@article {pmid39196112,
year = {2024},
author = {Padmanabhan, A and de Soysa, TY and Pelonero, A and Sapp, V and Shah, PP and Wang, Q and Li, L and Lee, CY and Sadagopan, N and Nishino, T and Ye, L and Yang, R and Karnay, A and Poleshko, A and Bolar, N and Linares-Saldana, R and Ranade, SS and Alexanian, M and Morton, SU and Jain, M and Haldar, SM and Srivastava, D and Jain, R},
title = {A genome-wide CRISPR screen identifies BRD4 as a regulator of cardiomyocyte differentiation.},
journal = {Nature cardiovascular research},
volume = {3},
number = {3},
pages = {317-331},
pmid = {39196112},
issn = {2731-0590},
support = {K08HL157700//U.S. Department of Health & Human Services | NIH | National Heart, Lung, and Blood Institute (NHLBI)/ ; P01 HL146366/HL/NHLBI NIH HHS/United States ; R01 HL057181/HL/NHLBI NIH HHS/United States ; R01 HL127240/HL/NHLBI NIH HHS/United States ; R01 HL015100//U.S. Department of Health & Human Services | NIH | National Heart, Lung, and Blood Institute (NHLBI)/ ; },
mesh = {*Myocytes, Cardiac/metabolism/cytology ; *Transcription Factors/genetics/metabolism ; Animals ; *Cell Differentiation/genetics ; *Induced Pluripotent Stem Cells/metabolism/cytology ; Humans ; *CRISPR-Cas Systems/genetics ; Cell Cycle Proteins/genetics/metabolism ; Mice ; Mouse Embryonic Stem Cells/metabolism/cytology ; Nuclear Proteins/genetics/metabolism ; Gene Expression Regulation, Developmental ; Cell Lineage/genetics ; Cells, Cultured ; Single-Cell Analysis ; Bromodomain Containing Proteins ; },
abstract = {Human induced pluripotent stem cell (hiPSC) to cardiomyocyte (CM) differentiation has reshaped approaches to studying cardiac development and disease. In this study, we employed a genome-wide CRISPR screen in a hiPSC to CM differentiation system and reveal here that BRD4, a member of the bromodomain and extraterminal (BET) family, regulates CM differentiation. Chemical inhibition of BET proteins in mouse embryonic stem cell (mESC)-derived or hiPSC-derived cardiac progenitor cells (CPCs) results in decreased CM differentiation and persistence of cells expressing progenitor markers. In vivo, BRD4 deletion in second heart field (SHF) CPCs results in embryonic or early postnatal lethality, with mutants demonstrating myocardial hypoplasia and an increase in CPCs. Single-cell transcriptomics identified a subpopulation of SHF CPCs that is sensitive to BRD4 loss and associated with attenuated CM lineage-specific gene programs. These results highlight a previously unrecognized role for BRD4 in CM fate determination during development and a heterogenous requirement for BRD4 among SHF CPCs.},
}

*Myocytes, Cardiac/metabolism/cytology
*Transcription Factors/genetics/metabolism
Animals
*Cell Differentiation/genetics
*Induced Pluripotent Stem Cells/metabolism/cytology
Humans
*CRISPR-Cas Systems/genetics
Cell Cycle Proteins/genetics/metabolism
Mice
Mouse Embryonic Stem Cells/metabolism/cytology
Nuclear Proteins/genetics/metabolism
Gene Expression Regulation, Developmental
Cell Lineage/genetics
Cells, Cultured
Single-Cell Analysis
Bromodomain Containing Proteins

@article {pmid39194596,
year = {2024},
author = {Hu, K and Yin, W and Bai, Y and Zhang, J and Yin, J and Zhu, Q and Mu, Y},
title = {CRISPR-Based Biosensors for Medical Diagnosis: Readout from Detector-Dependence Detection Toward Naked Eye Detection.},
journal = {Biosensors},
volume = {14},
number = {8},
pages = {},
doi = {10.3390/bios14080367},
pmid = {39194596},
issn = {2079-6374},
support = {No.32071481//National Natural Science Foundation of China/ ; No. LQ22F010005//Zhejiang Provincial Natural Science Foundation of China under Grant/ ; },
mesh = {*Biosensing Techniques ; *CRISPR-Cas Systems ; Humans ; Biomarkers ; },
abstract = {The detection of biomarkers (such as DNA, RNA, and protein) plays a vital role in medical diagnosis. The CRISPR-based biosensors utilize the CRISPR/Cas system for biometric recognition of targets and use biosensor strategy to read out biological signals without the employment of professional operations. Consequently, the CRISPR-based biosensors demonstrate great potential for the detection of biomarkers with high sensitivity and specificity. However, the signal readout still relies on specialized detectors, limiting its application in on-site detection for medical diagnosis. In this review, we summarize the principles and advances of the CRISPR-based biosensors with a focus on medical diagnosis. Then, we review the advantages and progress of CRISPR-based naked eye biosensors, which can realize diagnosis without additional detectors for signal readout. Finally, we discuss the challenges and further prospects for the development of CRISPR-based biosensors.},
}

*Biosensing Techniques
*CRISPR-Cas Systems
Humans
Biomarkers

@article {pmid39194140,
year = {2024},
author = {Domingues, W and Folgosi, VÂ and Sanabani, SS and Leite Junior, PD and Assone, T and Casseb, J},
title = {Novel approaches for HTLV-1 therapy: innovative applications of CRISPR-Cas9.},
journal = {Revista do Instituto de Medicina Tropical de Sao Paulo},
volume = {66},
number = {},
pages = {e48},
doi = {10.1590/S1678-9946202466048},
pmid = {39194140},
issn = {1678-9946},
mesh = {Humans ; *CRISPR-Cas Systems ; *Human T-lymphotropic virus 1/genetics ; *Gene Editing/methods ; *HTLV-I Infections/therapy/genetics/virology ; Genetic Therapy ; Paraparesis, Tropical Spastic/therapy/genetics/virology ; },
abstract = {The human T-cell lymphotropic virus type 1 (HTLV-1) is a single-stranded positive-sense RNA virus that belongs to the Retroviridae family, genus Deltaretro, and infects approximately five to 10 million people worldwide. Although a significant number of individuals living with HTLV-1 remain asymptomatic throughout their lives, some develop one or more severe clinical conditions, such as HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), a progressive and debilitating disease, and/or a subtype of non-Hodgkin's lymphoma with a more threatening course known as adult T-cell leukemia/lymphoma (ATLL). Moreover, current therapeutic options are limited and focus primarily on treating symptoms and controlling viral latency. CRISPR-Cas9 gene editing is proposed as a promising tool to address the intricate links associated with HTLV-1. By targeting or silencing key genes during initial infection and dysregulating immune signaling pathways, CRISPR-Cas9 offers potential intervention opportunities. In this review, we address the therapeutic potential of CRISPR-Cas9 gene editing, as well as examine the primary mechanisms involved in editing potential target genes and discuss the existing evidence in the current scientific literature.},
}

Humans
*CRISPR-Cas Systems
*Human T-lymphotropic virus 1/genetics
*Gene Editing/methods
*HTLV-I Infections/therapy/genetics/virology
Genetic Therapy
Paraparesis, Tropical Spastic/therapy/genetics/virology

@article {pmid39193213,
year = {2024},
author = {Kanapiya, A and Amanbayeva, U and Tulegenova, Z and Abash, A and Zhangazin, S and Dyussembayev, K and Mukiyanova, G},
title = {Recent advances and challenges in plant viral diagnostics.},
journal = {Frontiers in plant science},
volume = {15},
number = {},
pages = {1451790},
doi = {10.3389/fpls.2024.1451790},
pmid = {39193213},
issn = {1664-462X},
abstract = {Accurate and timely diagnosis of plant viral infections plays a key role in effective disease control and maintaining agricultural productivity. Recent advances in the diagnosis of plant viruses have significantly expanded our ability to detect and monitor viral pathogens in agricultural crops. This review discusses the latest advances in diagnostic technologies, including both traditional methods and the latest innovations. Conventional methods such as enzyme-linked immunosorbent assay and DNA amplification-based assays remain widely used due to their reliability and accuracy. However, diagnostics such as next-generation sequencing and CRISPR-based detection offer faster, more sensitive and specific virus detection. The review highlights the main advantages and limitations of detection systems used in plant viral diagnostics including conventional methods, biosensor technologies and advanced sequence-based techniques. In addition, it also discusses the effectiveness of commercially available diagnostic tools and challenges facing modern diagnostic techniques as well as future directions for improving informed disease management strategies. Understanding the main features of available diagnostic methodologies would enable stakeholders to choose optimal management strategies against viral threats and ensure global food security.},
}

@article {pmid39192133,
year = {2025},
author = {Wu, XM and Zheng, SY and Chang, MX},
title = {Zebrafish as a Model for Investigating Antiviral Innate Immunity.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2854},
number = {},
pages = {221-236},
pmid = {39192133},
issn = {1940-6029},
mesh = {Animals ; *Zebrafish/immunology/genetics/virology ; *Immunity, Innate/genetics ; *CRISPR-Cas Systems ; *Disease Models, Animal ; Virus Diseases/immunology/genetics ; Gene Knockout Techniques ; Animals, Genetically Modified ; },
abstract = {Zebrafish is a widely used model organism in genetics, developmental biology, pathology, and immunology research. Due to their fast reproduction, large numbers, transparent early embryos, and high genetic conservation with the human genome, zebrafish have been used as a model for studying human and fish viral diseases. In particular, the ability to easily perform forward and reverse genetics and lacking a functional adaptive immune response during the early period of development establish the zebrafish as a favored option to assess the functional implication of specific genes in the antiviral innate immune response and the pathogenesis of viral diseases. In this chapter, we detail protocols for the antiviral innate immunity analysis using the zebrafish model, including the generation of gene-overexpression zebrafish, generation of gene-knockout zebrafish by clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology, methods of viral infection in zebrafish larvae, analyzing the expression of antiviral genes in zebrafish larvae using qRT-PCR, Western blotting and transcriptome sequencing, and in vivo antiviral assays. These experimental protocols provide effective references for studying the antiviral immune response in the zebrafish model.},
}

Animals
*Zebrafish/immunology/genetics/virology
*Immunity, Innate/genetics
*CRISPR-Cas Systems
*Disease Models, Animal
Virus Diseases/immunology/genetics
Gene Knockout Techniques
Animals, Genetically Modified

@article {pmid39192119,
year = {2025},
author = {Shen, Y and Zhao, X and Zheng, C and Chen, Q},
title = {CRISPR-Mediated Construction of Gene-Knockout Mice for Investigating Antiviral Innate Immunity.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2854},
number = {},
pages = {61-74},
pmid = {39192119},
issn = {1940-6029},
mesh = {Animals ; *Immunity, Innate/genetics ; *CRISPR-Cas Systems ; Mice ; *Mice, Knockout ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Gene Knockout Techniques/methods ; Nucleotidyltransferases/genetics/metabolism ; Virus Diseases/immunology/genetics ; },
abstract = {With the rapid development of CRISPR-Cas9 technology, gene editing has become a powerful tool for studying gene function. Specifically, in the study of the mechanisms by which natural immune responses combat viral infections, gene knockout mouse models have provided an indispensable platform. This article describes a detailed protocol for constructing gene knockout mice using the CRISPR-Cas9 system. This field focuses on the design of single-guide RNAs (sgRNAs) targeting the antiviral immune gene cGAS, embryo microinjection, and screening and verification of gene editing outcomes. Furthermore, this study provides methods for using cGAS gene knockout mice to analyze the role of specific genes in natural immune responses. Through this protocol, researchers can efficiently generate specific gene knockout mouse models, which not only helps in understanding the functions of the immune system but also offers a powerful experimental tool for exploring the mechanisms of antiviral innate immunity.},
}

Animals
*Immunity, Innate/genetics
*CRISPR-Cas Systems
Mice
*Mice, Knockout
*RNA, Guide, CRISPR-Cas Systems/genetics
*Gene Editing/methods
Gene Knockout Techniques/methods
Nucleotidyltransferases/genetics/metabolism
Virus Diseases/immunology/genetics

@article {pmid39192118,
year = {2025},
author = {Shen, Y and Feng, Z and Zheng, C and Chen, Q},
title = {CRISPR-Mediated Library Screening of Gene-Knockout Cell Lines for Investigating Antiviral Innate Immunity.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2854},
number = {},
pages = {51-60},
pmid = {39192118},
issn = {1940-6029},
mesh = {*Immunity, Innate/genetics ; *CRISPR-Cas Systems/genetics ; Humans ; *Gene Knockout Techniques ; *Gene Library ; High-Throughput Nucleotide Sequencing/methods ; Host-Pathogen Interactions/immunology/genetics ; Cell Line ; Lentivirus/genetics ; },
abstract = {The application of CRISPR-mediated library screening has fundamentally transformed functional genomics by revealing the complexity of virus-host interactions. This protocol describes the use of CRISPR-mediated library screening to identify key functional genes regulating the innate immune response to PEDV infection. We detail a step-by-step process, starting from the design and construction of a customized CRISPR knockout library targeting genes involved in innate immunity to the effective delivery of these constructs into cells using lentiviral vectors. Subsequently, we outline the process of identifying functional genes postviral attack, including the use of next-generation sequencing (NGS), to analyze and identify knockout cells that exhibit altered responses to infection. This integrated approach provides researchers in immunology and virology with a resource and a robust framework for uncovering the genetic basis of host-pathogen interactions and the arsenal of the innate immune system against viral invasions.},
}

*Immunity, Innate/genetics
*CRISPR-Cas Systems/genetics
Humans
*Gene Knockout Techniques
*Gene Library
High-Throughput Nucleotide Sequencing/methods
Host-Pathogen Interactions/immunology/genetics
Cell Line
Lentivirus/genetics

@article {pmid39056144,
year = {2024},
author = {Stockhammer, A and Spalt, C and Klemt, A and Benz, LS and Harel, S and Natalia, V and Wiench, L and Freund, C and Kuropka, B and Bottanelli, F},
title = {When less is more - a fast TurboID knock-in approach for high-sensitivity endogenous interactome mapping.},
journal = {Journal of cell science},
volume = {137},
number = {16},
pages = {},
doi = {10.1242/jcs.261952},
pmid = {39056144},
issn = {1477-9137},
support = {278001972//Deutsche Forschungsgemeinschaft/ ; //Freie Universitat Berlin/ ; },
mesh = {Humans ; *CRISPR-Cas Systems ; Gene Knock-In Techniques ; Protein Interaction Mapping/methods ; HEK293 Cells ; },
abstract = {In recent years, proximity labeling has established itself as an unbiased and powerful approach to map the interactome of specific proteins. Although physiological expression of labeling enzymes is beneficial for the mapping of interactors, generation of the desired cell lines remains time-consuming and challenging. Using our established pipeline for rapid generation of C- and N-terminal CRISPR-Cas9 knock-ins (KIs) based on antibiotic selection, we were able to compare the performance of commonly used labeling enzymes when endogenously expressed. Endogenous tagging of the µ subunit of the adaptor protein (AP)-1 complex with TurboID allowed identification of known interactors and cargo proteins that simple overexpression of a labeling enzyme fusion protein could not reveal. We used the KI strategy to compare the interactome of the different AP complexes and clathrin and were able to assemble lists of potential interactors and cargo proteins that are specific for each sorting pathway. Our approach greatly simplifies the execution of proximity labeling experiments for proteins in their native cellular environment and allows going from CRISPR transfection to mass spectrometry analysis and interactome data in just over a month.},
}

Humans
*CRISPR-Cas Systems
Gene Knock-In Techniques
Protein Interaction Mapping/methods
HEK293 Cells

@article {pmid38973166,
year = {2024},
author = {Li, M and Guan, M and Lin, J and Zhu, K and Zhu, J and Guo, M and Li, Y and Chen, Y and Chen, Y and Zou, Y and Wu, D and Xu, J and Yi, W and Fan, Y and Ma, S and Chen, Y and Xu, J and Yang, L and Dai, J and Ye, T and Lu, Z and Chen, Y},
title = {Early blood immune molecular alterations in cynomolgus monkeys with a PSEN1 mutation causing familial Alzheimer's disease.},
journal = {Alzheimer's & dementia : the journal of the Alzheimer's Association},
volume = {20},
number = {8},
pages = {5492-5510},
doi = {10.1002/alz.14046},
pmid = {38973166},
issn = {1552-5279},
support = {2021YFE0203000//National Key R&D Program of China/ ; 2018YFA0801404//National Key R&D Program of China/ ; 2021YFC2500103//National Key R&D Program of China/ ; 32061160472//National Natural Science Foundation of China (NSFC)/RGC Joint Research Scheme/ ; 32100778//National Natural Science Foundation of China/ ; 81961128019//National Natural Science Foundation of China/ ; 82071187//National Natural Science Foundation of China/ ; 2019B1515130004//Guangdong Provincial Fund for Basic and Applied Basic Research/ ; 2022A1515011639//Guangdong Provincial Fund for Basic and Applied Basic Research/ ; 2018B030336001//Guangdong Provincial Key S&T Program/ ; F-2021-Z99-504979//Development and Reform Commission of Shenzhen/ ; JCYJ20220818100800001//Technology Innovation Commission of Shenzhen/ ; JCYJ20210324102006018//Technology Innovation Commission of Shenzhen/ ; JCYJ20220531100217038//Technology Innovation Commission of Shenzhen/ ; ZDSYS20200828154800001//Technology Innovation Commission of Shenzhen/ ; KQTD20221101093608028//Technology Innovation Commission of Shenzhen/ ; INNOHK18SC01//Innovation and Technology Commission of HKSAR/ ; },
mesh = {Animals ; *Macaca fascicularis ; *Alzheimer Disease/genetics/blood ; *Presenilin-1/genetics ; *Disease Models, Animal ; *Mutation/genetics ; Amyloid beta-Peptides/cerebrospinal fluid ; tau Proteins/genetics ; CRISPR-Cas Systems ; Exons/genetics ; Male ; Transcriptome ; Biomarkers/blood/cerebrospinal fluid ; },
abstract = {INTRODUCTION: More robust non-human primate models of Alzheimer's disease (AD) will provide new opportunities to better understand the pathogenesis and progression of AD.

METHODS: We designed a CRISPR/Cas9 system to achieve precise genomic deletion of exon 9 in cynomolgus monkeys using two guide RNAs targeting the 3' and 5' intron sequences of PSEN1 exon 9. We performed biochemical, transcriptome, proteome, and biomarker analyses to characterize the cellular and molecular dysregulations of this non-human primate model.

RESULTS: We observed early changes of AD-related pathological proteins (cerebrospinal fluid Aβ42 and phosphorylated tau) in PSEN1 mutant (ie, PSEN1-ΔE9) monkeys. Blood transcriptome and proteome profiling revealed early changes in inflammatory and immune molecules in juvenile PSEN1-ΔE9 cynomolgus monkeys.

DISCUSSION: PSEN1 mutant cynomolgus monkeys recapitulate AD-related pathological protein changes, and reveal early alterations in blood immune signaling. Thus, this model might mimic AD-associated pathogenesis and has potential utility for developing early diagnostic and therapeutic interventions.

HIGHLIGHTS: A dual-guide CRISPR/Cas9 system successfully mimics AD PSEN1-ΔE9 mutation by genomic excision of exon 9. PSEN1 mutant cynomolgus monkey-derived fibroblasts exhibit disrupted PSEN1 endoproteolysis and increased Aβ secretion. Blood transcriptome and proteome profiling implicate early inflammatory and immune molecular dysregulation in juvenile PSEN1 mutant cynomolgus monkeys. Cerebrospinal fluid from juvenile PSEN1 mutant monkeys recapitulates early changes of AD-related pathological proteins (increased Aβ42 and phosphorylated tau).},
}

Animals
*Macaca fascicularis
*Alzheimer Disease/genetics/blood
*Presenilin-1/genetics
*Disease Models, Animal
*Mutation/genetics
Amyloid beta-Peptides/cerebrospinal fluid
tau Proteins/genetics
CRISPR-Cas Systems
Exons/genetics
Male
Transcriptome
Biomarkers/blood/cerebrospinal fluid

@article {pmid39191394,
year = {2024},
author = {Gupta, V and Chitranshi, N and Gupta, VB},
title = {Genetic Risk, Inflammation, and Therapeutics: An Editorial Overview of Recent Advances in Aging Brains and Neurodegeneration.},
journal = {Aging and disease},
volume = {15},
number = {5},
pages = {1989-1993},
doi = {10.14336/AD.2024.0986},
pmid = {39191394},
issn = {2152-5250},
mesh = {Humans ; *Neurodegenerative Diseases/therapy/genetics/metabolism/pathology ; *Aging/pathology ; *Brain/pathology/metabolism ; Inflammation/genetics/therapy ; Oxidative Stress ; Neuroinflammatory Diseases/metabolism/genetics ; },
abstract = {Neurodegenerative disorders, including Dementia, Parkinson's disease, various Vision disorders, Multiple sclerosis, and transsynaptic degenerative changes represent a significant challenge in aging populations. This editorial synthesizes and discusses recent advancements in understanding the genetic and environmental factors contributing to these diseases. Central to these advancements is the role of neuroinflammation and oxidative stress, which exacerbate neuronal damage and accelerate disease progression. Emerging research underscores the significance of mitochondrial dysfunction and protein aggregation in neurodegenerative pathology, highlighting shared mechanisms across various disorders. Innovative therapeutic strategies, including gene therapy, CRISPR-Cas technology, and the use of naturally occurring antioxidant molecules, are being investigated to target and manage these conditions. Additionally, lifestyle interventions such as exercise and healthy diet have shown promise in enhancing brain plasticity and reducing neuroinflammation. Advances in neuroimaging and biomarker discovery are necessary to improve early diagnosis, while clinical and preclinical studies are essential for the translation of these novel treatments. This edition aims to bridge the gap between molecular mechanisms and therapeutic applications, offering insights into potential interventions to mitigate the impact of neurodegenerative diseases. By establishing a deeper understanding of these complex processes, we aim to move closer to effective prevention and treatment strategies, ultimately improving the quality of life for those affected by neurodegenerative disorders.},
}

Humans
*Neurodegenerative Diseases/therapy/genetics/metabolism/pathology
*Aging/pathology
*Brain/pathology/metabolism
Inflammation/genetics/therapy
Oxidative Stress
Neuroinflammatory Diseases/metabolism/genetics

@article {pmid39191105,
year = {2024},
author = {Song, SF and Zhang, XW and Chen, S and Shu, Y and Yu, YL and Wang, JH},
title = {CRISPR-based dual-aptamer proximity ligation coupled hybridization chain reaction for precise detection of tumor extracellular vesicles and cancer diagnosis.},
journal = {Talanta},
volume = {280},
number = {},
pages = {126780},
doi = {10.1016/j.talanta.2024.126780},
pmid = {39191105},
issn = {1873-3573},
abstract = {Tumor cell-derived extracellular vesicles (TEVs) contain numerous cellular molecules and are considered potential biomarkers for non-invasive liquid biopsy. However, due to the low abundance of TEVs secreted by tumor cells and their phenotypic heterogeneity, there is a lack of sensitive and specific methods to quantify TEVs. Here, we developed a dual-aptamer proximity ligation-coupled hybridization chain reaction (HCR) method for tracing TEVs, exploiting CRISPR to achieve highly sensitive detection. Taking advantage of the high binding affinity of aptamers, the two aptamers (AptEpCAM, AptHER2) exhibited the high selectivity for TEVs recognition. HCR generated long-repeated sequence containing multiple crRNA targetable barcodes, and the signals were further amplified by CRISPR upon recognizing the HCR sequences, thereby enhancing the sensitivity. Under optimal conditions, the developed method demonstrated a favorable linear relationship in the range of 2 × 10[3]-10[7] particles/μL, with a limit of detection (LOD) of 3.3 × 10[2] particles/μL. We directly applied our assay to clinical plasma analysis, achieving 100 % accuracy in cancer diagnosis, thus demonstrating the potential clinical applications of TEVs. Due to its simplicity and rapidity, excellent sensitivity and specificity, this method has broad applications in clinical medicine.},
}

@article {pmid39192117,
year = {2025},
author = {Feng, Z and Shen, Y and Zheng, C and Chen, Q},
title = {CRISPR-Mediated Viral Gene Knockout to Investigate Viral Evasion of Antiviral Innate Immunity.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2854},
number = {},
pages = {41-50},
pmid = {39192117},
issn = {1940-6029},
abstract = {The innate immune system relies on a variety of pathogen recognition receptors (PRRs) as the first line of defense against pathogenic invasions. Viruses have evolved multiple strategies to evade the host immune system through coevolution with hosts. The CRISPR-Cas system is an adaptive immune system in bacteria or archaea that defends against viral reinvasion by targeting nucleic acids for cleavage. Based on the characteristics of Cas proteins and their variants, the CRISPR-Cas system has been developed into a versatile gene-editing tool capable of gene knockout or knock-in operations to achieve genetic variations in organisms. It is now widely used in the study of viral immune evasion mechanisms. This chapter will introduce the use of the CRISPR-Cas9 system for editing herpes simplex virus 1 (HSV-1) genes to explore the mechanisms by which HSV-1 evades host innate immunity and the experimental procedures involved.},
}

@article {pmid39191721,
year = {2024},
author = {Zhang, L and Zhou, X and Aryal, S and Veasey, V and Zhang, P and Li, FJ and Luan, Y and Bhatia, R and Zhou, Y and Lu, R},
title = {CRISPR screen of venetoclax response-associated genes identifies transcription factor ZNF740 as a key functional regulator.},
journal = {Cell death & disease},
volume = {15},
number = {8},
pages = {627},
pmid = {39191721},
issn = {2041-4889},
support = {CA259480//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; RSG-22-036-01-DMC//American Cancer Society (American Cancer Society, Inc.)/ ; },
mesh = {*Sulfonamides/pharmacology ; Humans ; *Bridged Bicyclo Compounds, Heterocyclic/pharmacology ; *Leukemia, Myeloid, Acute/genetics/drug therapy/metabolism/pathology ; Animals ; Cell Line, Tumor ; Proto-Oncogene Proteins c-bcl-2/metabolism/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Mice ; Drug Resistance, Neoplasm/genetics ; Antineoplastic Agents/pharmacology/therapeutic use ; Transcription Factors/metabolism/genetics ; CRISPR-Cas Systems/genetics ; },
abstract = {BCL-2 inhibitors such as venetoclax offer therapeutic promise in acute myeloid leukemia (AML) and other cancers, but drug resistance poses a significant challenge. It is crucial to understand the mechanisms that regulate venetoclax response. While correlative studies have identified numerous genes linked to venetoclax sensitivity, their direct impact on the drug response remains unclear. In this study, we targeted around 1400 genes upregulated in venetoclax-sensitive primary AML samples and carried out a CRISPR knockout screen to evaluate their direct effects on venetoclax response. Our screen identified the transcription factor ZNF740 as a critical regulator, with its expression consistently predicting venetoclax sensitivity across subtypes of the FAB classification. ZNF740 depletion leads to increased resistance to ventoclax, while its overexpression enhances sensitivity to the drug. Mechanistically, our integrative transcriptomic and genomic analysis identifies NOXA as a direct target of ZNF740, which negatively regulates MCL-1 protein stability. Loss of ZNF740 downregulates NOXA and increases the steady state protein levels of MCL-1 in AML cells. Restoring NOXA expression in ZNF740-depleted cells re-sensitizes AML cells to venetoclax treatment. Furthermore, we demonstrated that dual targeting of MCL-1 and BCL-2 effectively treats ZNF740-deficient AML in vivo. Together, our work systematically elucidates the causal relationship between venetoclax response signature genes and establishes ZNF740 as a novel transcription factor regulating venetoclax sensitivity.},
}

*Sulfonamides/pharmacology
Humans
*Bridged Bicyclo Compounds, Heterocyclic/pharmacology
*Leukemia, Myeloid, Acute/genetics/drug therapy/metabolism/pathology
Animals
Cell Line, Tumor
Proto-Oncogene Proteins c-bcl-2/metabolism/genetics
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Mice
Drug Resistance, Neoplasm/genetics
Antineoplastic Agents/pharmacology/therapeutic use
Transcription Factors/metabolism/genetics
CRISPR-Cas Systems/genetics

@article {pmid39190135,
year = {2024},
author = {Li, J and Wang, D and Zhao, P and Chen, T and Ma, L and Zhou, Y},
title = {Engineering broad-spectrum resistance to rice bacterial blight by editing the OsETR susceptible haplotype using CRISPR/Cas9.},
journal = {Plant cell reports},
volume = {43},
number = {9},
pages = {222},
pmid = {39190135},
issn = {1432-203X},
support = {ZDYF2022XDNY180//the Key Research and Development Program of Hainan Province/ ; 31661143009//Natural Science Foundation for Young Scientists of Shanxi Province/ ; 31571632//Innovative Research Group Project of the National Natural Science Foundation of China/ ; },
mesh = {*Oryza/genetics/microbiology ; *CRISPR-Cas Systems ; *Disease Resistance/genetics ; *Plant Diseases/microbiology/genetics/immunology ; *Gene Editing/methods ; *Haplotypes/genetics ; Plant Proteins/genetics/metabolism ; Xanthomonas/pathogenicity/physiology ; Plants, Genetically Modified/genetics ; },
abstract = {CRISPR/Cas9-mediated knockout of the susceptible haplotype of OsETR, encoding an embryogenesis transmembrane protein, confers broad-spectrum resistance to bacterial leaf blight in a susceptible rice cultivar without yield penalty.},
}

*Oryza/genetics/microbiology
*CRISPR-Cas Systems
*Disease Resistance/genetics
*Plant Diseases/microbiology/genetics/immunology
*Gene Editing/methods
*Haplotypes/genetics
Plant Proteins/genetics/metabolism
Xanthomonas/pathogenicity/physiology
Plants, Genetically Modified/genetics

@article {pmid39189452,
year = {2024},
author = {Rahimi, S and Balusamy, SR and Perumalsamy, H and Ståhlberg, A and Mijakovic, I},
title = {CRISPR-Cas target recognition for sensing viral and cancer biomarkers.},
journal = {Nucleic acids research},
volume = {},
number = {},
pages = {},
doi = {10.1093/nar/gkae736},
pmid = {39189452},
issn = {1362-4962},
support = {NNF20OC0064547//Novo Nordisk Foundation/ ; C 2021-1705//Kristina Stenborgs foundation for scientific research/ ; 2020-04733//Vinnova-Swelifes, Medtech4Healths/ ; 2023-01315//FORMAS/ ; 23-299//ÅForsk/ ; 22-2080//Swedish Cancer Society/ ; 2022-0030//Swedish Childhood Cancer Foundation/ ; 2021-01008//Swedish Research Council/ ; //Swedish state/ ; 965065//ALF/ ; 2020-04141//Sweden's Innovation Agency/ ; //Sjöberg Foundation/ ; //Chalmers library/ ; },
abstract = {Nucleic acid-based diagnostics is a promising venue for detection of pathogens causing infectious diseases and mutations related to cancer. However, this type of diagnostics still faces certain challenges, and there is a need for more robust, simple and cost-effective methods. Clustered regularly interspaced short palindromic repeats (CRISPRs), the adaptive immune systems present in the prokaryotes, has recently been developed for specific detection of nucleic acids. In this review, structural and functional differences of CRISPR-Cas proteins Cas9, Cas12 and Cas13 are outlined. Thereafter, recent reports about applications of these Cas proteins for detection of viral genomes and cancer biomarkers are discussed. Further, we highlight the challenges associated with using these technologies to replace the current diagnostic approaches and outline the points that need to be considered for designing an ideal Cas-based detection system for nucleic acids.},
}

@article {pmid39185796,
year = {2024},
author = {Yang, C and Wei, Y and Li, X and Xu, K and Huo, X and Chen, G and Zhao, H and Wang, J and Wei, T and Qing, Y and Guo, J and Zhao, H and Zhang, X and Jiao, D and Xiong, Z and Jamal, MA and Zhao, HY and Wei, HJ},
title = {Production of Four-Gene (GTKO/hCD55/hTBM/hCD39)-Edited Donor Pigs and Kidney Xenotransplantation.},
journal = {Xenotransplantation},
volume = {31},
number = {4},
pages = {e12881},
doi = {10.1111/xen.12881},
pmid = {39185796},
issn = {1399-3089},
support = {2019YFA0110700//National Key R&D Program of China/ ; 202102AA310047//Major Science and Technology Project of Yunnan Province/ ; },
mesh = {Animals ; *Transplantation, Heterologous/methods ; *Kidney Transplantation/methods ; Swine ; *Gene Editing/methods ; *Animals, Genetically Modified ; *Galactosyltransferases/genetics ; CRISPR-Cas Systems ; Macaca mulatta ; Nuclear Transfer Techniques ; Heterografts ; Humans ; Graft Survival/immunology ; Graft Rejection/immunology ; Apyrase ; Antigens, CD ; },
abstract = {BACKGROUND: The number of multigene-modified donor pigs for xenotransplantation is increasing with the advent of gene-editing technologies. However, it remains unclear which gene combination is suitable for specific organ transplantation.

METHODS: In this study, we utilized CRISPR/Cas9 gene editing technology, piggyBac transposon system, and somatic cell cloning to construct GTKO/hCD55/hTBM/hCD39 four-gene-edited cloned (GEC) pigs and performed kidney transplantation from pig to rhesus monkey to evaluate the effectiveness of these GEC pigs.

RESULTS: First, 107 cell colonies were obtained through drug selection, of which seven were 4-GE colonies. Two colonies were selected for somatic cell nuclear transfer (SCNT), resulting in seven fetuses, of which four were GGTA1 biallelic knockout. Out of these four, two fetuses had higher expression of hCD55, hTBM, and hCD39. Therefore, these two fetuses were selected for two consecutive rounds of cloning, resulting in 97 live piglets. After phenotype identification, the GGTA1 gene of these pigs was inactivated, and hCD55, hTBM, and hCD39 were expressed in cells and multiple tissues. Furthermore, the numbers of monkey IgM and IgG binding to the peripheral blood mononuclear cells (PBMCs) of the 4-GEC pigs were markedly reduced. Moreover, 4-GEC porcine PBMCs had greater survival rates than those from wild-type pigs through complement-mediated cytolysis assays. In pig-to-monkey kidney xenotransplantation, the kidney xenograft successfully survived for 11 days. All physiological and biochemical indicators were normal, and no hyperacute rejection or coagulation abnormalities were found after transplantation.

CONCLUSION: These results indicate that the GTKO/hCD55/hTBM/hCD39 four-gene modification effectively alleviates immune rejection, and the pig kidney can functionally support the recipient monkey's life.},
}

Animals
*Transplantation, Heterologous/methods
*Kidney Transplantation/methods
Swine
*Gene Editing/methods
*Animals, Genetically Modified
*Galactosyltransferases/genetics
CRISPR-Cas Systems
Macaca mulatta
Nuclear Transfer Techniques
Heterografts
Humans
Graft Survival/immunology
Graft Rejection/immunology
Apyrase
Antigens, CD

@article {pmid39184070,
year = {2024},
author = {Maxim, DS and Wu, DW and Johnson, NS and Charu, V and Carter, JN and Anand, S and Church, GM and Bhalla, V},
title = {EditABLE: A Simple Web Application for Designing Genome Editing Experiments.},
journal = {Research square},
volume = {},
number = {},
pages = {},
doi = {10.21203/rs.3.rs-4775705/v1},
pmid = {39184070},
issn = {2693-5015},
abstract = {CRISPR-Cas genome editing is transformative; however, there is no simple tool available for determining the optimal genome editing technology to create specific mutations for experimentation or to correct mutations as a curative therapy for specific diseases. We developed editABLE, an online resource (editable-app.stanford.edu) to provide computationally validated CRISPR editors and guide RNAs based on user provided sequence data. We demonstrate the utility of editABLE by applying it to one of the most common monogenic disorders, autosomal dominant polycystic kidney disease (ADPKD), identifying specific editing tools across the landscape of ADPKD mutations.},
}

@article {pmid39146367,
year = {2024},
author = {Matoušková, M and Plachý, J and Kučerová, D and Pecnová, Ľ and Reinišová, M and Geryk, J and Karafiát, V and Hron, T and Hejnar, J},
title = {Rapid adaptive evolution of avian leukosis virus subgroup J in response to biotechnologically induced host resistance.},
journal = {PLoS pathogens},
volume = {20},
number = {8},
pages = {e1012468},
doi = {10.1371/journal.ppat.1012468},
pmid = {39146367},
issn = {1553-7374},
mesh = {Animals ; *Avian Leukosis Virus/genetics/physiology ; *Chickens/virology ; *Avian Leukosis/virology/genetics ; Poultry Diseases/virology/genetics ; Disease Resistance/genetics ; CRISPR-Cas Systems ; Gene Editing ; Chick Embryo ; Evolution, Molecular ; Viral Envelope Proteins/genetics/metabolism ; Fibroblasts/virology/metabolism ; },
abstract = {Genetic editing of the germline using CRISPR/Cas9 technology has made it possible to alter livestock traits, including the creation of resistance to viral diseases. However, virus adaptability could present a major obstacle in this effort. Recently, chickens resistant to avian leukosis virus subgroup J (ALV-J) were developed by deleting a single amino acid, W38, within the ALV-J receptor NHE1 using CRISPR/Cas9 genome editing. This resistance was confirmed both in vitro and in vivo. In vitro resistance of W38-/- chicken embryonic fibroblasts to all tested ALV-J strains was shown. To investigate the capacity of ALV-J for further adaptation, we used a retrovirus reporter-based assay to select adapted ALV-J variants. We assumed that adaptive mutations overcoming the cellular resistance would occur within the envelope protein. In accordance with this assumption, we isolated and sequenced numerous adapted virus variants and found within their envelope genes eight independent single nucleotide substitutions. To confirm the adaptive capacity of these substitutions, we introduced them into the original retrovirus reporter. All eight variants replicated effectively in W38-/- chicken embryonic fibroblasts in vitro while in vivo, W38-/- chickens were sensitive to tumor induction by two of the variants. Importantly, receptor alleles with more extensive modifications have remained resistant to the virus. These results demonstrate an important strategy in livestock genome engineering towards antivirus resistance and illustrate that cellular resistance induced by minor receptor modifications can be overcome by adapted virus variants. We conclude that more complex editing will be necessary to attain robust resistance.},
}

Animals
*Avian Leukosis Virus/genetics/physiology
*Chickens/virology
*Avian Leukosis/virology/genetics
Poultry Diseases/virology/genetics
Disease Resistance/genetics
CRISPR-Cas Systems
Gene Editing
Chick Embryo
Evolution, Molecular
Viral Envelope Proteins/genetics/metabolism
Fibroblasts/virology/metabolism

@article {pmid39043964,
year = {2024},
author = {Tran, NT and Graf, R and Acevedo-Ochoa, E and Trombke, J and Weber, T and Sommermann, T and Salomon, C and Kühn, R and Rajewsky, K and Chu, VT},
title = {In vivo CRISPR/Cas9-mediated screen reveals a critical function of TFDP1 and E2F4 transcription factors in hematopoiesis.},
journal = {Leukemia},
volume = {38},
number = {9},
pages = {2003-2015},
pmid = {39043964},
issn = {1476-5551},
support = {CH 1968/1-1//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; ZT-0027//Helmholtz Association/ ; },
mesh = {*Hematopoiesis/genetics ; Animals ; *CRISPR-Cas Systems ; *Hematopoietic Stem Cells/metabolism/cytology ; Mice ; *E2F4 Transcription Factor/genetics/metabolism ; *Transcription Factor DP1/genetics/metabolism ; Cell Proliferation ; Humans ; Cell Cycle/genetics ; Cell Differentiation/genetics ; Mice, Inbred C57BL ; },
abstract = {Hematopoiesis is a continuous process of blood cell production driven by hematopoietic stem and progenitor cells (HSPCs) in the bone marrow. Proliferation and differentiation of HSPCs are regulated by complex transcriptional networks. In order to identify transcription factors with key roles in HSPC-mediated hematopoietic reconstitution, we developed an efficient and robust CRISPR/Cas9-based in vivo genetic screen. Using this experimental system, we identified the TFDP1 transcription factor to be essential for HSPC proliferation and post-transplant hematopoiesis. We further discovered that E2F4, an E2F transcription factor, serves as a binding partner of TFDP1 and is required for HSPC proliferation. Deletion of TFDP1 caused downregulation of genes associated with the cell cycle, with around 50% of these genes being identified as direct targets of TFDP1 and E2F4. Thus, our study expands the transcriptional network governing hematopoietic development through an in vivo CRISPR/Cas9-based genetic screen and identifies TFDP1/E2F4 as positive regulators of cell cycle genes in HSPCs.},
}

*Hematopoiesis/genetics
Animals
*CRISPR-Cas Systems
*Hematopoietic Stem Cells/metabolism/cytology
Mice
*E2F4 Transcription Factor/genetics/metabolism
*Transcription Factor DP1/genetics/metabolism
Cell Proliferation
Humans
Cell Cycle/genetics
Cell Differentiation/genetics
Mice, Inbred C57BL

@article {pmid39011887,
year = {2024},
author = {Yang, Y and Rocamonde-Lago, I and Shen, B and Berzina, I and Zipf, J and Högberg, B},
title = {Re-engineered guide RNA enables DNA loops and contacts modulating repression in E. coli.},
journal = {Nucleic acids research},
volume = {52},
number = {15},
pages = {9328-9339},
doi = {10.1093/nar/gkae591},
pmid = {39011887},
issn = {1362-4962},
support = {KAW 2017.0114//Knut and Alice Wallenberg Foundation/ ; //Göran Gustafsson Foundation for Research in Natural Sciences and Medicine/ ; 2019-01474//Swedish Research Council/ ; 341 908//Academy of Finland/ ; 724 872/ERC_/European Research Council/International ; },
mesh = {*Escherichia coli/genetics/metabolism ; *RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; Nucleic Acid Conformation ; CRISPR-Cas Systems ; DNA/metabolism/chemistry/genetics ; DNA, Bacterial/metabolism/genetics ; Aptamers, Nucleotide/chemistry/genetics/metabolism ; CRISPR-Associated Protein 9/metabolism/genetics ; },
abstract = {RNA serves as information media as well as molecular scaffold in nature and synthetic systems. The single guide RNA (sgRNA) widely applied in CRISPR techniques exemplifies both functions, with a guide region bearing DNA base-pairing information, and a structural motif for Cas9 protein scaffolding. The scaffold region has been modified by fusing RNA aptamers to the tetra-stem loop. The guide region is typically not regarded as a pluggable module as it encodes the essential function of DNA sequence recognition. Here, we investigate a chimera of two sgRNAs, with distinct guide sequences joined by an RNA linker (dgRNA), regarding its DNA binding function and loop induction capability. First, we studied the sequence bi-specificity of the dgRNA and discovered that the RNA linker allows distal parts of double-stranded DNA to be brought into proximity. To test the activity of the dgRNA in organisms, we used the LacZ gene as a reporter and recapitulated the loop-mediated gene inhibition by LacI in E. coli. We found that the dgRNA can be applied to target distal genomic regions with comparable levels of inhibition. The capability of dgRNA to induce DNA contacts solely requires dCas9 and RNA, making it a minimal system to remodel chromosomal conformation in various organisms.},
}

*Escherichia coli/genetics/metabolism
*RNA, Guide, CRISPR-Cas Systems/genetics/metabolism
Nucleic Acid Conformation
CRISPR-Cas Systems
DNA/metabolism/chemistry/genetics
DNA, Bacterial/metabolism/genetics
Aptamers, Nucleotide/chemistry/genetics/metabolism
CRISPR-Associated Protein 9/metabolism/genetics

@article {pmid38995679,
year = {2024},
author = {Wang, J and Liao, Z and Jin, X and Liao, L and Zhang, Y and Zhang, R and Zhao, X and Qin, H and Chen, J and He, Y and Zhuang, C and Tang, J and Huang, S},
title = {Xanthomonas oryzae pv. oryzicola effector Tal10a directly activates rice OsHXK5 expression to facilitate pathogenesis.},
journal = {The Plant journal : for cell and molecular biology},
volume = {119},
number = {5},
pages = {2423-2436},
doi = {10.1111/tpj.16929},
pmid = {38995679},
issn = {1365-313X},
support = {2020GXNSFDA297026//Guangxi Natural Science Foundation/ ; SKLCUSA-a202009//Independent project of State Key Laboratory State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources/ ; 31860032//National Natural Science Foundation of China/ ; },
mesh = {*Oryza/microbiology/genetics ; *Xanthomonas/pathogenicity/physiology/genetics ; *Plant Diseases/microbiology/genetics ; *Plant Proteins/genetics/metabolism ; *Gene Expression Regulation, Plant ; *Bacterial Proteins/genetics/metabolism ; Transcription Activator-Like Effectors/genetics/metabolism ; Virulence/genetics ; Promoter Regions, Genetic/genetics ; Disease Resistance/genetics ; CRISPR-Cas Systems ; Gene Editing ; Plants, Genetically Modified ; },
abstract = {Bacterial leaf streak (BLS), caused by Xanthomonas oryzae pv. oryzicola (Xoc), is a major bacterial disease in rice. Transcription activator-like effectors (TALEs) from Xanthomonas can induce host susceptibility (S) genes and facilitate infection. However, knowledge of the function of Xoc TALEs in promoting bacterial virulence is limited. In this study, we demonstrated the importance of Tal10a for the full virulence of Xoc. Through computational prediction and gene expression analysis, we identified the hexokinase gene OsHXK5 as a host target of Tal10a. Tal10a directly binds to the gene promoter region and activates the expression of OsHXK5. CRISPR/Cas9-mediated gene editing in the effector binding element (EBE) of OsHXK5 significantly increases rice resistance to Xoc, while OsHXK5 overexpression enhances the susceptibility of rice plants and impairs rice defense responses. Moreover, simultaneous editing of the promoters of OsSULTR3;6 and OsHXK5 confers robust resistance to Xoc in rice. Taken together, our findings highlight the role of Tal10a in targeting OsHXK5 to promote infection and suggest that OsHXK5 represents a potential target for engineering rice resistance to Xoc.},
}

*Oryza/microbiology/genetics
*Xanthomonas/pathogenicity/physiology/genetics
*Plant Diseases/microbiology/genetics
*Plant Proteins/genetics/metabolism
*Gene Expression Regulation, Plant
*Bacterial Proteins/genetics/metabolism
Transcription Activator-Like Effectors/genetics/metabolism
Virulence/genetics
Promoter Regions, Genetic/genetics
Disease Resistance/genetics
CRISPR-Cas Systems
Gene Editing
Plants, Genetically Modified

@article {pmid38953163,
year = {2024},
author = {Schep, R and Trauernicht, M and Vergara, X and Friskes, A and Morris, B and Gregoricchio, S and Manzo, SG and Zwart, W and Beijersbergen, RL and Medema, RH and van Steensel, B},
title = {Chromatin context-dependent effects of epigenetic drugs on CRISPR-Cas9 editing.},
journal = {Nucleic acids research},
volume = {52},
number = {15},
pages = {8815-8832},
doi = {10.1093/nar/gkae570},
pmid = {38953163},
issn = {1362-4962},
support = {91 215 067//ZonMW TOP/ ; //The Dutch Cancer Society/ ; //EU/MUR MSCA Young Researcher Fellowship/ ; 694466/ERC_/European Research Council/International ; //KWF Dutch Cancer Society/ ; },
mesh = {Humans ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Epigenesis, Genetic/drug effects ; *Chromatin/metabolism/genetics ; *Histone Deacetylase Inhibitors/pharmacology ; DNA Repair ; CRISPR-Associated Protein 9/metabolism/genetics ; Heterochromatin/metabolism/genetics ; Cell Line ; Histones/metabolism ; Euchromatin/genetics ; DNA Breaks, Double-Stranded/drug effects ; },
abstract = {The efficiency and outcome of CRISPR/Cas9 editing depends on the chromatin state at the cut site. It has been shown that changing the chromatin state can influence both the efficiency and repair outcome, and epigenetic drugs have been used to improve Cas9 editing. However, because the target proteins of these drugs are not homogeneously distributed across the genome, the efficacy of these drugs may be expected to vary from locus to locus. Here, we systematically analyzed this chromatin context-dependency for 160 epigenetic drugs. We used a human cell line with 19 stably integrated reporters to induce a double-stranded break in different chromatin environments. We then measured Cas9 editing efficiency and repair pathway usage by sequencing the mutational signatures. We identified 58 drugs that modulate Cas9 editing efficiency and/or repair outcome dependent on the local chromatin environment. For example, we find a subset of histone deacetylase inhibitors that improve Cas9 editing efficiency throughout all types of heterochromatin (e.g. PCI-24781), while others were only effective in euchromatin and H3K27me3-marked regions (e.g. apicidin). In summary, this study reveals that most epigenetic drugs alter CRISPR editing in a chromatin-dependent manner, and provides a resource to improve Cas9 editing more selectively at the desired location.},
}

Humans
*CRISPR-Cas Systems
*Gene Editing/methods
*Epigenesis, Genetic/drug effects
*Chromatin/metabolism/genetics
*Histone Deacetylase Inhibitors/pharmacology
DNA Repair
CRISPR-Associated Protein 9/metabolism/genetics
Heterochromatin/metabolism/genetics
Cell Line
Histones/metabolism
Euchromatin/genetics
DNA Breaks, Double-Stranded/drug effects

@article {pmid38924638,
year = {2024},
author = {Chen, M and Huang, X and Shi, Y and Wang, W and Huang, Z and Tong, Y and Zou, X and Xu, Y and Dai, Z},
title = {CRISPR/Pepper-tDeg: A Live Imaging System Enables Non-Repetitive Genomic Locus Analysis with One Single-Guide RNA.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {11},
number = {32},
pages = {e2402534},
doi = {10.1002/advs.202402534},
pmid = {38924638},
issn = {2198-3844},
support = {22274169//National Natural Science Foundation of China/ ; 2023A1515012027//Guangdong Basic and Applied Basic Research Foundation/ ; A2303001//Shenzhen Medical Research Fund/ ; JCYJ20210324124003008//Shenzhen Fundamental Research Program/ ; 2023B03J1380//Science and Technology Program of Guangzhou City/ ; 202103000003//Science and Technology Program of Guangzhou City/ ; 2020B1212060077//Guangdong Science and Technology Plan Project Grant/ ; },
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Genomics/methods ; Telomere/genetics ; },
abstract = {CRISPR-based genomic-imaging systems have been utilized for spatiotemporal imaging of the repetitive genomic loci in living cells, but they are still challenged by limited signal-to-noise ratio (SNR) at a non-repetitive genomic locus. Here, an efficient genomic-imaging system is proposed, termed CRISPR/Pepper-tDeg, by engineering the CRISPR sgRNA scaffolds with the degron-binding Pepper aptamers for binding fluorogenic proteins fused with Tat peptide derived degron domain (tDeg). The target-dependent stability switches of both sgRNA and fluorogenic protein allow this system to image repetitive telomeres sensitively with a 5-fold higher SNR than conventional CRISPR/MS2-MCP system using "always-on" fluorescent protein tag. Subsequently, CRISPR/Pepper-tDeg is applied to simultaneously label and track two different genomic loci, telomeres and centromeres, in living cells by combining two systems. Given a further improved SNR by the split fluorescent protein design, CRISPR/Pepper-tDeg system is extended to non-repetitive sequence imaging using only one sgRNA with two aptamer insertions. Neither complex sgRNA design nor difficult plasmid construction is required, greatly reducing the technical barriers to define spatiotemporal organization and dynamics of both repetitive and non-repetitive genomic loci in living cells, and thus demonstrating the large application potential of this genomic-imaging system in biological research, clinical diagnosis and therapy.},
}

Humans
*CRISPR-Cas Systems/genetics
*RNA, Guide, CRISPR-Cas Systems/genetics
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Genomics/methods
Telomere/genetics

@article {pmid38923275,
year = {2024},
author = {Zhang, H and Jiang, W and Song, T and Song, M and Liu, S and Zhou, J and Cheng, H and Ding, Y},
title = {Lipid-Polymer Nanoparticles Mediate Compartmentalized Delivery of Cas9 and sgRNA for Glioblastoma Vasculature and Immune Reprogramming.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {11},
number = {32},
pages = {e2309314},
doi = {10.1002/advs.202309314},
pmid = {38923275},
issn = {2198-3844},
support = {82073401//National Natural Science Foundation of China/ ; 82104097//National Natural Science Foundation of China/ ; 82372113//National Natural Science Foundation of China/ ; BK20231018//Natural Science Foundation of Jiangsu Province/ ; 2632023TD05//Fundamental Research Funds for the Central Universities/ ; 2632023GR18//Fundamental Research Funds for the Central Universities/ ; //National Ten Thousand Talents Program for Young Top-notch Talents/ ; TJ-2021-002//Young Talent Support Project of Jiangsu Association for Science and Technology/ ; [2022]3-16-190//Jiangsu 333 High-level Talent Training Project/ ; CPU2022QZ18//Double First-Rate Construction Plan of China Pharmaceutical University/ ; QJHKY[2022]387//Project of the State Key Laboratory of Functions and Applications of Medicinal Plants, Guizhou Medical University/ ; },
mesh = {*Glioblastoma/metabolism/genetics/drug therapy ; *Nanoparticles/chemistry ; Mice ; Animals ; *Polymers/chemistry ; Humans ; Lipids/chemistry ; RNA, Guide, CRISPR-Cas Systems/genetics ; Disease Models, Animal ; Brain Neoplasms/genetics/drug therapy/metabolism ; Cell Line, Tumor ; Blood-Brain Barrier/metabolism ; STAT3 Transcription Factor/metabolism/genetics ; CRISPR-Associated Protein 9/genetics/metabolism ; CRISPR-Cas Systems ; },
abstract = {Hypervascularized glioblastoma is naturally sensitive to anti-angiogenesis but suffers from low efficacy of transient vasculature normalization. In this study, a lipid-polymer nanoparticle is synthesized to execute compartmentalized Cas9 and sgRNA delivery for a permanent vasculature editing strategy by knocking out the signal transducer and activator of transcription 3 (STAT3). The phenylboronic acid branched cationic polymer is designed to condense sgRNA electrostatically (inner compartment) and patch Cas9 coordinatively (outer compartment), followed by liposomal hybridization with angiopep-2 decoration for blood-brain barrier (BBB) penetration. The lipid-polymer nanoparticles can reach glioblastoma within 2 h post intravenous administration, and hypoxia in tumor cells triggers charge-elimination and degradation of the cationic polymer for burst release of Cas9 and sgRNA, accompanied by instant Cas9 RNP assembly, yielding ≈50% STAT3 knockout. The downregulation of downstream vascular endothelial growth factor (VEGF) reprograms vasculature normalization to improve immune infiltration, collaborating with interleukin-6 (IL-6) and interleukin-10 (IL-10) reduction to develop anti-glioblastoma responses. Collectively, the combinational assembly for compartmentalized Cas9/sgRNA delivery provides a potential solution in glioblastoma therapy.},
}

*Glioblastoma/metabolism/genetics/drug therapy
*Nanoparticles/chemistry
Mice
Animals
*Polymers/chemistry
Humans
Lipids/chemistry
RNA, Guide, CRISPR-Cas Systems/genetics
Disease Models, Animal
Brain Neoplasms/genetics/drug therapy/metabolism
Cell Line, Tumor
Blood-Brain Barrier/metabolism
STAT3 Transcription Factor/metabolism/genetics
CRISPR-Associated Protein 9/genetics/metabolism
CRISPR-Cas Systems

@article {pmid38908026,
year = {2024},
author = {Wu, P and Yao, M and Wang, W},
title = {Differential impact of quiescent non-coding loci on chromatin entropy.},
journal = {Nucleic acids research},
volume = {52},
number = {15},
pages = {8778-8799},
doi = {10.1093/nar/gkae535},
pmid = {38908026},
issn = {1362-4962},
support = {RB5-07012//California Institute for Regenerative Medicine/ ; R01HG009626/NH/NIH HHS/United States ; R01HG009626/NH/NIH HHS/United States ; },
mesh = {Humans ; *Chromatin/metabolism/genetics ; *Entropy ; *Genome, Human ; Machine Learning ; Epigenesis, Genetic ; Gene Regulatory Networks ; CRISPR-Cas Systems ; Genetic Loci ; },
abstract = {Non-coding regions of the human genome are important for functional regulations, but their mechanisms remain elusive. We used machine learning to guide a CRISPR screening on hubs (i.e. non-coding loci forming many 3D contacts) and significantly increased the discovery rate of hubs essential for cell growth. We found no clear genetic or epigenetic differences between essential and nonessential hubs, but we observed that some neighboring hubs in the linear genome have distinct spatial contacts and opposite effects on cell growth. One such pair in an epigenetically quiescent region showed different impacts on gene expression, chromatin accessibility and chromatin organization. We also found that deleting the essential hub altered the genetic network activity and increased the entropy of chromatin accessibility, more severe than that caused by deletion of the nonessential hub, suggesting that they are critical for maintaining an ordered chromatin structure. Our study reveals new insights into the system-level roles of non-coding regions in the human genome.},
}

Humans
*Chromatin/metabolism/genetics
*Entropy
*Genome, Human
Machine Learning
Epigenesis, Genetic
Gene Regulatory Networks
CRISPR-Cas Systems
Genetic Loci

@article {pmid38692586,
year = {2024},
author = {Noguchi, Y and Matsui, R and Suh, J and Dou, Y and Suzuki, J},
title = {Genome-Wide Screening Approaches for Biochemical Reactions Independent of Cell Growth.},
journal = {Annual review of genomics and human genetics},
volume = {25},
number = {1},
pages = {51-76},
doi = {10.1146/annurev-genom-121222-115958},
pmid = {38692586},
issn = {1545-293X},
mesh = {Humans ; *CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; Flow Cytometry/methods ; Animals ; Genome-Wide Association Study ; Gene Editing/methods ; Gene Library ; },
abstract = {Genome-wide screening is a potent approach for comprehensively understanding the molecular mechanisms of biological phenomena. However, despite its widespread use in the past decades across various biological targets, its application to biochemical reactions with temporal and reversible biological outputs remains a formidable challenge. To uncover the molecular machinery underlying various biochemical reactions, we have recently developed the revival screening method, which combines flow cytometry-based cell sorting with library reconstruction from collected cells. Our refinements to the traditional genome-wide screening technique have proven successful in revealing the molecular machinery of biochemical reactions of interest. In this article, we elucidate the technical basis of revival screening, focusing on its application to CRISPR-Cas9 single guide RNA (sgRNA) library screening. Finally, we also discuss the future of genome-wide screening while describing recent achievements from in vitro and in vivo screening.},
}

Humans
*CRISPR-Cas Systems
RNA, Guide, CRISPR-Cas Systems/genetics/metabolism
Flow Cytometry/methods
Animals
Genome-Wide Association Study
Gene Editing/methods
Gene Library

@article {pmid39181868,
year = {2024},
author = {Mittal, P and Myers, JA and Carter, RD and Radko-Juettner, S and Malone, HA and Rosikiewicz, W and Robertson, AN and Zhu, Z and Narayanan, IV and Hansen, BS and Parrish, M and Bhanu, NV and Mobley, RJ and Rehg, JE and Xu, B and Drosos, Y and Pruett-Miller, SM and Ljungman, M and Garcia, BA and Wu, G and Partridge, JF and Roberts, CWM},
title = {PHF6 cooperates with SWI/SNF complexes to facilitate transcriptional progression.},
journal = {Nature communications},
volume = {15},
number = {1},
pages = {7303},
pmid = {39181868},
issn = {2041-1723},
support = {R01 CA113794/CA/NCI NIH HHS/United States ; R01 CA172152/CA/NCI NIH HHS/United States ; R01 CA273455/CA/NCI NIH HHS/United States ; },
mesh = {Humans ; *SMARCB1 Protein/metabolism/genetics ; *Rhabdoid Tumor/genetics/metabolism/pathology ; *Micrognathism/genetics/metabolism ; Cell Line, Tumor ; *Transcription Factors/metabolism/genetics ; *Repressor Proteins/metabolism/genetics ; *Promoter Regions, Genetic/genetics ; Face/abnormalities ; Chromatin/metabolism ; Intellectual Disability/genetics/metabolism ; Chromatin Assembly and Disassembly ; Mutation ; Foot Deformities, Congenital/genetics/metabolism ; Chromosomal Proteins, Non-Histone/metabolism/genetics ; CRISPR-Cas Systems ; Transcription, Genetic ; Animals ; Mice ; Abnormalities, Multiple ; Hand Deformities, Congenital ; Neck/abnormalities ; },
abstract = {Genes encoding subunits of SWI/SNF (BAF) chromatin remodeling complexes are mutated in nearly 25% of cancers. To gain insight into the mechanisms by which SWI/SNF mutations drive cancer, we contributed ten rhabdoid tumor (RT) cell lines mutant for SWI/SNF subunit SMARCB1 to a genome-scale CRISPR-Cas9 depletion screen performed across 896 cell lines. We identify PHF6 as specifically essential for RT cell survival and demonstrate that dependency on Phf6 extends to Smarcb1-deficient cancers in vivo. As mutations in either SWI/SNF or PHF6 can cause the neurodevelopmental disorder Coffin-Siris syndrome, our findings of a dependency suggest a previously unrecognized functional link. We demonstrate that PHF6 co-localizes with SWI/SNF complexes at promoters, where it is essential for maintenance of an active chromatin state. We show that in the absence of SMARCB1, PHF6 loss disrupts the recruitment and stability of residual SWI/SNF complex members, collectively resulting in the loss of active chromatin at promoters and stalling of RNA Polymerase II progression. Our work establishes a mechanistic basis for the shared syndromic features of SWI/SNF and PHF6 mutations in CSS and the basis for selective dependency on PHF6 in SMARCB1-mutant cancers.},
}

Humans
*SMARCB1 Protein/metabolism/genetics
*Rhabdoid Tumor/genetics/metabolism/pathology
*Micrognathism/genetics/metabolism
Cell Line, Tumor
*Transcription Factors/metabolism/genetics
*Repressor Proteins/metabolism/genetics
*Promoter Regions, Genetic/genetics
Face/abnormalities
Chromatin/metabolism
Intellectual Disability/genetics/metabolism
Chromatin Assembly and Disassembly
Mutation
Foot Deformities, Congenital/genetics/metabolism
Chromosomal Proteins, Non-Histone/metabolism/genetics
CRISPR-Cas Systems
Transcription, Genetic
Animals
Mice
Abnormalities, Multiple
Hand Deformities, Congenital
Neck/abnormalities

@article {pmid39180405,
year = {2024},
author = {Kim, DY and Lee, SY and Ha, HJ and Park, HH},
title = {Structural basis of Cas3 activation in type I-C CRISPR-Cas system.},
journal = {Nucleic acids research},
volume = {},
number = {},
pages = {},
doi = {10.1093/nar/gkae723},
pmid = {39180405},
issn = {1362-4962},
support = {NRF-2021R1A2C3003331//National Research Foundation of Korea/ ; },
abstract = {CRISPR-Cas systems function as adaptive immune mechanisms in bacteria and archaea and offer protection against phages and other mobile genetic elements. Among many types of CRISPR-Cas systems, Type I CRISPR-Cas systems are most abundant, with target interference depending on a multi-subunit, RNA-guided complex known as Cascade that recruits a transacting helicase nuclease, Cas3, to degrade the target. While structural studies on several other types of Cas3 have been conducted long ago, it was only recently that the structural study of Type I-C Cas3 in complex with Cascade was revealed, shedding light on how Cas3 achieve its activity in the Cascade complex. In the present study, we elucidated the first structure of standalone Type I-C Cas3 from Neisseria lactamica (NlaCas3). Structural analysis revealed that the histidine-aspartate (HD) nuclease active site of NlaCas3 was bound to two Fe2+ ions that inhibited its activity. Moreover, NlaCas3 could cleave both single-stranded and double-stranded DNA in the presence of Ni2+ or Co2+, showing the highest activity in the presence of both Ni2+ and Mg2+ ions. By comparing the structural studies of various Cas3 proteins, we determined that our NlaCas3 stays in an inactive conformation, allowing us to understand the structural changes associated with its activation and their implication.},
}

@article {pmid39067682,
year = {2024},
author = {Yu, J and Ji, S and Tao, H and Shan, X and Yan, Y and Sun, X and Tu, X and Li, L and Deng, C},
title = {Cell-specific expression of Cre recombinase in rat noradrenergic neurons via CRISPR-Cas9 system.},
journal = {Neuroscience},
volume = {556},
number = {},
pages = {31-41},
doi = {10.1016/j.neuroscience.2024.07.032},
pmid = {39067682},
issn = {1873-7544},
mesh = {Animals ; *Integrases/genetics/metabolism ; *Adrenergic Neurons/metabolism ; *CRISPR-Cas Systems ; *Rats, Transgenic ; Rats ; *Dopamine beta-Hydroxylase/genetics/metabolism ; Locus Coeruleus/metabolism ; Male ; Rats, Sprague-Dawley ; },
abstract = {Noradrenergic neurons play a crucial role in the functioning of the nervous system. They formed compact small clusters in the central nervous system. To target noradrenergic neurons in combination with viral tracing and achieve cell-type specific functional manipulation using chemogenetic or optogenetic tools, new transgenic animal lines are needed, especially rat models for their advantages in large body size with facilitating easy operation, physiological parameter monitoring, and accommodating complex behavioral and cognitive studies. In this study, we successfully generated a transgenic rat strain capable of expressing Cre recombinase under the control of the dopamine beta-hydroxylase (DBH) gene promoter using the CRISPR-Cas9 system. Our validation process included co-immunostaining with Cre and DBH antibodies, confirming the specific expression of Cre recombinase. Furthermore, stereotaxic injection of a fluorescence-labeled AAV-DIO virus illustrated the precise Cre-loxP-mediated recombination activity in noradrenergic neurons within the locus coeruleus (LC). Through crossbreeding with the LSL-fluorescence reporter rat line, DBH-Cre rats proved instrumental in delineating the position and structure of noradrenergic neuron clusters A1, A2, A6 (LC), and A7 in rats. Additionally, our specific activation of the LC noradrenergic neurons showed effective behavioral readout using chemogenetics of this rat line. Our results underscore the effectiveness and specificity of Cre recombinase in noradrenergic neurons, serving as a robust tool for cell-type specific targeting of small-sized noradrenergic nuclei. This approach enhances our understanding of their anatomical, physiological, and pathological roles, contributing to a more profound comprehension of noradrenergic neuron function in the nervous system.},
}

Animals
*Integrases/genetics/metabolism
*Adrenergic Neurons/metabolism
*CRISPR-Cas Systems
*Rats, Transgenic
Rats
*Dopamine beta-Hydroxylase/genetics/metabolism
Locus Coeruleus/metabolism
Male
Rats, Sprague-Dawley

@article {pmid39029255,
year = {2024},
author = {Yu, Z and Shao, Y and Shi, D and Dong, Y and Zhang, Y and Cheng, F and Wang, Z and Tu, J and Qi, K and Song, X},
title = {A rapid, ultrasensitive, and highly specific method for detecting fowl adenovirus serotype 4 based on the LAMP-CRISPR/Cas12a system.},
journal = {Poultry science},
volume = {103},
number = {9},
pages = {104048},
pmid = {39029255},
issn = {1525-3171},
mesh = {*Adenoviridae Infections/veterinary/virology/diagnosis ; *Poultry Diseases/virology/diagnosis ; Animals ; *Chickens ; *Aviadenovirus/genetics/classification/isolation & purification ; *Nucleic Acid Amplification Techniques/veterinary/methods ; *CRISPR-Cas Systems ; *Sensitivity and Specificity ; Serogroup ; Molecular Diagnostic Techniques/veterinary/methods ; },
abstract = {Fowl adenovirus serotype 4 (FAdV-4) is the causative agent of hydropericardium hepatitis syndrome in chickens, which causes severe economic impact to the poultry industry. A simple, swift and reliable detection is crucial for timely identification of FAdV-4 infection, promoting effective viral prevention and control measures. Herein, the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 12a (Cas12a) system detection platform based on loop-mediated isothermal amplification (LAMP) was studied. The CRISPR RNA (crRNA) and LAMP primers were designed and screened based on the highly conserved region of the FAdV-4 hexon gene. The parameters were then optimized individually to achieve the ideal reaction performance. The platform could lead visual detection of FAdV-4 to achieve as low as 1 copy in less than 40 min without the need for specialized instrumentation or complex equipment. Moreover, it was greatly specific, and did not cross-react with other common avian viruses. Following the validation of 30 clinical samples of suspected FAdV-4 infection, the results LAMP-CRISPR/Cas12a method generated showed fully concordance with which of the gold standard quantitative real-time PCR. To summarize, this study presented a novel, swift, expedient and inexpensive detection platform for FAdV-4, which is beneficial to viral inchoate diagnosis and point-of-care testing.},
}

*Adenoviridae Infections/veterinary/virology/diagnosis
*Poultry Diseases/virology/diagnosis
Animals
*Chickens
*Aviadenovirus/genetics/classification/isolation & purification
*Nucleic Acid Amplification Techniques/veterinary/methods
*CRISPR-Cas Systems
*Sensitivity and Specificity
Serogroup
Molecular Diagnostic Techniques/veterinary/methods

@article {pmid39179961,
year = {2024},
author = {Wu, X and Yang, Y and Ru, Y and Hao, R and Zhao, D and Ren, R and Lu, B and Li, Y and Sun, S and Zheng, H and Wang, W},
title = {Knockout of the WD40 domain of ATG16L1 enhances foot and mouth disease virus replication.},
journal = {BMC genomics},
volume = {25},
number = {1},
pages = {796},
pmid = {39179961},
issn = {1471-2164},
support = {2021YFD1800300//National Key Research and Development Program of China/ ; 2022SDZG02//The open competition program of top ten critical priorities of Agricultural Science and Technology Innovation for the 14th Five-Year Plan of Guangdong Province, China/ ; 21ZD3NA001-5//Key Technologies R&D Program of Gansu Province, China/ ; NCTIP-XD/C03//Project of National Center of Technology Innovation for Pigs, China/ ; },
mesh = {*Virus Replication ; *Foot-and-Mouth Disease Virus/genetics/physiology ; *Autophagy-Related Proteins/genetics/metabolism ; Animals ; *Gene Knockout Techniques ; *Autophagy/genetics ; Cell Line ; WD40 Repeats/genetics ; CRISPR-Cas Systems ; Foot-and-Mouth Disease/virology ; },
abstract = {The WD40 domain is one of the most abundant domains and is among the top interacting domains in eukaryotic genomes. The WD40 domain of ATG16L1 is essential for LC3 recruitment to endolysosomal membranes during non-canonical autophagy, but dispensable for canonical autophagy. Canonical autophagy was utilized by FMDV, while the relationship between FMDV and non-canonical autophagy is still elusive. In the present study, WD40 knockout (KO) PK15 cells were successfully generated via CRISPR/cas9 technology as a tool for studying the effect of non-canonical autophagy on FMDV replication. The results of growth curve analysis, morphological observation and karyotype analysis showed that the WD40 knockout cell line was stable in terms of growth and morphological characteristics. After infection with FMDV, the expression of viral protein, viral titers, and the number of copies of viral RNA in the WD40-KO cells were significantly greater than those in the wild-type PK15 cells. Moreover, RNA‒seq technology was used to sequence WD40-KO cells and wild-type cells infected or uninfected with FMDV. Differentially expressed factors such as Mx1, RSAD2, IFIT1, IRF9, IFITM3, GBP1, CXCL8, CCL5, TNFRSF17 were significantly enriched in the autophagy, NOD-like receptor signaling pathway, RIG-I-like receptor signaling pathway, Toll-like receptor signaling pathway, cytokine-cytokine receptor interaction and TNF signaling pathway, etc. The expression levels of differentially expressed genes were detected via qRT‒PCR, which was consistent with the RNA‒seq data. Here, we experimentally demonstrate for the first time that knockout of the WD40 domain of ATG16L1 enhances FMDV replication by downregulation innate immune factors. In addition, this result also indicates non-canonical autophagy inhibits FMDV replication. In total, our results play an essential role in regulating the replication level of FMDV and providing new insights into virus-host interactions and potential antiviral strategies.},
}

*Virus Replication
*Foot-and-Mouth Disease Virus/genetics/physiology
*Autophagy-Related Proteins/genetics/metabolism
Animals
*Gene Knockout Techniques
*Autophagy/genetics
Cell Line
WD40 Repeats/genetics
CRISPR-Cas Systems
Foot-and-Mouth Disease/virology

@article {pmid39179646,
year = {2024},
author = {Benites-Pariente, JS and Samolski, I and Ludeña, Y and Villena, GK},
title = {CRISPR/Cas9 mediated targeted knock-in of eglA gene to improve endoglucanase activity of Aspergillus fumigatus LMB-35Aa.},
journal = {Scientific reports},
volume = {14},
number = {1},
pages = {19661},
pmid = {39179646},
issn = {2045-2322},
support = {177-2015-CONCYTEC-FONDECYT-DE//Consejo Nacional de Ciencia, Tecnología e Innovación Tecnológica -Peru (/ ; Grant No 144-PROINNOVATE-CETF2-2022//PROINNOVATE-Ministry of Production of Peru/ ; },
mesh = {*CRISPR-Cas Systems ; *Aspergillus fumigatus/genetics/enzymology ; *Cellulase/genetics/metabolism ; *Gene Editing/methods ; *Fungal Proteins/genetics/metabolism ; Gene Knock-In Techniques ; Biofilms/growth & development ; Fermentation ; },
abstract = {Bioeconomy goals for using biomass feedstock for biofuels and bio-based production has arisen the demand for fungal strains and enzymes for biomass processing. Despite well-known Trichoderma and Aspergillus commercial strains, continuous bioprospecting has revealed the fungal biodiversity potential for production of biomass degrading enzymes. The strain Aspergillus fumigatus LMB-35Aa has revealed a great potential as source of lignocellulose-degrading enzymes. Nevertheless, genetic improvement should be considered to increase its biotechnological potential. Molecular manipulation based on homologous direct recombination (HDR) in filamentous fungi poses a challenge since its low recombination rate. Currently, CRISPR/Cas9-mediated mutagenesis can enable precise and efficient editing of filamentous fungi genomes. In this study, a CRISPR/Cas9-mediated gene editing strategy for improving endoglucanase activity of A. fumigatus LMB-35Aa strain was successfully used, which constitutes the first report of heterologous cellulase production in filamentous fungi using this technology. For this, eglA gene from A. niger ATCC 10,864 was integrated into conidial melanin pksP gene locus, which facilitated the selection of edited events discerned by the emergence of albino colonies. Heterologous production of the EglA enzyme in a biofilm fermentation system resulted in a 40% improvement in endoglucanase activity of the mutant strain compared to the wild type.},
}

*CRISPR-Cas Systems
*Aspergillus fumigatus/genetics/enzymology
*Cellulase/genetics/metabolism
*Gene Editing/methods
*Fungal Proteins/genetics/metabolism
Gene Knock-In Techniques
Biofilms/growth & development
Fermentation

@article {pmid39179566,
year = {2024},
author = {Guo, J and Gong, L and Yu, H and Li, M and An, Q and Liu, Z and Fan, S and Yang, C and Zhao, D and Han, J and Xiang, H},
title = {Engineered minimal type I CRISPR-Cas system for transcriptional activation and base editing in human cells.},
journal = {Nature communications},
volume = {15},
number = {1},
pages = {7277},
pmid = {39179566},
issn = {2041-1723},
support = {32100499//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32150020//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32230061//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32150020//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {Humans ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Transcriptional Activation ; HEK293 Cells ; CRISPR-Associated Protein 9/metabolism/genetics ; Genetic Engineering/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Type I CRISPR-Cas systems are widespread and have exhibited high versatility and efficiency in genome editing and gene regulation in prokaryotes. However, due to the multi-subunit composition and large size, their application in eukaryotes has not been thoroughly investigated. Here, we demonstrate that the type I-F2 Cascade, the most compact among type I systems, with a total gene size smaller than that of SpCas9, can be developed for transcriptional activation in human cells. The efficiency of the engineered I-F2 tool can match or surpass that of dCas9. Additionally, we create a base editor using the I-F2 Cascade, which induces a considerably wide editing window (~30 nt) with a bimodal distribution. It can expand targetable sites, which is useful for disrupting functional sequences and genetic screening. This research underscores the application of compact type I systems in eukaryotes, particularly in the development of a base editor with a wide editing window.},
}

Humans
*CRISPR-Cas Systems
*Gene Editing/methods
*Transcriptional Activation
HEK293 Cells
CRISPR-Associated Protein 9/metabolism/genetics
Genetic Engineering/methods
RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid39179547,
year = {2024},
author = {Gholami, A and Mohkam, M and Soleimanian, S and Sadraeian, M and Lauto, A},
title = {Bacterial nanotechnology as a paradigm in targeted cancer therapeutic delivery and immunotherapy.},
journal = {Microsystems & nanoengineering},
volume = {10},
number = {1},
pages = {113},
pmid = {39179547},
issn = {2055-7434},
abstract = {Cancer, a multifaceted and diverse ailment, presents formidable obstacles to traditional treatment modalities. Nanotechnology presents novel prospects for surmounting these challenges through its capacity to facilitate meticulous and regulated administration of therapeutic agents to malignant cells while concurrently modulating the immune system to combat neoplasms. Bacteria and their derivatives have emerged as highly versatile and multifunctional platforms for cancer nanotherapy within the realm of nanomaterials. This comprehensive review delves into the multifaceted and groundbreaking implementations of bacterial nanotechnology within cancer therapy. This review encompasses four primary facets: the utilization of bacteria as living conveyors of medicinal substances, the employment of bacterial components as agents that stimulate the immune system, the deployment of bacterial vectors as tools for delivering genetic material, and the development of bacteria-derived nano-drugs as intelligent nano-medications. Furthermore, we elucidate the merits and modalities of operation pertaining to these bacterial nano-systems, along with their capacity to synergize with other cutting-edge nanotechnologies, such as CRISPR-Cas systems. Additionally, we offer insightful viewpoints regarding the forthcoming trajectories and prospects within this expanding domain. It is our deduction that bacterial nanotechnology embodies a propitious and innovative paradigm in the realm of cancer therapy, which has the potential to provide numerous advantages and synergistic effects in enhancing the outcomes and quality of life for individuals afflicted with cancer.},
}

@article {pmid39179542,
year = {2024},
author = {Kantor, B and O'Donovan, B and Rittiner, J and Hodgson, D and Lindner, N and Guerrero, S and Dong, W and Zhang, A and Chiba-Falek, O},
title = {The therapeutic implications of all-in-one AAV-delivered epigenome-editing platform in neurodegenerative disorders.},
journal = {Nature communications},
volume = {15},
number = {1},
pages = {7259},
pmid = {39179542},
issn = {2041-1723},
support = {R01 AG057522/AG/NIA NIH HHS/United States ; R41 AG077992/AG/NIA NIH HHS/United States ; },
mesh = {*Gene Editing/methods ; *Dependovirus/genetics ; *CRISPR-Cas Systems/genetics ; Humans ; Animals ; *Genetic Vectors/genetics ; Mice ; Neurodegenerative Diseases/genetics/therapy ; Genetic Therapy/methods ; Epigenome ; HEK293 Cells ; Methyl-CpG-Binding Protein 2/genetics/metabolism ; Epigenesis, Genetic ; Alzheimer Disease/genetics/therapy ; Apolipoproteins E/genetics ; Staphylococcus aureus/genetics ; },
abstract = {Safely and efficiently controlling gene expression is a long-standing goal of biomedical research, and CRISPR/Cas system can be harnessed to create powerful tools for epigenetic editing. Adeno-associated-viruses (AAVs) represent the delivery vehicle of choice for therapeutic platform. However, their small packaging capacity isn't suitable for large constructs including most CRISPR/dCas9-effector vectors. Thus, AAV-based CRISPR/Cas systems have been delivered via two separate viral vectors. Here we develop a compact CRISPR/dCas9-based repressor system packaged in AAV as a single optimized vector. The system comprises the small Staphylococcus aureus (Sa)dCas9 and an engineered repressor molecule, a fusion of MeCP2's transcription repression domain (TRD) and KRAB. The dSaCas9-KRAB-MeCP2(TRD) vector platform repressed robustly and sustainably the expression of multiple genes-of-interest, in vitro and in vivo, including ApoE, the strongest genetic risk factor for late onset Alzheimer's disease (LOAD). Our platform broadens the CRISPR/dCas9 toolset available for transcriptional manipulation of gene expression in research and therapeutic settings.},
}

*Gene Editing/methods
*Dependovirus/genetics
*CRISPR-Cas Systems/genetics
Humans
Animals
*Genetic Vectors/genetics
Mice
Neurodegenerative Diseases/genetics/therapy
Genetic Therapy/methods
Epigenome
HEK293 Cells
Methyl-CpG-Binding Protein 2/genetics/metabolism
Epigenesis, Genetic
Alzheimer Disease/genetics/therapy
Apolipoproteins E/genetics
Staphylococcus aureus/genetics

@article {pmid39179185,
year = {2024},
author = {Tharp, O and Sansbury, BM and Kmiec, EB},
title = {CRISPR-directed gene-editing induces genetic rearrangement within the human globin gene locus.},
journal = {Gene},
volume = {},
number = {},
pages = {148879},
doi = {10.1016/j.gene.2024.148879},
pmid = {39179185},
issn = {1879-0038},
abstract = {CRISPR-Cas is a revolutionary technology but has already demonstrated significant feasibility for clinical and non-clinical applications. While the efficiency and precision of this remarkable genetic tool is unprecedented, unfortunately, a series of collateral genetic rearrangement have been reported in response to double-stranded DNA breakage. Once these molecular scissions occur, the cascade of DNA repair reactions can lead to genomic rearrangements especially if breakage takes place within a family of sequence related genes. Here, we demonstrate that CRISPR- directed gene editing near the sickle cell mutation site generates a curious genetic outcome; a footprint of the δ globin gene proximal to the CRISPR/Cas cut site(s). This rearrangement is not dependent on the presence of an exogenously added DNA template but is apparently dependent on a double strand break. Our results the highlight recombinational capacity of double strand breaks in human chromosomes where the aim is to edit a human gene.},
}

@article {pmid39178725,
year = {2024},
author = {Hu, X and Enbar, T and Tang, L},
title = {Delivery approaches of immunomodulatory nucleic acids for cancer therapy.},
journal = {Current opinion in biotechnology},
volume = {89},
number = {},
pages = {103182},
doi = {10.1016/j.copbio.2024.103182},
pmid = {39178725},
issn = {1879-0429},
abstract = {Messenger RNA (mRNA) vaccines have made remarkable public health contributions during the pandemic and initiated a new era for nucleic acid-based therapeutics. With the unique strength of nucleic acids, including not only mRNA but also DNA, microRNA, small interfering RNA (siRNA), and other nucleic acids, either in tuning off genes or introducing function, nucleic acid therapeutics have been regarded as potential candidates for the treatment of many different diseases, especially for the immunomodulation in cancer. However, the scope of the applications was limited by the challenges in delivery due to intrinsic properties of nucleic acids including low stability, immunogenicity, and toxicity. Bioengineering approaches toward efficient and targeted delivery of therapeutic nucleic acids have gained momentum in clinical applications in the past few decades. Recent advances in the biotechnological approaches for the delivery of mRNA, siRNA, and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas for immunomodulatory are promising alternatives in designing future cancer immunotherapy.},
}

@article {pmid39176473,
year = {2024},
author = {Zhang, L and Hou, L and Cai, HH and Sun, B and Han, DM and Chen, FZ},
title = {Cascading CRISPR/Cas and Nanozyme for Enhanced Organic Photoelectrochemical Transistor Detection with Triple Signal Amplification.},
journal = {Analytical chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.analchem.4c03220},
pmid = {39176473},
issn = {1520-6882},
abstract = {Innovative signal amplification and transduction play pivotal roles in bioanalysis. Herein, cascading CRISPR/Cas and the nanozyme are integrated with electronic amplification in an organic photoelectrochemical transistor (OPECT) to enable triple signal amplification, which is exemplified by the miRNA-triggered CRISPR/Cas13a system and polyoxometalate nanozyme for OPECT detection of miRNA-21. The CRISPR/Cas13a-enabled release of glucose oxidase could synergize with peroxidase-like SiW12 to induce catalytic precipitation on the photogate, inhibiting the interfacial mass transfer and thus the significant suppression of the channel current. The as-developed OPECT sensor demonstrates good sensitivity and selectivity for miRNA-21 detection, with a linear range from 1 fM to 10 nM and an ultralow detection limit of 0.53 fM. This study features the integration of bio- and nanoenzyme cascade and electronic triple signal amplification for OPECT detection.},
}

@article {pmid39121652,
year = {2024},
author = {Schuster, J and Fatima, A and Papadopoulos, N and de Guidi, C and Sobol, M and Dahl, N},
title = {Generation of a ZEB2 deficient human iPSC line (KICRi002A-4).},
journal = {Stem cell research},
volume = {80},
number = {},
pages = {103521},
doi = {10.1016/j.scr.2024.103521},
pmid = {39121652},
issn = {1876-7753},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *Zinc Finger E-box Binding Homeobox 2/metabolism/genetics ; Cell Line ; Cell Differentiation ; CRISPR-Cas Systems ; },
abstract = {The transcription factor ZEB2 is essential for early embryonic development. Using CRISPR/Cas9, we generated a ZEB2 deficient human iPSC cell line (KICRi002A-4), carrying a homozygous 790 bp deletion in ZEB2 that involves part of exon 5, intron 5 and part of exon 6. The deletion leads to markedly reduced levels of a truncated ZEB2 transcript. No ZEB2 protein was detected by immunopreciptation. The iPSC line expressed pluripotency markers and showed a capacity to differentiate into the three germ layers in vitro. Assessment of genomic integrity revealed a normal karyotype without detectable OFF-target editing. The iPSC line KICRi002A-4 thus offers a valuable resource to study the role of ZEB2 for the commitment and differentiation of various human cell lineages.},
}

Humans
*Induced Pluripotent Stem Cells/metabolism/cytology
*Zinc Finger E-box Binding Homeobox 2/metabolism/genetics
Cell Line
Cell Differentiation
CRISPR-Cas Systems

@article {pmid39121651,
year = {2024},
author = {Oh, JY and Yoo, DH and Im, YS and Kim, YO},
title = {Generation and characterization of a human iPSC line expressing EGFP-tagged CDH1, KSCBi002-A-3.},
journal = {Stem cell research},
volume = {80},
number = {},
pages = {103510},
doi = {10.1016/j.scr.2024.103510},
pmid = {39121651},
issn = {1876-7753},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *Cadherins/metabolism/genetics ; *Green Fluorescent Proteins/metabolism/genetics ; Cell Line ; CRISPR-Cas Systems ; Antigens, CD/metabolism/genetics ; Cell Differentiation ; },
abstract = {E-cadherin, a transmembrane protein, is essential for maintaining the integrity and structure of human pluripotent stem cells (hPSCs) by facilitating strong cell-cell adhesion and communication, which is crucial for their colony formation and pluripotency. Here, we used the CRISPR/Cas9 system to introduce the enhanced green fluorescent protein (EGFP)-tagged CDH1 into the AAVS1 locus, a safe harbour site, of human induced pluripotent stem cells (hiPSCs). The engineered cell line, KSCBi002-A-3, expressed functional CDH1-EGFP fusion protein, exhibited normal cell morphology, maintained a normal karyotype, and retained pluripotent state.},
}

Humans
*Induced Pluripotent Stem Cells/metabolism/cytology
*Cadherins/metabolism/genetics
*Green Fluorescent Proteins/metabolism/genetics
Cell Line
CRISPR-Cas Systems
Antigens, CD/metabolism/genetics
Cell Differentiation

@article {pmid39111000,
year = {2024},
author = {Lim, SW and Lee, KI and Cui, S and Fang, X and Shin, YJ and Lee, H and Lee, JY and Chung, BH and Yang, CW},
title = {Generation of green fluorescent protein reporter knock-in iPSC line at the 3'UTR region of the KLOTHO locus.},
journal = {Stem cell research},
volume = {80},
number = {},
pages = {103499},
doi = {10.1016/j.scr.2024.103499},
pmid = {39111000},
issn = {1876-7753},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *Klotho Proteins ; *3' Untranslated Regions ; *Green Fluorescent Proteins/metabolism/genetics ; *Glucuronidase/metabolism/genetics ; Cell Line ; CRISPR-Cas Systems ; Genes, Reporter ; Cell Differentiation ; Gene Knock-In Techniques/methods ; Genetic Loci ; },
abstract = {We generated a human induced pluripotent stem cell (hiPSC) line (CMCi014-A-78) expressing a GFP reporter in the 3'-UTR region of the KLOTHO locus using CRISPR/Cas9-mediated homologous recombination to screen for candidates regulating KLOTHO. The established cell line exhibits a normal karyotype, typical stem cell morphology, expression of pluripotency markers, and the ability to differentiate into the three germ layers. Consequently, this hiPSC line could serve as a valuable resource for screening KLOTHO regulators in hiPSC-derived target cells or organoids.},
}

Humans
*Induced Pluripotent Stem Cells/metabolism/cytology
*Klotho Proteins
*3' Untranslated Regions
*Green Fluorescent Proteins/metabolism/genetics
*Glucuronidase/metabolism/genetics
Cell Line
CRISPR-Cas Systems
Genes, Reporter
Cell Differentiation
Gene Knock-In Techniques/methods
Genetic Loci

@article {pmid39098170,
year = {2024},
author = {Fatima, N and Dillen, L and Hommersom, MP and Çepni, E and Fatima, F and van Beusekom, E and Albert, S and Ali Khan, A and de Brouwer, APM and van Bokhoven, H},
title = {Generation of induced pluripotent stem cell line (UCSFi001-A-77) carrying a biallelic frameshift variant in exon 4 of SGIP1 through CRISPR/Cas9.},
journal = {Stem cell research},
volume = {80},
number = {},
pages = {103511},
doi = {10.1016/j.scr.2024.103511},
pmid = {39098170},
issn = {1876-7753},
mesh = {*Induced Pluripotent Stem Cells/metabolism ; Humans ; *CRISPR-Cas Systems ; *Frameshift Mutation ; *Exons ; Cell Line ; Carrier Proteins/genetics/metabolism ; Cell Differentiation ; },
abstract = {SGIP1 encodes a protein Src homology 3-domain growth factor receptor-bound 2-like endophilin interacting protein 1. It is involved in the regulation of clathrin-mediated endocytosis along with having a role in energy homeostasis in neuronal systems. We generated an isogenic human induced pluripotent stem cell (iPSC) line with a biallelic frameshift variant in SGIP1. This exon has been shown to be subject to alternative splicing, leading to an isoform lacking 24 amino acids that are present in the longest SGIP isoform. The newly generated iPSC line will be helpful to dissect the differential properties of the two SGIP isoforms.},
}

*Induced Pluripotent Stem Cells/metabolism
Humans
*CRISPR-Cas Systems
*Frameshift Mutation
*Exons
Cell Line
Carrier Proteins/genetics/metabolism
Cell Differentiation

@article {pmid39096898,
year = {2024},
author = {Xue, N and Hong, D and Zhang, D and Wang, Q and Zhang, S and Yang, L and Chen, X and Li, Y and Han, H and Hu, C and Liu, M and Song, G and Guan, Y and Wang, L and Zhu, Y and Li, D},
title = {Engineering IscB to develop highly efficient miniature editing tools in mammalian cells and embryos.},
journal = {Molecular cell},
volume = {84},
number = {16},
pages = {3128-3140.e4},
doi = {10.1016/j.molcel.2024.07.007},
pmid = {39096898},
issn = {1097-4164},
mesh = {Animals ; *Gene Editing/methods ; Mice ; Humans ; *CRISPR-Cas Systems ; Embryo, Mammalian/metabolism ; HEK293 Cells ; Protein Engineering/methods ; },
abstract = {The IscB proteins, as the ancestors of Cas9 endonuclease, hold great promise due to their small size and potential for diverse genome editing. However, their activity in mammalian cells is unsatisfactory. By introducing three residual substitutions in IscB, we observed an average 7.5-fold increase in activity. Through fusing a sequence-non-specific DNA-binding protein domain, the eIscB-D variant achieved higher editing efficiency, with a maximum of 91.3%. Moreover, engineered ωRNA was generated with a 20% reduction in length and slightly increased efficiency. The engineered eIscB-D/eωRNA system showed an average 20.2-fold increase in activity compared with the original IscB. Furthermore, we successfully adapted eIscB-D for highly efficient cytosine and adenine base editing. Notably, eIscB-D is highly active in mouse cell lines and embryos, enabling the efficient generation of disease models through mRNA/ωRNA injection. Our study suggests that these miniature genome-editing tools have great potential for diverse applications.},
}

Animals
*Gene Editing/methods
Mice
Humans
*CRISPR-Cas Systems
Embryo, Mammalian/metabolism
HEK293 Cells
Protein Engineering/methods

@article {pmid39059534,
year = {2024},
author = {You, D and Xu, T and Huang, BZ and Zhu, L and Wu, F and Deng, LS and Liu, ZY and Duan, JQ and Wang, YM and Ge, LP and Liu, ZH and Sun, J and Zeng, X and Lang, LQ and Zhou, YC and Chen, DS and Lai, SY and Ai, YR and Huang, JB and Xu, ZW},
title = {Rapid, sensitive, and visual detection of swine Japanese encephalitis virus with a one-pot RPA-CRISPR/EsCas13d-based dual readout portable platform.},
journal = {International journal of biological macromolecules},
volume = {277},
number = {Pt 1},
pages = {134151},
doi = {10.1016/j.ijbiomac.2024.134151},
pmid = {39059534},
issn = {1879-0003},
mesh = {*Encephalitis Virus, Japanese/isolation & purification/genetics ; Animals ; *Nucleic Acid Amplification Techniques/methods ; Encephalitis, Japanese/diagnosis/virology ; Molecular Diagnostic Techniques/methods ; Swine ; CRISPR-Cas Systems ; Sensitivity and Specificity ; RNA, Viral/genetics/analysis ; },
abstract = {Japanese encephalitis (JE), a mosquito-borne zoonotic disease caused by the Japanese encephalitis virus (JEV), poses a serious threat to global public health. The low viremia levels typical in JEV infections make RNA detection challenging, necessitating early and rapid diagnostic methods for effective control and prevention. This study introduces a novel one-pot detection method that combines recombinant enzyme polymerase isothermal amplification (RPA) with CRISPR/EsCas13d targeting, providing visual fluorescence and lateral flow assay (LFA) results. Our portable one-pot RPA-EsCas13d platform can detect as few as two copies of JEV nucleic acid within 1 h, without cross-reactivity with other pathogens. Validation against clinical samples showed 100 % concordance with real-time PCR results, underscoring the method's simplicity, sensitivity, and specificity. This efficacy confirms the platform's suitability as a novel point-of-care testing (POCT) solution for detecting and monitoring the JE virus in clinical and vector samples, especially valuable in remote and resource-limited settings.},
}

*Encephalitis Virus, Japanese/isolation & purification/genetics
Animals
*Nucleic Acid Amplification Techniques/methods
Encephalitis, Japanese/diagnosis/virology
Molecular Diagnostic Techniques/methods
Swine
CRISPR-Cas Systems
Sensitivity and Specificity
RNA, Viral/genetics/analysis

@article {pmid39059080,
year = {2024},
author = {Jiang, S and Dai, T and Li, Q and Xu, T and Zhang, W and Sun, J and Liu, H},
title = {Generation of ASCL1-mCherry knock-in reporter in human embryonic stem cell line, WAe001-A-2E, using CRISPR/Cas9-based gene targeting.},
journal = {Stem cell research},
volume = {80},
number = {},
pages = {103500},
doi = {10.1016/j.scr.2024.103500},
pmid = {39059080},
issn = {1876-7753},
mesh = {Humans ; *CRISPR-Cas Systems ; *Human Embryonic Stem Cells/metabolism/cytology ; *Basic Helix-Loop-Helix Transcription Factors/metabolism/genetics ; Cell Line ; *Gene Targeting ; Gene Knock-In Techniques ; Genes, Reporter ; Cell Differentiation ; Red Fluorescent Protein ; },
abstract = {Achaete-Scute Complex Homolog 1 (ASCL1) is a key regulator in the development and function of the nervous system, particularly in the process of neuronal and neuroendocrine cell differentiation. By employing the CRISPR/Cas9 system, we successfully established an ASCL1-mCherry knock-in human embryonic stem cell (hESC) line by inserting a P2A-mCherry fragment at the ASCL1 locus. The mCherry reporter effectively demonstrated the expression level of endogenous ASCL1 during the process of inducing pulmonary neuroendocrine cells (PNECs) from hESC. This reporter cell line holds significant value as a research tool for investigating the process of lung neuroendocrine cell differentiation, conducting drug screening, and exploring the underlying mechanisms of lung diseases associated with PNECs dysfunction.},
}

Humans
*CRISPR-Cas Systems
*Human Embryonic Stem Cells/metabolism/cytology
*Basic Helix-Loop-Helix Transcription Factors/metabolism/genetics
Cell Line
*Gene Targeting
Gene Knock-In Techniques
Genes, Reporter
Cell Differentiation
Red Fluorescent Protein

@article {pmid38702575,
year = {2024},
author = {Pandey, P and Vavilala, SL},
title = {From Gene Editing to Biofilm Busting: CRISPR-CAS9 Against Antibiotic Resistance-A Review.},
journal = {Cell biochemistry and biophysics},
volume = {82},
number = {2},
pages = {549-560},
pmid = {38702575},
issn = {1559-0283},
mesh = {*CRISPR-Cas Systems ; *Biofilms/drug effects ; *Gene Editing ; Anti-Bacterial Agents/pharmacology ; Drug Resistance, Microbial/genetics ; Humans ; Bacteria/genetics/drug effects ; Drug Resistance, Bacterial/genetics ; },
abstract = {In recent decades, the development of novel antimicrobials has significantly slowed due to the emergence of antimicrobial resistance (AMR), intensifying the global struggle against infectious diseases. Microbial populations worldwide rapidly develop resistance due to the widespread use of antibiotics, primarily targeting drug-resistant germs. A prominent manifestation of this resistance is the formation of biofilms, where bacteria create protective layers using signaling pathways such as quorum sensing. In response to this challenge, the CRISPR-Cas9 method has emerged as a ground-breaking strategy to counter biofilms. Initially identified as the "adaptive immune system" of bacteria, CRISPR-Cas9 has evolved into a state-of-the-art genetic engineering tool. Its exceptional precision in altering specific genes across diverse microorganisms positions it as a promising alternative for addressing antibiotic resistance by selectively modifying genes in diverse microorganisms. This comprehensive review concentrates on the historical background, discovery, developmental stages, and distinct components of CRISPR Cas9 technology. Emphasizing its role as a widely used genome engineering tool, the review explores how CRISPR Cas9 can significantly contribute to the targeted disruption of genes responsible for biofilm formation, highlighting its pivotal role in reshaping strategies to combat antibiotic resistance and mitigate the challenges posed by biofilm-associated infectious diseases.},
}

*CRISPR-Cas Systems
*Biofilms/drug effects
*Gene Editing
Anti-Bacterial Agents/pharmacology
Drug Resistance, Microbial/genetics
Humans
Bacteria/genetics/drug effects
Drug Resistance, Bacterial/genetics

@article {pmid39176261,
year = {2024},
author = {Liu, T and Liu, Q and Chen, F and Shi, Y and Maimaiti, G and Yang, Z and Zheng, S and Lu, X and Li, H and Chen, Z},
title = {An accurate and convenient method for Mycoplasma pneumoniae via one-step LAMP-CRISPR/Cas12b detection platform.},
journal = {Frontiers in cellular and infection microbiology},
volume = {14},
number = {},
pages = {1409078},
doi = {10.3389/fcimb.2024.1409078},
pmid = {39176261},
issn = {2235-2988},
mesh = {*Mycoplasma pneumoniae/genetics/isolation & purification ; *Nucleic Acid Amplification Techniques/methods ; Humans ; *Sensitivity and Specificity ; *Pneumonia, Mycoplasma/diagnosis/microbiology ; *Molecular Diagnostic Techniques/methods ; *CRISPR-Cas Systems ; Child ; Limit of Detection ; },
abstract = {INTRODUCTION: Mycoplasma pneumoniae (MP) is the major cause of respiratory infections that threaten the health of children and adolescents worldwide. Therefore, an early, simple, and accurate detection approach for MP is critical to prevent outbreaks of MP-induced community-acquired pneumonia.

METHODS: Here, we explored a simple and accurate method for MP identification that combines loop-mediated isothermal amplification (LAMP) with the CRISPR/Cas12b assay in a one-pot reaction.

RESULTS: In the current study, the whole reaction was completed within 1 h at a constant temperature of 57°C. The limit of detection of this assay was 33.7 copies per reaction. The specificity of the LAMP-CRISPR/Cas12b method was 100%, without any cross-reactivity with other pathogens. Overall, 272 clinical samples were used to evaluate the clinical performance of LAMP-CRISPR/Cas12b. Compared with the gold standard results from real-time PCR, the present method provided a sensitivity of 88.11% (126/143), specificity of 100% (129/129), and consistency of 93.75% (255/272).

DISCUSSION: Taken together, our preliminary results illustrate that the LAMP-CRISPR/Cas12b method is a simple and reliable tool for MP diagnosis that can be performed in resource-limited regions.},
}

*Mycoplasma pneumoniae/genetics/isolation & purification
*Nucleic Acid Amplification Techniques/methods
Humans
*Sensitivity and Specificity
*Pneumonia, Mycoplasma/diagnosis/microbiology
*Molecular Diagnostic Techniques/methods
*CRISPR-Cas Systems
Child
Limit of Detection

@article {pmid39174599,
year = {2024},
author = {Geng, Z and Tai, YT and Wang, Q and Gao, Z},
title = {AUTS2 disruption causes neuronal differentiation defects in human cerebral organoids through hyperactivation of the WNT/β-catenin pathway.},
journal = {Scientific reports},
volume = {14},
number = {1},
pages = {19522},
pmid = {39174599},
issn = {2045-2322},
support = {R35GM133496/GM/NIGMS NIH HHS/United States ; },
mesh = {Humans ; *Organoids/metabolism ; *Wnt Signaling Pathway ; *Neurons/metabolism ; *Cell Differentiation ; *Transcription Factors/metabolism/genetics ; *Cytoskeletal Proteins/metabolism/genetics ; CRISPR-Cas Systems ; beta Catenin/metabolism/genetics ; Human Embryonic Stem Cells/metabolism ; Brain/metabolism/pathology ; },
abstract = {Individuals with the Autism Susceptibility Candidate 2 (AUTS2) gene disruptions exhibit symptoms such as intellectual disability, microcephaly, growth retardation, and distinct skeletal and facial differences. The role of AUTS2 in neurodevelopment has been investigated using animal and embryonic stem cell models. However, the precise molecular mechanisms of how AUTS2 influences neurodevelopment, particularly in humans, are not thoroughly understood. Our study employed a 3D human cerebral organoid culture system, in combination with genetic, genomic, cellular, and molecular approaches, to investigate how AUTS2 impacts neurodevelopment through cellular signaling pathways. We used CRISPR/Cas9 technology to create AUTS2-deficient human embryonic stem cells and then generated cerebral organoids with these cells. Our transcriptomic analyses revealed that the absence of AUTS2 in cerebral organoids reduces the populations of cells committed to the neuronal lineage, resulting in an overabundance of cells with a transcription profile resembling that of choroid plexus (ChP) cells. Intriguingly, we found that AUTS2 negatively regulates the WNT/β-catenin signaling pathway, evidenced by its overactivation in AUTS2-deficient cerebral organoids and in luciferase reporter cells lacking AUTS2. Importantly, treating the AUTS2-deficient cerebral organoids with a WNT inhibitor reversed the overexpression of ChP genes and increased the downregulated neuronal gene expression. This study offers new insights into the role of AUTS2 in neurodevelopment and suggests potential targeted therapies for neurodevelopmental disorders.},
}

Humans
*Organoids/metabolism
*Wnt Signaling Pathway
*Neurons/metabolism
*Cell Differentiation
*Transcription Factors/metabolism/genetics
*Cytoskeletal Proteins/metabolism/genetics
CRISPR-Cas Systems
beta Catenin/metabolism/genetics
Human Embryonic Stem Cells/metabolism
Brain/metabolism/pathology

@article {pmid39078660,
year = {2024},
author = {Gao, Y and Ang, YS and Yung, LL},
title = {One-Pot Detection of Proteins Using a Two-Way Extension-Based Assay with Cas12a.},
journal = {ACS sensors},
volume = {9},
number = {8},
pages = {3928-3937},
doi = {10.1021/acssensors.4c00370},
pmid = {39078660},
issn = {2379-3694},
mesh = {*CRISPR-Cas Systems ; Immunoassay/methods ; Humans ; Aptamers, Nucleotide/chemistry ; CRISPR-Associated Proteins ; Nucleic Acid Amplification Techniques/methods ; Becaplermin/analysis ; Endodeoxyribonucleases/chemistry ; Bacterial Proteins/chemistry ; },
abstract = {Protein biomarkers are an important class of biomarkers in disease diagnosis and are traditionally detected by enzyme-linked immunosorbent assay and mass spectrometry, which involve multiple steps and a complex workflow. In recent years, many CRISPR-Cas12a-based methods for protein detection have been developed; however, most of them have not overcome the workflow complications observed in traditional assays, limiting their applicability in point-of-care testing. In this work, we designed a single-step, one-pot, and proximity-based isothermal immunoassay integrating CRISPR Cas12a for homogeneous protein target detection with a simplified workflow and high sensitivity. Probes consisting of different binders (small molecule, aptamer, and antibody) conjugated with oligonucleotides undergo two-way extension upon binding to the protein targets, leading to downstream DNA amplification by a pair of nicking enzymes and polymerases to generate target sequences for Cas12a signal generation. We used the streptavidin-biotin model to demonstrate the design of our assay and proved that all three elements of protein detection (target protein binding, DNA amplification, and Cas12a signal generation) could coexist in one pot and proceed isothermally in a single buffer system at a low reaction volume of 10 μL. The plug-and-play applicability of our assay has been successfully demonstrated using four different protein targets, streptavidin, PDGF-BB, antidigoxigenin antibody, and IFNγ, with the limit of detection ranging from fM to pM.},
}

*CRISPR-Cas Systems
Immunoassay/methods
Humans
Aptamers, Nucleotide/chemistry
CRISPR-Associated Proteins
Nucleic Acid Amplification Techniques/methods
Becaplermin/analysis
Endodeoxyribonucleases/chemistry
Bacterial Proteins/chemistry

@article {pmid39013507,
year = {2024},
author = {Li, P and Wei, Y and Shi, J and Wu, J and Wu, Y and Yan, J and Liu, S and Tan, X and Huang, KJ},
title = {CRISPR/Cas12a-triggered ordered concatemeric DNA probes signal-on/off multifunctional analytical sensing system for ultrasensitive detection of thalassemia.},
journal = {International journal of biological macromolecules},
volume = {276},
number = {Pt 1},
pages = {133884},
doi = {10.1016/j.ijbiomac.2024.133884},
pmid = {39013507},
issn = {1879-0003},
mesh = {Humans ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems/genetics ; *DNA Probes/chemistry/genetics ; *Thalassemia/diagnosis/genetics ; Glucose Oxidase/chemistry/metabolism ; Electrochemical Techniques/methods ; Limit of Detection ; Electrodes ; },
abstract = {Based on CRISPR/Cas12a triggered ordered concatemeric DNA probes, a "on/off" self-powered biosensor is developed to achieve highly sensitive detection of thalassemia gene CD142 through open-circuit potential-assisted visual signal output. The ingeniously constructed glucose oxidase (GOD)-functionalized ordered concatemeric DNA probe structure can significantly amplify signal output, while the coupled CRISPR/Cas12a system is served as a "signal switch" with excellent signal-transducing capabilities. When the ordered concatemeric DNA probe structure is anchored on electrode, the response signal of the sensing system is in the "signal on" mode. While, the presence of the target activates the non-specific cleavage activity of the CRISPR/Cas12a system, causing the sensing system to switch to the "signal off" mode. In the detection system, GOD catalyzes the oxidation of glucose to produce hydrogen peroxide, which further catalyzes the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) to form a color product, enabling visual signal of the target through naked-eye color contrast. By employing a multifunctional analytical mode combining electrochemical and visual signal outputs, accurate determination of the target is achieved, with linear ranges of 0.0001-100 pM, and detection limits of 48.1 aM (S/N = 3). This work provides a reference method for sensitive detection of thalassemia genes and holds great diagnostic potential in biomedical applications.},
}

Humans
*Biosensing Techniques/methods
*CRISPR-Cas Systems/genetics
*DNA Probes/chemistry/genetics
*Thalassemia/diagnosis/genetics
Glucose Oxidase/chemistry/metabolism
Electrochemical Techniques/methods
Limit of Detection
Electrodes

@article {pmid39174527,
year = {2024},
author = {Chen, R and Shi, X and Yao, X and Gao, T and Huang, G and Ning, D and Cao, Z and Xu, Y and Liang, W and Tian, SZ and Zhu, Q and Fang, L and Zheng, M and Hu, Y and Cui, H and Chen, W},
title = {Specific multivalent molecules boost CRISPR-mediated transcriptional activation.},
journal = {Nature communications},
volume = {15},
number = {1},
pages = {7222},
pmid = {39174527},
issn = {2041-1723},
mesh = {*CRISPR-Cas Systems ; *Transcriptional Activation ; Humans ; Promoter Regions, Genetic ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; HEK293 Cells ; Binding Sites ; Chromatin/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats ; Enhancer Elements, Genetic ; },
abstract = {CRISPR/Cas-based transcriptional activators can be enhanced by intrinsically disordered regions (IDRs). However, the underlying mechanisms are still debatable. Here, we examine 12 well-known IDRs by fusing them to the dCas9-VP64 activator, of which only seven can augment activation, albeit independently of their phase separation capabilities. Moreover, modular domains (MDs), another class of multivalent molecules, though ineffective in enhancing dCas9-VP64 activity on their own, show substantial enhancement in transcriptional activation when combined with dCas9-VP64-IDR. By varying the number of gRNA binding sites and fusing dCas9-VP64 with different IDRs/MDs, we uncover that optimal, rather than maximal, cis-trans cooperativity enables the most robust activation. Finally, targeting promoter-enhancer pairs yields synergistic effects, which can be further amplified via enhancing chromatin interactions. Overall, our study develops a versatile platform for efficient gene activation and sheds important insights into CRIPSR-based transcriptional activators enhanced with multivalent molecules.},
}

*CRISPR-Cas Systems
*Transcriptional Activation
Humans
Promoter Regions, Genetic
RNA, Guide, CRISPR-Cas Systems/genetics/metabolism
HEK293 Cells
Binding Sites
Chromatin/metabolism
Clustered Regularly Interspaced Short Palindromic Repeats
Enhancer Elements, Genetic

@article {pmid39173952,
year = {2024},
author = {Jung, H and Inaba, Y and Banta, S},
title = {CRISPR/dCas12a knock-down of Acidithiobacillus ferrooxidans electron transport chain bc1 complexes enables enhanced metal sulfide bioleaching.},
journal = {The Journal of biological chemistry},
volume = {},
number = {},
pages = {107703},
doi = {10.1016/j.jbc.2024.107703},
pmid = {39173952},
issn = {1083-351X},
abstract = {Acidithiobacillus ferrooxidans is an acidophilic chemolithoautotroph that plays an important role in biogeochemical iron and sulfur cycling and is a member of the consortia used in industrial hydrometallurgical processing of copper. Metal sulfide bioleaching is catalyzed by the regeneration of ferric iron, however, bioleaching of chalcopyrite, the dominant unmined form of copper on Earth, is inhibited by surface passivation. Here, we report the implementation of CRISPR interference (CRISPRi) using the catalytically inactive Cas12a (dCas12a) in A. ferrooxidans to knockdown the expression of genes in the petI and petII operons. These operons encode bc1 complex proteins and knockdown of these genes enabled the manipulation (enhancement or repression) of iron oxidation. The petB2 gene knockdown strain enhanced iron oxidation, leading to enhanced pyrite and chalcopyrite oxidation, which correlated with reduced biofilm formation and decreased surface passivation of the minerals. These findings highlight the utility of CRISPRi/dCas12a technology for engineering A. ferrooxidans while unveiling a new strategy to manipulate and improve bioleaching efficiency.},
}

@article {pmid39173188,
year = {2024},
author = {Zhang, L and Wang, H and Yang, S and Liu, J and Li, J and Lu, Y and Cheng, J and Xu, Y},
title = {High-Throughput and Integrated CRISPR/Cas12a-Based Molecular Diagnosis Using a Deep Learning Enabled Microfluidic System.},
journal = {ACS nano},
volume = {},
number = {},
pages = {},
doi = {10.1021/acsnano.4c05734},
pmid = {39173188},
issn = {1936-086X},
abstract = {CRISPR/Cas-based molecular diagnosis demonstrates potent potential for sensitive and rapid pathogen detection, notably in SARS-CoV-2 diagnosis and mutation tracking. Yet, a major hurdle hindering widespread practical use is its restricted throughput, limited integration, and complex reagent preparation. Here, a system, microfluidic multiplate-based ultrahigh throughput analysis of SARS-CoV-2 variants of concern using CRISPR/Cas12a and nonextraction RT-LAMP (mutaSCAN), is proposed for rapid detection of SARS-CoV-2 and its variants with limited resource requirements. With the aid of the self-developed reagents and deep-learning enabled prototype device, our mutaSCAN system can detect SARS-CoV-2 in mock swab samples below 30 min as low as 250 copies/mL with the throughput up to 96 per round. Clinical specimens were tested with this system, the accuracy for routine and mutation testing (22 wildtype samples, 26 mutational samples) was 98% and 100%, respectively. No false-positive results were found for negative (n = 24) samples.},
}

@article {pmid39173055,
year = {2024},
author = {Gong, M and Peng, C and Yang, C and Wang, Z and Qian, H and Hu, X and Zhou, P and Shan, C and Ding, Q},
title = {Genome-wide CRISPR/Cas9 screen identifies SLC39A9 and PIK3C3 as crucial entry factors for Ebola virus infection.},
journal = {PLoS pathogens},
volume = {20},
number = {8},
pages = {e1012444},
doi = {10.1371/journal.ppat.1012444},
pmid = {39173055},
issn = {1553-7374},
mesh = {*Virus Internalization ; *Ebolavirus/genetics/physiology/metabolism ; Humans ; *CRISPR-Cas Systems ; *Hemorrhagic Fever, Ebola/virology/metabolism/genetics ; Cation Transport Proteins/metabolism/genetics ; Class III Phosphatidylinositol 3-Kinases/metabolism/genetics ; Virus Replication ; Animals ; HEK293 Cells ; },
abstract = {The Ebola virus (EBOV) has emerged as a significant global health concern, notably during the 2013-2016 outbreak in West Africa. Despite the clinical approval of two EBOV antibody drugs, there is an urgent need for more diverse and effective antiviral drugs, along with comprehensive understanding of viral-host interactions. In this study, we harnessed a biologically contained EBOVΔVP30-EGFP cell culture model which could recapitulate the entire viral life cycle, to conduct a genome-wide CRISPR/Cas9 screen. Through this, we identified PIK3C3 (phosphatidylinositide 3-kinase) and SLC39A9 (zinc transporter) as crucial host factors for EBOV infection. Genetic depletion of SLC39A9 and PIK3C3 lead to reduction of EBOV entry, but not impact viral genome replication, suggesting that SLC39A9 and PIK3C3 act as entry factors, facilitating viral entry into host cells. Moreover, PIK3C3 kinase activity is indispensable for the internalization of EBOV virions, presumably through the regulation of endocytic and autophagic membrane traffic, which has been previously recognized as essential for EBOV internalization. Notably, our study demonstrated that PIK3C3 kinase inhibitor could effectively block EBOV infection, underscoring PIK3C3 as a promising drug target. Furthermore, biochemical analysis showed that recombinant SLC39A9 protein could directly bind viral GP protein, which further promotes the interaction of viral GP protein with cellular receptor NPC1. These findings suggests that SLC39A9 plays dual roles in EBOV entry. Initially, it serves as an attachment factor during the early entry phase by engaging with the viral GP protein. Subsequently, SLC39A9 functions an adaptor protein, facilitating the interaction between virions and the NPC1 receptor during the late entry phase, prior to cathepsin cleavage on the viral GP. In summary, this study offers novel insights into virus-host interactions, contributing valuable information for the development of new therapies against EBOV infection.},
}

*Virus Internalization
*Ebolavirus/genetics/physiology/metabolism
Humans
*CRISPR-Cas Systems
*Hemorrhagic Fever, Ebola/virology/metabolism/genetics
Cation Transport Proteins/metabolism/genetics
Class III Phosphatidylinositol 3-Kinases/metabolism/genetics
Virus Replication
Animals
HEK293 Cells

@article {pmid39172826,
year = {2024},
author = {Aguirre Rivera, J and Mao, G and Sabantsev, A and Panfilov, M and Hou, Q and Lindell, M and Chanez, C and Ritort, F and Jinek, M and Deindl, S},
title = {Massively parallel analysis of single-molecule dynamics on next-generation sequencing chips.},
journal = {Science (New York, N.Y.)},
volume = {385},
number = {6711},
pages = {892-898},
doi = {10.1126/science.adn5371},
pmid = {39172826},
issn = {1095-9203},
mesh = {*High-Throughput Nucleotide Sequencing/methods ; *Single Molecule Imaging/methods ; *DNA/chemistry/genetics ; *Microscopy, Fluorescence/methods ; CRISPR-Associated Protein 9 ; Sequence Analysis, DNA/methods ; Gene Library ; CRISPR-Cas Systems ; },
abstract = {Single-molecule techniques are ideally poised to characterize complex dynamics but are typically limited to investigating a small number of different samples. However, a large sequence or chemical space often needs to be explored to derive a comprehensive understanding of complex biological processes. Here we describe multiplexed single-molecule characterization at the library scale (MUSCLE), a method that combines single-molecule fluorescence microscopy with next-generation sequencing to enable highly multiplexed observations of complex dynamics. We comprehensively profiled the sequence dependence of DNA hairpin properties and Cas9-induced target DNA unwinding-rewinding dynamics. The ability to explore a large sequence space for Cas9 allowed us to identify a number of target sequences with unexpected behaviors. We envision that MUSCLE will enable the mechanistic exploration of many fundamental biological processes.},
}

*High-Throughput Nucleotide Sequencing/methods
*Single Molecule Imaging/methods
*DNA/chemistry/genetics
*Microscopy, Fluorescence/methods
CRISPR-Associated Protein 9
Sequence Analysis, DNA/methods
Gene Library
CRISPR-Cas Systems

@article {pmid39172289,
year = {2024},
author = {Wang, L and Cui, J and Zhang, N and Wang, X and Su, J and Vallés, MP and Wu, S and Yao, W and Chen, X and Chen, D},
title = {OsIPK1 frameshift mutations disturb phosphorus homeostasis and impair starch synthesis during grain filling in rice.},
journal = {Plant molecular biology},
volume = {114},
number = {5},
pages = {91},
pmid = {39172289},
issn = {1573-5028},
support = {21JCYBJC00010//Natural Science Foundation of Tianjin/ ; 60//Fundamental Research Funds for the Central Universities, Nankai University/ ; 32070349//National Natural Science Foundation of China/ ; ITTRRS20211000-02//Rice Industry Technological System of Tianjin/ ; },
mesh = {*Frameshift Mutation ; *Oryza/genetics/metabolism/growth & development ; *Starch/biosynthesis/metabolism ; *Phosphorus/metabolism ; *Plant Proteins/genetics/metabolism ; *Gene Expression Regulation, Plant ; *Homeostasis ; Seeds/genetics/metabolism/growth & development ; CRISPR-Cas Systems ; Edible Grain/genetics/metabolism ; Phosphotransferases (Alcohol Group Acceptor)/genetics/metabolism ; Plants, Genetically Modified ; Phytic Acid/metabolism/biosynthesis ; },
abstract = {Inositol 1,3,4,5,6-pentakisphosphate 2-kinase (IPK1) catalyzes the final step in phytic acid (InsP6) synthesis. In this study, the effects of OsIPK1 mutations on InsP6 synthesis, grain filling and their underlying mechanisms were investigated. Seven gRNAs were designed to disrupt the OsIPK1 gene via CRISPR/CAS9 system. Only 4 of them generated 29 individual insertion or deletion T0 plants, in which nine biallelic or heterozygous genotypes were identified. Segregation analysis revealed that OsIPK1 frameshift mutants are homozygous lethality. The biallelic and heterozygous frameshift mutants exhibited significant reduction in yield-related traits, particularly in the seed-setting rate and yield per plant. Despite a notable decline in pollen viability, the male and female gametes had comparable transmission rates to their progenies in the mutants. A significant number of the filling-aborted (FA) grains was observed in mature grains of these heterozygous frameshift mutants. These grains exhibited a nearly complete blockage of InsP6 synthesis, resulting in a pronounced increase in Pi content. In contrast, a slight decline in InsP6 content was observed in the plump grains. During the filling stage, owing to the excessive accumulation of Pi, starch synthesis was significantly impaired, and the endosperm development-specific gene expression was nearly abolished. Consistently, the activity of whereas AGPase, a key enzyme in starch synthesis, was significantly decreased and Pi transporter gene expression was upregulated in the FA grains. Taken together, these results demonstrate that OsIPK1 frameshift mutations result in excessive Pi accumulation, decreased starch synthesis, and ultimately leading to lower yields in rice.},
}

*Frameshift Mutation
*Oryza/genetics/metabolism/growth & development
*Starch/biosynthesis/metabolism
*Phosphorus/metabolism
*Plant Proteins/genetics/metabolism
*Gene Expression Regulation, Plant
*Homeostasis
Seeds/genetics/metabolism/growth & development
CRISPR-Cas Systems
Edible Grain/genetics/metabolism
Phosphotransferases (Alcohol Group Acceptor)/genetics/metabolism
Plants, Genetically Modified
Phytic Acid/metabolism/biosynthesis

@article {pmid39171866,
year = {2024},
author = {Alalmaie, A and Khashan, R},
title = {Mechanistic Insight Into the Conformational Changes of Cas8 Upon Binding to Different PAM Sequences in the Transposon-Encoded Type I-F CRISPR-Cas System.},
journal = {Proteins},
volume = {},
number = {},
pages = {},
doi = {10.1002/prot.26730},
pmid = {39171866},
issn = {1097-0134},
abstract = {The INTEGRATE system is a gene-editing approach that offers advantages over the widely used CRISPR-Cas9 system. It does not introduce double strand breaks in the target DNA but rather integrates the desired DNA sequence directly into it. The first step in the integration process is PAM recognition, which is critical to understanding and optimizing the system. Experimental testing revealed varying integration efficiencies of different PAM mutants, and computational simulations were carried out to gain mechanistic insight into the conformational changes of Cas8 during PAM recognition. Our results showed that the interaction between Arg246 and guanine at position (-1) of the target strand is critical for PAM recognition. We found that unfavorable interactions in the 5'-AC-3' PAM mutant disrupted this interaction and may be responsible for its 0% integration efficiency. Additionally, we discovered that PAM sequences not only initiate the integration process but also regulate it through an allosteric mechanism that connects the N-terminal domain and the helical bundle of Cas8. This allosteric regulation was present in all PAMs tested, even those with lower integration efficiencies, such as 5'-TC-3' and 5'-AC-3'. We identified the Cas8 residues that are involved in this regulation. Our findings provide valuable insights into PAM recognition mechanisms in the INTEGRATE system and can help improve the gene-editing technology.},
}

@article {pmid39168259,
year = {2024},
author = {Zanganeh, S and Zahedi, AM and Bardsiri, MS and Bazi, A and Bastanifard, M and Shool, S and Kouhbananinejad, SM and Farsinejad, A and Afgar, A and Shahabi, A and Mirzaei-Parsa, MJ},
title = {Recent advances and applications of the CRISPR-Cas system in the gene therapy of blood disorders.},
journal = {Gene},
volume = {},
number = {},
pages = {148865},
doi = {10.1016/j.gene.2024.148865},
pmid = {39168259},
issn = {1879-0038},
}

@article {pmid39089555,
year = {2024},
author = {Chen, X and Moran Torres, JP and Tedjai, SVK and Lugones, LG and Wösten, HAB},
title = {Functional analysis of FlbA-regulated transcription factor genes in Aspergillus niger using a multiplexed CRISPRoff system.},
journal = {International journal of biological macromolecules},
volume = {277},
number = {Pt 2},
pages = {134326},
doi = {10.1016/j.ijbiomac.2024.134326},
pmid = {39089555},
issn = {1879-0003},
mesh = {*Aspergillus niger/genetics ; *Transcription Factors/genetics/metabolism ; *Gene Expression Regulation, Fungal ; *Fungal Proteins/genetics ; CRISPR-Cas Systems/genetics ; Spores, Fungal/genetics ; Phenotype ; Promoter Regions, Genetic/genetics ; },
abstract = {FlbA of Aspergillus niger (indirectly) regulates 36 transcription factor (TF) genes. As a result, it promotes sporulation and represses vegetative growth, protein secretion and lysis. In this study, the functions of part of the FlbA-regulated TF genes were studied by using CRISPRoff. This system was recently introduced as an epigenetic tool for modulating gene expression in A. niger. A plasmid encompassing an optimized CRISPRoff system as well as a library of sgRNA genes that target the promoters of the 36 FlbA-regulated TF genes was introduced in A. niger. Out of 24 transformants that exhibited a sporulation phenotype, 12 and 18 strains also showed a biomass and secretion phenotype, respectively. The transforming sgRNAs, and thus the genes responsible for the phenotypes, were identified from five of the transformants. The results show that the genes dofA, dofB, dofC, dofD, and socA are involved in sporulation and extracellular enzyme activity, while dofA and socA also play roles in biomass formation. Overall, this study shows that the multiplexed CRISPRoff system can be effectively used for functional analysis of genes in a fungus.},
}

*Aspergillus niger/genetics
*Transcription Factors/genetics/metabolism
*Gene Expression Regulation, Fungal
*Fungal Proteins/genetics
CRISPR-Cas Systems/genetics
Spores, Fungal/genetics
Phenotype
Promoter Regions, Genetic/genetics

@article {pmid39019219,
year = {2024},
author = {Yun, S and Noh, M and Yu, J and Kim, HJ and Hui, CC and Lee, H and Son, JE},
title = {Unlocking biological mechanisms with integrative functional genomics approaches.},
journal = {Molecules and cells},
volume = {47},
number = {8},
pages = {100092},
doi = {10.1016/j.mocell.2024.100092},
pmid = {39019219},
issn = {0219-1032},
mesh = {Humans ; *Genomics/methods ; CRISPR-Cas Systems ; Epigenesis, Genetic ; Chromatin/metabolism/genetics ; High-Throughput Nucleotide Sequencing/methods ; },
abstract = {Reverse genetics offers precise functional insights into genes through the targeted manipulation of gene expression followed by phenotypic assessment. While these approaches have proven effective in model organisms such as Saccharomyces cerevisiae, large-scale genetic manipulations in human cells were historically unfeasible due to methodological limitations. However, recent advancements in functional genomics, particularly clustered regularly interspaced short palindromic repeats (CRISPR)-based screening technologies and next-generation sequencing platforms, have enabled pooled screening technologies that allow massively parallel, unbiased assessments of biological phenomena in human cells. This review provides a comprehensive overview of cutting-edge functional genomic screening technologies applicable to human cells, ranging from short hairpin RNA screens to modern CRISPR screens. Additionally, we explore the integration of CRISPR platforms with single-cell approaches to monitor gene expression, chromatin accessibility, epigenetic regulation, and chromatin architecture following genetic perturbations at the omics level. By offering an in-depth understanding of these genomic screening methods, this review aims to provide insights into more targeted and effective strategies for genomic research and personalized medicine.},
}

Humans
*Genomics/methods
CRISPR-Cas Systems
Epigenesis, Genetic
Chromatin/metabolism/genetics
High-Throughput Nucleotide Sequencing/methods

@article {pmid38878231,
year = {2024},
author = {Dalgleish, R},
title = {Re: Identification of a family with van der Hoeve's syndrome harboring a novel COL1A1 mutation and generation of patient-derived iPSC lines and CRISPR/Cas9-corrected isogenic iPSCs.},
journal = {Human cell},
volume = {37},
number = {5},
pages = {1610-1611},
pmid = {38878231},
issn = {1749-0774},
mesh = {Humans ; *Induced Pluripotent Stem Cells ; *CRISPR-Cas Systems/genetics ; *Mutation/genetics ; *Collagen Type I, alpha 1 Chain ; *Collagen Type I/genetics/metabolism ; Cell Line ; },
}

Humans
*Induced Pluripotent Stem Cells
*CRISPR-Cas Systems/genetics
*Mutation/genetics
*Collagen Type I, alpha 1 Chain
*Collagen Type I/genetics/metabolism
Cell Line

@article {pmid39167979,
year = {2024},
author = {Lei, H and Xiong, W and Li, M and Qi, Q and Liu, X and Wang, S and Tian, T and Zhou, X},
title = {Enhanced control of RNA modification and CRISPR-Cas activity through redox-triggered disulfide cleavage.},
journal = {Bioorganic & medicinal chemistry},
volume = {112},
number = {},
pages = {117878},
doi = {10.1016/j.bmc.2024.117878},
pmid = {39167979},
issn = {1464-3391},
abstract = {Chemical RNA modification has emerged as a flexible approach for post-synthetic modifications in chemical biology research. Guide RNA (gRNA) plays a crucial role in the clustered regularly interspaced short palindromic repeats and associated protein system (CRISPR-Cas). Several toolkits have been developed to regulate gene expression and editing through modifications of gRNA. However, conditional regulation strategies to control gene editing in cells as required are still lacking. In this context, we introduce a strategy employing a cyclic disulfide-substituted acylating agent to randomly acylate the 2'-OH group on the gRNA strand. The CRISPR-Cas systems demonstrate off-on transformation activity driven by redox-triggered disulfide cleavage and undergo intramolecular cyclization, which releases the functionalized gRNA. Dithiothreitol (DTT) exhibits superior reductive capabilities in cleaving disulfides compared to glutathione (GSH), requiring fewer reductants. This acylation method with cyclic disulfides enables conditional control of CRISPR-Cas9, CRISPR-Cas13a, RNA hybridization, and aptamer folding. Our strategy facilitates precise in vivo control of gene editing, making it particularly valuable for targeted applications.},
}

@article {pmid39166823,
year = {2024},
author = {He, Y and Gao, M and Zhu, X and Peng, W and Zhou, Y and Cheng, J and Bai, L and Bao, J},
title = {Large-Scale Formation and Long-Term Culture of Hepatocyte Organoids From Streamlined In Vivo Genome-Edited GGTA1[-/-] Pigs for Bioartificial Liver Applications.},
journal = {Xenotransplantation},
volume = {31},
number = {4},
pages = {e12878},
doi = {10.1111/xen.12878},
pmid = {39166823},
issn = {1399-3089},
support = {82070640//National Natural Science Foundation of China/ ; ZYIC21014//West China Hospital Sichuan University/ ; },
mesh = {Animals ; *Hepatocytes ; *Galactosyltransferases/genetics ; Swine ; *Transplantation, Heterologous/methods ; *Organoids/metabolism ; *Liver, Artificial ; *Gene Editing/methods ; Humans ; Animals, Genetically Modified ; CRISPR-Cas Systems ; Gene Knockout Techniques/methods ; Coculture Techniques/methods ; },
abstract = {Hepatocyte transplantation and bioartificial liver (BAL) systems hold significant promise as less invasive alternatives to traditional transplantation, providing crucial temporary support for patients with acute and chronic liver failure. Although human hepatocytes are ideal, their use is limited by ethical concerns and donor availability, leading to the use of porcine hepatocytes in BAL systems due to their functional similarities. Recent advancements in gene-editing technology have improved porcine organ xenotransplantation clinical trials by addressing immune rejection issues. Gene-edited pigs, such as alpha-1,3-galactosyltransferase (GGTA1) knockout pigs, offer a secure source of primary cells for BAL systems. Our research focuses on optimizing the safety and functionality of porcine primary hepatocytes during large-scale cultivation. We achieved this by creating GGTA1 knockout pigs through one-step delivery of CRISPR/Cas9 to pig zygotes via oviduct injection of rAAV, and enhancing hepatocyte viability and function by co-culturing hepatocytes with Roof plate-specific spondin 1 overexpressing HUVECs (R-HUVECs). Using a Rocker culture system, approximately 10[10] primary porcine hepatocytes and R-HUVECs rapidly formed organoids with a diameter of 92.1 ± 28.1 µm within 24 h. These organoids not only maintained excellent functionality but also supported partial hepatocyte self-renewal during long-term culture over 28 days. Gene-edited primary porcine hepatocyte organoids will significantly advance the applications of hepatocyte transplantation and BAL systems.},
}

Animals
*Hepatocytes
*Galactosyltransferases/genetics
Swine
*Transplantation, Heterologous/methods
*Organoids/metabolism
*Liver, Artificial
*Gene Editing/methods
Humans
Animals, Genetically Modified
CRISPR-Cas Systems
Gene Knockout Techniques/methods
Coculture Techniques/methods

@article {pmid39166476,
year = {2024},
author = {Binder, SC and Schneberger, N and Schmitz, M and Engeser, M and Geyer, M and Rouillon, C and Hagelueken, G},
title = {The SAVED domain of the type III CRISPR protease CalpL is a ring nuclease.},
journal = {Nucleic acids research},
volume = {},
number = {},
pages = {},
doi = {10.1093/nar/gkae676},
pmid = {39166476},
issn = {1362-4962},
support = {HA-6805/6-1//German Research Foundation/ ; EXC2151-390873048//Germany's Excellence Strategy/ ; //University of Bonn/ ; },
abstract = {Prokaryotic CRISPR-Cas immune systems detect and cleave foreign nucleic acids. In type III CRISPR-Cas systems, the Cas10 subunit of the activated recognition complex synthesizes cyclic oligoadenylates (cOAs), second messengers that activate downstream ancillary effector proteins. Once the viral attack has been weathered, elimination of extant cOA is essential to limit the antiviral response and to allow cellular recovery. Various families of ring nucleases have been identified, specializing in the degradation of cOAs either as standalone enzymes or as domains of effector proteins. Here we describe the ring nuclease activity inherent in the SAVED domain of the cA4-activated CRISPR Lon protease CalpL. We characterize the kinetics of cA4 cleavage and identify key catalytic residues. We demonstrate that cA4-induced oligomerization of CalpL is essential not only for activation of the protease, but is also required for nuclease activity. Further, the nuclease activity of CalpL poses a limitation to the protease reaction, indicating a mechanism for regulation of the CalpL/T/S signaling cascade. This work is the first demonstration of a catalytic SAVED domain and gives new insights into the dynamics of transcriptional adaption in CRISPR defense systems.},
}

@article {pmid39166136,
year = {2024},
author = {Gholami, A and Mohkam, M and Soleimanian, S and Sadraeian, M and Lauto, A},
title = {Bacterial nanotechnology as a paradigm in targeted cancer therapeutic delivery and immunotherapy.},
journal = {Microsystems & nanoengineering},
volume = {10},
number = {},
pages = {113},
pmid = {39166136},
issn = {2055-7434},
abstract = {Cancer, a multifaceted and diverse ailment, presents formidable obstacles to traditional treatment modalities. Nanotechnology presents novel prospects for surmounting these challenges through its capacity to facilitate meticulous and regulated administration of therapeutic agents to malignant cells while concurrently modulating the immune system to combat neoplasms. Bacteria and their derivatives have emerged as highly versatile and multifunctional platforms for cancer nanotherapy within the realm of nanomaterials. This comprehensive review delves into the multifaceted and groundbreaking implementations of bacterial nanotechnology within cancer therapy. This review encompasses four primary facets: the utilization of bacteria as living conveyors of medicinal substances, the employment of bacterial components as agents that stimulate the immune system, the deployment of bacterial vectors as tools for delivering genetic material, and the development of bacteria-derived nano-drugs as intelligent nano-medications. Furthermore, we elucidate the merits and modalities of operation pertaining to these bacterial nano-systems, along with their capacity to synergize with other cutting-edge nanotechnologies, such as CRISPR-Cas systems. Additionally, we offer insightful viewpoints regarding the forthcoming trajectories and prospects within this expanding domain. It is our deduction that bacterial nanotechnology embodies a propitious and innovative paradigm in the realm of cancer therapy, which has the potential to provide numerous advantages and synergistic effects in enhancing the outcomes and quality of life for individuals afflicted with cancer.},
}

@article {pmid39165749,
year = {2024},
author = {Hwang, S and Lee, W and Lee, Y},
title = {Development of a nucleic acid detection method based on the CRISPR-Cas13 for point-of-care testing of bovine viral diarrhea virus-1b.},
journal = {Journal of animal science and technology},
volume = {66},
number = {4},
pages = {781-791},
pmid = {39165749},
issn = {2055-0391},
abstract = {Bovine viral diarrhea (BVD) is a single-stranded, positive-sense ribonucleic acid (RNA) virus belonging to the genus Pestivirus of the Flaviviridae family. BVD frequently causes economic losses to farmers. Among bovine viral diarrhea virus (BVDV) strains, BVDV-1b is predominant and widespread in Hanwoo calves. Reverse-transcription polymerase chain reaction (RT-PCR) is an essential method for diagnosing BVDV-1b and has become the gold standard for diagnosis in the Republic of Korea. However, this diagnostic method is time-consuming and requires expensive equipment. Therefore, Clustered regularly interspaced short palindromic repeats-Cas (CRISPR-Cas) systems have been used for point-of-care (POC) testing of viruses. Developing a sensitive and specific method for POC testing of BVDV-1b would be advantageous for controlling the spread of infection. Thus, this study aimed to develop a novel nucleic acid detection method using the CRISPR-Cas13 system for POC testing of BVDV-1b. The sequence of the BVD virus was extracted from National Center for Biotechnology Information (NC_001461.1), and the 5' untranslated region, commonly used for detection, was selected. CRISPR RNA (crRNA) was designed using the Cas13 design program and optimized for the expression and purification of the LwCas13a protein. Madin Darby bovine kidney (MDBK) cells were infected with BVDV-1b, incubated, and the viral RNA was extracted. To enable POC viral detection, the compatibility of the CRISPR-Cas13 system was verified with a paper-based strip through collateral cleavage activity. Finally, a colorimetric assay was used to evaluate the detection of BVDV-1b by combining the previously obtained crRNA and Cas13a protein on a paper strip. In conclusion, the CRISPR-Cas13 system is highly sensitive, specific, and capable of nucleic acid detection, making it an optimal system for the early point-of-care testing of BVDV-1b.},
}

@article {pmid39165171,
year = {2024},
author = {Liaghat, A and Yang, J and Whitaker, R and Pascual, M},
title = {Punctuated virus-driven succession generates dynamical alternations in CRISPR-mediated microbe-virus coevolution.},
journal = {Journal of the Royal Society, Interface},
volume = {21},
number = {217},
pages = {20240195},
pmid = {39165171},
issn = {1742-5662},
support = {//Division of Biological Infrastructure/ ; //Gordon and Betty Moore Foundation/ ; },
mesh = {*CRISPR-Cas Systems ; Host-Pathogen Interactions/genetics ; Models, Biological ; Viruses/genetics ; Evolution, Molecular ; Biological Evolution ; },
abstract = {The coevolutionary dynamics of lytic viruses and microbes with CRISPR-Cas immunity exhibit alternations between sustained host control of viral proliferation and major viral epidemics in previous computational models. These alternating dynamics have yet to be observed in other host-pathogen systems. Here, we address the breakdown of control and transition to large outbreaks with a stochastic eco-evolutionary model. We establish the role of host density-dependent competition in punctuated virus-driven succession and associated diversity trends that concentrate escape pathways during control phases. Using infection and escape networks, we derive the viral emergence probability whose fluctuations of increasing size and frequency characterize the approach to large outbreaks. We explore alternation probabilities as a function of non-dimensional parameters related to the probability of viral escape and host competition. Our results demonstrate how emergent feedbacks between host competition and viral diversification render the host immune structure fragile, potentiating a dynamical transition to large epidemics.},
}

*CRISPR-Cas Systems
Host-Pathogen Interactions/genetics
Models, Biological
Viruses/genetics
Evolution, Molecular
Biological Evolution

@article {pmid39163906,
year = {2024},
author = {Sabrina Freitas-Alves, N and Moreira-Pinto, CE and Távora, FTPK and Paes-de-Melo, B and Arraes, FBM and Lourenço-Tessutti, IT and Moura, SM and Oliveira, AC and Morgante, CV and Qi, Y and Fatima Grossi-de-Sa, M},
title = {CRISPR/Cas genome editing in soybean: Challenges and new insights to overcome existing bottlenecks.},
journal = {Journal of advanced research},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.jare.2024.08.024},
pmid = {39163906},
issn = {2090-1224},
abstract = {BACKGROUND: Soybean is a worldwide-cultivated crop due to its applications in the food, feed, and biodiesel industries. Genome editing in soybean began with ZFN and TALEN technologies; however, CRISPR/Cas has emerged and shortly became the preferable approach for soybean genome manipulation since it is more precise, easy to handle, and cost-effective. Recent reports have focused on the conventional Cas9 nuclease, Cas9 nickase (nCas9) derived base editors, and Cas12a (formally Cpf1) as the most commonly used genome editors in soybean. Nonetheless, several challenges in the complex plant genetic engineering pipeline need to be overcome to effectively edit the genome of an elite soybean cultivar. These challenges include (1) optimizing CRISPR cassette design (i.e., gRNA and Cas promoters, gRNA design and testing, number of gRNAs, and binary vector), (2) improving transformation frequency, (3) increasing the editing efficiency ratio of targeted plant cells, and (4) improving soybean crop production.

AIM OF REVIEW: This review provides an overview of soybean genome editing using CRISPR/Cas technology, discusses current challenges, and highlights theoretical (insights) and practical suggestions to overcome the existing bottlenecks.

The CRISPR/Cas system was discovered as part of the bacterial innate immune system. It has been used as a biotechnological tool for genome editing and efficiently applied in soybean to unveil gene function, improve agronomic traits such as yield and nutritional grain quality, and enhance biotic and abiotic stress tolerance. To date, the editing efficiency has been validated using protoplasts and hairy root assays, while stable plant transformation relies on Agrobacterium-mediated and particle bombardment methods. Nevertheless, most steps of the CRISPR/Cas workflow require optimizations to achieve a more effective genome editing in soybean plants.},
}

@article {pmid39134876,
year = {2024},
author = {Abdallah, K and Huys, I and Claes, KJ and Simoens, S},
title = {Budget Impact of Disease-Modifying Treatments and a CRISPR Gene-Edited Therapy for Sickle Cell Disease.},
journal = {Clinical drug investigation},
volume = {44},
number = {8},
pages = {611-627},
pmid = {39134876},
issn = {1179-1918},
support = {G0B9819N//Fonds Wetenschappelijk Onderzoek/ ; },
mesh = {Humans ; *Anemia, Sickle Cell/genetics/drug therapy/economics/therapy ; *Antibodies, Monoclonal, Humanized/economics/therapeutic use ; *Budgets ; Belgium ; Gene Editing/methods/economics ; CRISPR-Cas Systems ; Genetic Therapy/economics/methods ; Cost-Benefit Analysis ; },
abstract = {BACKGROUND AND OBJECTIVE: Treatment of sickle cell disease (SCD) has traditionally focused on symptomatic and preventative care. Recent advances in novel therapeutic developments, likely to be orphan-designated, are anticipated to carry a substantial price tag. This study assesses the potential budget impact of adopting disease-modifying treatments, crizanlizumab and voxelotor, and pioneering CRISPR gene-edited therapy, CTX001, in the Belgian healthcare system.

METHODS: The perspective of the Belgian healthcare payer (RIZIV-INAMI including patient copayments), a 5-year horizon with a 2-10% uptake of disease-modifying interventions, and a 2% uptake of CTX001 were considered. Data, encompassing target population, current (chronic and acute management, curative hematopoietic stem cell transplantation) and new (crizanlizumab, voxelotor, and CTX001) interventions, clinical effectiveness, adverse events, healthcare resource utilization, and associated costs, were gathered through a comprehensive literature review (first phase) and two Delphi panels involving hematologists (second phase). The cost difference between a "world with and without crizanlizumab, voxelotor, and CTX001" was calculated to obtain the budget impact. Three scenario analyses were conducted: a 5-13% and 4% uptake analysis, a 10-18% and 8% uptake analysis, respectively for disease-modifying treatments (crizanlizumab and voxelotor) and CTX001, and a 0% crizanlizumab uptake and managed entry agreements analysis . A ± 20% univariate sensitivity analysis was performed to test the robustness of the analysis.

RESULTS: The total five-year cumulative budget impact was estimated at €30,024,968, with 91% attributed to drug acquisition costs. The largest budget impact share was for CTX001 (€25,575,150), while crizanlizumab (€2,301,095) and voxelotor (€2,148,723) was relatively small. In scenarios one and three, a two-fold increase of the cumulative budget impact to €60,731,772 and a four-fold increase to €120,846,256 from the base case was observed. In scenario three, this budget impact decreased by 63% to €11,212,766. Patient population size, number of treated patients, and drug costs influenced the analysis the most, while discontinuation, acute crisis, and adverse event rates had comparatively minimal impact.

CONCLUSIONS: Belgian decision-makers may consider alternative financing models, such as outcome-based risk-sharing agreements or annuities, to ensure sustainable coverage of these treatments. This study adheres to recommended practices for assessing budget impact of orphan drugs, distinguishing it from earlier studies with potentially weaker methodologies.},
}

Humans
*Anemia, Sickle Cell/genetics/drug therapy/economics/therapy
*Antibodies, Monoclonal, Humanized/economics/therapeutic use
*Budgets
Belgium
Gene Editing/methods/economics
CRISPR-Cas Systems
Genetic Therapy/economics/methods
Cost-Benefit Analysis

@article {pmid38987595,
year = {2024},
author = {Brödel, AK and Charpenay, LH and Galtier, M and Fuche, FJ and Terrasse, R and Poquet, C and Havránek, J and Pignotti, S and Krawczyk, A and Arraou, M and Prevot, G and Spadoni, D and Yarnall, MTN and Hessel, EM and Fernandez-Rodriguez, J and Duportet, X and Bikard, D},
title = {In situ targeted base editing of bacteria in the mouse gut.},
journal = {Nature},
volume = {632},
number = {8026},
pages = {877-884},
pmid = {38987595},
issn = {1476-4687},
mesh = {Animals ; Mice ; *Escherichia coli/genetics ; *Gene Editing/methods ; *Klebsiella pneumoniae/genetics ; *beta-Lactamases/genetics/metabolism ; *Gastrointestinal Microbiome ; Female ; Bacteriophages/genetics ; CRISPR-Cas Systems/genetics ; Male ; Gastrointestinal Tract/microbiology ; Genes, Bacterial/genetics ; Genetic Vectors/genetics ; },
abstract = {Microbiome research is now demonstrating a growing number of bacterial strains and genes that affect our health[1]. Although CRISPR-derived tools have shown great success in editing disease-driving genes in human cells[2], we currently lack the tools to achieve comparable success for bacterial targets in situ. Here we engineer a phage-derived particle to deliver a base editor and modify Escherichia coli colonizing the mouse gut. Editing of a β-lactamase gene in a model E. coli strain resulted in a median editing efficiency of 93% of the target bacterial population with a single dose. Edited bacteria were stably maintained in the mouse gut for at least 42 days following treatment. This was achieved using a non-replicative DNA vector, preventing maintenance and dissemination of the payload. We then leveraged this approach to edit several genes of therapeutic relevance in E. coli and Klebsiella pneumoniae strains in vitro and demonstrate in situ editing of a gene involved in the production of curli in a pathogenic E. coli strain. Our work demonstrates the feasibility of modifying bacteria directly in the gut, offering a new avenue to investigate the function of bacterial genes and opening the door to the design of new microbiome-targeted therapies.},
}

Animals
Mice
*Escherichia coli/genetics
*Gene Editing/methods
*Klebsiella pneumoniae/genetics
*beta-Lactamases/genetics/metabolism
*Gastrointestinal Microbiome
Female
Bacteriophages/genetics
CRISPR-Cas Systems/genetics
Male
Gastrointestinal Tract/microbiology
Genes, Bacterial/genetics
Genetic Vectors/genetics

@article {pmid38894572,
year = {2024},
author = {Bai, Y and Nan, Y and Wu, T and Zhu, A and Xie, X and Sun, Y and Deng, Y and Dou, Z and Hu, X and Zhou, R and Xu, S and Zhang, Y and Fan, J and Ju, D},
title = {Lipid Nanoparticle-Mediated Delivery of CRISPR-Cas9 Against Rubicon Ameliorates NAFLD by Modulating CD36 Along with Glycerophospholipid Metabolism.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {11},
number = {31},
pages = {e2400493},
pmid = {38894572},
issn = {2198-3844},
support = {2023YFC3404000//National Key Research and Development Program of China/ ; 82371781//National Natural Science Foundation of China/ ; 82073752//National Natural Science Foundation of China/ ; 32070935//National Natural Science Foundation of China/ ; 21QB1401800//Shanghai Rising-Star Program/ ; 20S11904700//Shanghai Science and Technology Development Foundation/ ; },
mesh = {Animals ; *Non-alcoholic Fatty Liver Disease/genetics/metabolism/therapy ; Mice ; *Glycerophospholipids/metabolism ; *Nanoparticles ; *CRISPR-Cas Systems/genetics ; *CD36 Antigens/genetics/metabolism ; *Disease Models, Animal ; Mice, Inbred C57BL ; Male ; Lipids ; Lipid Metabolism/genetics ; Humans ; Liposomes ; },
abstract = {Non-alcoholic fatty liver disease (NAFLD) is a prominent cause of various chronic metabolic hepatic diseases with limited therapeutics. Rubicon, an essential regulator in lysosomal degradation, is reported to exacerbate hepatic steatosis in NAFLD mice and patients, indicating its probability of being a therapeutic target for NAFLD treatment. In this study, the therapeutic potential of Rubicon blockage is investigated. Lipid nanoparticles carrying Rubicon-specific CRISPR-Cas9 components exhibited liver accumulation, cell internalization, and Rubicon knockdown. A single administration of the nanoparticles results in attenuated lipid deposition and hepatic steatosis, with lower circulating lipid levels and decreased adipocyte size in NAFLD mice. Furthermore, the increase of phosphatidylcholine and phosphatidylethanolamine levels can be observed in the NAFLD mice livers after Rubicon silencing, along with regulatory effects on metabolism-related genes such as CD36, Gpcpd1, Chka, and Lpin2. The results indicate that knockdown of Rubicon improves glycerophospholipid metabolism and thereby ameliorates the NAFLD progression, which provides a potential strategy for NAFLD therapy via the restoration of Rubicon.},
}

Animals
*Non-alcoholic Fatty Liver Disease/genetics/metabolism/therapy
Mice
*Glycerophospholipids/metabolism
*Nanoparticles
*CRISPR-Cas Systems/genetics
*CD36 Antigens/genetics/metabolism
*Disease Models, Animal
Mice, Inbred C57BL
Male
Lipids
Lipid Metabolism/genetics
Humans
Liposomes

@article {pmid38881503,
year = {2024},
author = {Zhang, G and Song, Z and Huang, S and Wang, Y and Sun, J and Qiao, L and Li, G and Feng, Y and Han, W and Tang, J and Chen, Y and Huang, X and Liu, F and Wang, X and Liu, J},
title = {nCas9 Engineering for Improved Target Interaction Presents an Effective Strategy to Enhance Base Editing.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {11},
number = {31},
pages = {e2405426},
pmid = {38881503},
issn = {2198-3844},
support = {2021YFF1000704//National Key R&D Program of China/ ; 2019YFA0802802//National Key R&D Program of China/ ; 2023ZD0405104//Sci-Tech Innovation 2030 Key Program/ ; 32272848//National Natural Science Foundation of China/ ; 2023TQ0329//China Postdoctoral Science Foundation/ ; 2023M743261//China Postdoctoral Science Foundation/ ; 2022ZB708//Excellent Postdoctoral Fellow of Jiangsu Province/ ; ZDBS-ZRKJZ-TLC008//Key Research Program of Chinese Academy of Sciences/ ; 2022GD-TSLD-46//Double chain fusion project of Shaanxi/ ; },
mesh = {*Gene Editing/methods ; Humans ; *CRISPR-Cas Systems/genetics ; *CRISPR-Associated Protein 9/genetics/metabolism ; HEK293 Cells ; },
abstract = {Base editors (BEs) are a recent generation of genome editing tools that couple a cytidine or adenosine deaminase activity to a catalytically impaired Cas9 moiety (nCas9) to enable specific base conversions at the targeted genomic loci. Given their strong application potential, BEs are under active developments toward greater levels of efficiency and safety. Here, a previously overlooked nCas9-centric strategy is explored for enhancement of BE. Based on a cytosine BE (CBE), 20 point mutations associated with nCas9-target interaction are tested. Subsequently, from the initial positive X-to-arginine hits, combinatorial modifications are applied to establish further enhanced CBE variants (1.1-1.3). Parallel nCas9 modifications in other versions of CBEs including A3A-Y130F-BE4max, YEE-BE4max, CGBE, and split-AncBE4max, as well as in the context of two adenine BEs (ABE), likewise enhance their respective activities. The same strategy also substantially improves the efficiencies of high-fidelity nCas9/BEs. Further evidence confirms that the stabilization of nCas9-substrate interactions underlies the enhanced BE activities. In support of their translational potential, the engineered CBE and ABE variants respectively enable 82% and 25% higher rates of editing than the controls in primary human T-cells. This study thus demonstrates a highly adaptable strategy for enhancing BE, and for optimizing other forms of Cas9-derived tools.},
}

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

@article {pmid39163781,
year = {2024},
author = {Shi, X and Zhang, J and Ding, Y and Li, H and Yao, S and Hu, T and Zhao, C and Wang, J},
title = {Ultrasensitive detection platform for Staphylococcus aureus based on DNAzyme tandem blocking CRISPR/Cas12a system.},
journal = {Biosensors & bioelectronics},
volume = {264},
number = {},
pages = {116671},
doi = {10.1016/j.bios.2024.116671},
pmid = {39163781},
issn = {1873-4235},
abstract = {Detection methods based on CRISPR/Cas12a have been widely developed in the application of pathogenic microorganisms to guarantee food safety and public health. For sensitive detection, the CRISPR-based strategies are often in tandem with amplification methods. However, that may increase the detection time and the process may introduce nucleic acid contamination resulting in non-specific amplification. Herein, we established a sensitive S. aureus detection strategy based on the CRISPR/Cas12a system combined with DNAzyme. The activity of Cas12a is blocked by extending the spacer of crRNA (bcrRNA) and can be reactivated by Mn[2+]. NH2-modified S. aureus-specific aptamer was loaded on the surface of Fe3O4 MNPs (apt-Fe3O4 MNPs) and MnO2 NPs (apt-MnO2 NPs) by EDC/NHS chemistry. The S. aureus was captured to form apt-Fe3O4 MNPs/S. aureus/apt-MnO2 NPs complex and then MnO2 NPs were etched to release Mn[2+] to activate DNAzyme. The active DNAzyme can cleave the hairpin structure in bcrRNA to recover the activity of the CRISPR/Cas system. By initiating the whole detection process by generating Mn[2+] through nanoparticle etching, we established a rapid detection assay without nucleic acid extraction and amplification process. The proposed strategy has been applied in the ultrasensitive quantitative detection of S. aureus and has shown good performance with an LOD of 5 CFU/mL in 29 min. Besides, the proposed method can potentially be applied to other targets by simply changing the recognition element and has the prospect of developing a universal detection strategy.},
}

@article {pmid39163294,
year = {2024},
author = {Kim, CS and Cairns, J and Quarantotti, V and Kaczkowski, B and Wang, Y and Konings, P and Zhang, X},
title = {A statistical simulation model to guide the choices of analytical methods in arrayed CRISPR screen experiments.},
journal = {PloS one},
volume = {19},
number = {8},
pages = {e0307445},
doi = {10.1371/journal.pone.0307445},
pmid = {39163294},
issn = {1932-6203},
mesh = {*CRISPR-Cas Systems ; *Models, Statistical ; Gene Editing/methods ; Humans ; Computer Simulation ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {An arrayed CRISPR screen is a high-throughput functional genomic screening method, which typically uses 384 well plates and has different gene knockouts in different wells. Despite various computational workflows, there is currently no systematic way to find what is a good workflow for arrayed CRISPR screening data analysis. To guide this choice, we developed a statistical simulation model that mimics the data generating process of arrayed CRISPR screening experiments. Our model is flexible and can simulate effects on phenotypic readouts of various experimental factors, such as the effect size of gene editing, as well as biological and technical variations. With two examples, we showed that the simulation model can assist making principled choice of normalization and hit calling method for the arrayed CRISPR data analysis. This simulation model is implemented in an R package and can be downloaded from Github.},
}

*CRISPR-Cas Systems
*Models, Statistical
Gene Editing/methods
Humans
Computer Simulation
Clustered Regularly Interspaced Short Palindromic Repeats/genetics

@article {pmid39162876,
year = {2024},
author = {Li, G and Li, S and Li, X and He, W and Tan, X and Liang, J and Zhou, Z},
title = {A novel electrochemical aptasensor based on NrGO-H-Mn3O4 NPs integrated CRISPR/Cas12a system for ultrasensitive low-density lipoprotein determination.},
journal = {Mikrochimica acta},
volume = {191},
number = {9},
pages = {547},
pmid = {39162876},
issn = {1436-5073},
support = {Nos. 62161009//National Natural Science Foundation of China/ ; YCSW2023301//Innovation Project of Guangxi Graduate Education/ ; RC-XJ2022000401//Projects of Talents Recruitment of GDUPT/ ; 2024GXNSFAA010440//the Natural Science Foundation of Guangxi Zhuang Autonomous Region/ ; No. GuikeAA 24011005//the Guangxi Science and Technology Major Program/ ; },
mesh = {*Manganese Compounds/chemistry ; *Lipoproteins, LDL/blood/chemistry ; Humans ; *Electrochemical Techniques/methods ; *Oxides/chemistry ; *Graphite/chemistry ; *CRISPR-Cas Systems ; *Aptamers, Nucleotide/chemistry ; *Hemin/chemistry ; *Biosensing Techniques/methods ; *Limit of Detection ; DNA, Single-Stranded/chemistry ; Nanoparticles/chemistry ; },
abstract = {Atherosclerosis cardiovascular disease (ASCVD) has become one of the leading death causes in humans. Low-density lipoprotein (LDL) is an important biomarker for assessing ASCVD risk level. Thus, monitoring LDL levels can be an important means for early diagnosis of ASCVD. Herein, a novel electrochemical aptasensor for determination LDL was designed based on nitrogen-doped reduced graphene oxide-hemin-manganese oxide nanoparticles (NrGO-H-Mn3O4 NPs) integrated with clustered regularly interspaced short palindromic repeats and associated proteins (CRISPR/Cas12a) system. NrGO-H-Mn3O4 NPs not only have a large surface area and remarkable enhanced electrical conductivity but also the interconversion of different valence states of iron in hemin can provide an electrical signal. Nonspecific single-stranded DNA (ssDNA) was bound to NrGO-H-Mn3O4 NPs to form a signaling probe and was immobilized on the electrode surface. The CRISPR/Cas12a system has excellent trans-cleavage activity, which can be used to cleave ssDNA, thus detaching the NrGO-H-Mn3O4 NPs from the sensing interface and attenuating the electrical signal. Significant signal change triggered by the target was ultimately obtained, thus achieving sensitive detection of the LDL in range from 0.005 to 1000.0 nM with the detection limit of 0.005 nM. The proposed sensor exhibited good stability, selectivity, and stability and achieved reliable detection of LDL in serum samples, demonstrating its promising application prospects for the diagnostic application of LDL.},
}

*Manganese Compounds/chemistry
*Lipoproteins, LDL/blood/chemistry
Humans
*Electrochemical Techniques/methods
*Oxides/chemistry
*Graphite/chemistry
*CRISPR-Cas Systems
*Aptamers, Nucleotide/chemistry
*Hemin/chemistry
*Biosensing Techniques/methods
*Limit of Detection
DNA, Single-Stranded/chemistry
Nanoparticles/chemistry

@article {pmid39162556,
year = {2024},
author = {Ma, Z and Qian, C and Yao, Z and Tang, M and Chen, K and Zhao, D and Hu, P and Zhou, T and Cao, J},
title = {Coexistence of plasmid-mediated tmexCD2-toprJ2, blaIMP-4, and blaNDM-1 in Klebsiella quasipneumoniae.},
journal = {Microbiology spectrum},
volume = {},
number = {},
pages = {e0387423},
doi = {10.1128/spectrum.03874-23},
pmid = {39162556},
issn = {2165-0497},
abstract = {Klebsiella quasipneumoniae is a potential pathogen that has not been studied comprehensively. The emergence of multidrug-resistant (MDR) K. quasipneumoniae, specifically strains resistant to tigecycline and carbapenem, presents a significant challenge to clinical treatment. This investigation aimed to characterize MDR K. quasipneumoniae strain FK8966, co-carrying tmexCD2-toprJ2, blaIMP-4, and blaNDM-1 by plasmids. It was observed that FK8966's MDR was primarily because of the IncHI1B-like plasmid co-carrying tmexCD2-toprJ2 and blaIMP-4, and an IncFIB(K)/IncFII(K) plasmid harboring blaNDM-1. Furthermore, the phylogenetic analysis revealed that IncHI1B-like plasmids carrying tmexCD2-toprJ2 were disseminated among different bacteria, specifically in China. Additionally, according to the comparative genomic analysis, the MDR regions indicated that the tmexCD2-toprJ2 gene cluster was inserted into the umuC gene, while blaIMP-4 was present in transposon TnAs3 linked to the class 1 integron (IntI1). It was also observed that an ΔTn3000 insertion with blaNDM-1 made a novel blaNDM-1 harboring IncFIB(K)/IncFII(K) plasmid. The antimicrobial resistance prevalence and phylogenetic analyses of K. quasipneumoniae strains indicated that FK8966 is a distinct MDR branch of K. quasipneumoniae. Furthermore, CRISPR-Cas system analysis showed that many K. quasipneumoniae CRISPR-Cas systems lacked spacers matching the two aforementioned novel resistance plasmids, suggesting that these resistance plasmids have the potential to disseminate within K. quasipneumoniae. Therefore, the spread of MDR K. quasipneumoniae and plasmids warrants further attention.IMPORTANCEThe emergence of multidrug-resistant K. quasipneumoniae poses a great threat to clinical care, and the situation is exacerbated by the dissemination of tigecycline- and carbapenem-resistant genes. Therefore, monitoring these pathogens and their resistance plasmids is urgent and crucial. This study identified tigecycline- and carbapenem-resistant K. quasipneumoniae strain, FK8966. Furthermore, it is the first study to report the coexistence of tmexCD2-toprJ2, blaIMP-4, and blaNDM-1 in K. quasipneumoniae. Moreover, the CRISPR-Cas system of many K. quasipneumoniae lacks spacers that match the plasmids carried by FK8966, which are crucial for mediating resistance against tigecycline and carbapenems, indicating their potential to disseminate within K. quasipneumoniae.},
}

@article {pmid39162172,
year = {2024},
author = {Markley, HC and Helms, KJ and Maar, M and Zentner, GE and Wade, MJ and Zelhof, AC},
title = {Generating and testing the efficacy of reagents for CRISPR/Cas9 homology directed repair-based manipulations in Tribolium.},
journal = {Journal of insect science (Online)},
volume = {24},
number = {4},
pages = {},
doi = {10.1093/jisesa/ieae082},
pmid = {39162172},
issn = {1536-2442},
support = {2019-33522-30064//NIFA/ ; 2019-33522-30064//USDA/ ; IOS-1928781//NSF/ ; },
mesh = {*Tribolium/genetics ; Animals ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Recombinational DNA Repair ; Gene Drive Technology/methods ; },
abstract = {CRISPR/Cas9 manipulations are possible in many insects and ever expanding. Nonetheless, success in one species and techniques developed for it are not necessarily applicable to other species. As such, the development and expansion of CRISPR-based (clustered regularly interspaced short palindromic repeats) genome-editing tools and methodologies are dependent upon direct experimentation. One useful technique is Cas9-dependent homologous recombination, which is a critical tool for studying gene function but also for developing pest related applications like gene drive. Here, we report our attempts to induce Cas9 homology directed repair (HDR) and subsequent gene drive in Tribolium castaneum (Herbst; Insecta: Coleoptera: Tenebrionidae). Utilizing constructs containing 1 or 2 target gRNAs in combination with Cas9 under 2 different promoters and corresponding homology arms, we found a high incidence of CRISPR/Cas9 induced mutations but no evidence of homologous recombination. Even though the generated constructs provide new resources for CRISPR/Cas9 modification of the Tribolium genome, our results suggest that additional modifications and increased sample sizes will be necessary to increase the potential and detection for HDR of the Tribolium genome.},
}

*Tribolium/genetics
Animals
*CRISPR-Cas Systems
*Gene Editing/methods
Recombinational DNA Repair
Gene Drive Technology/methods

@article {pmid39161182,
year = {2024},
author = {Hu, R and Guo, C and Liu, C and Zhang, Q and Zhang, X and Chen, Y and Liu, Y},
title = {From Lab to Home: Ultrasensitive Rapid Detection of SARS-CoV-2 with a Cascade CRISPR/Cas13a-Cas12a System Based Lateral Flow Assay.},
journal = {Analytical chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.analchem.4c02726},
pmid = {39161182},
issn = {1520-6882},
abstract = {Currently, CRISPR/Cas-based molecular diagnostic techniques usually rely on the introduction of nucleic acid amplification to improve their sensitivity, which is usually more time-consuming, susceptible to aerosol contamination, and therefore not suitable for at-home molecular testing. In this research, we developed an advanced CRISPR/Cas13a-Cas12a-based lateral flow assay that facilitated the ultrasensitive and rapid detection of SARS-CoV-2 RNA directly from samples, without the need for nucleic acid amplification. This method was called CRISPR LFA enabling at-home RNA testing (CLEAR). CLEAR used a novel cascade mechanism with specially designed probes that fold into hairpin structures, enabling visual detection of SARS-CoV-2 sequences down to 1 aM sensitivity levels. More importantly, CLEAR had a positive coincidence rate of 100% and a negative coincidence rate of 100% for clinical nasopharyngeal swabs from 16 patients. CLEAR was particularly suitable for at-home molecular testing, providing a low-cost, user-friendly solution that can efficiently distinguish between different SARS-CoV-2 variants. CLEAR overcame the common limitations of high sensitivity and potential contamination associated with traditional PCR-based systems, making it a promising tool for widespread public health application, especially in environments with limited access to laboratory resources.},
}

@article {pmid39160220,
year = {2024},
author = {Liu, F and Zhang, X and Yang, Y},
title = {Simulation of CRISPR-Cas9 editing on evolving barcode and accuracy of lineage tracing.},
journal = {Scientific reports},
volume = {14},
number = {1},
pages = {19213},
pmid = {39160220},
issn = {2045-2322},
support = {R01 CA251950/CA/NCI NIH HHS/United States ; R01 CA251950/CA/NCI NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; *Computer Simulation ; Algorithms ; DNA Barcoding, Taxonomic/methods ; Cell Lineage/genetics ; INDEL Mutation ; Mutation ; },
abstract = {We designed a simulation program that mimics the CRISPR-Cas9 editing on evolving barcode and double strand break repair procedure along with cell divisions. Emerging barcode mutations tend to build upon previously existing mutations, occurring sequentially with each generation. This process results in a unique mutation profile in each cell. We sample the barcodes in leaf cells and reconstruct the lineage, comparing it to the original lineage tree to test algorithm accuracy under different parameter settings. Our computational simulations validate the reasonable assumptions deduced from experimental observations, emphasizing that factors such as sampling size, barcode length, multiple barcodes, indel probabilities, and Cas9 activity are critical for accurate and successful lineage tracing. Among the many factors we found that sampling size and indel probabilities are two major ones that affect lineage tracing accuracy. Large segment deletions in early generations could greatly impact lineage accuracy. These simulation results offer insightful recommendations for enhancing the design and analysis of Cas9-mediated molecular barcodes in actual experiments.},
}

*CRISPR-Cas Systems
*Gene Editing/methods
*Computer Simulation
Algorithms
DNA Barcoding, Taxonomic/methods
Cell Lineage/genetics
INDEL Mutation
Mutation

@article {pmid39121862,
year = {2024},
author = {Yoshimi, K and Kuno, A and Yamauchi, Y and Hattori, K and Taniguchi, H and Mikamo, K and Iida, R and Ishida, S and Goto, M and Takeshita, K and Ito, R and Takahashi, R and Takahashi, S and Mashimo, T},
title = {Genome editing using type I-E CRISPR-Cas3 in mice and rat zygotes.},
journal = {Cell reports methods},
volume = {4},
number = {8},
pages = {100833},
doi = {10.1016/j.crmeth.2024.100833},
pmid = {39121862},
issn = {2667-2375},
mesh = {Animals ; *Zygote/metabolism ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Rats ; Mice ; Female ; },
abstract = {The type I CRISPR system has recently emerged as a promising tool, especially for large-scale genomic modification, but its application to generate model animals by editing zygotes had not been established. In this study, we demonstrate genome editing in zygotes using the type I-E CRISPR-Cas3 system, which efficiently generates deletions of several thousand base pairs at targeted loci in mice with 40%-70% editing efficiency without off-target mutations. To overcome the difficulties associated with detecting the variable deletions, we used a newly long-read sequencing-based multiplex genotyping approach. Demonstrating remarkable versatility, our Cas3-based technique was successfully extended to rats as well as mice, even by zygote electroporation methods. Knockin for SNP exchange and genomic replacement with a donor plasmid were also achieved in mice. This pioneering work with the type I CRISPR zygote editing system offers increased flexibility and broader applications in genetic engineering across different species.},
}

Animals
*Zygote/metabolism
*Gene Editing/methods
*CRISPR-Cas Systems/genetics
Rats
Mice
Female

@article {pmid39054057,
year = {2024},
author = {Jansen, G and Gebert, D and Kumar, TR and Simmons, E and Murphy, S and Teixeira, FK},
title = {Tolerance thresholds underlie responses to DNA damage during germline development.},
journal = {Genes & development},
volume = {38},
number = {13-14},
pages = {631-654},
doi = {10.1101/gad.351701.124},
pmid = {39054057},
issn = {1549-5477},
mesh = {Animals ; *Germ Cells ; *DNA Breaks, Double-Stranded ; *DNA Transposable Elements/genetics ; CRISPR-Cas Systems/genetics ; DNA Damage/genetics ; Drosophila melanogaster/genetics ; Female ; Oogenesis/genetics ; },
abstract = {Selfish DNA modules like transposable elements (TEs) are particularly active in the germline, the lineage that passes genetic information across generations. New TE insertions can disrupt genes and impair the functionality and viability of germ cells. However, we found that in P-M hybrid dysgenesis in Drosophila, a sterility syndrome triggered by the P-element DNA transposon, germ cells harbor unexpectedly few new TE insertions despite accumulating DNA double-strand breaks (DSBs) and inducing cell cycle arrest. Using an engineered CRISPR-Cas9 system, we show that generating DSBs at silenced P-elements or other noncoding sequences is sufficient to induce germ cell loss independently of gene disruption. Indeed, we demonstrate that both developing and adult mitotic germ cells are sensitive to DSBs in a dosage-dependent manner. Following the mitotic-to-meiotic transition, however, germ cells become more tolerant to DSBs, completing oogenesis regardless of the accumulated genome damage. Our findings establish DNA damage tolerance thresholds as crucial safeguards of genome integrity during germline development.},
}

Animals
*Germ Cells
*DNA Breaks, Double-Stranded
*DNA Transposable Elements/genetics
CRISPR-Cas Systems/genetics
DNA Damage/genetics
Drosophila melanogaster/genetics
Female
Oogenesis/genetics

@article {pmid39048074,
year = {2024},
author = {Guan, Q and Wang, Z and Zhang, K and Liu, Z and Zhou, H and Cao, D and Mao, X},
title = {CRISPR/Cas9-mediated neuronal deletion of 5-lipoxygenase alleviates deficits in mouse models of epilepsy.},
journal = {Journal of advanced research},
volume = {63},
number = {},
pages = {73-90},
doi = {10.1016/j.jare.2024.07.018},
pmid = {39048074},
issn = {2090-1224},
mesh = {Animals ; *CRISPR-Cas Systems ; Mice ; *Disease Models, Animal ; *Arachidonate 5-Lipoxygenase/metabolism/genetics ; *Neurons/metabolism ; *Hippocampus/metabolism ; *Epilepsy/genetics ; Male ; Mice, Inbred C57BL ; Gene Deletion ; Epilepsy, Temporal Lobe/genetics ; Dependovirus/genetics ; Pilocarpine ; },
abstract = {INTRODUCTION: Our previous work reveals a critical role of activation of neuronal Alox5 in exacerbating brain injury post seizures. However, whether neuronal Alox5 impacts the pathological process of epilepsy remains unknown.

OBJECTIVES: To prove the feasibility of neuron-specific deletion of Alox5 via CRISPR-Cas9 in the blockade of seizure onset and epileptic progression.

METHODS: Here, we employed a Clustered regularly interspaced short-palindromic repeat-associated proteins 9 system (CRISPR/Cas9) system delivered by adeno-associated virus (AAV) to specifically delete neuronal Alox5 gene in the hippocampus to explore its therapeutic potential in various epilepsy mouse models and possible mechanisms.

RESULTS: Neuronal depletion of Alox5 was successfully achieved in the brain. AAV delivery of single guide RNA of Alox5 in hippocampus resulted in reducing seizure severity, delaying epileptic progression and improving epilepsy-associated neuropsychiatric comorbidities especially anxiety, cognitive deficit and autistic-like behaviors in pilocarpine- and kainic acid-induced temporal lobe epilepsy (TLE) models. In addition, neuronal Alox5 deletion also reversed neuron loss, neurodegeneration, astrogliosis and mossy fiber sprouting in TLE model. Moreover, a battery of tests including analysis of routine blood test, hepatic function, renal function, routine urine test and inflammatory factors demonstrated no noticeable toxic effect, suggesting that Alox5 deletion possesses the satisfactory biosafety. Mechanistically, the anti-epileptic effect of Alox5 deletion might be associated with reduction of glutamate level to restore excitatory/inhibitory balance by reducing CAMKII-mediated phosphorylation of Syn I[Ser603].

CONCLUSION: Our findings showed the translational potential of AAV-mediated delivery of CRISPR-Cas9 system including neuronal Alox5 gene for an alternative promising therapeutic approach to treat epilepsy.},
}

Animals
*CRISPR-Cas Systems
Mice
*Disease Models, Animal
*Arachidonate 5-Lipoxygenase/metabolism/genetics
*Neurons/metabolism
*Hippocampus/metabolism
*Epilepsy/genetics
Male
Mice, Inbred C57BL
Gene Deletion
Epilepsy, Temporal Lobe/genetics
Dependovirus/genetics
Pilocarpine

@article {pmid39160035,
year = {2024},
author = {Gao, R and Liu, X and Xiong, Z and Wang, G and Ai, L},
title = {Research progress on detection of foodborne pathogens: The more rapid and accurate answer to food safety.},
journal = {Food research international (Ottawa, Ont.)},
volume = {193},
number = {},
pages = {114767},
doi = {10.1016/j.foodres.2024.114767},
pmid = {39160035},
issn = {1873-7145},
mesh = {*Foodborne Diseases/microbiology/prevention & control ; *Food Microbiology/methods ; *Food Safety/methods ; Humans ; *Biosensing Techniques/methods ; CRISPR-Cas Systems ; Food Contamination/analysis ; },
abstract = {In recent years, foodborne diseases have posed a serious threat to human health, and rapid detection of foodborne pathogens is particularly crucial for the prevention and control of such diseases. This article offers a detailed overview of the development of detection techniques for foodborne pathogens, transitioning from traditional microbiological culture methods to the current array of techniques, including immunological, molecular biological, and biosensor-based methods. It summarizes the technical principles, advantages, disadvantages, and research progress of these diverse methods. Furthermore, the article demonstrates that the combination of different methods enhances the efficiency and accuracy of pathogens detection. Specifically, the article focuses on the application and advantages of combining CRISPR/Cas systems with other detection methods in the detection of foodborne pathogens. CRISPR/Cas systems, with their high specificity, sensitivity, and ease of operation, show great potential in the field of foodborne pathogens detection. When integrated with other detection techniques such as immunological detection techniques, molecular biology detection techniques, and biosensors, the accuracy and efficiency of detection can be further improved. By fully utilizing these tools, early detection and control of foodborne diseases can be achieved, enhancing public health and preventing disease outbreaks. This article serves as a valuable reference for exploring more convenient, accurate, and sensitive field detection methods for foodborne pathogens, promoting the application of rapid detection techniques, and ensuring food safety and human health.},
}

*Foodborne Diseases/microbiology/prevention & control
*Food Microbiology/methods
*Food Safety/methods
Humans
*Biosensing Techniques/methods
CRISPR-Cas Systems
Food Contamination/analysis

@article {pmid39159817,
year = {2024},
author = {Madaan, V and Kollara, A and Spaner, D and Brown, TJ},
title = {ISGylation enhances double-stranded RNA-induced interferon response and NFκB signaling in fallopian tube epithelial cells.},
journal = {The Journal of biological chemistry},
volume = {},
number = {},
pages = {107686},
doi = {10.1016/j.jbc.2024.107686},
pmid = {39159817},
issn = {1083-351X},
abstract = {Heritable mutations in BRCA1 associate with increased risk of high-grade serous tubo-ovarian cancer (HGSTOC). Non-genetic risk factors associated with this cancer, which arises from fallopian tube epithelial (FTE) cells, suggests a role for repetitive ovulation wherein FTE cells are exposed to inflammatory signaling molecules within follicular fluid. We previously reported increased NFκB and EGFR signaling in BRCA1-deficient primary FTE cells, with follicular fluid exposure further increasing abundance of interferon-stimulated gene (ISG) transcripts, including the ubiquitin-like protein ISG15 and other ISGylation pathway members. Both NFκB and type I interferon signaling are upregulated by stimulation of cGAS-STING or MDA5 and RIGI pattern recognition receptors (PRRs). Since some PRRs and their signal transduction pathway members are ISGylated, we tested the impact of ISG15 and ISGylation on IRF3 and NFκB signaling through cGAS-STING or RIGI and MDA5 activation. Expression of ISG15 or UBA7, the E1-like ISG15 activating enzyme, in immortalized FTE cells was disrupted by CRISPR gene editing. Activation of IRF3 by RIGI or MDA5 but not cGAS-STING was attenuated by loss of either ISG15 or UBA7 and this was reflected by a similar effect on NFκB activation and downstream targets. Loss of ISGylation decreased levels of both MDA5 and RIGI, with knock-down of RIGI but not MDA5, decreasing IRF3 and NFκB activation in parental cells. These finding indicate that ISGylation enhances the ability of dsRNA to activate cytokine release and pro-inflammatory signaling. Further work to explore ISGylation as a target for prevention of HGSTOC in BRCA1 mutation carriers is warranted.},
}

@article {pmid39158453,
year = {2024},
author = {Ishida, S and Sawai, T},
title = {From CRISPR to Conscience: Ethical Dilemmas in Gene Editing and Genetic Selection.},
journal = {The American journal of bioethics : AJOB},
volume = {24},
number = {8},
pages = {67-70},
doi = {10.1080/15265161.2024.2361900},
pmid = {39158453},
issn = {1536-0075},
mesh = {Humans ; *Gene Editing/ethics ; *Conscience ; Selection, Genetic ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
}

Humans
*Gene Editing/ethics
*Conscience
Selection, Genetic
CRISPR-Cas Systems
Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid39158272,
year = {2024},
author = {Upreti, C and Kumar, P and Durso, LM and Palmer, KL},
title = {CRISPR-Cas inhibits plasmid transfer and immunizes bacteria against antibiotic resistance acquisition in manure.},
journal = {Applied and environmental microbiology},
volume = {},
number = {},
pages = {e0087624},
doi = {10.1128/aem.00876-24},
pmid = {39158272},
issn = {1098-5336},
abstract = {The horizontal transfer of antibiotic resistance genes among bacteria is a pressing global issue. The bacterial defense system clustered regularly interspaced short palindromic repeats (CRISPR)-Cas acts as a barrier to the spread of antibiotic resistance plasmids, and CRISPR-Cas-based antimicrobials can be effective to selectively deplete antibiotic-resistant bacteria. While significant surveillance efforts monitor the spread of antibiotic-resistant bacteria in the clinical context, a major, often overlooked aspect of the issue is resistance emergence in agriculture. Farm animals are commonly treated with antibiotics, and antibiotic resistance in agriculture is on the rise. Yet, CRISPR-Cas efficacy has not been investigated in this setting. Here, we evaluate the prevalence of CRISPR-Cas in agricultural Enterococcus faecalis strains and its antiplasmid efficacy in an agricultural niche: manure. Analyzing 1,986 E. faecalis genomes from human and animal hosts, we show that the prevalence of CRISPR-Cas subtypes is similar between clinical and agricultural E. faecalis strains. Using plasmid conjugation assays, we found that CRISPR-Cas is a significant barrier against resistance plasmid transfer in manure. Finally, we used a CRISPR-based antimicrobial approach to cure resistant E. faecalis of erythromycin resistance, but this was limited by delivery efficiency of the CRISPR antimicrobial in manure. However, immunization of bacteria against resistance gene acquisition in manure was highly effective. Together, our results show that E. faecalis CRISPR-Cas is prevalent and effective in an agricultural setting and has the potential to be utilized for depleting antibiotic-resistant populations. Our work has broad implications for tackling antibiotic resistance in the increasingly relevant agricultural setting, in line with a One Health approach.IMPORTANCEAntibiotic resistance is a growing global health crisis in human and veterinary medicine. Previous work has shown technologies based on CRISPR-Cas-a bacterial defense system-to be effective in tackling antibiotic resistance. Here we test if CRISPR-Cas is present and effective in agricultural niches, specifically in the ubiquitously present bacterium, Enterococcus faecalis. We show that CRISPR-Cas is both prevalent and functional in manure and has the potential to be used to specifically kill bacteria carrying antibiotic resistance genes. This study demonstrates the utility of CRISPR-Cas-based strategies for control of antibiotic resistance in agricultural settings.},
}

@article {pmid39156766,
year = {2024},
author = {Liu, F and Li, R and Zhu, Z and Yang, Y and Lu, F},
title = {Current developments of gene therapy in human diseases.},
journal = {MedComm},
volume = {5},
number = {9},
pages = {e645},
pmid = {39156766},
issn = {2688-2663},
abstract = {Gene therapy has witnessed substantial advancements in recent years, becoming a constructive tactic for treating various human diseases. This review presents a comprehensive overview of these developments, with a focus on their diverse applications in different disease contexts. It explores the evolution of gene delivery systems, encompassing viral (like adeno-associated virus; AAV) and nonviral approaches, and evaluates their inherent strengths and limitations. Moreover, the review delves into the progress made in targeting specific tissues and cell types, spanning the eye, liver, muscles, and central nervous system, among others, using these gene technologies. This targeted approach is crucial in addressing a broad spectrum of genetic disorders, such as inherited lysosomal storage diseases, neurodegenerative disorders, and cardiovascular diseases. Recent clinical trials and successful outcomes in gene therapy, particularly those involving AAV and the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated proteins, are highlighted, illuminating the transformative potentials of this approach in disease treatment. The review summarizes the current status of gene therapy, its prospects, and its capacity to significantly ameliorate patient outcomes and quality of life. By offering comprehensive analysis, this review provides invaluable insights for researchers, clinicians, and stakeholders, enriching the ongoing discourse on the trajectory of disease treatment.},
}

@article {pmid39155782,
year = {2024},
author = {Lin, L and Tao, J and Meng, Y and Gan, Y and He, X and Li, S and Zhang, J and Gao, F and Xin, D and Wang, L and Fan, Y and Chen, B and Lu, Z and Xu, Y},
title = {Genome-wide CRISPR screening identifies critical role of phosphatase and tensin homologous (PTEN) in sensitivity of acute myeloid leukemia to chemotherapy.},
journal = {Journal of Zhejiang University. Science. B},
volume = {25},
number = {8},
pages = {700-710},
doi = {10.1631/jzus.B2300555},
pmid = {39155782},
issn = {1862-1783},
support = {LY21H080005//the Zhejiang Provincial Natural Science Foundation of China/ ; 81572920 and 82100171//the National Natural Science Foundation of China/ ; },
mesh = {Humans ; *PTEN Phosphohydrolase/metabolism/genetics ; *Leukemia, Myeloid, Acute/drug therapy/genetics ; *Apoptosis/drug effects ; Cell Line, Tumor ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Cell Proliferation/drug effects ; Signal Transduction ; TOR Serine-Threonine Kinases/metabolism ; Proto-Oncogene Proteins c-akt/metabolism ; Antineoplastic Agents/pharmacology/therapeutic use ; Cell Survival/drug effects ; Drug Resistance, Neoplasm ; CRISPR-Cas Systems ; },
abstract = {Although significant progress has been made in the development of novel targeted drugs for the treatment of acute myeloid leukemia (AML) in recent years, chemotherapy still remains the mainstay of treatment and the overall survival is poor in most patients. Here, we demonstrated the antileukemia activity of a novel small molecular compound NL101, which is formed through the modification on bendamustine with a suberanilohydroxamic acid (SAHA) radical. NL101 suppresses the proliferation of myeloid malignancy cells and primary AML cells. It induces DNA damage and caspase 3-mediated apoptosis. A genome-wide clustered regularly interspaced short palindromic repeats (CRISPR) library screen revealed that phosphatase and tensin homologous (PTEN) gene is critical for the regulation of cell survival upon NL101 treatment. The knockout or inhibition of PTEN significantly reduced NL101-induced apoptosis in AML and myelodysplastic syndrome (MDS) cells, accompanied by the activation of protein kinase B (AKT) signaling pathway. The inhibition of mammalian target of rapamycin (mTOR) by rapamycin enhanced the sensitivity of AML cells to NL101-induced cell death. These findings uncover PTEN protein expression as a major determinant of chemosensitivity to NL101 and provide a novel strategy to treat AML with the combination of NL101 and rapamycin.},
}

Humans
*PTEN Phosphohydrolase/metabolism/genetics
*Leukemia, Myeloid, Acute/drug therapy/genetics
*Apoptosis/drug effects
Cell Line, Tumor
*Clustered Regularly Interspaced Short Palindromic Repeats
Cell Proliferation/drug effects
Signal Transduction
TOR Serine-Threonine Kinases/metabolism
Proto-Oncogene Proteins c-akt/metabolism
Antineoplastic Agents/pharmacology/therapeutic use
Cell Survival/drug effects
Drug Resistance, Neoplasm
CRISPR-Cas Systems

@article {pmid39155100,
year = {2024},
author = {Wang, T and Ding, K and Wang, X and Wang, Z and Liu, G and Zang, Y and Lin, S and Zhou, H and Wang, Q},
title = {Dual amplification dynamic DNA network system for CRISPR/Cas12a based p53 gene detection.},
journal = {Analytica chimica acta},
volume = {1321},
number = {},
pages = {343048},
doi = {10.1016/j.aca.2024.343048},
pmid = {39155100},
issn = {1873-4324},
mesh = {*Tumor Suppressor Protein p53/genetics ; *CRISPR-Cas Systems/genetics ; Humans ; Nucleic Acid Amplification Techniques ; Biosensing Techniques/methods ; DNA/chemistry/genetics ; Limit of Detection ; Genes, p53 ; Nucleic Acid Hybridization ; },
abstract = {BACKGROUND: It is estimated that over 50 % of human cancers are caused by mutations in the p53 gene. Early sensitive and accurate detection of the p53 gene is important for diagnosis of cancers in the early stage. However, conventional detection techniques often suffer from strict reaction conditions, or unsatisfied sensitivity, so we need to develop a new strategy for accurate detection of p53 gene with smart designability, multiple signal amplification in mild reaction conditions.

RESULTS: In this study, CRISPR/Cas system is exploited in entropy-driven catalysis (EDC) and hybridization chain reaction (CHA) dual signal amplification sensing strategies. The products of both reactions can efficiently and separately activate CRISPR/Cas12a which greatly amplifies the fluorescent signal. The method has good linearity in p53 detection with the concentration ranged from 0.1 fM to 0.5 pM with ultra-low detection limit of 0.096 fM. It also showed good performance in serum, offering potentials for early disease detection.

SIGNIFICANCE: The designed dual amplification dynamic DNA network system exhibits an ultra-sensitive fluorescence biosensing for p53 gene identification. The method is simple to operate and requires only one buffer for the experiment, and meanwhile shows smart designability which could be used for a wide range of markers. Thus, we believe the present work will provide a potential tool for the construction and development of sensitive fluorescent biosensors for diseases.},
}

*Tumor Suppressor Protein p53/genetics
*CRISPR-Cas Systems/genetics
Humans
Nucleic Acid Amplification Techniques
Biosensing Techniques/methods
DNA/chemistry/genetics
Limit of Detection
Genes, p53
Nucleic Acid Hybridization

@article {pmid39058317,
year = {2024},
author = {Chaumont, L and Peruzzi, M and Huetz, F and Raffy, C and Le Hir, J and Minke, J and Boudinot, P and Collet, B},
title = {Salmonid Double-stranded RNA-Dependent Protein Kinase Activates Apoptosis and Inhibits Protein Synthesis.},
journal = {Journal of immunology (Baltimore, Md. : 1950)},
volume = {213},
number = {5},
pages = {700-717},
doi = {10.4049/jimmunol.2400076},
pmid = {39058317},
issn = {1550-6606},
support = {871108//EU/ ; 817923//EU/ ; 301083//RCN/ ; 2020/0646//Association Nationale de la Recherche et de la Technologie (ANRT)/ ; },
mesh = {Animals ; *Apoptosis/immunology ; *eIF-2 Kinase/metabolism/genetics ; *Salmon/immunology ; Cell Line ; *Protein Biosynthesis/immunology ; Immunity, Innate ; Novirhabdovirus/physiology/immunology ; Fish Proteins/genetics/immunology/metabolism ; RNA, Double-Stranded/immunology ; Fish Diseases/immunology ; CRISPR-Cas Systems ; },
abstract = {dsRNA-dependent protein kinase R (PKR) is a key factor of innate immunity. It is involved in translation inhibition, apoptosis, and enhancement of the proinflammatory and IFN responses. However, how these antiviral functions are conserved during evolution remains largely unknown. Overexpression and knockout studies in a Chinook salmon (Oncorhynchus tshawytscha) cell line were conducted to assess the role of salmonid PKR in the antiviral response. Three distinct mRNA isoforms from a unique pkr gene, named pkr-fl (full length), pkr-ml (medium length) and pkr-sl (short length), were cloned and a pkr-/- clonal fish cell line was developed using CRISPR/Cas9 genome editing. PKR-FL includes an N-terminal dsRNA-binding domain and a C-terminal kinase domain, whereas PKR-ML and PKR-SL display a truncated or absent kinase domain, respectively. PKR-FL is induced during IFNA2 stimulation but not during viral hemorrhagic septicemia virus (VHSV) infection. Overexpression experiments showed that only PKR-FL possesses antiviral functions, including activation of apoptosis and inhibition of de novo protein synthesis. Knockout experiments confirmed that PKR is involved in apoptosis activation during the late stage of VHSV infection. Endogenous PKR also plays a critical role in translation inhibition upon poly(I:C) transfection after IFNA2 treatment. It is, however, not involved in translational arrest during VHSV infection. Extra- and intracellular titrations showed that endogenous PKR does not directly inhibit viral replication but apparently favors virion release into the supernatant, likely by triggering late apoptosis. Altogether, our data confirm that salmonid PKR has conserved molecular functions that VHSV appears to bypass with subversion strategies.},
}

Animals
*Apoptosis/immunology
*eIF-2 Kinase/metabolism/genetics
*Salmon/immunology
Cell Line
*Protein Biosynthesis/immunology
Immunity, Innate
Novirhabdovirus/physiology/immunology
Fish Proteins/genetics/immunology/metabolism
RNA, Double-Stranded/immunology
Fish Diseases/immunology
CRISPR-Cas Systems

@article {pmid39051914,
year = {2024},
author = {Liu, Y and Gou, S and Qiu, L and Xu, Z and Yang, H and Yang, S and Zhao, Y},
title = {A CRISPR/Cas12a-powered gold/nickel foam surface-enhanced Raman spectroscopy biosensor for nucleic acid specific detection in foods.},
journal = {The Analyst},
volume = {149},
number = {17},
pages = {4343-4350},
doi = {10.1039/d4an00778f},
pmid = {39051914},
issn = {1364-5528},
mesh = {*Spectrum Analysis, Raman/methods ; *Biosensing Techniques/methods ; *Gold/chemistry ; *CRISPR-Cas Systems ; *Limit of Detection ; *Metal Nanoparticles/chemistry ; Drinking Water/analysis ; Silver/chemistry ; DNA, Single-Stranded/chemistry ; Food Analysis/methods ; Endodeoxyribonucleases/chemistry ; CRISPR-Associated Proteins/genetics ; Food Contamination/analysis ; Bacterial Proteins/genetics ; },
abstract = {Food is a necessary source of energy, but it also serves as a pathway for transmitting infectious pathogens, making food safety a matter of great concern. Rapid, accurate, and specific detection methods for foodborne viruses are crucial. Surface-Enhanced Raman Scattering (SERS), due to its superior sensitivity and characteristic fingerprint spectra, holds enormous potential. However, due to the limitations of SERS, it requires specific conditions to achieve specificity. In order to enhance the specificity and accuracy of nucleic acid detection based on SERS, we have developed a CRISPR-Cas12a-mediated SERS technique to identify target DNA, harnessing the targeting recognition capability of CRISPR-Cas12a and ultra-sensitive SERS tags and successfully addressing SERS' lack of specific detection capability. This system includes a gold/nickel foam substrate (Au-NFs) and a reporter (ssDNA-ROX). The phenomenon of colloidal gold/silver nano-aggregation due to magnesium ions, which is commonly encountered in CRISPR-SERS, was simultaneously solved using AuNFs. The qualitative and quantitative analysis of target DNA in drinking water was performed by monitoring the intensity change of ROX Raman reporter molecules. The results showed that the sensor detected DNA within 30 min and the limit of detection (LOD) was 8.23 fM. This is expected to become one of the alternative methods for nucleic acid detection for its rapid detection and high specificity.},
}

*Spectrum Analysis, Raman/methods
*Biosensing Techniques/methods
*Gold/chemistry
*CRISPR-Cas Systems
*Limit of Detection
*Metal Nanoparticles/chemistry
Drinking Water/analysis
Silver/chemistry
DNA, Single-Stranded/chemistry
Food Analysis/methods
Endodeoxyribonucleases/chemistry
CRISPR-Associated Proteins/genetics
Food Contamination/analysis
Bacterial Proteins/genetics

@article {pmid39011640,
year = {2024},
author = {Gong, S and Song, K and Pan, W and Li, N and Tang, B},
title = {CRISPR-Cas12a-based ultrasensitive assay for visual detection of SARS-CoV-2 RNA.},
journal = {The Analyst},
volume = {149},
number = {17},
pages = {4418-4424},
doi = {10.1039/d4an00479e},
pmid = {39011640},
issn = {1364-5528},
mesh = {*SARS-CoV-2/genetics/isolation & purification ; *CRISPR-Cas Systems/genetics ; *RNA, Viral/analysis/genetics ; Humans ; *Limit of Detection ; *Nucleic Acid Amplification Techniques/methods ; *COVID-19/diagnosis/virology ; COVID-19 Nucleic Acid Testing/methods/instrumentation ; Magnetite Nanoparticles/chemistry ; Endodeoxyribonucleases/chemistry/genetics ; CRISPR-Associated Proteins/genetics ; DNA, Single-Stranded/chemistry/genetics ; Bacterial Proteins ; },
abstract = {The development of ultrasensitive and visual methods is of great significance for molecular diagnosis at the point-of-care. In this study, we have integrated recombinase polymerase amplification (RPA) with the CRISPR-Cas12a system to design an ultrasensitive strategy for visual nucleic acid testing. RPA is utilized to amplify the target nucleic acid, producing amplicons that activate the single-stranded DNase property of CRISPR-Cas12a. The activated CRISPR-Cas12a then degrades the single-stranded DNA on magnetic nanoparticles (MNPs), releasing immobilized GOx from the MNPs which catalyses the chromogenic substrate. The developed method exhibits remarkable sensitivity, successfully detecting as low as 10 aM (∼6 copies per μL) of the target nucleic acid by visual colour changes in solution. The instrumental limit of detection is calculated to be 2.86 aM (∼2 copies per μL), comparable to the sensitivity of polymerase chain reaction (PCR). Importantly, this approach only requires isothermal incubation operation and does not involve costly instruments. The method has been validated by visually detecting the SARS-CoV-2 RNA gene fragment within 50 minutes. With its ultrasensitivity, simplicity of operation, and potential for integration into a point-of-care detection kit, this strategy holds great promise for nucleic acid testing in various settings.},
}

*SARS-CoV-2/genetics/isolation & purification
*CRISPR-Cas Systems/genetics
*RNA, Viral/analysis/genetics
Humans
*Limit of Detection
*Nucleic Acid Amplification Techniques/methods
*COVID-19/diagnosis/virology
COVID-19 Nucleic Acid Testing/methods/instrumentation
Magnetite Nanoparticles/chemistry
Endodeoxyribonucleases/chemistry/genetics
CRISPR-Associated Proteins/genetics
DNA, Single-Stranded/chemistry/genetics
Bacterial Proteins

@article {pmid38803114,
year = {2024},
author = {Wei, Y and Zhang, H and Fan, J and Cai, Q and Zhang, Z and Wang, J and Zhang, M and Yan, F and Jiang, J and Xie, H and Luo, X and Wei, L and Lin, Y and He, W and Qu, M and Zhang, X and Zhu, Y and Xie, H and Zhang, J},
title = {Multiplex-genome-editing based rapid directional improvement of complex traits in rice.},
journal = {Plant biotechnology journal},
volume = {22},
number = {9},
pages = {2624-2628},
pmid = {38803114},
issn = {1467-7652},
support = {2020J011354//The Natural Science Foundation of Fujian Province/ ; CARS-01-20//The National Rice Industry Technology System of Modern Agriculture for China/ ; KJZD202401//Key Science and Technology Projects of Fujian Academy of Agricultural Sciences/ ; XTCXGC2021001//The "5511" Collaborative Innovation project for High-quality Development and Surpasses of Agriculture between Government of Fujian and Chinese Academy of Agricultural Sciences/ ; 2020NZ08016//Key Program of Science and Technology in Fujian Province/ ; GBMUC-2019-002//Open Project Fund of Key Laboratory of Ministry of Education for Genetics, Breeding, and Multiple Utilization of Crops of Fujian Agricultrue and Forestry University/ ; },
mesh = {*Oryza/genetics/growth & development ; *Gene Editing/methods ; *Genome, Plant/genetics ; Plant Breeding/methods ; Phenotype ; CRISPR-Cas Systems/genetics ; Plants, Genetically Modified/genetics ; },
abstract = {Although thousands of genes have been identified or cloned in rice (Oryza sativa) in the last two decades, the majority of them have only been separately characterized in specific varieties or single-gene modified backgrounds, thus limiting their practical application. We developed an optimized multiplex genome editing (MGE) toolbox that can efficiently assemble and stably express up to twelve sgRNA targets in a single plant expression vector. In this study, we established the MGE-based Rapid Directional Improvement (MRDI) strategy for directional improvement of complex agronomic traits in one small-scale rice transformation. This approach provides a rapid and practical procedure, encompassing sgRNA assembly, transgene-free screening and the creation of promising germplasm, by combining the precision of gene editing with phenotype-based field breeding. The MRDI strategy was used to generate the full diversity of twelve main agronomic genes in rice cultivar FXZ for the directional improvement of its growth duration and plant architecture. After applying the MRDI to FXZ, ideal plants with the desired traits of early heading date reduced plant height, and more effective panicles were generated without compromising yield, blast resistance and grain quality. Furthermore, the results of whole-genome sequencing (WGS), including the analysis of structural variations (SVs) and single nucleotide variations (SNVs) in the MGE plants, confirmed the high specificity and low frequency of unwanted mutations associated with this strategy. The MRDI breeding strategy would be a robust approach for exploring and applying crucial agronomic genes, as well as for generating novel elite germplasm in the future.},
}

*Oryza/genetics/growth & development
*Gene Editing/methods
*Genome, Plant/genetics
Plant Breeding/methods
Phenotype
CRISPR-Cas Systems/genetics
Plants, Genetically Modified/genetics

@article {pmid38713743,
year = {2024},
author = {He, Y and Han, Y and Ma, Y and Liu, S and Fan, T and Liang, Y and Tang, X and Zheng, X and Wu, Y and Zhang, T and Qi, Y and Zhang, Y},
title = {Expanding plant genome editing scope and profiles with CRISPR-FrCas9 systems targeting palindromic TA sites.},
journal = {Plant biotechnology journal},
volume = {22},
number = {9},
pages = {2488-2503},
pmid = {38713743},
issn = {1467-7652},
support = {32072045//National Natural Science Foundation of China/ ; 32101205//National Natural Science Foundation of China/ ; 32270433//National Natural Science Foundation of China/ ; 2023ZD04074//STI 2030-Major Projects/ ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; *Genome, Plant/genetics ; *Oryza/genetics ; CRISPR-Associated Protein 9/metabolism/genetics ; },
abstract = {CRISPR-Cas9 is widely used for genome editing, but its PAM sequence requirements limit its efficiency. In this study, we explore Faecalibaculum rodentium Cas9 (FrCas9) for plant genome editing, especially in rice. FrCas9 recognizes a concise 5'-NNTA-3' PAM, targeting more abundant palindromic TA sites in plant genomes than the 5'-NGG-3' PAM sites of the most popular SpCas9. FrCas9 shows cleavage activities at all tested 5'-NNTA-3' PAM sites with editing outcomes sharing the same characteristics of a typical CRISPR-Cas9 system. FrCas9 induces high-efficiency targeted mutagenesis in stable rice lines, readily generating biallelic mutants with expected phenotypes. We augment FrCas9's ability to generate larger deletions through fusion with the exonuclease, TREX2. TREX2-FrCas9 generates much larger deletions than FrCas9 without compromise in editing efficiency. We demonstrate TREX2-FrCas9 as an efficient tool for genetic knockout of a microRNA gene. Furthermore, FrCas9-derived cytosine base editors (CBEs) and adenine base editors (ABE) are developed to produce targeted C-to-T and A-to-G base edits in rice plants. Whole-genome sequencing-based off-target analysis suggests that FrCas9 is a highly specific nuclease. Expression of TREX2-FrCas9 in plants, however, causes detectable guide RNA-independent off-target mutations, mostly as single nucleotide variants (SNVs). Together, we have established an efficient CRISPR-FrCas9 system for targeted mutagenesis, large deletions, C-to-T base editing, and A-to-G base editing in plants. The simple palindromic TA motif in the PAM makes the CRISPR-FrCas9 system a promising tool for genome editing in plants with an expanded targeting scope.},
}

*Gene Editing/methods
*CRISPR-Cas Systems
*Genome, Plant/genetics
*Oryza/genetics
CRISPR-Associated Protein 9/metabolism/genetics

@article {pmid38709858,
year = {2024},
author = {Wang, X and Fang, T and Lu, J and Tripathi, L and Qi, Y},
title = {Broad range plastid genome editing with monomeric TALE-linked cytosine and dual base editors.},
journal = {Plant biotechnology journal},
volume = {22},
number = {9},
pages = {2441-2443},
pmid = {38709858},
issn = {1467-7652},
support = {22010332//United States Agency for International Development/ ; MD-PSLA-243089//Maryland Agricultural Experiment Station/ ; IOS-2029889//National Science Fundation/ ; IOS-2132693//National Science Fundation/ ; },
mesh = {*Gene Editing/methods ; *Cytosine/metabolism ; *Genome, Plastid/genetics ; Transcription Activator-Like Effector Nucleases/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Plastids/genetics ; },
}

*Gene Editing/methods
*Cytosine/metabolism
*Genome, Plastid/genetics
Transcription Activator-Like Effector Nucleases/genetics/metabolism
CRISPR-Cas Systems/genetics
Plastids/genetics

@article {pmid38643514,
year = {2024},
author = {Niu, Q and Xie, H and Cao, X and Song, M and Wang, X and Li, S and Pang, K and Zhang, Y and Zhu, JK and Zhu, J},
title = {Engineering soybean with high levels of herbicide resistance with a Cas12-SF01-based cytosine base editor.},
journal = {Plant biotechnology journal},
volume = {22},
number = {9},
pages = {2435-2437},
pmid = {38643514},
issn = {1467-7652},
support = {32270367//National Natural Science Foundation of China/ ; NK2022010301//National Key Research and Development Program of China/ ; 2021LZGC003//Seed-Industrialized Development Program in Shandong Province, China/ ; 2021LZGC012//Key Research & Development Program of Shandong Province, China/ ; },
mesh = {*Glycine max/genetics/metabolism/drug effects ; *Herbicide Resistance/genetics ; *Cytosine/metabolism ; Gene Editing ; CRISPR-Cas Systems/genetics ; Herbicides/pharmacology ; Plants, Genetically Modified/genetics ; },
}

*Glycine max/genetics/metabolism/drug effects
*Herbicide Resistance/genetics
*Cytosine/metabolism
Gene Editing
CRISPR-Cas Systems/genetics
Herbicides/pharmacology
Plants, Genetically Modified/genetics

@article {pmid38623687,
year = {2024},
author = {Zhang, S and Wu, S and Jia, Z and Zhang, J and Li, Y and Ma, X and Fan, B and Wang, P and Gao, Y and Ye, Z and Wang, W},
title = {Exploring the influence of a single-nucleotide mutation in EIN4 on tomato fruit firmness diversity through fruit pericarp microstructure.},
journal = {Plant biotechnology journal},
volume = {22},
number = {9},
pages = {2379-2394},
pmid = {38623687},
issn = {1467-7652},
support = {31902024//National Natural Science Foundation of China/ ; 212102110413//Scientific and Technological Project of Henan Province/ ; 30500495//Research fund for the Doctoral program of Henan Agricultural University/ ; },
mesh = {*Solanum lycopersicum/genetics/growth & development ; *Fruit/genetics/growth & development ; *Plant Proteins/genetics/metabolism ; *Polymorphism, Single Nucleotide/genetics ; *Receptors, Cell Surface/genetics/metabolism ; *Genome-Wide Association Study ; Gene Expression Regulation, Plant ; Gene Editing ; CRISPR-Cas Systems ; },
abstract = {Tomato (Solanum lycopersicum) stands as one of the most valuable vegetable crops globally, and fruit firmness significantly impacts storage and transportation. To identify genes governing tomato firmness, we scrutinized the firmness of 266 accessions from core collections. Our study pinpointed an ethylene receptor gene, SlEIN4, located on chromosome 4 through a genome-wide association study (GWAS) of fruit firmness in the 266 tomato core accessions. A single-nucleotide polymorphism (SNP) (A → G) of SlEIN4 distinguished lower (AA) and higher (GG) fruit firmness genotypes. Through experiments, we observed that overexpression of SlEIN4[AA] significantly delayed tomato fruit ripening and dramatically reduced fruit firmness at the red ripe stage compared with the control. Conversely, gene editing of SlEIN4[AA] with CRISPR/Cas9 notably accelerated fruit ripening and significantly increased fruit firmness at the red ripe stage compared with the control. Further investigations revealed that fruit firmness is associated with alterations in the microstructure of the fruit pericarp. Additionally, SlEIN4[AA] positively regulates pectinase activity. The transient transformation assay verified that the SNP (A → G) on SlEIN4 caused different genetic effects, as overexpression of SlEIN4[GG] increased fruit firmness. Moreover, SlEIN4 exerts a negative regulatory role in tomato ripening by impacting ethylene evolution through the abundant expression of ethylene pathway regulatory genes. This study presents the first evidence of the role of ethylene receptor genes in regulating fruit firmness. These significant findings will facilitate the effective utilization of firmness and ripening traits in tomato improvement, offering promising opportunities for enhancing tomato storage and transportation capabilities.},
}

*Solanum lycopersicum/genetics/growth & development
*Fruit/genetics/growth & development
*Plant Proteins/genetics/metabolism
*Polymorphism, Single Nucleotide/genetics
*Receptors, Cell Surface/genetics/metabolism
*Genome-Wide Association Study
Gene Expression Regulation, Plant
Gene Editing
CRISPR-Cas Systems

@article {pmid35271349,
year = {2024},
author = {Fu, X and Duan, Z and Lu, X and Zhu, Y and Ren, Y and Zhang, W and Sun, X and Ge, L and Yang, J},
title = {SND1 Promotes Radioresistance in Cervical Cancer Cells by Targeting the DNA Damage Response.},
journal = {Cancer biotherapy & radiopharmaceuticals},
volume = {39},
number = {6},
pages = {425-434},
doi = {10.1089/cbr.2021.0371},
pmid = {35271349},
issn = {1557-8852},
mesh = {Humans ; *Uterine Cervical Neoplasms/genetics/radiotherapy/pathology/metabolism ; *Radiation Tolerance/genetics ; *DNA Damage ; Female ; HeLa Cells ; *Endonucleases/genetics/metabolism ; Apoptosis/genetics ; DNA Repair ; CRISPR-Cas Systems ; Ataxia Telangiectasia Mutated Proteins/genetics/metabolism ; },
abstract = {Background: Radiotherapy is one of the most effective therapeutic strategies for cervical cancer patients, although radioresistance-mediated residual and recurrent tumors are the main cause of treatment failure. However, the mechanism of tumor radioresistance is still elusive. DNA damage response pathways are key determinants of radioresistance. The purpose of this study was to investigate the role and mechanism of SND1 in radioresistance of cervical cancer. Methods: A stable HeLa cell line with SND1 knockout (HeLa-KO) was generated through a modified CRISPR/Cas9 double-nicking gene editing system. The stable CaSki cell lines with SND1 knockdown (CaSki-Ctrl, CaSki-SND1-sh-1, CaSki-SND1-sh-2) were constructed through lentivirus transfection with the pSil-SND1-sh-1 and pSil-SND1-sh-2 plasmids. Results: It was observed that SND1 deficiency significantly increased the radiosensitivity of cervical cancer cells. It was also found that silencing SND1 promotes radiation-induced apoptosis. Significantly, the cells with a loss of SND1 function exhibited inefficient ataxia telangiectasia mutated pathway activation, subsequently impairing DNA repair and G2/M checkpoint arrest. In addition, threonine 103 is an important phosphorylation site of SND1 under DNA damaging stress. Conclusion: Collectively, the results of this study reveal a potent radiosensitizing effect of silencing SND1 or T103 mutation on cervical cancer cells, providing novel insights into potential therapeutic strategies for cervical cancer treatment.},
}

Humans
*Uterine Cervical Neoplasms/genetics/radiotherapy/pathology/metabolism
*Radiation Tolerance/genetics
*DNA Damage
Female
HeLa Cells
*Endonucleases/genetics/metabolism
Apoptosis/genetics
DNA Repair
CRISPR-Cas Systems
Ataxia Telangiectasia Mutated Proteins/genetics/metabolism

@article {pmid39155053,
year = {2024},
author = {Koller, U and Bauer, JW},
title = {Emerging DNA & RNA editing strategies for the treatment of epidermolysis bullosa.},
journal = {The Journal of dermatological treatment},
volume = {35},
number = {1},
pages = {2391452},
doi = {10.1080/09546634.2024.2391452},
pmid = {39155053},
issn = {1471-1753},
mesh = {Humans ; *Epidermolysis Bullosa/therapy/genetics ; *Genetic Therapy ; *Gene Editing ; *CRISPR-Cas Systems ; RNA Editing ; Quality of Life ; },
abstract = {Background: Epidermolysis bullosa (EB) is a clinically-heterogeneous genodermatosis with severe manifestations in the skin and other organs. The significant burden this condition places on patients justifies the development of gene therapeutic strategies targeting the genetic cause of the disease. Methods: Emerging RNA and DNA editing tools have shown remarkable advances in efficiency and safety. Applicable both in ex vivo- and in vivo settings, these gene therapeutics based on gene replacement or editing are either at the pre-clinical or clinical stage. Results: The recent landmark FDA approvals for gene editing based on CRISPR/Cas9, along with the first FDA-approved redosable in vivo gene replacement therapy for EB, will invigorate ongoing research efforts, increasing the likelihood of achieving local cure via CRISPR-based technologies in the near future. Conclusions: This review discusses the status quo of current gene therapeutics that act at the level of RNA or DNA, all with the common aim of improving the quality of life for EB patients.},
}

Humans
*Epidermolysis Bullosa/therapy/genetics
*Genetic Therapy
*Gene Editing
*CRISPR-Cas Systems
RNA Editing
Quality of Life

@article {pmid39153653,
year = {2024},
author = {Razipour, M and Jamali, Z and Khorsand, M and Zargar, M and Maghsudlu, M and Ghadami, E and Shakoori, A},
title = {Circular RNAs in laryngeal cancer.},
journal = {Clinica chimica acta; international journal of clinical chemistry},
volume = {},
number = {},
pages = {119916},
doi = {10.1016/j.cca.2024.119916},
pmid = {39153653},
issn = {1873-3492},
abstract = {Laryngeal cancer remains a significant global health concern, with poor prognosis for advanced-stage disease highlighting the need for novel diagnostic, prognostic, and therapeutic approaches. Circular RNAs (circRNAs), a class of covalently closed non-coding RNAs, have emerged as important regulators of gene expression and cellular processes in various cancers, including laryngeal cancer. This review summarizes the current understanding of circRNAs in laryngeal cancer, covering their biogenesis, regulatory mechanisms, and potential clinical applications. We explore the diverse functions of circRNAs, including their roles as miRNA sponges, protein interactors, and direct mRNA regulators, and their influence on key cellular processes such as proliferation, invasion, and metastasis. The review highlights promising circRNAs as diagnostic and prognostic biomarkers, as well as potential therapeutic targets. We also outline current strategies for circRNA modulation, including suppression techniques like RNA interference and CRISPR/Cas systems, and overexpression methods using vectors and synthetic circRNAs.},
}