
title = {Molecular mechanisms underlying glucose-dependent insulinotropic polypeptide secretion in human duodenal organoids.},
journal = {Diabetologia},
volume = {68},
number = {1},
pages = {217-230},
pmid = {39441374},
issn = {1432-0428},
support = {100574/Z/12/Z/WT_/Wellcome Trust/United Kingdom ; 220271/Z/20/Z/WT_/Wellcome Trust/United Kingdom ; MR/M009041/1/MRC_/Medical Research Council/United Kingdom ; MRC_MC_UU_00014/5/MRC_/Medical Research Council/United Kingdom ; MRC_MC_UU_12012/3/MRC_/Medical Research Council/United Kingdom ; },
mesh = {Humans ; *Gastric Inhibitory Polypeptide/metabolism ; *Organoids/metabolism ; *Duodenum/metabolism ; *Receptors, G-Protein-Coupled/metabolism/genetics ; *Enteroendocrine Cells/metabolism ; Glucose/metabolism ; Receptors, Calcium-Sensing/metabolism/genetics ; Calcium/metabolism ; CRISPR-Cas Systems ; Cyclic AMP/metabolism ; },
abstract = {AIMS/HYPOTHESIS: Glucose-dependent insulinotropic polypeptide (GIP) is an incretin hormone secreted by enteroendocrine K cells in the proximal small intestine. This study aimed to explore the function of human K cells at the molecular and cellular levels.

METHODS: CRISPR-Cas9 homology-directed repair was used to insert transgenes encoding a yellow fluorescent protein (Venus) or an Epac-based cAMP sensor (Epac-S-H187) in the GIP locus in human duodenal-derived organoids. Fluorescently labelled K cells were purified by FACS for RNA-seq and peptidomic analysis. GIP reporter organoids were employed for GIP secretion assays, live-cell imaging of Ca[2+] using Fura-2 and cAMP using Epac-S-H187, and basic electrophysiological characterisation. The G protein-coupled receptor genes GPR142 and CASR were knocked out to evaluate roles in amino acid sensing.

RESULTS: RNA-seq of human duodenal K cells revealed enrichment of several G protein-coupled receptors involved in nutrient sensing, including FFAR1, GPBAR1, GPR119, CASR and GPR142. Glucose induced action potential firing and cytosolic Ca[2+] elevation and caused a 1.8-fold increase in GIP secretion, which was inhibited by the sodium glucose co-transporter 1/2 (SGLT1/2) blocker sotagliflozin. Activation of the long-chain fatty acid receptor free fatty acid receptor 1 (FFAR1) induced a 2.7-fold increase in GIP secretion, while tryptophan and phenylalanine stimulated secretion by 2.8- and 2.1-fold, respectively. While CASR knockout blunted intracellular Ca[2+] responses, a CASR/GPR142 double knockout was needed to reduce GIP secretory responses to aromatic amino acids.

CONCLUSIONS/INTERPRETATION: The newly generated human organoid K cell model enables transcriptomic and functional characterisation of nutrient-sensing pathways involved in human GIP secretion. Both calcium-sensing receptor (CASR) and G protein-coupled receptor 142 (GPR142) contribute to protein-stimulated GIP secretion. This model will be further used to identify potential targets for modulation of native GIP secretion in diabetes and obesity.},
}

Humans
*Gastric Inhibitory Polypeptide/metabolism
*Organoids/metabolism
*Duodenum/metabolism
*Receptors, G-Protein-Coupled/metabolism/genetics
*Enteroendocrine Cells/metabolism
Glucose/metabolism
Receptors, Calcium-Sensing/metabolism/genetics
Calcium/metabolism
CRISPR-Cas Systems
Cyclic AMP/metabolism

@article {pmid39702333,
year = {2024},
author = {Liang, QZ and Chen, W and Liu, RC and Fu, QL and Fu, GH and Cheng, LF and Chen, HM and Jiang, NS and Zhu, T and Huang, Y},
title = {CRISPR/Cas12a and recombinase polymerase amplification-based rapid on-site nucleic acid detection of duck circovirus.},
journal = {Virology journal},
volume = {21},
number = {1},
pages = {322},
pmid = {39702333},
issn = {1743-422X},
support = {ZYTS202423//the Freedom Explore Program of Fujian Academy of Agricultural Sciences/ ; 2023J01363//the Natural Science Foundation Project of Fujian Province/ ; CARS-42//the National Waterfowl Industry Technology System of Modern Agriculture for China/ ; },
mesh = {*Circovirus/genetics/isolation & purification ; Animals ; *Ducks/virology ; *Circoviridae Infections/veterinary/diagnosis/virology ; *CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods ; *Sensitivity and Specificity ; Poultry Diseases/virology/diagnosis ; Recombinases/metabolism/genetics ; CRISPR-Associated Proteins/genetics ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {BACKGROUND: Duck circovirus (DuCV) infections commonly induce immunosuppression and secondary infections in ducks, resulting in significant economic losses in the duck breeding industry. Currently, effective vaccines and treatments for DuCV have been lacking. Therefore, rapid, specific, and sensitive detection methods are crucial for preventing and controlling DuCV.

METHODS: A lateral flow strip (LFS) detection method was developed using recombinase polymerase amplification (RPA) and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 12a (Cas12a). The RPA-CRISPR/Cas12a-LFS targeted the DuCV replication protein (Rep) and was operated at 37 ℃ and allowed for visual interpretation without requiring sophisticated equipment.

RESULTS: The results revealed that the reaction time of RPA-CRISPR/Cas12a-LFS is only 45 min. This method achieved a low detection limit of 2.6 gene copies. Importantly, this method demonstrated high specificity and no cross-reactivity with six other avian viruses. In a study involving 97 waterfowl samples, the Rep RPA-CRISPR/Cas12a-LFS showed 100% consistency and agreement with real-time quantitative polymerase chain reaction.

CONCLUSION: These findings underscored the potential of this user-friendly, rapid, sensitive, and accurate detection method for on-site DuCV detection.},
}

*Circovirus/genetics/isolation & purification
Animals
*Ducks/virology
*Circoviridae Infections/veterinary/diagnosis/virology
*CRISPR-Cas Systems
*Nucleic Acid Amplification Techniques/methods
*Sensitivity and Specificity
Poultry Diseases/virology/diagnosis
Recombinases/metabolism/genetics
CRISPR-Associated Proteins/genetics
Bacterial Proteins
Endodeoxyribonucleases

@article {pmid39701024,
year = {2024},
author = {Lee, J and Jeong, C},
title = {Single-molecule perspectives of CRISPR/Cas systems: target search, recognition, and cleavage.},
journal = {BMB reports},
volume = {},
number = {},
pages = {},
pmid = {39701024},
issn = {1976-670X},
abstract = {CRISPR/Cas systems have emerged as powerful tools for gene editing, nucleic acid detection, and therapeutic applications. Recent advances in single-molecule techniques have provided new insights into the DNA-targeting mechanisms of CRISPR/Cas systems, in particular, Types I, II, and V. Here, we review how single-molecule approaches have expanded our understanding of key processes, namely target search, recognition, and cleavage. Furthermore, we focus on the dynamic behavior of Cas proteins, including PAM site recognition and R-loop formation, which are crucial to ensure specificity and efficiency in gene editing. Additionally, we discuss the conformational changes and interactions that drive precise DNA cleavage by different Cas proteins. This mini review provides a comprehensive overview of CRISPR/Cas molecular dynamics, offering conclusive insights into their broader potential for genome editing and biotechnological applications.},
}

@article {pmid39700796,
year = {2024},
author = {Wang, X and Ding, H and Sun, Y and Ma, Y and Wang, G and Chen, J and Choo, J and Chen, L},
title = {CRISPR/HCR-powered ratiometric fluorescence aptasensor for ochratoxin A detection.},
journal = {Food chemistry},
volume = {468},
number = {},
pages = {142437},
doi = {10.1016/j.foodchem.2024.142437},
pmid = {39700796},
issn = {1873-7072},
abstract = {To address the need for highly sensitive and reliable detection of trace ochratoxin A (OTA) in food matrices, we developed a ratiometric fluorescent aptasensor by integrating CRISPR/Cas12a, hybridization chain reaction (HCR), and horseradish peroxidase (HRP)-induced inner filter effect (IFE). The mechanism involves OTA releasing an activator that initiates CRISPR/Cas12a trans-cleavage, blocking HCR assembly. This reduces HRP levels, limiting the conversion of o-phenylenediamine (OPD) to fluorescent 2,3-diaminophenazine (DAP) (emitting at 562 nm) while maintaining strong emission from 2-amino terephthalic acid (BDC-NH2) at 426 nm. The F426/F562 ratio serves as a "signal-on" indicator, enabling sensitive OTA detection over 0.1 pM to 10 nM, with a detection limit of 0.0417 pM. The method exhibits excellent reproducibility, with intra-day and inter-day relative standard deviations (RSDs) of 1.91 %-3.87 % and 1.79 %, respectively, along with recovery rates of 90.1 %-110.6 % in real samples. These advantages highlight its significant potential for CRISPR/Cas-based OTA detection.},
}

@article {pmid39700179,
year = {2024},
author = {Bowland, K and Lai, J and Skaist, A and Zhang, Y and Teh, SSK and Roberts, NJ and Thompson, E and Wheelan, SJ and Hruban, RH and Karchin, R and Bailey, MH and Iacobuzio-Donahue, CA and Eshleman, JR},
title = {Islands of genomic stability in the face of genetically unstable metastatic cancer.},
journal = {PloS one},
volume = {19},
number = {12},
pages = {e0298490},
pmid = {39700179},
issn = {1932-6203},
mesh = {Humans ; *Genomic Instability ; *Pancreatic Neoplasms/genetics/pathology ; *CRISPR-Cas Systems/genetics ; *Neoplasm Metastasis/genetics ; Carcinoma, Pancreatic Ductal/genetics/pathology/secondary ; Mutation ; Male ; Whole Genome Sequencing ; Female ; },
abstract = {INTRODUCTION: Metastatic cancer affects millions of people worldwide annually and is the leading cause of cancer-related deaths. Most patients with metastatic disease are not eligible for surgical resection, and current therapeutic regimens have varying success rates, some with 5-year survival rates below 5%. Here, we test the hypothesis that metastatic cancer can be genetically targeted by exploiting single base substitution mutations unique to individual cells that occur as part of normal aging prior to transformation. These mutations are targetable because ~10% of them form novel tumor-specific "NGG" protospacer adjacent motif (PAM) sites targetable by CRISPR-Cas9.

METHODS: Whole genome sequencing was performed on five rapid autopsy cases of patient-matched primary tumor, normal and metastatic tissue from pancreatic ductal adenocarcinoma decedents. CRISPR-Cas9 PAM targets were determined by bioinformatic tumor-normal subtraction for each patient and verified in metastatic samples by high-depth capture-based sequencing.

RESULTS: We found that 90% of PAM targets were maintained between primary carcinomas and metastases overall. We identified rules that predict PAM loss or retention, where PAMs located in heterozygous regions in the primary tumor can be lost in metastases (private LOH), but PAMs occurring in regions of loss of heterozygosity (LOH) in the primary tumor were universally conserved in metastases.

CONCLUSIONS: Regions of truncal LOH are strongly retained in the presence of genetic instability and, therefore, represent genetic vulnerabilities in pancreatic adenocarcinomas. A CRISPR-based gene therapy approach targeting these regions may be a novel way to genetically target metastatic cancer.},
}

Humans
*Genomic Instability
*Pancreatic Neoplasms/genetics/pathology
*CRISPR-Cas Systems/genetics
*Neoplasm Metastasis/genetics
Carcinoma, Pancreatic Ductal/genetics/pathology/secondary
Mutation
Male
Whole Genome Sequencing
Female

@article {pmid39700052,
year = {2024},
author = {Li, X and Chen, B and Xie, Y and Luo, Y and Zhu, D and Wang, L and Su, S},
title = {Polyvalent Aptamers Structure-Mediated Fluorescent Aptasensor for the Early Diagnosis of Alzheimer's Disease by Coupling with HCR and CRISPR-Cas System.},
journal = {Analytical chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.analchem.4c05329},
pmid = {39700052},
issn = {1520-6882},
abstract = {The early diagnosis of Alzheimer's disease (AD) plays a vital role in slowing the progression of AD and improving the quality of human life. However, it is still a challenge in the medical field. Herein, an ultrasensitive fluorescent aptasensor was designed for the detection of special phosphorylated tau181 (P-tau181) by coupling with polyvalent aptamers (PAs) structure, hybridization chain reaction (HCR), and the CRISPR-Cas system. Coupling with the signal amplification strategy, the specific recognition ability of the aptamer, and the high cleavage activity of Cas12a protein, the designed aptasensor showed a wide linear range (0.1-10[6] pg/mL), a low detection limit (0.069 pg/mL), high selectivity, and excellent anti-interference ability for the detection of P-tau181. Moreover, the aptasensor can efficiently analyze P-tau181 in artificial cerebro spinal fluid (aCSF) and serum, proving that it has a promising application in the early diagnosis of AD.},
}

@article {pmid39699265,
year = {2024},
author = {Li, X and Wang, L and Lin, J and Gu, Y and Liu, Z and Hu, J},
title = {Detection of CRISPR‒Cas and type I R-M systems in Klebsiella pneumoniae of human and animal origins and their relationship to antibiotic resistance and virulence.},
journal = {Microbiology spectrum},
volume = {},
number = {},
pages = {e0000924},
doi = {10.1128/spectrum.00009-24},
pmid = {39699265},
issn = {2165-0497},
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR)‒CRISPR-associated protein (Cas) and restriction‒modification (R-M) systems are important immune systems in bacteria. Information about the distributions of these two systems in Klebsiella pneumoniae from different hosts and their mutual effect on antibiotic resistance and virulence is still limited. In this study, the whole genomes of 520 strains of K. pneumoniae from GenBank, including 325 from humans and 195 from animals, were collected for CRISPR‒Cas systems and type I R-M systems, virulence genes, antibiotic resistance genes, and multilocus sequence typing detection. The results showed that host origin had no obvious influence on the distributions of the two systems (CRISPR‒Cas systems in 29.8% and 24.1%, type I R-M systems in 9.8% and 11.8% of human-origin and animal-origin strains, respectively) in K. pneumoniae. Identical spacer sequences from different hosts demonstrated there was a risk of human-animal transmission. All virulence genes (yersiniabactin, colibactin, aerobactin, salmochelin, rmpADC, and rmpA2) detection rates were higher when only the CRISPR‒Cas systems were present but were all reduced when coexisting with type I R-M systems. However, a lower prevalence of most antibiotic-resistance genes was found when the CRISPR‒Cas systems were alone, and when type I R-M systems were coexisting, some of the antibiotic resistance gene incidence rates were even lower (aminoglycosides, clindamycins, rifampicin-associated resistance genes, sulfonamides, methotrexates, beta-lactamases, and ultrabroad-spectrum beta-lactamases), and some of them were higher instead (quinolones, macrolides, tetracyclines, and carbapenems). The synergistic and opposed effects of the two systems on virulence and antibiotic-resistance genes need further study.IMPORTANCEK. pneumoniae is an important opportunistic pathogen responsible for both human and animal infections, and the emergence of hypervirulent and multidrug-resistant K. pneumoniae has made it difficult to control this pathogen worldwide. Here, we find that CRISPR‒Cas and restriction-modification systems, which function as adaptive and innate immune systems in bacteria, have synergistic and opposed effects on virulence and antibiotic resistance genes in K. pneumoniae. Moreover, this study provides insights into the distributions of the two systems in K. pneumoniae from different hosts, and there is no significant difference in the prevalence of the two systems among K. pneumoniae spp. In addition, this study also characterizes the CRISPR arrays of K. pneumoniae from different hosts, suggesting that the strains sharing the same spacer sequences have the potential to spread between humans and animals.},
}

@article {pmid39699033,
year = {2024},
author = {Paggi, RA and Ferrari, MC and Cerletti, M and Giménez, MI and Schwarz, TS and Marchfelder, A and De Castro, RE},
title = {Practical laboratory class to assess gene silencing using CRISPR interference (CRISPRi) technology in the archaeon Haloferax volcanii.},
journal = {Biochemistry and molecular biology education : a bimonthly publication of the International Union of Biochemistry and Molecular Biology},
volume = {},
number = {},
pages = {},
doi = {10.1002/bmb.21872},
pmid = {39699033},
issn = {1539-3429},
support = {//Universidad Nacional de Mar del Plata/ ; PICT 02228//Fondo para la Investigación Científica y Tecnológica (FONCYT)/ ; },
abstract = {Perturbation of gene expression using RNA interference (RNAi) or CRISPR interference (CRISPRi) is a useful strategy to explore the function of essential genes. In the archaeon Haloferax volcanii, the CRISPR-Cas system has been adapted as a CRISPRi tool to silence the expression of specific genes. We developed a laboratory class (LC) to conceptualize gene silencing through inactivation of the H. volcanii LonB protease gene, a negative regulator of carotenoid pigments biosynthesis, using CRISPRi. This LC has been successfully applied in the Biology and Biochemistry of Microorganisms course for undergraduate students of Biology in 2022 and 2023. The following objectives were proposed: (a) generate H. volcanii mutant strains with reduced expression of the lonB gene using CRISPRi; (b) examine the effect of lonB gene silencing on cell pigmentation and growth rate; (c) assess lonB gene repression by Western blotting (WB). This LC allows students to obtain and screen CRISPRi silenced-mutants by means of simple procedures using a non-pathogenic organism as well as handle basic microbiology, biochemistry and molecular biology protocols. Additionally, the LC fosters social actions through collaborative work (experimental work), the interpretation and discussion of data and the ability to communicate outcomes orally and in a written format (scientific report).},
}

@article {pmid39602282,
year = {2025},
author = {Lim, RM and Lu, A and Chuang, BM and Anaraki, C and Chu, B and Halbrook, CJ and Edinger, AL},
title = {CARMIL1-AA selectively inhibits macropinocytosis while sparing autophagy.},
journal = {Molecular biology of the cell},
volume = {36},
number = {1},
pages = {ar4},
doi = {10.1091/mbc.E24-09-0434},
pmid = {39602282},
issn = {1939-4586},
mesh = {*Pinocytosis/physiology ; Humans ; *Autophagy/physiology ; Cell Line, Tumor ; Sodium-Hydrogen Exchangers/metabolism/genetics ; Amiloride/pharmacology/analogs & derivatives ; Cell Proliferation ; CRISPR-Cas Systems ; },
abstract = {Macropinocytosis is reported to fuel tumor growth and drug resistance by allowing cancer cells to scavenge extracellular macromolecules. However, accurately defining the role of macropinocytosis in cancer depends on our ability to selectively block this process. 5-(N-ethyl-N-isopropyl)-amiloride (EIPA) is widely used to inhibit macropinocytosis but affects multiple Na[+]/H[+] exchangers (NHE) that regulate cytoplasmic and organellar pH. Consistent with this, we report that EIPA slows proliferation to a greater extent than can be accounted for by macropinocytosis inhibition and triggers conjugation of ATG8 to single membranes (CASM). Knocking down only NHE1 would not avoid macropinocytosis-independent effects on pH. Moreover, contrary to published reports, NHE1 loss did not block macropinocytosis in multiple cell lines. Knocking down CARMIL1 with CRISPR-Cas9 editing limited macropinocytosis, but only by 50%. In contrast, expressing the CARMIL1-AA mutant inhibits macropinocytosis induced by a wide range of macropinocytic stimuli to a similar extent as EIPA. CARMIL1-AA expression did not inhibit proliferation, highlighting the shortcomings of EIPA as a macropinocytosis inhibitor. Importantly, autophagy, another actin dependent, nutrient-producing process, was not affected by CARMIL1-AA expression. In sum, constitutive or inducible CARMIL1-AA expression reduced macropinocytosis without affecting proliferation, RAC activation, or autophagy, other processes that drive tumor initiation and progression.},
}

*Pinocytosis/physiology
Humans
*Autophagy/physiology
Cell Line, Tumor
Sodium-Hydrogen Exchangers/metabolism/genetics
Amiloride/pharmacology/analogs & derivatives
Cell Proliferation
CRISPR-Cas Systems

@article {pmid39565688,
year = {2024},
author = {Asano, K and Yoshimi, K and Takeshita, K and Mitsuhashi, S and Kochi, Y and Hirano, R and Tingyu, Z and Ishida, S and Mashimo, T},
title = {CRISPR Diagnostics for Quantification and Rapid Diagnosis of Myotonic Dystrophy Type 1 Repeat Expansion Disorders.},
journal = {ACS synthetic biology},
volume = {13},
number = {12},
pages = {3926-3935},
doi = {10.1021/acssynbio.4c00265},
pmid = {39565688},
issn = {2161-5063},
mesh = {*Myotonic Dystrophy/genetics/diagnosis ; Humans ; *CRISPR-Cas Systems/genetics ; *Trinucleotide Repeat Expansion/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Sensitivity and Specificity ; },
abstract = {Repeat expansion disorders, exemplified by myotonic dystrophy type 1 (DM1), present challenges in diagnostic quantification because of the variability and complexity of repeat lengths. Traditional diagnostic methods, including PCR and Southern blotting, exhibit limitations in sensitivity and specificity, necessitating the development of innovative approaches for precise and rapid diagnosis. Here, we introduce a CRISPR-based diagnostic method, REPLICA (repeat-primed locating of inherited disease by Cas3), for the quantification and rapid diagnosis of DM1. This method, using in vitro-assembled CRISPR-Cas3, demonstrates superior sensitivity and specificity in quantifying CTG repeat expansion lengths, correlated with disease severity. We also validate the robustness and accuracy of CRISPR diagnostics in quantitatively diagnosing DM1 using patient genomes. Furthermore, we optimize a REPLICA-based assay for point-of-care-testing using lateral flow test strips, facilitating rapid screening and detection. In summary, REPLICA-based CRISPR diagnostics offer precise and rapid detection of repeat expansion disorders, promising personalized treatment strategies.},
}

*Myotonic Dystrophy/genetics/diagnosis
Humans
*CRISPR-Cas Systems/genetics
*Trinucleotide Repeat Expansion/genetics
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Sensitivity and Specificity

@article {pmid39499900,
year = {2024},
author = {Liu, Z and Chen, S and Lo, CH and Wang, Q and Sun, Y},
title = {All-in-one AAV-mediated Nrl gene inactivation rescues retinal degeneration in Pde6a mice.},
journal = {JCI insight},
volume = {9},
number = {24},
pages = {},
doi = {10.1172/jci.insight.178159},
pmid = {39499900},
issn = {2379-3708},
mesh = {Animals ; *Cyclic Nucleotide Phosphodiesterases, Type 6/genetics ; Mice ; *Dependovirus/genetics ; *Genetic Therapy/methods ; *Retinal Degeneration/therapy/genetics/pathology ; *Gene Editing/methods ; *Disease Models, Animal ; *Eye Proteins/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Retinitis Pigmentosa/genetics/therapy/pathology ; Retina/pathology/metabolism ; Humans ; Mutation ; Basic-Leucine Zipper Transcription Factors ; },
abstract = {Retinitis pigmentosa (RP) is a complex group of inherited retinal diseases characterized by progressive death of photoreceptor cells and eventual blindness. Pde6a, which encodes a cGMP-specific phosphodiesterase, is a crucial pathogenic gene for autosomal recessive RP (RP43); there is no effective therapy for this form of RP. The compact CRISPR/Staphylococcus aureus Cas9 (CRISPR/SaCas9) system, which can be packaged into a single adeno-associated virus (AAV), holds promise for simplifying effective gene therapy. Here, we demonstrated that all-in-one AAV-SaCas9-mediated Nrl gene inactivation can efficiently prevent retinal degeneration in a RP mouse model with Pde6anmf363/nmf363 mutation. We screened single-guide RNAs capable of efficiently editing the mouse Nrl gene in N2a cells and then achieved effective gene editing by using a single AAV to codeliver SaCas9 and an optimal Nrl-sg2 into the mouse retina. Excitingly, in vivo inactivation of Nrl improved photoreceptor cell survival and rescued retinal function in treated Pde6a-deficient mice. Thus, we showed that a practical, gene-independent method, AAV-SaCas9-mediated Nrl inactivation, holds promise for future therapeutic applications in patients with RP.},
}

Animals
*Cyclic Nucleotide Phosphodiesterases, Type 6/genetics
Mice
*Dependovirus/genetics
*Genetic Therapy/methods
*Retinal Degeneration/therapy/genetics/pathology
*Gene Editing/methods
*Disease Models, Animal
*Eye Proteins/genetics/metabolism
CRISPR-Cas Systems/genetics
Retinitis Pigmentosa/genetics/therapy/pathology
Retina/pathology/metabolism
Humans
Mutation
Basic-Leucine Zipper Transcription Factors

@article {pmid39440484,
year = {2024},
author = {Wong, PK and Syafruddin, SE and Cheah, FC and Azmi, N and Ng, PY and Chua, EW},
title = {Introduction of a single-nucleotide variant, rs16851030, into the ADORA1 gene increased cellular susceptibility to hypoxia.},
journal = {Personalized medicine},
volume = {21},
number = {6},
pages = {353-366},
doi = {10.1080/17410541.2024.2412514},
pmid = {39440484},
issn = {1744-828X},
support = {FRGS/1/2019/SKK08/UKM/03/4//Fundamental Research Grant Scheme/ ; },
mesh = {Humans ; *Polymorphism, Single Nucleotide/genetics ; HEK293 Cells ; *Receptor, Adenosine A1/genetics ; *CRISPR-Cas Systems/genetics ; Cell Hypoxia/genetics ; Genetic Predisposition to Disease/genetics ; Gene Editing/methods ; Hypoxia/genetics ; Caffeine/pharmacology ; 3' Untranslated Regions/genetics ; },
abstract = {Aim: Rs16851030, a single-nucleotide variant located in the 3'-untranslated region of the ADORA1 gene, has been proposed as a potential marker of caffeine sensitivity in apnea of prematurity. Besides, it is associated with aspirin-induced asthma and the development of acute chest syndrome. However, its functional significance is still unconfirmed. This study aimed to elucidate the functional impact of rs16851030 by using CRISPR/Cas9 approach to induce the DNA variant and attendant physiological changes.Methods: Rs16851030 was introduced into HEK293 cells via homology-directed repair (HDR). Edited cells were fluorescence-enriched, sorted, isolated, and expanded into single-cell-derived clones. The edit was confirmed by Sanger sequencing. RNA sequencing was used to analyze affected pathways.Results: Rs16851030-mutant cells showed increased susceptibility to hypoxia, a condition related to apnea of prematurity. After 24 h of hypoxia, the viability of mutant clones 1 and 2 was low compared with wild-type cells (75.45% and 74.47% vs. 96.34%). RNA sequencing revealed transcriptomic changes linked to this increased vulnerability.Conclusion: Rs16851030 impairs cellular resistance to hypoxia, suggesting its role in conditions like apnea of prematurity. Further research should investigate the molecular mechanisms and transcriptomic alterations caused by rs16851030 under hypoxic conditions.},
}

Humans
*Polymorphism, Single Nucleotide/genetics
HEK293 Cells
*Receptor, Adenosine A1/genetics
*CRISPR-Cas Systems/genetics
Cell Hypoxia/genetics
Genetic Predisposition to Disease/genetics
Gene Editing/methods
Hypoxia/genetics
Caffeine/pharmacology
3' Untranslated Regions/genetics

@article {pmid39192154,
year = {2025},
author = {Zhang, H and Zhu, JK},
title = {Epigenetic gene regulation in plants and its potential applications in crop improvement.},
journal = {Nature reviews. Molecular cell biology},
volume = {26},
number = {1},
pages = {51-67},
pmid = {39192154},
issn = {1471-0080},
mesh = {*Epigenesis, Genetic/genetics ; *DNA Methylation/genetics ; *Crops, Agricultural/genetics ; *Gene Expression Regulation, Plant/genetics ; Gene Editing/methods ; Plant Breeding/methods ; Plants/genetics/metabolism ; CRISPR-Cas Systems/genetics ; },
abstract = {DNA methylation, also known as 5-methylcytosine, is an epigenetic modification that has crucial functions in plant growth, development and adaptation. The cellular DNA methylation level is tightly regulated by the combined action of DNA methyltransferases and demethylases. Protein complexes involved in the targeting and interpretation of DNA methylation have been identified, revealing intriguing roles of methyl-DNA binding proteins and molecular chaperones. Structural studies and in vitro reconstituted enzymatic systems have provided mechanistic insights into RNA-directed DNA methylation, the main pathway catalysing de novo methylation in plants. A better understanding of the regulatory mechanisms will enable locus-specific manipulation of the DNA methylation status. CRISPR-dCas9-based epigenome editing tools are being developed for this goal. Given that DNA methylation patterns can be stably transmitted through meiosis, and that large phenotypic variations can be contributed by epimutations, epigenome editing holds great promise in crop breeding by creating additional phenotypic variability on the same genetic material.},
}

*Epigenesis, Genetic/genetics
*DNA Methylation/genetics
*Crops, Agricultural/genetics
*Gene Expression Regulation, Plant/genetics
Gene Editing/methods
Plant Breeding/methods
Plants/genetics/metabolism
CRISPR-Cas Systems/genetics

@article {pmid38377059,
year = {2024},
author = {Shamjetsabam, ND and Rana, R and Malik, P and Ganguly, NK},
title = {CRISPR/Cas9: an overview of recent developments and applications in cancer research.},
journal = {International journal of surgery (London, England)},
volume = {110},
number = {10},
pages = {6198-6213},
pmid = {38377059},
issn = {1743-9159},
mesh = {*CRISPR-Cas Systems ; Humans ; *Neoplasms/genetics/therapy ; *Gene Editing/methods ; Genetic Therapy/methods ; Biomedical Research ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated protein 9 (Cas9) has risen as a potent gene editing method with vast potential across numerous domains, including its application in cancer research and therapy. This review article provides an extensive overview of the research that has been done so far on CRISPR-Cas9 with an emphasis on how it could be utilized in the treatment of cancer. The authors go into the underlying ideas behind CRISPR-Cas9, its mechanisms of action, and its application for the study of cancer biology. Furthermore, the authors investigate the various uses of CRISPR-Cas9 in cancer research, spanning from the discovery of genes and the disease to the creation of novel therapeutic approaches. The authors additionally discuss the challenges and limitations posed by CRISPR-Cas9 technology and offer insights into the potential applications and future directions of this cutting-edge field of research. The article intends to consolidate the present understanding and stimulate more research into CRISPR-Cas9's promise as a game-changing tool for cancer research and therapy.},
}

*CRISPR-Cas Systems
Humans
*Neoplasms/genetics/therapy
*Gene Editing/methods
Genetic Therapy/methods
Biomedical Research

@article {pmid39698811,
year = {2024},
author = {Soczek, KM and Cofsky, JC and Tuck, OT and Shi, H and Doudna, JA},
title = {CRISPR-Cas12a bends DNA to destabilize base pairs during target interrogation.},
journal = {Nucleic acids research},
volume = {},
number = {},
pages = {},
doi = {10.1093/nar/gkae1192},
pmid = {39698811},
issn = {1362-4962},
support = {U19AI171110/NH/NIH HHS/United States ; U19NS132303/NS/NINDS NIH HHS/United States ; R21HL173710/HB/NHLBI NIH HHS/United States ; 2334028//National Science Foundation/ ; /HHMI/Howard Hughes Medical Institute/United States ; //Panattoni Family Foundation/ ; //National Science Foundation Graduate Research Fellowship/ ; //Helen Hay Whitney Foundation/ ; //HHMI Fellow of The Jane Coffin Childs Fund for Medical Research/ ; DE-AC02-05CH11231//Department of Energy/ ; 24 180//Apple Tree Partners/ ; //Lawrence Livermore National Laboratory/ ; //UCB-Hampton University Summer Program/ ; //Mr. Li Ka Shing/ ; //Koret-Berkeley-TAU/ ; //Innovative Genomics Institute/ ; },
abstract = {RNA-guided endonucleases are involved in processes ranging from adaptive immunity to site-specific transposition and have revolutionized genome editing. CRISPR-Cas9, -Cas12 and related proteins use guide RNAs to recognize ∼20-nucleotide target sites within genomic DNA by mechanisms that are not yet fully understood. We used structural and biochemical methods to assess early steps in DNA recognition by Cas12a protein-guide RNA complexes. We show here that Cas12a initiates DNA target recognition by bending DNA to induce transient nucleotide flipping that exposes nucleobases for DNA-RNA hybridization. Cryo-EM structural analysis of a trapped Cas12a-RNA-DNA surveillance complex and fluorescence-based conformational probing show that Cas12a-induced DNA helix destabilization enables target discovery and engagement. This mechanism of initial DNA interrogation resembles that of CRISPR-Cas9 despite distinct evolutionary origins and different RNA-DNA hybridization directionality of these enzyme families. Our findings support a model in which RNA-mediated DNA interference begins with local helix distortion by transient CRISPR-Cas protein binding.},
}

@article {pmid39698318,
year = {2024},
author = {Cao, S and Ma, D and Xie, J and Wu, Z and Yan, H and Ji, S and Zhou, M and Zhu, S},
title = {Point-of-care testing diagnosis of African swine fever virus by targeting multiple genes with enzymatic recombinase amplification and CRISPR/Cas12a System.},
journal = {Frontiers in cellular and infection microbiology},
volume = {14},
number = {},
pages = {1474825},
pmid = {39698318},
issn = {2235-2988},
mesh = {*African Swine Fever Virus/genetics/isolation & purification ; Animals ; *CRISPR-Cas Systems ; Swine ; *African Swine Fever/diagnosis/virology ; *Point-of-Care Testing ; *Sensitivity and Specificity ; *Recombinases/metabolism/genetics ; Nucleic Acid Amplification Techniques/methods ; CRISPR-Associated Proteins/genetics ; Endodeoxyribonucleases/genetics ; Molecular Diagnostic Techniques/methods ; Bacterial Proteins ; },
abstract = {African swine fever virus (ASFV) infection is causing devastating outbreaks globally; pig farming has suffered severe economic losses due to the ASFV. Currently, strict biosecurity control measures can mitigate the incidence of ASF. Rapid, cost-effective, and sensitive detection of ASFV can significantly reduce disease transmission and mortality. CRISPR/Cas-associated proteins can detect polymorphisms with high specificity and sensitivity, making them ideal for detecting pathogens. In this study, based on CRISPR/Cas12a integrated with enzymatic recombinase amplification (ERA) technology, a CRISPR/Cas12a detection system capable of identifying ASFV E183L, K205R, and C962R gene sequences has been developed. The ERA-CRISPR/Cas12a detection system detected ASFV precisely without cross-reactivity with other porcine pathogen templates and with a sensitivity detection limit of 10 copies per reaction; it takes 60 minutes to complete the detection process. In combination with this integrated ERA pre-amplification and Cas12a/crRNA cutting assay, it provides a rapid, straightforward, sensitive, and specific method for ASFV detection in the field.},
}

*African Swine Fever Virus/genetics/isolation & purification
Animals
*CRISPR-Cas Systems
Swine
*African Swine Fever/diagnosis/virology
*Point-of-Care Testing
*Sensitivity and Specificity
*Recombinases/metabolism/genetics
Nucleic Acid Amplification Techniques/methods
CRISPR-Associated Proteins/genetics
Endodeoxyribonucleases/genetics
Molecular Diagnostic Techniques/methods
Bacterial Proteins

@article {pmid39696686,
year = {2024},
author = {Izadifar, M and Massumi, M and Prentice, KJ and Oussenko, T and Li, B and Elbaz, J and Puri, M and Wheeler, MB and Nagy, A},
title = {Microfluidic chip systems for characterizing glucose-responsive insulin-secreting cells equipped with FailSafe kill-switch.},
journal = {Stem cell research & therapy},
volume = {15},
number = {1},
pages = {486},
pmid = {39696686},
issn = {1757-6512},
support = {143231//Canadian Institutes of Health Research Foundation Grant/ ; 950-230422//Canada Research Chairs/ ; },
mesh = {*Insulin-Secreting Cells/metabolism/drug effects ; Animals ; Mice ; *Glucose/metabolism ; *Thymidine Kinase/genetics/metabolism ; Ganciclovir/pharmacology ; CRISPR-Cas Systems ; CDC2 Protein Kinase/metabolism/genetics ; Lab-On-A-Chip Devices ; Insulin/metabolism ; Homeodomain Proteins/metabolism/genetics ; Trans-Activators ; },
abstract = {BACKGROUND: Pluripotent cell-derived islet replacement therapy offers promise for treating Type 1 diabetes (T1D), but concerns about uncontrolled cell proliferation and tumorigenicity present significant safety challenges. To address the safety concern, this study aims to establish a proof-of-concept for a glucose-responsive, insulin-secreting cell line integrated with a built-in FailSafe kill-switch.

METHOD: We generated β cell-induced progenitor-like cells (βiPLCs) from primary mouse pancreatic β cells through interrupted reprogramming. Then, we transcriptionally linked our FailSafe (FS) kill-switch, HSV-thymidine kinase (TK), to Cdk1 gene using a CRISPR/Cas9 knock-in strategy, resulting in a FailSafe βiPLC line, designated as FSβiPLCs. Subsequently we evaluated and confirmed the functionality of the drug-inducible kill-switch in FSβiPLCs at different ganciclovir (GCV) concentrations using our PDMS-based transcapillary microfluidic system. Finally, we assessed the functionality of FSβiPLCs by characterizing the dynamics of insulin secretion in response to changes in glucose concentration using our microfluidic perfusion glucose-stimulated insulin secretion (GSIS) assay-on- chip.

RESULTS: The βiPLCs exhibited Ins1, Pdx1 and Nkx6.1 expression, and glucose responsive insulin secretion, the essential properties of pancreatic beta cells. The βiPLCs were amenable to genome editing which allowed for the insertion of the kill-switch into the 3'UTR of Cdk1, confirmed by PCR genotyping. Our transcapillary microfluidic system confirmed the functionality of the drug-inducible kill-switch in FSβiPLCs, showing an effective cell ablation of dividing cells from a heterogeneous cell population at different ganciclovir (GCV) concentrations. The Ki67 expression assessment further confirmed that slow- or non-dividing cells in the FSβiPLC population were resistant to GCV. Our perfusion glucose-stimulated insulin secretion (GSIS) assay-on-chip revealed that the resistant non-dividing FSβiPLCs exhibited higher levels of insulin secretion and glucose responsiveness compared to their proliferating counterparts.

CONCLUSIONS: This study establishes a proof-of-concept for the integration of a FailSafe kill-switch system into a glucose-responsive, insulin-secreting cell line to address the safety concerns in stem cell-derived cell replacement treatment for T1D. The microfluidic systems provided valuable insights into the functionality and safety of these engineered cells, demonstrating the potential of the kill-switch to reduce the risk of tumorigenicity in pluripotent cell-derived insulin-secreting cells.},
}

*Insulin-Secreting Cells/metabolism/drug effects
Animals
Mice
*Glucose/metabolism
*Thymidine Kinase/genetics/metabolism
Ganciclovir/pharmacology
CRISPR-Cas Systems
CDC2 Protein Kinase/metabolism/genetics
Lab-On-A-Chip Devices
Insulin/metabolism
Homeodomain Proteins/metabolism/genetics
Trans-Activators

@article {pmid39696608,
year = {2024},
author = {Lin, Z and Yao, Q and Lai, K and Jiao, K and Zeng, X and Lei, G and Zhang, T and Dai, H},
title = {Cas12f1 gene drives propagate efficiently in herpesviruses and induce minimal resistance.},
journal = {Genome biology},
volume = {25},
number = {1},
pages = {311},
pmid = {39696608},
issn = {1474-760X},
mesh = {*CRISPR-Cas Systems ; *Herpesvirus 1, Human/genetics ; Gene Drive Technology/methods ; Animals ; CRISPR-Associated Protein 9/metabolism ; CRISPR-Associated Proteins/metabolism/genetics ; Humans ; },
abstract = {BACKGROUND: Synthetic CRISPR-Cas9 gene drive has been developed to control harmful species. However, resistance to Cas9 gene drive can be acquired easily when DNA repair mechanisms patch up the genetic insults introduced by Cas9 and incorporate mutations to the sgRNA target. Although many strategies to reduce the occurrence of resistance have been developed so far, they are difficult to implement and not always effective.

RESULTS: Here, Cas12f1, a recently developed CRISPR-Cas system with minimal potential for causing mutations within target sequences, has been explored as a potential platform for yielding low-resistance in gene drives. We construct Cas9 and Cas12f1 gene drives in a fast-replicating DNA virus, HSV1. Cas9 and Cas12f1 gene drives are able to spread among the HSV1 population with specificity towards their target sites, and their transmission among HSV1 viruses is not significantly affected by the reduced fitness incurred by the viral carriers. Cas12f1 gene drives spread similarly as Cas9 gene drives at high introduction frequency but transmit more slowly than Cas9 gene drives at low introduction frequency. However, Cas12f1 gene drives outperform Cas9 gene drives because they reach higher penetration and induce lower resistance than Cas9 gene drives in all cases.

CONCLUSIONS: Due to lower resistance and higher penetration, Cas12f1 gene drives could potentially supplant Cas9 gene drives for population control.},
}

*CRISPR-Cas Systems
*Herpesvirus 1, Human/genetics
Gene Drive Technology/methods
Animals
CRISPR-Associated Protein 9/metabolism
CRISPR-Associated Proteins/metabolism/genetics
Humans

@article {pmid39696488,
year = {2024},
author = {Koonin, EV and Makarova, KS},
title = {CRISPR in mobile genetic elements: counter-defense, inter-element competition and RNA-guided transposition.},
journal = {BMC biology},
volume = {22},
number = {1},
pages = {295},
pmid = {39696488},
issn = {1741-7007},
mesh = {*DNA Transposable Elements/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; CRISPR-Cas Systems ; Interspersed Repetitive Sequences ; RNA, Guide, CRISPR-Cas Systems/genetics ; Bacteria/genetics/virology ; Plasmids/genetics ; },
abstract = {CRISPR are adaptive immunity systems that protect bacteria and archaea from viruses and other mobile genetic elements (MGE) via an RNA-guided interference mechanism. However, in the course of the host-parasite co-evolution, CRISPR systems have been recruited by MGE themselves for counter-defense or other functions. Some bacteriophages encode fully functional CRISPR systems that target host defense systems, and many others recruited individual components of CRISPR systems, such as single repeat units that inhibit host CRISPR systems and CRISPR mini-arrays that target related viruses contributing to inter-virus competition. Many plasmids carry type IV or subtype V-M CRISPR systems that appear to be involved in inter-plasmid competition. Numerous Tn7-like and Mu-like transposons encode CRISPR-associated transposases (CASTs) in which interference-defective CRISPR systems of type I or type V mediate RNA-guided, site-specific transposition. The recruitment of CRISPR systems and their components by MGE is a manifestation of extensive gene shuttling between host immune systems and MGE, a major trend in the coevolution of MGE with their hosts.},
}

*DNA Transposable Elements/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
CRISPR-Cas Systems
Interspersed Repetitive Sequences
RNA, Guide, CRISPR-Cas Systems/genetics
Bacteria/genetics/virology
Plasmids/genetics

@article {pmid39696352,
year = {2024},
author = {Zheng, S and Luo, M and Huang, H and Huang, X and Peng, Z and Zheng, S and Tan, J},
title = {New insights into the role of mitophagy related gene affecting the metastasis of osteosarcoma through scRNA-seq and CRISPR-Cas9 genome editing.},
journal = {Cell communication and signaling : CCS},
volume = {22},
number = {1},
pages = {592},
pmid = {39696352},
issn = {1478-811X},
support = {82460548//National Natural Science Foundation of China/ ; 20242BAB20368//Natural Science Foundation of Jiangxi Province of China/ ; },
mesh = {*Osteosarcoma/genetics/pathology ; *Mitophagy/genetics ; Humans ; *CRISPR-Cas Systems/genetics ; *Gene Editing ; Animals ; *Bone Neoplasms/genetics/pathology/metabolism ; Neoplasm Metastasis/genetics ; Cell Line, Tumor ; Mice ; Single-Cell Analysis ; Single-Cell Gene Expression Analysis ; Mitochondrial Precursor Protein Import Complex Proteins ; },
abstract = {BACKGROUND: Osteosarcoma (OSA), the most common primary bone malignancy, poses significant challenges due to its aggressive nature and propensity for metastasis, especially in adolescents. Mitophagy analysis can help identify new therapeutic targets and combined treatment strategies.

METHODS: This study integrates single-cell sequencing (scRNA-seq) data and bulk-seq to identify mitophagy-related genes (MRGs) associated with the progression of OSA metastasis and analyze their clinical significance. scRNA-seq data elucidates the relationship between mitophagy and OSA metastasis, employing "CellChat" R package to explore intercellular communications and report on hundreds of ligand-receptor interactions. Subsequently, the combination of bulk-seq and CRISPR-Cas9 gene editing identifies mitophagy-related biomarker associated with metastatic prognosis. Finally, validation of the relationship between mitophagy and OSA metastasis is achieved through cellular biology experiments and animal studies.

RESULTS: The distinct mitophagy activity of various mitochondria manifests in diverse spatial localization, cellular developmental trajectories, and intercellular interactions. OSA tissue exhibits notable heterogeneity in mitophagy within osteoblastic OSA cells. However, high mitophagy activity correlates consistently with high metastatic potential. Subsequently, we identified three critical genes associated with mitophagy in OSA, namely RPS27A, TOMM20 and UBB. According to the aforementioned queue of genes, we have constructed a mitophagy_score (MIP_score). We observed that it consistently predicts patient prognosis in both internal and external datasets, demonstrating strong robustness and stability. Furthermore, we have found that MIP_score can also guide chemotherapy, with varying sensitivities to chemotherapeutic agents based on different MIP_score. It is noteworthy that, through the integration of CRISPR-Cas9 genome-wide screening and validation via cellular and animal experiments, we have identified RPS27A as a potential novel biomarker for OSA.

CONCLUSIONS: Our comprehensive analysis elucidated the profile of mitophagy throughout the OSA metastasis process, forming the basis for a mitophagy-related prognostic model that addresses clinical outcomes and drug sensitivity following OSA metastasis. Additionally, an online interactive platform was established to assist clinicians in decision-making (https://mip-score.shinyapps.io/labtan/). These findings lay the groundwork for developing targeted therapies aimed at improving the prognosis of OSA patients.},
}

*Osteosarcoma/genetics/pathology
*Mitophagy/genetics
Humans
*CRISPR-Cas Systems/genetics
*Gene Editing
Animals
*Bone Neoplasms/genetics/pathology/metabolism
Neoplasm Metastasis/genetics
Cell Line, Tumor
Mice
Single-Cell Analysis
Single-Cell Gene Expression Analysis
Mitochondrial Precursor Protein Import Complex Proteins

@article {pmid39695426,
year = {2024},
author = {Prado, MB and Coelho, BP and Iglesia, RP and Alves, RN and Boccacino, JM and Fernandes, CFL and Melo-Escobar, MI and Ayyadhury, S and Cruz, MC and Santos, TG and Beraldo, FH and Fan, J and Ferreira, FM and Nakaya, HI and Prado, MAM and Prado, VF and Duennwald, ML and Lopes, MH},
title = {Prion protein regulates invasiveness in glioblastoma stem cells.},
journal = {BMC cancer},
volume = {24},
number = {1},
pages = {1539},
pmid = {39695426},
issn = {1471-2407},
support = {2017/26158‑0//Fundação de Amparo à Pesquisa do Estado de São Paulo/ ; 2019/14952‑0//Fundação de Amparo à Pesquisa do Estado de São Paulo/ ; 2020/03714-8//Fundação de Amparo à Pesquisa do Estado de São Paulo/ ; 2022/08198-3//Fundação de Amparo à Pesquisa do Estado de São Paulo/ ; 2020/07450‑5//Fundação de Amparo à Pesquisa do Estado de São Paulo/ ; 2019/14741‑9//Fundação de Amparo à Pesquisa do Estado de São Paulo/ ; 2019/11097‑1//Fundação de Amparo à Pesquisa do Estado de São Paulo/ ; 2017/20271‑0//Fundação de Amparo à Pesquisa do Estado de São Paulo/ ; 101796/2020‑0//Conselho Nacional de Desenvolvimento Científico e Tecnológico/ ; 409941/2021‑2//Conselho Nacional de Desenvolvimento Científico e Tecnológico/ ; 03592-2021 RGPIN//Natural Sciences and Engineering Research Council of Canada/ ; },
mesh = {*Glioblastoma/pathology/metabolism/genetics ; Humans ; *Neoplastic Stem Cells/metabolism/pathology ; Cell Line, Tumor ; *Neoplasm Invasiveness ; *Brain Neoplasms/pathology/metabolism/genetics ; Cell Proliferation ; Gene Knockout Techniques ; PrPC Proteins/metabolism/genetics ; Prion Proteins/metabolism/genetics ; Gene Expression Regulation, Neoplastic ; Cell Movement ; CRISPR-Cas Systems ; },
abstract = {BACKGROUND: Glioblastoma (GBM) is an aggressive brain tumor driven by glioblastoma stem cells (GSCs), which represent an appealing target for therapeutic interventions. The cellular prion protein (PrP[C]), a scaffold protein involved in diverse cellular processes, interacts with various membrane and extracellular matrix molecules, influencing tumor biology. Herein, we investigate the impact of PrP[C] expression on GBM.

METHODS: To address this goal, we employed CRISPR-Cas9 technology to generate PrP[C] knockout (KO) glioblastoma cell lines, enabling detailed loss-of-function studies. Bulk RNA sequencing followed by differentially expressed gene and pathway enrichment analyses between U87 or U251 PrP[C]-wild-type (WT) cells and PrP[C]-knockout (KO) cells were used to identify pathways regulated by PrP[C]. Immunofluorescence assays were used to evaluate cellular morphology and protein distribution. For assessment of protein levels, Western blot and flow cytometry assays were employed. Transwell and growth curve assays were used to determine the impact of loss-of-PrP[C] in GBM invasiveness and proliferation, respectively. Single-cell RNA sequencing analysis of data from patient tumors from The Cancer Genome Atlas (TCGA) and the Broad Institute of Single-Cell Data Portal were used to evaluate the correspondence between our in vitro results and patient samples.

RESULTS: Transcriptome analysis of PrP[C]-KO GBM cell lines revealed altered expression of genes associated with crucial tumor progression pathways, including migration, proliferation, and stemness. These findings were corroborated by assays that revealed impaired invasion, migration, proliferation, and self-renewal in PrP[C]-KO GBM cells, highlighting its critical role in sustaining tumor growth. Notably, loss-of-PrP[C] disrupted the expression and localization of key stemness markers, particularly CD44. Additionally, the modulation of PrP[C] levels through CD44 overexpression further emphasizes their regulatory role in these processes.

CONCLUSIONS: These findings establish PrP[C] as a modulator of essential molecules on the cell surface of GSCs, highlighting its potential as a therapeutic target for GBM.},
}

*Glioblastoma/pathology/metabolism/genetics
Humans
*Neoplastic Stem Cells/metabolism/pathology
Cell Line, Tumor
*Neoplasm Invasiveness
*Brain Neoplasms/pathology/metabolism/genetics
Cell Proliferation
Gene Knockout Techniques
PrPC Proteins/metabolism/genetics
Prion Proteins/metabolism/genetics
Gene Expression Regulation, Neoplastic
Cell Movement
CRISPR-Cas Systems

@article {pmid39694744,
year = {2024},
author = {Zhu, Y and Yu, X and Wu, J},
title = {CRISPR/Cas: a toolkit for plant disease diagnostics.},
journal = {Trends in plant science},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.tplants.2024.11.011},
pmid = {39694744},
issn = {1878-4372},
abstract = {Genetic factors and infectious pathogens that cause plant diseases have a major impact on agricultural production. In recent years, the potential of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system in nucleic acid analysis and plant disease diagnostics has been demonstrated. We highlight progress of CRISPR/Cas technology that is significant for monitoring plant growth and preventing diseases.},
}

@article {pmid39694477,
year = {2024},
author = {Harding, KR and Malone, LM and Kyte, NAP and Jackson, SA and Smith, LM and Fineran, PC},
title = {Genome-wide identification of bacterial genes contributing to nucleus-forming jumbo phage infection.},
journal = {Nucleic acids research},
volume = {},
number = {},
pages = {},
doi = {10.1093/nar/gkae1194},
pmid = {39694477},
issn = {1362-4962},
support = {//Marsden Fund/ ; //Royal Society of New Zealand/ ; //Bioprotection Aotearoa/ ; //James Cook Research Fellowship/ ; //University of Otago Doctoral Scholarships/ ; //Division of Health Sciences Career Development Postdoctoral Fellowship/ ; //EMBO Postdoctoral Fellowship/ ; },
abstract = {The Chimalliviridae family of bacteriophages (phages) form a proteinaceous nucleus-like structure during infection of their bacterial hosts. This phage 'nucleus' compartmentalises phage DNA replication and transcription, and shields the phage genome from DNA-targeting defence systems such as CRISPR-Cas and restriction-modification. Their insensitivity to DNA-targeting defences makes nucleus-forming jumbo phages attractive for phage therapy. However, little is known about the bacterial gene requirements during the infectious cycle of nucleus-forming phages or how phage resistance may emerge. To address this, we used the Serratia nucleus-forming jumbo phage PCH45 and exploited a combination of high-throughput transposon mutagenesis and deep sequencing (Tn-seq), and CRISPR interference (CRISPRi). We identified over 90 host genes involved in nucleus-forming phage infection, the majority of which were either involved in the biosynthesis of the primary receptor, flagella, or influenced swimming motility. In addition, the bacterial outer membrane lipopolysaccharide contributed to PCH45 adsorption. Other unrelated Serratia-flagellotropic phages used similar host genes as the nucleus-forming phage, indicating that phage resistance can lead to cross-resistance against diverse phages. Our findings demonstrate that resistance to nucleus-forming jumbo phages can readily emerge via bacterial surface receptor mutation and this should be a major factor when designing strategies for their use in phage therapy.},
}

@article {pmid39693940,
year = {2024},
author = {Baranova, SV and Zhdanova, PV and Golyshev, VM and Lomzov, AA and Pestryakov, PE and Chernonosov, AA and Koval, VV},
title = {Thermodynamic parameters obtained for the formation of the Cas12a-RNA/DNA complex.},
journal = {Biochemical and biophysical research communications},
volume = {743},
number = {},
pages = {151176},
doi = {10.1016/j.bbrc.2024.151176},
pmid = {39693940},
issn = {1090-2104},
abstract = {The thermodynamics of interactions between Cas12a, RNA, and DNA are important to understanding the molecular mechanisms governing CRISPR-Cas12a's specificity and function. In this study, we employed isothermal titration calorimetry (ITC) and molecular dynamics (MD) simulations to investigate the binding properties and energetic contributions of Cas12a-crRNA complexes with single-stranded (ssDNA) and double-stranded (dsDNA) DNA substrates. ITC analyses revealed significant thermal effects during the interaction of Cas12a-crRNA with ssDNA but no detectable effects with dsDNA. The binding to ssDNA was characterized by an enthalpy change (ΔH°) of -243 ± 18 kcal/mol and a stoichiometry of ∼0.3, indicating partial binding due to structural hindrances such as intramolecular secondary structures in RNA and DNA. MD simulations further supported these findings, highlighting the stability and dynamic behavior of Cas12a-crRNA complexes with both DNA substrates. Binding free energy calculations (MM-GBSA) revealed stronger stabilization of the Cas12a-crRNA complex by dsDNA compared to ssDNA, likely driven by additional electrostatic interactions and protein-DNA contacts. However, these interactions did not produce measurable heat effects in ITC experiments. The combined experimental and computational findings demonstrate that the CRISPR-Cas12a system's interactions with nucleic acids are predominantly governed by their structural characteristics and conformational flexibility. These results deepen our understanding of the thermodynamic and structural principles underlying Cas12a-mediated target recognition and cleavage.},
}

@article {pmid39693439,
year = {2024},
author = {Wang, Q and Xu, X and Chen, S and Lu, R and Li, L and Lo, CH and Liu, Z and Ning, K and Li, T and Kowal, TJ and Wang, B and Hartnett, ME and Wang, S and Qi, LS and Sun, Y},
title = {dCasMINI-mediated therapy rescues photoreceptors degeneration in a mouse model of retinitis pigmentosa.},
journal = {Science advances},
volume = {10},
number = {51},
pages = {eadn7540},
doi = {10.1126/sciadv.adn7540},
pmid = {39693439},
issn = {2375-2548},
mesh = {Animals ; *Retinitis Pigmentosa/therapy/genetics/pathology/metabolism ; *Disease Models, Animal ; *Cyclic Nucleotide Phosphodiesterases, Type 6/genetics/metabolism ; Mice ; *Genetic Therapy/methods ; *Dependovirus/genetics ; Electroretinography ; Retinal Degeneration/therapy/genetics/pathology ; CRISPR-Cas Systems ; Humans ; Retinal Rod Photoreceptor Cells/metabolism/pathology ; Eye Proteins/metabolism/genetics ; Retinal Cone Photoreceptor Cells/metabolism/pathology ; },
abstract = {Retinitis pigmentosa (RP) is characterized by degeneration of rod and cone photoreceptors that progresses to irreversible blindness. Now, there are no mutation-agnostic approaches to treat RP. Here, we utilized a single adeno-associated virus (AAV)-based CRISPR activation system to activate phosphodiesterase 6B (Pde6b) to mitigate the severe degeneration in Pde6a[nmf363] mice. We demonstrate that transcriptional activation of Pde6b can rescue the loss of Pde6a, with preservation of retinal structure, restoration of electroretinography responses, and improvement of visual function as assessed by optokinetic response and looming-induced escape behaviors. These findings demonstrate the therapeutic potential of a dCasMINI-mediated activation strategy that provides a mutation-independent treatment for retinal degeneration. This study offers a promising therapeutic approach for RP and potentially other forms of genetic diseases.},
}

Animals
*Retinitis Pigmentosa/therapy/genetics/pathology/metabolism
*Disease Models, Animal
*Cyclic Nucleotide Phosphodiesterases, Type 6/genetics/metabolism
Mice
*Genetic Therapy/methods
*Dependovirus/genetics
Electroretinography
Retinal Degeneration/therapy/genetics/pathology
CRISPR-Cas Systems
Humans
Retinal Rod Photoreceptor Cells/metabolism/pathology
Eye Proteins/metabolism/genetics
Retinal Cone Photoreceptor Cells/metabolism/pathology

@article {pmid39693429,
year = {2024},
author = {Shembrey, C and Yang, R and Casan, J and Hu, W and Chen, H and Singh, GJ and Sadras, T and Prasad, K and Shortt, J and Johnstone, RW and Trapani, JA and Ekert, PG and Fareh, M},
title = {Principles of CRISPR-Cas13 mismatch intolerance enable selective silencing of point-mutated oncogenic RNA with single-base precision.},
journal = {Science advances},
volume = {10},
number = {51},
pages = {eadl0731},
doi = {10.1126/sciadv.adl0731},
pmid = {39693429},
issn = {2375-2548},
mesh = {*CRISPR-Cas Systems ; Humans ; RNA, Guide, CRISPR-Cas Systems/genetics ; Point Mutation ; Gene Silencing ; Gene Editing/methods ; Polymorphism, Single Nucleotide ; Proto-Oncogene Proteins p21(ras)/genetics ; Base Pair Mismatch ; Membrane Proteins/genetics ; Oncogenes ; RNA, Neoplasm/genetics/metabolism ; GTP Phosphohydrolases ; Proto-Oncogene Proteins B-raf ; },
abstract = {Single-nucleotide variants (SNVs) are extremely prevalent in human cancers, although most of these remain clinically unactionable. The programmable RNA nuclease CRISPR-Cas13 has been deployed to specifically target oncogenic RNAs. However, silencing oncogenic SNVs with single-base precision remains extremely challenging due to the intrinsic mismatch tolerance of Cas13. Here, we show that introducing synthetic mismatches at precise positions of the spacer sequence enables de novo design of guide RNAs [CRISPR RNAs (crRNAs)] with strong preferential silencing of point-mutated transcripts. We applied these design principles to effectively silence the oncogenic KRAS G12 hotspot, NRAS G12D and BRAF V600E transcripts with minimal off-target silencing of the wild-type transcripts, underscoring the adaptability of this platform to silence various SNVs. Unexpectedly, the SNV-selective crRNAs harboring mismatched nucleotides reduce the promiscuous collateral activity of the RfxCas13d ortholog. These findings demonstrate that the CRISPR-Cas13 system can be reprogrammed to target mutant transcripts with single-base precision, showcasing the tremendous potential of this tool in personalized transcriptome editing.},
}

*CRISPR-Cas Systems
Humans
RNA, Guide, CRISPR-Cas Systems/genetics
Point Mutation
Gene Silencing
Gene Editing/methods
Polymorphism, Single Nucleotide
Proto-Oncogene Proteins p21(ras)/genetics
Base Pair Mismatch
Membrane Proteins/genetics
Oncogenes
RNA, Neoplasm/genetics/metabolism
GTP Phosphohydrolases
Proto-Oncogene Proteins B-raf

@article {pmid39693343,
year = {2024},
author = {Casagrande Raffi, G and Chen, J and Feng, X and Chen, Z and Lieftink, C and Deng, S and Mo, J and Zeng, C and Steur, M and Wang, J and Bleijerveld, OB and Hoekman, L and van der Wel, N and Wang, F and Beijersbergen, R and Zheng, J and Bernards, R and Wang, L},
title = {An antibiotic that mediates immune destruction of senescent cancer cells.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {121},
number = {52},
pages = {e2417724121},
doi = {10.1073/pnas.2417724121},
pmid = {39693343},
issn = {1091-6490},
support = {787925//EC | ERC | HORIZON EUROPE European Research Council (ERC)/ ; LSH-TKI-LSHM20083//Health Holland/ ; 19-051-ASP//Mark Foundation For Cancer Research (The Mark Foundation for Cancer Research)/ ; 12539//Dutch Cancer Society KWF/ ; W2432050; 82372695//National Natural Science Foundation of China-Guangdong Joint Fund (NSFC)/ ; 2024A04J6484//Guangdong Basic and Applied Basic Research Foundation/ ; },
mesh = {Humans ; *Cellular Senescence/drug effects/immunology ; *Pyrans/pharmacology ; *Killer Cells, Natural/immunology/drug effects/metabolism ; Cell Line, Tumor ; Reactive Oxygen Species/metabolism ; Necroptosis/drug effects ; Neoplasms/immunology/drug therapy/metabolism ; CD8-Positive T-Lymphocytes/immunology/drug effects/metabolism ; Anti-Bacterial Agents/pharmacology ; Receptors, TNF-Related Apoptosis-Inducing Ligand/metabolism/genetics ; Pyroptosis/drug effects ; Apoptosis/drug effects ; Animals ; CRISPR-Cas Systems ; Polyether Polyketides ; },
abstract = {Drugs that eliminate senescent cells, senolytics, can be powerful when combined with prosenescence cancer therapies. Using a CRISPR/Cas9-based genetic screen, we identify here SLC25A23 as a vulnerability of senescent cancer cells. Suppressing SLC25A23 disrupts cellular calcium homeostasis, impairs oxidative phosphorylation, and interferes with redox signaling, leading to death of senescent cells. These effects can be replicated by salinomycin, a cation ionophore antibiotic. Salinomycin prompts a pyroptosis-apoptosis-necroptosis (PAN)optosis-like cell death in senescent cells, including apoptosis and two forms of immunogenic cell death: necroptosis and pyroptosis. Notably, we observed that salinomycin treatment or SLC25A23 suppression elevates reactive oxygen species, upregulating death receptor 5 via Jun N-terminal protein kinase (JNK) pathway activation. We show that a combination of a death receptor 5 (DR5) agonistic antibody and salinomycin is a robust senolytic cocktail. We provide evidence that this drug combination provokes a potent natural killer (NK) and CD8+ T cell-mediated immune destruction of senescent cancer cells, mediated by the pyroptotic cytokine interleukin 18 (IL18).},
}

Humans
*Cellular Senescence/drug effects/immunology
*Pyrans/pharmacology
*Killer Cells, Natural/immunology/drug effects/metabolism
Cell Line, Tumor
Reactive Oxygen Species/metabolism
Necroptosis/drug effects
Neoplasms/immunology/drug therapy/metabolism
CD8-Positive T-Lymphocytes/immunology/drug effects/metabolism
Anti-Bacterial Agents/pharmacology
Receptors, TNF-Related Apoptosis-Inducing Ligand/metabolism/genetics
Pyroptosis/drug effects
Apoptosis/drug effects
Animals
CRISPR-Cas Systems
Polyether Polyketides

@article {pmid39667940,
year = {2024},
author = {Yang, B and Wu, C and Teng, Y and Chou, KJ and Guarnieri, MT and Xiong, W},
title = {Tailoring microbial fitness through computational steering and CRISPRi-driven robustness regulation.},
journal = {Cell systems},
volume = {15},
number = {12},
pages = {1133-1147.e4},
doi = {10.1016/j.cels.2024.11.012},
pmid = {39667940},
issn = {2405-4720},
mesh = {*CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Editing/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; Computer Simulation ; Genetic Fitness/genetics ; Escherichia coli/genetics/metabolism ; Organisms, Genetically Modified ; },
abstract = {The widespread application of genetically modified microorganisms (GMMs) across diverse sectors underscores the pressing need for robust strategies to mitigate the risks associated with their potential uncontrolled escape. This study merges computational modeling with CRISPR interference (CRISPRi) to refine GMM metabolic robustness. Utilizing ensemble modeling, we achieved high-throughput in silico screening for enzymatic targets susceptible to expression alterations. Translating these insights, we developed functional CRISPRi, boosting fitness control via multiplexed gene knockdown. Our method, enhanced by an insulator-improved gRNA structure and an off-switch circuit controlling a compact Cas12m, resulted in rationally engineered strains with escape frequencies below National Institutes of Health standards. The effectiveness of this approach was confirmed under various conditions, showcasing its ability for secure GMM management. This research underscores the resilience of microbial metabolism, strategically modifying key nodes to halt growth without provoking significant resistance, thereby enabling more reliable and precise GMM control. A record of this paper's transparent peer review process is included in the supplemental information.},
}

*CRISPR-Cas Systems/genetics
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Gene Editing/methods
RNA, Guide, CRISPR-Cas Systems/genetics
Computer Simulation
Genetic Fitness/genetics
Escherichia coli/genetics/metabolism
Organisms, Genetically Modified

@article {pmid39589913,
year = {2024},
author = {Wang, Q and Yang, G and Jia, R and Wang, F and Wang, G and Xu, Z and Li, J and Li, B and Yu, L and Zhang, Y and Alariqi, M and Cao, J and Liang, S and Zhang, X and Nie, X and Jin, S},
title = {Utilizing the mutant library to investigate the functional characterization of GhGLR3.4 regulating jasmonic acid to defense pest infestation.},
journal = {The Plant journal : for cell and molecular biology},
volume = {120},
number = {6},
pages = {2889-2903},
doi = {10.1111/tpj.17152},
pmid = {39589913},
issn = {1365-313X},
support = {32272128//National Natural Science Foundation of China/ ; 32401780//National Natural Science Foundation of China/ ; 027Y2022-021//Science and Technology Project of Hubei Province of China National Tobacco Corporation/ ; 2022YFF1001403//National Key Research and Development Program of China/ ; 32325039//National Natural Science Fund of China for Distinguished Young Scholars/ ; },
mesh = {*Cyclopentanes/metabolism ; *Oxylipins/metabolism ; *Gossypium/genetics/parasitology ; *Plant Proteins/genetics/metabolism ; Animals ; Receptors, Glutamate/genetics/metabolism ; Gene Expression Regulation, Plant ; Signal Transduction ; Plant Growth Regulators/metabolism ; Arabidopsis/genetics/physiology ; Mutation ; CRISPR-Cas Systems ; Plant Diseases/parasitology/genetics/immunology ; },
abstract = {The glutamate receptor (GLR) serves as a ligand-gated ion channel that plays a vital role in plant growth, development, and stress response. Nevertheless, research on GLRs in cotton is still very limited. The present study conducted a comprehensive analysis of GLRs gene family in cotton. In total, 41 members of the GLR family were identified in cotton unveiling distinct subgroups in comparison to Arabidopsis. Among these members, the third subgroup highlights its pivotal role in cotton's defense against insect infestation. Furthermore, the CRISPR/Cas9 system was utilized to create a mutant library of GLR members, which consisted of a total of 135 independent mutant lines, resulting in the production of novel cotton materials with valuable breeding potential for pest control. Further, this study elucidates the influence of GhGLR3.4 on jasmonic acid (JA) pathway signal transduction and demonstrated its participation in the influx of intracellular Ca[2+], which regulates "calcium transients" following stimulation, thereby influencing multiple intracellular reactions. The study also found that GhGLR3.4 influences the synthesis of the JA pathway and actively partakes in long-distance signal transmission among plants, facilitating the transfer of defense signals to neighbor leaves and thereby triggering systemic defense. Consequently, this research advances our knowledge of plants' comprehensive defense mechanism against insect pest infestation.},
}

*Cyclopentanes/metabolism
*Oxylipins/metabolism
*Gossypium/genetics/parasitology
*Plant Proteins/genetics/metabolism
Animals
Receptors, Glutamate/genetics/metabolism
Gene Expression Regulation, Plant
Signal Transduction
Plant Growth Regulators/metabolism
Arabidopsis/genetics/physiology
Mutation
CRISPR-Cas Systems
Plant Diseases/parasitology/genetics/immunology

@article {pmid39581190,
year = {2025},
author = {Egawa, M and Uno, N and Komazaki, R and Ohkame, Y and Yamazaki, K and Yoshimatsu, C and Ishizu, Y and Okano, Y and Miyamoto, H and Osaki, M and Suzuki, T and Hosomichi, K and Aizawa, Y and Kazuki, Y and Tomizuka, K},
title = {Generation of Monosomy 21q Human iPS Cells by CRISPR/Cas9-Mediated Interstitial Megabase Deletion.},
journal = {Genes to cells : devoted to molecular & cellular mechanisms},
volume = {30},
number = {1},
pages = {e13184},
doi = {10.1111/gtc.13184},
pmid = {39581190},
issn = {1365-2443},
support = {21-101//Exploratory Research Center on Life and Living Systems, National Institutes of Natural Sciences/ ; JP24bm1123038//Japan Agency for Medical Research and Development/ ; JP24ama121046//Japan Agency for Medical Research and Development/ ; JP24gm0010010//Japan Agency for Medical Research and Development/ ; JPMJCR18S4//CREST, Japan Science and Technology Agency/ ; JPMJSP2134//SPRING, Japan Science and Technology Agency/ ; },
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *CRISPR-Cas Systems ; *Chromosomes, Human, Pair 21/genetics ; *Chromosome Deletion ; Monosomy/genetics ; Cell Line ; },
abstract = {Missing an entire chromosome or chromosome arm in normal diploid cells has a deleterious impact on cell viability, which may contribute to the development of specific birth defects. Nevertheless, the effects of chromosome loss in human cells have remained unexplored due to the lack of suitable model systems. Here, we developed an efficient, selection-free approach to generate partial monosomy in human induced pluripotent stem cells (iPSCs). The introduction of Cas9 proteins and a pair of gRNAs induces over megabase-sized interstitial chromosomal deletions. Using human chromosome 21 (HSA21) as a model, partial monosomy 21q (PM21q) iPSC lines with deletions ranging from 4.5 to 27.9 Mb were isolated. A 33.6 Mb deletion, encompassing all protein-coding genes on 21q, was also achieved, establishing the first 21q monosomy human iPSC line. Transcriptome and proteome analyses revealed that the abundances of mRNA and protein encoded by the majority of genes in the monosomic regions are half of the diploid expression level, indicating an absence of dosage compensation. The ability to generate customized partial monosomy cell lines on an isogenic, karyotypically normal background should facilitate the gain of novel insights into the impact of chromosome loss on cellular fitness.},
}

Humans
*Induced Pluripotent Stem Cells/metabolism/cytology
*CRISPR-Cas Systems
*Chromosomes, Human, Pair 21/genetics
*Chromosome Deletion
Monosomy/genetics
Cell Line

@article {pmid39569987,
year = {2024},
author = {Yamamoto, S and Afifi, OA and Lam, LPY and Takeda-Kimura, Y and Osakabe, Y and Osakabe, K and Bartley, LE and Umezawa, T and Tobimatsu, Y},
title = {Disruption of aldehyde dehydrogenase decreases cell wall-bound p-hydroxycinnamates and improves cell wall digestibility in rice.},
journal = {The Plant journal : for cell and molecular biology},
volume = {120},
number = {6},
pages = {2828-2845},
doi = {10.1111/tpj.17148},
pmid = {39569987},
issn = {1365-313X},
support = {Exploratory Research on Humanosphere Science 2021//Research Institute for Sustainable Humanosphere, Kyoto University/ ; Exploratory Research on Humanosphere Science 2023//Research Institute for Sustainable Humanosphere, Kyoto University/ ; DE-SC0021126//Office of Science/ ; #20H03044//Japan Society for the Promotion of Science/ ; #22J13457//Japan Society for the Promotion of Science/ ; #24K01827//Japan Society for the Promotion of Science/ ; #1015621//National Institute of Food and Agriculture/ ; },
mesh = {*Cell Wall/metabolism ; *Oryza/genetics/metabolism/enzymology ; *Coumaric Acids/metabolism ; *Lignin/metabolism ; *Plant Proteins/genetics/metabolism ; Aldehyde Dehydrogenase/metabolism/genetics ; Xylans/metabolism ; Gene Expression Regulation, Plant ; CRISPR-Cas Systems ; },
abstract = {In grass cell walls, ferulic acid (FA) serves as an important cross-linker between cell wall polymers, such as arabinoxylan (AX) and lignin, affecting the physicochemical properties of the cell walls as well as the utilization properties of grass lignocellulose for biorefinering. Here, we demonstrate that hydroxycinnamaldehyde dehydrogenase (HCALDH) plays a crucial role in the biosynthesis of the FA used for cell wall feruloylation in rice (Oryza sativa). Bioinformatic and gene expression analyses of aldehyde dehydrogenases (ALDHs) identified two rice ALDH subfamily 2C members, OsHCALDH2 (OsALDH2C2) and OsHCALDH3 (OsALDH2C3), potentially involved in cell wall feruloylation in major vegetative tissues of rice. CRISPR-Cas9 genome editing of OsHCALDH2 and OsHCALDH3 revealed that the contents of AX-bound ferulate were reduced by up to ~45% in the cell walls of the HCALDH-edited mutants, demonstrating their roles in cell wall feruloylation. The abundance of hemicellulosic sugars including arabinosyl units on AX was notably reduced in the cell walls of the HCALDH-edited mutants, whereas cellulose and lignin contents remained unaffected. In addition to reducing cell wall-bound ferulate, the loss of OsHCALDH2 and/or OsHCALDH3 also partially reduced cell wall-bound p-coumarate and sinapate in the vegetative tissues of rice, whereas it did not cause detectable changes in the amount of γ-oryzanol (feruloyl sterols) in rice seeds. Furthermore, the HCALDH-edited mutants exhibited improved cell wall saccharification efficiency, both with and without alkaline pretreatment, plausibly due to the reduction in cell wall cross-linking FA. Overall, HCALDH appears to present a potent bioengineering target for enhancing utilization properties of grass lignocellulose.},
}

*Cell Wall/metabolism
*Oryza/genetics/metabolism/enzymology
*Coumaric Acids/metabolism
*Lignin/metabolism
*Plant Proteins/genetics/metabolism
Aldehyde Dehydrogenase/metabolism/genetics
Xylans/metabolism
Gene Expression Regulation, Plant
CRISPR-Cas Systems

@article {pmid39560384,
year = {2024},
author = {Steiner, S and Roy, CR},
title = {CRISPR-Cas9-based approaches for genetic analysis and epistatic interaction studies in Coxiella burnetii.},
journal = {mSphere},
volume = {9},
number = {12},
pages = {e0052324},
doi = {10.1128/msphere.00523-24},
pmid = {39560384},
issn = {2379-5042},
support = {R21AI171333,R56AI182383//HHS | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ ; },
mesh = {*Coxiella burnetii/genetics ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Epistasis, Genetic ; Bacterial Proteins/genetics ; Humans ; Animals ; Phenotype ; },
abstract = {UNLABELLED: Coxiella burnetii is an obligate intracellular bacterial pathogen that replicates to high numbers in an acidified lysosome-derived vacuole. Intracellular replication requires the Dot/Icm type IVB secretion system, which translocates over 100 different effector proteins into the host cell. Screens employing random transposon mutagenesis have identified several C. burnetii effectors that play an important role in intracellular replication; however, the difficulty in conducting directed mutagenesis has been a barrier to the systematic analysis of effector mutants and to the construction of double mutants to assess epistatic interactions between effectors. Here, two CRISPR-Cas9 technology-based approaches were developed to study C. burnetii phenotypes resulting from targeted gene disruptions. CRISPRi was used to silence gene expression and demonstrated that silencing of effectors or Dot/Icm system components resulted in phenotypes similar to those of transposon insertion mutants. A CRISPR-Cas9-mediated cytosine base editing protocol was developed to generate targeted loss-of-function mutants through the introduction of premature stop codons into C. burnetii genes. Cytosine base editing successfully generated double mutants in a single step. A double mutant deficient in both cig57 and cig2 had a robust and additive intracellular replication defect when compared to either single mutant, which is consistent with Cig57 and Cig2 functioning in independent pathways that both contribute to a vacuole that supports C. burnetii replication. Thus, CRISPR-Cas9-based technologies expand the genetic toolbox for C. burnetii and will facilitate genetic studies aimed at investigating the mechanisms this pathogen uses to replicate inside host cells.

IMPORTANCE: Understanding the genetic mechanisms that enable C. burnetii to replicate in mammalian host cells has been hampered by the difficulty in making directed mutations. Here, a reliable and efficient system for generating targeted loss-of-function mutations in C. burnetii using a CRISPR-Cas9-assisted base editing approach is described. This technology was applied to make double mutants in C. burnetii that enabled the genetic analysis of two genes that play independent roles in promoting the formation of vacuoles that support intracellular replication. This advance will accelerate the discovery of mechanisms important for C. burnetii host infection and disease.},
}

*Coxiella burnetii/genetics
*CRISPR-Cas Systems
*Gene Editing/methods
*Epistasis, Genetic
Bacterial Proteins/genetics
Humans
Animals
Phenotype

@article {pmid39546384,
year = {2024},
author = {Birchler, JA and Kelly, J and Singh, J and Liu, H and Zhang, Z and Char, SN and Sharma, M and Yang, H and Albert, PS and Yang, B},
title = {Synthetic minichromosomes in plants: past, present, and promise.},
journal = {The Plant journal : for cell and molecular biology},
volume = {120},
number = {6},
pages = {2356-2366},
doi = {10.1111/tpj.17142},
pmid = {39546384},
issn = {1365-313X},
support = {PGRP 2221891//National Science Foundation/ ; },
mesh = {*Chromosomes, Plant/genetics ; *Chromosomes, Artificial/genetics ; *Centromere/genetics ; *CRISPR-Cas Systems ; Gene Editing/methods ; Genetic Engineering/methods ; Plants/genetics ; Telomere/genetics ; Plants, Genetically Modified ; },
abstract = {The status of engineered mini-chromosomes/artificial chromosomes/synthetic chromosomes in plants is summarized. Their promise is that they provide a means to accumulate foreign genes on an independent entity other than the normal chromosomes, which would facilitate stacking of novel traits in a way that would not be linked to endogenous genes and that would facilitate transfer between lines. Centromeres in plants are epigenetic, and therefore the isolation of DNA underlying centromeres and reintroduction into plant cells will not establish a functional kinetochore, which obviates this approach for in vitro assembly of plant artificial chromosomes. This issue was bypassed by using telomere-mediated chromosomal truncation to produce mini-chromosomes with little more than an endogenous centromere that could in turn be used as a foundation to build synthetic chromosomes. Site-specific recombinases and various iterations of CRISPR-Cas9 editing provide many tools for the development and re-engineering of synthetic chromosomes.},
}

*Chromosomes, Plant/genetics
*Chromosomes, Artificial/genetics
*Centromere/genetics
*CRISPR-Cas Systems
Gene Editing/methods
Genetic Engineering/methods
Plants/genetics
Telomere/genetics
Plants, Genetically Modified

@article {pmid39521313,
year = {2025},
author = {Levis, H and Lewis, C and Fainor, M and Lawal, A and Stockham, E and Weston, J and Farhang, N and Gullbrand, SE and Bowles, RD},
title = {Targeted CRISPR regulation of ZNF865 enhances stem cell cartilage deposition, tissue maturation rates, and mechanical properties in engineered intervertebral discs.},
journal = {Acta biomaterialia},
volume = {191},
number = {},
pages = {276-291},
doi = {10.1016/j.actbio.2024.11.007},
pmid = {39521313},
issn = {1878-7568},
support = {R01 AR074998/AR/NIAMS NIH HHS/United States ; R01 AR083990/AR/NIAMS NIH HHS/United States ; },
mesh = {Humans ; *Intervertebral Disc/metabolism/cytology ; *Tissue Engineering/methods ; *Stem Cells/metabolism/cytology ; Cartilage/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats ; CRISPR-Cas Systems ; Biomechanical Phenomena ; },
abstract = {Cell and tissue engineering based approaches have garnered significant interest for treating intervertebral disc degeneration and associated low back pain due to the substantial limitations of currently available clinical treatments. Herein we present a clustered regularly interspaced short palindromic repeats (CRISPR)-guided gene modulation strategy to improve the therapeutic potential of cell and tissue engineering therapies for treating intervertebral disc disease. Recently, we discovered a zinc finger (ZNF) protein, ZNF865 (BLST), which is associated with no in-depth publications and has not been functionally characterized. Utilizing CRISPR-guided gene modulation, we show that ZNF865 regulates cell cycle progression and protein processing. As a result, regulating this gene acts as a primary titratable regulator of cell activity. We also demonstrate that targeted ZNF865 regulation can enhance protein production and fibrocartilage-like matrix deposition in human adipose-derived stem cells (hASCs). Furthermore, we demonstrate CRISPR-engineered hASCs ability to increase GAG and collagen II matrix deposition in human-size tissue-engineered discs by 8.5-fold and 88.6-fold, respectively, while not increasing collagen X expression compared to naive hASCs dosed with growth factors. With this increased tissue deposition, we observe significant improvements in compressive mechanical properties, generating a stiffer and more robust tissue. Overall, we present novel biology on ZNF865 and display the power of CRISPR-cell engineering to enhance strategies treating musculoskeletal disease. STATEMENT OF SIGNIFICANCE: This work reports on a novel gene, ZNF865 (also known as BLST), that when regulated with CRISPRa, improves cartilagenous tissue deposition in human sized tissue engineering constructs. Producing tissue engineering constructs at human scale has proven difficult, and this strategy presents a broadly applicable tool to enhance a cells ability to produce tissue at these scales, as we saw an ∼8-88 fold increase in tissue deposition and significantly improved biomechanics in large tissue engineered intervertebral disc compared to traditional growth factor differentiation methods. Additionally, this work begins to elucidate the biology of this novel zinc finger protein, which appears to be critical in regulating cell function and activity.},
}

Humans
*Intervertebral Disc/metabolism/cytology
*Tissue Engineering/methods
*Stem Cells/metabolism/cytology
Cartilage/metabolism
Clustered Regularly Interspaced Short Palindromic Repeats
CRISPR-Cas Systems
Biomechanical Phenomena

@article {pmid39504234,
year = {2024},
author = {Xie, Y and Xu, Y and Jia, H and Wang, K and Chen, S and Ma, T and Deng, Y and Lang, Z and Niu, Q},
title = {Tomato MADS-RIN regulates GAME5 expression to promote non-bitter glycoalkaloid biosynthesis in fruit.},
journal = {The Plant journal : for cell and molecular biology},
volume = {120},
number = {6},
pages = {2500-2514},
doi = {10.1111/tpj.17125},
pmid = {39504234},
issn = {1365-313X},
support = {2022AH020061//the Science Fund for Distinguished Young Scholars of Anhui Province/ ; 32270367; 32400275//the National Natural Science Foundation of China/ ; NK2022010301//the National Key Research and Development Program of China/ ; jxsq2020101081//the Double Thousand Plan of Jiangxi Province/ ; jxsq2020101082//the Double Thousand Plan of Jiangxi Province/ ; 2022AH010056//the Innovative research team of Anhui Education/ ; },
mesh = {*Solanum lycopersicum/genetics/metabolism ; *Fruit/genetics/metabolism ; *Plant Proteins/genetics/metabolism ; *Gene Expression Regulation, Plant ; MADS Domain Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Alkaloids/metabolism/biosynthesis ; Gene Editing ; Sapogenins ; },
abstract = {A well-known defense-associated steroidal glycoalkaloid (SGA) metabolic shift eliminates the bitterness and toxicity of ripe tomato fruits. This study was conducted to clarify the effects of MADS-RIN (RIN) and its cofactors on SGA metabolism in tomato fruits. Using a CRISPR/Cas9-based gene-editing system, we mutated RIN and two cofactor genes (FUL1 and FUL2). The observed changes to fruit color and size in the mutants reflected the overlapping and distinct effects of RIN, FUL1, and FUL2 on fruit ripening. According to a UPLC-MS/MS analysis, the RIN and cofactor mutants had decreased levels of the relatively non-toxic metabolite esculeoside A, but they accumulated toxic SGA pathway intermediates, suggesting RIN and its cofactors are directly involved in esculeoside A biosynthesis. Transcriptome and qPCR analyses detected the downregulated expression of GAME5, which encodes a key enzyme mediating esculeoside A biosynthesis. ChIP-seq and ChIP-qPCR analyses confirmed GAME5 is targeted by RIN. RIN was observed to activate GAME5 transcription by binding to two non-canonical CArG-boxes in the GAME5 promoter. Additionally, RIN promotes SGA metabolism independently of ethylene. Collectively, these findings enhance our understanding of the molecular mechanism governing tomato fruit ripening and SGA biosynthesis. Furthermore, they may be useful for improving tomato fruit quality and safety.},
}

*Solanum lycopersicum/genetics/metabolism
*Fruit/genetics/metabolism
*Plant Proteins/genetics/metabolism
*Gene Expression Regulation, Plant
MADS Domain Proteins/genetics/metabolism
CRISPR-Cas Systems
Alkaloids/metabolism/biosynthesis
Gene Editing
Sapogenins

@article {pmid39697359,
year = {2022},
author = {Hegeman, CV and de Jong, OG and Lorenowicz, MJ},
title = {A kaleidoscopic view of extracellular vesicles in lysosomal storage disorders.},
journal = {Extracellular vesicles and circulating nucleic acids},
volume = {3},
number = {4},
pages = {393-421},
pmid = {39697359},
issn = {2767-6641},
abstract = {Extracellular vesicles (EVs) are a heterogeneous population of stable lipid membrane particles that play a critical role in the regulation of numerous physiological and pathological processes. EV cargo, which includes lipids, proteins, and RNAs including miRNAs, is affected by the metabolic status of the parental cell. Concordantly, abnormalities in the autophagic-endolysosomal pathway, as seen in lysosomal storage disorders (LSDs), can affect EV release as well as EV cargo. LSDs are a group of over 70 inheritable diseases, characterized by lysosomal dysfunction and gradual accumulation of undigested molecules. LSDs are caused by single gene mutations that lead to a deficiency of a lysosomal protein or lipid. Lysosomal dysfunction sets off a cascade of alterations in the endolysosomal pathway that can affect autophagy and alter calcium homeostasis, leading to energy imbalance, oxidative stress, and apoptosis. The pathophysiology of these diseases is very heterogenous, complex, and currently incompletely understood. LSDs lead to progressive multisystemic symptoms that often include neurological deficits. In this review, a kaleidoscopic overview will be given on the roles of EVs in LSDs, from their contribution to pathology and diagnostics to their role as drug delivery vehicles. Furthermore, EV cargo and surface engineering strategies will be discussed to show the potential of EVs in future LSD treatment, both in the context of enzyme replacement therapy, as well as future gene editing strategies like CRISPR/Cas. The use of engineered EVs as drug delivery vehicles may mask therapeutic cargo from the immune system and protect it from degradation, improving circulation time and targeted delivery.},
}

@article {pmid39693337,
year = {2024},
author = {Li, LL and Xiao, Y and Wang, B and Zhuang, Y and Chen, Y and Lu, J and Lou, Y and Li, R},
title = {A frameshift mutation in JAZ10 resolves the growth versus defense dilemma in rice.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {121},
number = {52},
pages = {e2413564121},
doi = {10.1073/pnas.2413564121},
pmid = {39693337},
issn = {1091-6490},
support = {32372551//MOST | National Natural Science Foundation of China (NSFC)/ ; 32402385//MOST | National Natural Science Foundation of China (NSFC)/ ; 2021YFD1401100//MOST | National Key Research and Development Program of China (NKPs)/ ; NA//Max Planck Partner Group Program/ ; 226-2024-00159//MOE | Fundamental Research Funds for the Central Universities (Fundamental Research Fund for the Central Universities)/ ; },
mesh = {*Oryza/genetics/growth & development/parasitology/metabolism ; *Frameshift Mutation ; *Cyclopentanes/metabolism ; *Plant Proteins/genetics/metabolism ; *Oxylipins/metabolism ; CRISPR-Cas Systems ; Gene Expression Regulation, Plant ; Animals ; Plant Diseases/parasitology/genetics ; Hemiptera/genetics/growth & development/metabolism ; Gene Editing ; Signal Transduction ; Disease Resistance/genetics ; Plants, Genetically Modified/genetics ; Gibberellins/metabolism ; },
abstract = {CRISPR-Cas9 genome editing systems have revolutionized plant gene functional studies by enabling the targeted introduction of insertion-deletions (INDELs) via the nonhomologous end-joining (NHEJ) pathway. Frameshift-inducing INDELs can introduce a premature termination codon and, in other instances, can lead to the appearance of new proteins. Here, we found that mutations in the rice jasmonate (JA) signaling gene OsJAZ10 by CRISPR-Cas9-based genome editing did not affect canonical JA signaling. However, a type of mutant with an INDEL that yielded a novel frameshift protein named FJ10 (Frameshift mutation of JAZ10), exhibited enhanced rice growth and increased resistance to brown planthopper attacks. Overexpression of FJ10 in wild-type plants phenocopies OsJAZ10 frameshift mutants. Further characterization revealed that FJ10 interacts with Slender Rice 1 (OsSLR1) and F-box/Kelch 16 (OsFBK16). These interactions disrupt the function of OsSLR1 in suppressing gibberellin-mediated growth and the function of OsFBK16 in repressing lignin-mediated defense responses, respectively. Field experiments with FJ10-expressing plants demonstrate that this protein uncouples the growth-defense tradeoff, opening broad avenues to obtain cultivars with enhanced yield without compromised defenses.},
}

*Oryza/genetics/growth & development/parasitology/metabolism
*Frameshift Mutation
*Cyclopentanes/metabolism
*Plant Proteins/genetics/metabolism
*Oxylipins/metabolism
CRISPR-Cas Systems
Gene Expression Regulation, Plant
Animals
Plant Diseases/parasitology/genetics
Hemiptera/genetics/growth & development/metabolism
Gene Editing
Signal Transduction
Disease Resistance/genetics
Plants, Genetically Modified/genetics
Gibberellins/metabolism

@article {pmid39693330,
year = {2024},
author = {Perampalam, P and McDonald, JI and Dick, FA},
title = {GO-CRISPR: A highly controlled workflow to discover gene essentiality in loss-of-function screens.},
journal = {PloS one},
volume = {19},
number = {12},
pages = {e0315923},
doi = {10.1371/journal.pone.0315923},
pmid = {39693330},
issn = {1932-6203},
mesh = {Humans ; *Genes, Essential ; *CRISPR-Cas Systems ; *Workflow ; Ovarian Neoplasms/genetics/pathology ; Female ; Software ; Cell Line, Tumor ; RNA, Guide, CRISPR-Cas Systems/genetics ; Loss of Function Mutation ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {Genome-wide CRISPR screens are an effective discovery tool for genes that underlie diverse cellular mechanisms that can be scored through cell fitness. Loss-of-function screens are particularly challenging compared to gain-of-function because of the limited dynamic range of decreased sgRNA sequence detection. Here we describe Guide-Only control CRISPR (GO-CRISPR), an improved loss-of-function screening workflow, and its companion software package, Toolset for the Ranked Analysis of GO-CRISPR Screens (TRACS). We demonstrate a typical GO-CRISPR workflow in a non-proliferative 3D spheroid model of dormant high grade serous ovarian cancer and demonstrate superior performance to standard screening methods. The unique integration of the pooled sgRNA library quality and guide-only controls allows TRACS to identify novel molecular pathways that were previously unidentified in tumor dormancy and undetectable to analysis packages that lack the guide only controls. Together, GO-CRISPR and TRACS can robustly improve the discovery of essential genes in challenging biological scenarios such as growth arrested cells.},
}

Humans
*Genes, Essential
*CRISPR-Cas Systems
*Workflow
Ovarian Neoplasms/genetics/pathology
Female
Software
Cell Line, Tumor
RNA, Guide, CRISPR-Cas Systems/genetics
Loss of Function Mutation
Clustered Regularly Interspaced Short Palindromic Repeats/genetics

@article {pmid39692063,
year = {2024},
author = {Yuan, P and Usman, M and Liu, W and Adhikari, A and Zhang, C and Njiti, V and Xia, Y},
title = {Advancements in Plant Gene Editing Technology: From Construct Design to Enhanced Transformation Efficiency.},
journal = {Biotechnology journal},
volume = {19},
number = {12},
pages = {e202400457},
doi = {10.1002/biot.202400457},
pmid = {39692063},
issn = {1860-7314},
support = {OHO01511//USDA Hatch Project/ ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Plants, Genetically Modified/genetics ; Genome, Plant/genetics ; Transformation, Genetic ; Biotechnology/methods ; Plants/genetics ; },
abstract = {Plant gene editing technology has significantly advanced in recent years, thereby transforming both biotechnological research and agricultural practices. This review provides a comprehensive summary of recent advancements in this rapidly evolving field, showcasing significant discoveries from improved transformation efficiency to advanced construct design. The primary focus is on the maturation of the Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas)9 system, which has emerged as a powerful tool for precise gene editing in plants. Through a detailed exploration, we elucidate the intricacies of integrating genetic modifications into plant genomes, shedding light on transport mechanisms, transformation techniques, and optimization strategies specific to CRISPR constructs. Furthermore, we explore the initiatives aimed at extending the frontiers of gene editing to nonmodel plant species, showcasing the growing scope of this technology. Overall, this comprehensive review highlights the significant impact of recent advancements in plant gene editing, illuminating its transformative potential in driving agricultural innovation and biotechnological progress.},
}

*Gene Editing/methods
*CRISPR-Cas Systems/genetics
*Plants, Genetically Modified/genetics
Genome, Plant/genetics
Transformation, Genetic
Biotechnology/methods
Plants/genetics

@article {pmid39690340,
year = {2024},
author = {Dixit, Y and Yadav, P and Asnani, H and Sharma, AK},
title = {CRISPR/Cas9-Engineering for Increased Amylolytic Potential of Microbes for Sustainable Wastewater Treatment: A Review.},
journal = {Current microbiology},
volume = {82},
number = {1},
pages = {44},
pmid = {39690340},
issn = {1432-0991},
mesh = {*CRISPR-Cas Systems ; *Wastewater/microbiology ; *Amylases/genetics/metabolism ; Bacteria/genetics/metabolism/enzymology ; Gene Editing/methods ; Water Purification/methods ; Genetic Engineering/methods ; },
abstract = {Amylases are pivotal enzymes with extensive industrial applications, including food processing, textile manufacturing, pharmaceuticals, and biofuel production. Traditional methods for enhancing amylase production in microbial strains often lack precision and efficiency. The advent of CRISPR/Cas9 technology has revolutionized genetic engineering, offering precise and targeted modifications to microbial genomes. This review explores the potential of CRISPR/Cas9 for improving amylase production, highlighting its advantages over conventional methods. This review discusses the mechanism of CRISPR/Cas9, the identification and targeting of key genes involved in amylase synthesis and regulation, and the optimization of expression systems. Additionally, current review examines case studies demonstrating successful CRISPR/Cas9 applications in various microbial hosts. The review also delves into the integration of CRISPR/Cas9 in wastewater treatment, where genetically engineered amylolytic strains enhance the degradation of complex organic pollutants. Despite the promising prospects, challenges such as off-target effects and regulatory considerations remain. This review provides a comprehensive overview of the current advancements, challenges, and future directions in the application of CRISPR/Cas9 technology for amylase production and environmental biotechnology.},
}

*CRISPR-Cas Systems
*Wastewater/microbiology
*Amylases/genetics/metabolism
Bacteria/genetics/metabolism/enzymology
Gene Editing/methods
Water Purification/methods
Genetic Engineering/methods

@article {pmid39690326,
year = {2024},
author = {Barber, HM and Pater, AA and Gagnon, KT and Damha, MJ and O'Reilly, D},
title = {Chemical engineering of CRISPR-Cas systems for therapeutic application.},
journal = {Nature reviews. Drug discovery},
volume = {},
number = {},
pages = {},
pmid = {39690326},
issn = {1474-1784},
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR) technology has transformed molecular biology and the future of gene-targeted therapeutics. CRISPR systems comprise a CRISPR-associated (Cas) endonuclease and a guide RNA (gRNA) that can be programmed to guide sequence-specific binding, cleavage, or modification of complementary DNA or RNA. However, the application of CRISPR-based therapeutics is challenged by factors such as molecular size, prokaryotic or phage origins, and an essential gRNA cofactor requirement, which impact efficacy, delivery and safety. This Review focuses on chemical modification and engineering approaches for gRNAs to enhance or enable CRISPR-based therapeutics, emphasizing Cas9 and Cas12a as therapeutic paradigms. Issues that chemically modified gRNAs seek to address, including drug delivery, physiological stability, editing efficiency and off-target effects, as well as challenges that remain, are discussed.},
}

@article {pmid39688838,
year = {2024},
author = {Xu, X and Zhang, Y and Liu, J and Wei, S and Li, N and Yao, X and Wang, M and Su, X and Jing, G and Xu, J and Liu, Y and Lu, Y and Cheng, J and Xu, Y},
title = {Concurrent Detection of Protein and miRNA at the Single Extracellular Vesicle Level Using a Digital Dual CRISPR-Cas Assay.},
journal = {ACS nano},
volume = {},
number = {},
pages = {},
doi = {10.1021/acsnano.4c13557},
pmid = {39688838},
issn = {1936-086X},
abstract = {The simultaneous detection of proteins and microRNA (miRNA) at the single extracellular vesicle (EV) level shows great promise for precise disease profiling, owing to the heterogeneity and scarcity of tumor-derived EVs. However, a highly reliable method for multiple-target analysis of single EVs remains to be developed. In this study, a digital dual CRISPR-Cas-powered Single EV Evaluation (ddSEE) system was proposed to enable the concurrent detection of surface protein and inner miRNA of EVs at the single-molecule level. By optimizing simultaneous reaction conditions of CRISPR-Cas12a and CRISPR-Cas13a, the surface protein of EVs was detected by Cas12a using antibody-DNA conjugates to transfer the signal of the protein to DNA, while the inner miRNA was analyzed by Cas13a through EV-liposome fusion. A microfluidic chip containing 188,000 microwells was used to convert the CRISPR-Cas system into a digital assay format to enable the absolute quantification of miRNA/protein-positive EVs without bias through fluorescence imaging, which can detect as few as 214 EVs/μL. Finally, a total of 31 blood samples, 21 from breast cancer patients and 10 from healthy donors, were collected and tested, achieving a diagnostic accuracy of 92% in distinguishing patients with breast cancer from healthy donors. With its absolute quantification, ease of use, and multiplexed detection capability, the ddSEE system demonstrates its great potential for both EV research and clinical applications.},
}

@article {pmid39687096,
year = {2024},
author = {Karpenko, A and Shelenkov, A and Petrova, L and Gusarov, V and Zamyatin, M and Mikhaylova, Y and Akimkin, V},
title = {Two multidrug-resistant Proteus mirabilis clones carrying extended spectrum beta-lactamases revealed in a single hospital department by whole genome sequencing.},
journal = {Heliyon},
volume = {10},
number = {23},
pages = {e40821},
doi = {10.1016/j.heliyon.2024.e40821},
pmid = {39687096},
issn = {2405-8440},
abstract = {Proteus mirabilis bacteria is a component of normal intestinal microflora of humans and animals, but can also be found in hospital settings causing urinary tract infections and sepsis. The problem of treating such infections is complicated by multidrug-resistant isolates producing extended spectrum beta-lactamases (ESBL), and the number of ESBL-carrying P. mirabilis strains has significantly increased recently. This study presents a detailed analysis of 12 multidrug-resistant P. mirabilis isolates obtained from the wounds of different patients in one surgical department of a multidisciplinary hospital in Moscow, Russia, using the short- and long-read whole genome sequencing. The isolates under investigation divided into two clusters (clones) C1 and C2 based on their genomic profiles and carried antimicrobial resistance (AMR) genes corresponding well with phenotypic profiles, which was the first case of reporting two different P. mirabilis clones obtained simultaneously from the same specimens at one hospital, to the best of our knowledge. Some genes, including ESBL encoding ones, were specific for either C1 or C2 (aac(6')-Ib10, ant(2″)-Ia, qnrA1, bla VEB-6 and fosA3, bla CTX -M-65 , correspondingly). Additionally, the Salmonella genomic islands 1 were found that differed in composition of multiple antibiotic resistance regions between C1 and C2 groups. CRISPR-Cas system type I-E was revealed only in C2 isolates, while the same set of virulence factors was determined for both P. mirabilis clones. Diversity of all genetic factors found in case of simultaneous existence of two clones collected from the same source at one department indicates high pathogenic potential of P. mirabilis and poses a requirement of proper spreading monitoring. The data obtained will facilitate the understanding of AMR transfer and dynamics within clinical P. mirabilis isolates and contribute to epidemiological surveillance of this pathogen.},
}

@article {pmid39686519,
year = {2024},
author = {Hernandez, FJ},
title = {Nucleases: From Primitive Immune Defenders to Modern Biotechnology Tools.},
journal = {Immunology},
volume = {},
number = {},
pages = {},
doi = {10.1111/imm.13884},
pmid = {39686519},
issn = {1365-2567},
support = {2021-05641//Vetenskapsrådet/ ; //HORIZON-MSCA-2022-COFUND-101126600-SmartBRAIN3/ ; },
abstract = {The story of nucleases begins on the ancient battlefields of early Earth, where simple bacteria fought to survive against viral invaders. Nucleases are enzymes that degrade nucleic acids, with restriction endonucleases emerging as some of the earliest defenders, cutting foreign DNA to protect their bacteria hosts. However, bacteria sought more than just defence. They evolved the CRISPR-Cas system, an adaptive immune mechanism capable of remembering past invaders. The now-famous Cas9 nuclease, a key player in this system, has been harnessed for genome editing, revolutionising biotechnology. Over time, nucleases evolved from basic viral defence tools into complex regulators of immune function in higher organisms. In humans, DNases and RNases maintain immune balance by clearing cellular debris, preventing autoimmunity, and defending against pathogens. These enzymes have transformed from simple bacterial defenders to critical players in both human immunity and biotechnology. This review explores the evolutionary history of nucleases and their vital roles as protectors in the story of life's defence mechanisms.},
}

@article {pmid39684611,
year = {2024},
author = {Pavlova, SV and Shulgina, AE and Minina, JM and Zakian, SM and Dementyeva, EV},
title = {Generation of Isogenic iPSC Lines for Studying the Effect of the p.N515del (c.1543_1545delAAC) Variant on MYBPC3 Function and Hypertrophic Cardiomyopathy Pathogenesis.},
journal = {International journal of molecular sciences},
volume = {25},
number = {23},
pages = {},
pmid = {39684611},
issn = {1422-0067},
support = {22-15-00271//Russian Science Foundation/ ; },
mesh = {*Induced Pluripotent Stem Cells/metabolism/cytology ; *Cardiomyopathy, Hypertrophic/genetics/pathology/metabolism ; Humans ; *Myocytes, Cardiac/metabolism/cytology/pathology ; *Carrier Proteins/genetics/metabolism ; *Cell Differentiation/genetics ; *CRISPR-Cas Systems ; Cell Line ; Gene Editing ; },
abstract = {The clinical significance of numerous cardiovascular gene variants remains to be determined. CRISPR/Cas9 allows for the introduction and/or correction of a certain variant in induced pluripotent stem cells (iPSCs). The resulting isogenic iPSC lines can be differentiated into cardiomyocytes and used as a platform to assess the pathogenicity of the variant. In this study, isogenic iPSC lines were generated for a variant of unknown significance found previously in a patient with hypertrophic cardiomyopathy (HCM), p.N515del (c.1543_1545delAAC) in MYBPC3. The deletion was corrected with CRISPR/Cas9 in the patient-specific iPSCs. The iPSC lines with the corrected deletion in MYBPC3 maintained pluripotency and a normal karyotype and showed no off-target CRISPR/Cas9 activity. The isogenic iPSC lines, together with isogenic iPSC lines generated earlier via introducing the p.N515del (c.1543_1545delAAC) variant in MYBPC3 of iPSCs of a healthy donor, were differentiated into cardiomyocytes. The cardiomyocytes derived from both panels of the isogenic iPSCs showed an increased size in the presence of the deletion in MYBPC3, which is one of the HCM traits at the cellular level. This finding indicates the effectiveness of these iPSC lines for studying the impact of the variant on HCM development.},
}

*Induced Pluripotent Stem Cells/metabolism/cytology
*Cardiomyopathy, Hypertrophic/genetics/pathology/metabolism
Humans
*Myocytes, Cardiac/metabolism/cytology/pathology
*Carrier Proteins/genetics/metabolism
*Cell Differentiation/genetics
*CRISPR-Cas Systems
Cell Line
Gene Editing

@article {pmid39637860,
year = {2024},
author = {Ursch, LT and Müschen, JS and Ritter, J and Klermund, J and Bernard, BE and Kolb, S and Warmuth, L and Andrieux, G and Miller, G and Jiménez-Muñoz, M and Theis, FJ and Boerries, M and Busch, DH and Cathomen, T and Schumann, K},
title = {Modulation of TCR stimulation and pifithrin-α improves the genomic safety profile of CRISPR-engineered human T cells.},
journal = {Cell reports. Medicine},
volume = {5},
number = {12},
pages = {101846},
doi = {10.1016/j.xcrm.2024.101846},
pmid = {39637860},
issn = {2666-3791},
mesh = {Humans ; *T-Lymphocytes/immunology/drug effects ; *Receptors, Antigen, T-Cell/genetics/immunology/metabolism ; *CRISPR-Cas Systems/genetics ; *Benzothiazoles/pharmacology ; *Lymphocyte Activation/drug effects/immunology ; *Toluene/analogs & derivatives/pharmacology ; *Gene Editing/methods ; Cell Proliferation/drug effects ; Receptors, Chimeric Antigen/genetics/immunology ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {CRISPR-engineered chimeric antigen receptor (CAR) T cells are at the forefront of novel cancer treatments. However, several reports describe the occurrence of CRISPR-induced chromosomal aberrations. So far, measures to increase the genomic safety of T cell products focused mainly on the components of the CRISPR-Cas9 system and less on T cell-intrinsic features, such as their massive expansion after T cell receptor (TCR) stimulation. Here, we describe driving forces of indel formation in primary human T cells. Increased T cell activation and proliferation speed correlate with larger deletions. Editing of non-activated T cells reduces the risk of large deletions with the downside of reduced knockout efficiencies. Alternatively, the addition of the small-molecule pifithrin-α limits large deletions, chromosomal translocations, and aneuploidy in a p53-independent manner while maintaining the functionality of CRISPR-engineered T cells, including CAR T cells. Controlling T cell activation and pifithrin-α treatment are easily implementable strategies to improve the genomic integrity of CRISPR-engineered T cells.},
}

Humans
*T-Lymphocytes/immunology/drug effects
*Receptors, Antigen, T-Cell/genetics/immunology/metabolism
*CRISPR-Cas Systems/genetics
*Benzothiazoles/pharmacology
*Lymphocyte Activation/drug effects/immunology
*Toluene/analogs & derivatives/pharmacology
*Gene Editing/methods
Cell Proliferation/drug effects
Receptors, Chimeric Antigen/genetics/immunology
Clustered Regularly Interspaced Short Palindromic Repeats/genetics

@article {pmid39627502,
year = {2025},
author = {Ngoi, NYL and Gallo, D and Torrado, C and Nardo, M and Durocher, D and Yap, TA},
title = {Synthetic lethal strategies for the development of cancer therapeutics.},
journal = {Nature reviews. Clinical oncology},
volume = {22},
number = {1},
pages = {46-64},
pmid = {39627502},
issn = {1759-4782},
mesh = {Humans ; *Neoplasms/drug therapy/genetics ; *Synthetic Lethal Mutations ; Poly(ADP-ribose) Polymerase Inhibitors/therapeutic use ; CRISPR-Cas Systems ; Antineoplastic Agents/therapeutic use ; Drug Discovery/methods ; DNA Damage/drug effects ; },
abstract = {Synthetic lethality is a genetic phenomenon whereby the simultaneous presence of two different genetic alterations impairs cellular viability. Importantly, targeting synthetic lethal interactions offers potential therapeutic strategies for cancers with alterations in pathways that might otherwise be considered undruggable. High-throughput screening methods based on modern CRISPR-Cas9 technologies have emerged and become crucial for identifying novel synthetic lethal interactions with the potential for translation into biologically rational cancer therapeutic strategies as well as associated predictive biomarkers of response capable of guiding patient selection. Spurred by the clinical success of PARP inhibitors in patients with BRCA-mutant cancers, novel agents targeting multiple synthetic lethal interactions within DNA damage response pathways are in clinical development, and rational strategies targeting synthetic lethal interactions spanning alterations in epigenetic, metabolic and proliferative pathways have also emerged and are in late preclinical and/or early clinical testing. In this Review, we provide a comprehensive overview of established and emerging technologies for synthetic lethal drug discovery and development and discuss promising therapeutic strategies targeting such interactions.},
}

Humans
*Neoplasms/drug therapy/genetics
*Synthetic Lethal Mutations
Poly(ADP-ribose) Polymerase Inhibitors/therapeutic use
CRISPR-Cas Systems
Antineoplastic Agents/therapeutic use
Drug Discovery/methods
DNA Damage/drug effects

@article {pmid39626673,
year = {2024},
author = {Awasthi, S and Dobrolecki, LE and Sallas, C and Zhang, X and Li, Y and Khazaei, S and Ghosh, S and Jeter, CR and Liu, J and Mills, GB and Westin, SN and Lewis, MT and Peng, W and Sood, AK and Yap, TA and Yi, SS and McGrail, DJ and Sahni, N},
title = {UBA1 inhibition sensitizes cancer cells to PARP inhibitors.},
journal = {Cell reports. Medicine},
volume = {5},
number = {12},
pages = {101834},
doi = {10.1016/j.xcrm.2024.101834},
pmid = {39626673},
issn = {2666-3791},
mesh = {Humans ; *Poly(ADP-ribose) Polymerase Inhibitors/pharmacology ; *Ubiquitin-Activating Enzymes/antagonists & inhibitors/metabolism/genetics ; Animals ; Cell Line, Tumor ; Female ; Mice ; Xenograft Model Antitumor Assays ; Ovarian Neoplasms/drug therapy/pathology/genetics ; DNA Damage/drug effects ; CRISPR-Cas Systems/genetics ; Poly(ADP-ribose) Polymerases/metabolism/genetics ; },
abstract = {Therapeutic strategies targeting the DNA damage response, such as poly (ADP-ribose) polymerase (PARP) inhibitors (PARPi), have revolutionized cancer treatment in tumors deficient in homologous recombination (HR). However, overcoming innate and acquired resistance to PARPi remains a significant challenge. Here, we employ a genome-wide CRISPR knockout screen and discover that the depletion of ubiquitin-activating enzyme E1 (UBA1) enhances sensitivity to PARPi in HR-proficient ovarian cancer cells. We show that silencing or pharmacological inhibition of UBA1 sensitizes multiple cell lines and organoid models to PARPi. Mechanistic studies uncover that UBA1 inhibition not only impedes HR repair to sensitize cells to PARP inhibition but also increases PARylation, which may subsequently be targeted by PARP inhibition. In vivo experiments using patient-derived xenografts demonstrate that combining PARP and UBA1 inhibition provided significant survival benefit compared to individual therapies with no detectable signs of toxicity, establishing this combination approach as a promising strategy to extend PARPi benefit.},
}

Humans
*Poly(ADP-ribose) Polymerase Inhibitors/pharmacology
*Ubiquitin-Activating Enzymes/antagonists & inhibitors/metabolism/genetics
Animals
Cell Line, Tumor
Female
Mice
Xenograft Model Antitumor Assays
Ovarian Neoplasms/drug therapy/pathology/genetics
DNA Damage/drug effects
CRISPR-Cas Systems/genetics
Poly(ADP-ribose) Polymerases/metabolism/genetics

@article {pmid39505154,
year = {2024},
author = {Yu, X and Peng, J and Zhong, Q and Wu, A and Deng, X and Zhu, Y},
title = {Caspase-1 knockout disrupts pyroptosis and protects photoreceptor cells from photochemical damage.},
journal = {Molecular and cellular probes},
volume = {78},
number = {},
pages = {101991},
doi = {10.1016/j.mcp.2024.101991},
pmid = {39505154},
issn = {1096-1194},
mesh = {*Pyroptosis/genetics ; *Caspase 1/metabolism/genetics ; Animals ; *Gene Knockout Techniques ; CRISPR-Cas Systems/genetics ; Cell Line ; Mice ; Reactive Oxygen Species/metabolism ; Cell Proliferation ; Photoreceptor Cells/metabolism/radiation effects ; Light/adverse effects ; Cell Survival/radiation effects ; },
abstract = {AIM: Retinal photochemical damage (RPD) plays a significant role in the development of various ocular diseases, with Caspase-1 being a key contributor. This study investigates the protective effects of Caspase-1 gene-mediated pyroptosis against RPD.

METHODS: Differentially expressed genes (DEGs) associated with RPD were identified through the analysis of two expression profiles from the GEO database. Correlation analysis was used to pinpoint pyroptosis-related genes (PRGs) linked to RPD. A Caspase-1 knockout 661 W cell line was generated via CRISPR-Cas9 gene editing, and single-cell colonies were screened and purified. Validation of knockout cells was performed through RT-qPCR, gene sequencing, and Western blot analysis. Comparative assays on cell proliferation, intracellular reactive oxygen species (ROS), and cytotoxicity were conducted between wild-type and Caspase-1 knockout cells under light exposure. Further RT-qPCR and Western blot experiments examined changes in the mRNA and protein levels of key pyroptosis pathway components.

RESULTS: Significant alterations in Caspase-1 expression were observed among PRGs. Homozygous Caspase-1 knockout cell lines were confirmed through RT-qPCR, genomic PCR product sequencing, and Western blot analysis. Compared to wild-type 661 W cells, Caspase-1 knockout cells exhibited higher viability and proliferation rates after 24 h of light exposure, alongside reduced LDH release. The expression of downstream pyroptosis factors at both the mRNA and protein levels was markedly decreased in the knockout group.

CONCLUSION: CRISPR/Cas9-mediated Caspase-1 knockout enhanced the resistance of 661 W cells to photochemical damage, suggesting that Caspase-1 may serve as a potential therapeutic target for RPD-related diseases.},
}

*Pyroptosis/genetics
*Caspase 1/metabolism/genetics
Animals
*Gene Knockout Techniques
CRISPR-Cas Systems/genetics
Cell Line
Mice
Reactive Oxygen Species/metabolism
Cell Proliferation
Photoreceptor Cells/metabolism/radiation effects
Light/adverse effects
Cell Survival/radiation effects