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Trichostatin A for Efficient CRISPR-Cas9 Gene Editing of Human Pluripotent Stem Cells. 曲霉菌素A用于人多能干细胞的有效CRISPR-Cas9基因编辑。
IF 3.7 4区 生物学 Q2 GENETICS & HEREDITY Pub Date : 2023-10-01 Epub Date: 2023-09-07 DOI: 10.1089/crispr.2023.0033
Kaivalya Molugu, Namita Khajanchi, Cicera R Lazzarotto, Shengdar Q Tsai, Krishanu Saha

Genome-edited human-induced pluripotent stem cells (iPSCs) have broad applications in disease modeling, drug discovery, and regenerative medicine. Despite the development of clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system, the gene editing process is inefficient and can take several weeks to months to generate edited iPSC clones. We developed a strategy to improve the efficiency of the iPSC gene editing process via application of a small-molecule, trichostatin A (TSA), a Class I and II histone deacetylase inhibitor. We observed that TSA decreased global chromatin condensation and further resulted in increased gene-editing efficiency of iPSCs by twofold to fourfold while concurrently ensuring no increased off-target effects. The edited iPSCs could be clonally expanded while maintaining genomic integrity and pluripotency. The rapid generation of therapeutically relevant gene-edited iPSCs could be enabled by these findings.

基因组编辑的人类诱导多能干细胞在疾病建模、药物发现和再生医学中有着广泛的应用。尽管开发了聚集的规则间隔短回文重复序列(CRISPR)-Cas9系统,但基因编辑过程效率低下,可能需要几周到几个月才能生成编辑的iPSC克隆。我们开发了一种策略,通过应用小分子曲霉菌素a(TSA),一种I类和II类组蛋白脱乙酰酶抑制剂,来提高iPSC基因编辑过程的效率。我们观察到TSA减少了整体染色质浓缩,并进一步使iPSC的基因编辑效率提高了两到四倍,同时确保了不会增加脱靶效应。编辑后的iPSC可以克隆扩增,同时保持基因组完整性和多能性。这些发现可以快速产生治疗相关的基因编辑的iPSC。
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引用次数: 0
Pan-Coronavirus CRISPR-CasRx Effector System Significantly Reduces Viable Titer in HCoV-OC43, HCoV-229E, and SARS-CoV-2. 泛冠状病毒CRISPR-CasRx效应系统显著降低HCoV-OC43、HCoV-229E和SARS-CoV-2的活价
IF 3.7 4区 生物学 Q3 Biochemistry, Genetics and Molecular Biology Pub Date : 2023-08-01 DOI: 10.1089/crispr.2022.0095
Cathryn M Mayes, Joshua L Santarpia

CRISPR-based technology has become widely used as an antiviral strategy, including as a broad-spectrum human coronavirus (HCoV) therapeutic. In this work, we have designed a CRISPR-CasRx effector system with guide RNAs (gRNAs) that are cross-reactive among several HCoV species. We tested the efficacy of this pan-coronavirus effector system by evaluating the reduction in viral viability associated with different CRISPR targets in HCoV-OC43, HCoV-229E, and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We determined that several CRISPR targets significantly reduce viral titer, despite the presence of single nucleotide polymorphisms in the gRNA when compared with a non-targeting, negative control gRNA. CRISPR targets reduced viral titer between 85% and >99% in HCoV-OC43, between 78% and >99% in HCoV-229E, and between 70% and 94% in SARS-CoV-2 when compared with an untreated virus control. These data establish a proof-of-concept for a pan-coronavirus CRISPR effector system that is capable of reducing viable virus in both Risk Group 2 and Risk Group 3 HCoV pathogens.

基于crispr的技术已被广泛用于抗病毒策略,包括作为广谱人类冠状病毒(HCoV)的治疗方法。在这项工作中,我们设计了一个CRISPR-CasRx效应系统,其引导rna (grna)在几种HCoV物种中具有交叉反应性。我们通过评估不同CRISPR靶点在HCoV-OC43、HCoV-229E和严重急性呼吸综合征冠状病毒2 (SARS-CoV-2)中与病毒活力相关的降低来测试该泛冠状病毒效应系统的有效性。我们确定,与非靶向的阴性对照gRNA相比,尽管gRNA中存在单核苷酸多态性,但几种CRISPR靶标显著降低了病毒滴度。与未经治疗的病毒对照相比,CRISPR靶标使HCoV-OC43的病毒滴度降低85%至>99%,HCoV-229E的病毒滴度降低78%至>99%,SARS-CoV-2的病毒滴度降低70%至94%。这些数据为泛冠状病毒CRISPR效应系统建立了概念验证,该系统能够减少风险组2和风险组3 HCoV病原体中的活病毒。
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引用次数: 0
My CRISPR Story: Back to Brazil. 我的CRISPR故事:回到巴西。
IF 3.7 4区 生物学 Q3 Biochemistry, Genetics and Molecular Biology Pub Date : 2023-08-01 DOI: 10.1089/crispr.2023.0032
Daniel F M Monte
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引用次数: 0
Discovery and Characterization of Novel Type V Cas12f Nucleases with Diverse Protospacer Adjacent Motif Preferences. 具有不同原间隔基序邻近基序偏好的新型V型Cas12f核酸酶的发现和表征
IF 3.7 4区 生物学 Q3 Biochemistry, Genetics and Molecular Biology Pub Date : 2023-08-01 DOI: 10.1089/crispr.2023.0006
Allison Sharrar, Luisa Arake de Tacca, Trevor Collingwood, Zuriah Meacham, David Rabuka, Johanna Staples-Ager, Michael Schelle

Small Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated (Cas) effectors are key to developing gene editing therapies due to the packaging constraints of viral vectors. While Cas9 and Cas12a CRISPR-Cas effectors have advanced into select clinical applications, their size is prohibitive for efficient delivery of both nuclease and guide RNA in a single viral vector. Type V Cas12f effectors present a solution given their small size. In this study, we describe a novel set of miniature (<490AA) Cas12f nucleases that cleave double-stranded DNA in human cells. We determined their optimal trans-activating RNA empirically through rational modifications, which resulted in an optimal single guide RNA. We show that these nucleases have broad protospacer adjacent motif (PAM) preferences, allowing for expanded genome targeting. The unique characteristics of these novel nucleases add to the diversity of the miniature CRISPR-Cas toolbox while the expanded PAM allows for the editing of genomic locations that could not be accessed with existing Cas12f nucleases.

由于病毒载体的包装限制,小簇规则间隔短回文重复序列(CRISPR)-CRISPR相关(Cas)效应物是开发基因编辑疗法的关键。虽然Cas9和Cas12a CRISPR-Cas效应体已经进入特定的临床应用,但它们的大小对于在单个病毒载体中有效递送核酸酶和引导RNA来说是令人望而却步的。V型Cas12f效应器由于其小尺寸而提供了一种解决方案。在这项研究中,我们描述了一套新颖的微型(
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引用次数: 1
Predicting Mutations Generated by Cas9, Base Editing, and Prime Editing in Mammalian Cells. 预测哺乳动物细胞中Cas9、碱基编辑和引体编辑产生的突变。
IF 3.7 4区 生物学 Q3 Biochemistry, Genetics and Molecular Biology Pub Date : 2023-08-01 DOI: 10.1089/crispr.2023.0016
Juliane Weller, Ananth Pallaseni, Jonas Koeppel, Leopold Parts

The first fruits of the CRISPR-Cas revolution are starting to enter the clinic, with gene editing therapies offering solutions to previously incurable genetic diseases. The success of such applications hinges on control over the mutations that are generated, which are known to vary depending on the targeted locus. In this review, we present the current state of understanding and predicting CRISPR-Cas cutting, base editing, and prime editing outcomes in mammalian cells. We first provide an introduction to the basics of DNA repair and machine learning that the models rely on. We then overview the datasets and methods created for characterizing edits at scale, as well as the insights that have been derived from them. The predictions generated from these models serve as a foundation for designing efficient experiments across the broad contexts where these tools are applied.

CRISPR-Cas革命的第一批成果开始进入临床,基因编辑疗法为以前无法治愈的遗传疾病提供了解决方案。这种应用的成功取决于对所产生的突变的控制,这些突变是根据目标位点而变化的。在这篇综述中,我们介绍了对哺乳动物细胞中CRISPR-Cas切割、碱基编辑和初始编辑结果的理解和预测的现状。我们首先介绍了模型所依赖的DNA修复和机器学习的基础知识。然后,我们概述了为描述大规模编辑而创建的数据集和方法,以及从中获得的见解。从这些模型中产生的预测可以作为在应用这些工具的广泛背景下设计有效实验的基础。
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引用次数: 0
Rosalind Franklin Society Proudly Announces the 2022 Award Recipient for The CRISPR Journal. 罗莎琳德·富兰克林协会自豪地宣布了2022年CRISPR杂志的获奖者。
IF 3.7 4区 生物学 Q3 Biochemistry, Genetics and Molecular Biology Pub Date : 2023-08-01 DOI: 10.1089/crispr.2023.29162.rfs2022
Nicole F Brackett
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引用次数: 0
Fanzors: Mysterious TnpB-Like Bacterial Transposon-Related RNA-Guided DNA Nucleases of Eukaryotes. 神秘的tnpb样细菌转座子相关rna引导的真核生物DNA核酸酶。
IF 3.7 4区 生物学 Q3 Biochemistry, Genetics and Molecular Biology Pub Date : 2023-08-01 DOI: 10.1089/crispr.2023.29164.tka
Tautvydas Karvelis, Virginijus Siksnys
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引用次数: 0
CRISPR Milestones for Sustainable Agriculture and Forestry. 可持续农业和林业的CRISPR里程碑。
IF 3.7 4区 生物学 Q3 Biochemistry, Genetics and Molecular Biology Pub Date : 2023-08-01 DOI: 10.1089/crispr.2023.29163.editorial
Rodolphe Barrangou
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引用次数: 0
Multiplex Editing of the Nucleoredoxin1 Tandem Array in Poplar: From Small Indels to Translocations and Complex Inversions. 杨树核还原蛋白1串联阵列的多重编辑:从小索引到易位和复杂反转。
IF 3.7 4区 生物学 Q3 Biochemistry, Genetics and Molecular Biology Pub Date : 2023-08-01 DOI: 10.1089/crispr.2022.0096
Yen-Ho Chen, Shakuntala Sharma, William P Bewg, Liang-Jiao Xue, Cole R Gizelbach, Chung-Jui Tsai

The CRISPR-Cas9 system has been deployed for precision mutagenesis in an ever-growing number of species, including agricultural crops and forest trees. Its application to closely linked genes with extremely high sequence similarities has been less explored. In this study, we used CRISPR-Cas9 to mutagenize a tandem array of seven Nucleoredoxin1 (NRX1) genes spanning ∼100 kb in Populus tremula × Populus alba. We demonstrated efficient multiplex editing with one single guide RNA in 42 transgenic lines. The mutation profiles ranged from small insertions and deletions and local deletions in individual genes to large genomic dropouts and rearrangements spanning tandem genes. We also detected complex rearrangements including translocations and inversions resulting from multiple cleavage and repair events. Target capture sequencing was instrumental for unbiased assessments of repair outcomes to reconstruct unusual mutant alleles. The work highlights the power of CRISPR-Cas9 for multiplex editing of tandemly duplicated genes to generate diverse mutants with structural and copy number variations to aid future functional characterization.

CRISPR-Cas9系统已被用于越来越多的物种的精确诱变,包括农作物和森林树木。它在具有极高序列相似性的紧密连接基因中的应用研究较少。在这项研究中,我们使用CRISPR-Cas9诱变了白杨(Populus tremula × Populus alba)中7个NRX1基因的串联阵列,长度约为100 kb。我们在42个转基因品系中展示了使用单个引导RNA的高效多重编辑。突变谱范围从单个基因的小插入和缺失和局部缺失到跨串联基因的大基因组缺失和重排。我们还检测到复杂的重排,包括由多次裂解和修复事件引起的易位和倒位。靶捕获测序对于重建异常突变等位基因的修复结果的公正评估是有用的。这项工作强调了CRISPR-Cas9对串联复制基因进行多重编辑的能力,可以产生具有结构和拷贝数变化的多种突变体,以帮助未来的功能表征。
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引用次数: 0
Rapid and Technically Simple Detection of SARS-CoV-2 Variants Using CRISPR Cas12 and Cas13. 利用CRISPR Cas12和Cas13快速、技术简单地检测SARS-CoV-2变体
IF 3.7 4区 生物学 Q3 Biochemistry, Genetics and Molecular Biology Pub Date : 2023-08-01 DOI: 10.1089/crispr.2023.0007
Gabriel Lamothe, Julie Carbonneau, Charles Joly Beauparlant, Thierry Vincent, Patrik Quessy, Anthony Guedon, Gary Kobinger, Jean-Francois Lemay, Guy Boivin, Arnaud Droit, Nathalie Turgeon, Jacques P Tremblay

The worldwide proliferation of the SARS-CoV-2 virus in the past 3 years has allowed the virus to accumulate numerous mutations. Dangerous lineages have emerged one after another, each leading to a new wave of the pandemic. In this study, we have developed the THRASOS pipeline to rapidly discover lineage-specific mutation signatures and thus advise the development of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based diagnostic tests. We also optimized a strategy to modify loop-mediated isothermal amplification amplicons for downstream use with Cas12 and Cas13 for future multiplexing. The close ancestry of the BA.1 and BA.2 variants of SARS-CoV-2 (Omicron) made these excellent candidates for the development of a first test using this workflow. With a quick turnaround time and low requirements for laboratory equipment, the test we have created is ideally suited for settings such as mobile clinics lacking equipment such as Next-Generation Sequencers or Sanger Sequencers and the personnel to run these devices.

过去3年来,SARS-CoV-2病毒在全球范围内的扩散使该病毒积累了许多突变。危险的谱系一个接一个地出现,每一个都导致新一波大流行。在这项研究中,我们开发了THRASOS管道,以快速发现谱系特异性突变特征,从而建议开发基于集群规则间隔短回文重复序列(CRISPR)的诊断测试。我们还优化了一种策略,修改环介导的等温扩增扩增子,用于Cas12和Cas13的下游使用,以用于未来的多路复用。SARS-CoV-2 (Omicron)的ba - 1和ba -2变体的近亲使它们成为使用该工作流程开发第一个测试的优秀候选者。由于周转时间短,对实验室设备的要求低,我们创建的测试非常适合缺乏下一代测序仪或桑格测序仪等设备的移动诊所以及运行这些设备的人员等设置。
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CRISPR Journal
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