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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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引用次数: 0
CRISPR-Cas-Based Biomonitoring for Marine Environments: Toward CRISPR RNA Design Optimization Via Deep Learning. 基于CRISPR- cas的海洋环境生物监测:通过深度学习实现CRISPR RNA设计优化。
IF 3.7 4区 生物学 Q3 Biochemistry, Genetics and Molecular Biology Pub Date : 2023-08-01 DOI: 10.1089/crispr.2023.0019
Benjamín Durán-Vinet, Karla Araya-Castro, Anastasija Zaiko, Xavier Pochon, Susanna A Wood, Jo-Ann L Stanton, Gert-Jan Jeunen, Michelle Scriver, Anya Kardailsky, Tzu-Chiao Chao, Deependra K Ban, Maryam Moarefian, Kiana Aran, Neil J Gemmell

Almost all of Earth's oceans are now impacted by multiple anthropogenic stressors, including the spread of nonindigenous species, harmful algal blooms, and pathogens. Early detection is critical to manage these stressors effectively and to protect marine systems and the ecosystem services they provide. Molecular tools have emerged as a promising solution for marine biomonitoring. One of the latest advancements involves utilizing CRISPR-Cas technology to build programmable, rapid, ultrasensitive, and specific diagnostics. CRISPR-based diagnostics (CRISPR-Dx) has the potential to allow robust, reliable, and cost-effective biomonitoring in near real time. However, several challenges must be overcome before CRISPR-Dx can be established as a mainstream tool for marine biomonitoring. A critical unmet challenge is the need to design, optimize, and experimentally validate CRISPR-Dx assays. Artificial intelligence has recently been presented as a potential approach to tackle this challenge. This perspective synthesizes recent advances in CRISPR-Dx and machine learning modeling approaches, showcasing CRISPR-Dx potential to progress as a rising molecular tool candidate for marine biomonitoring applications.

现在,地球上几乎所有的海洋都受到多种人为压力因素的影响,包括非本地物种的传播、有害的藻华和病原体。早期发现对于有效管理这些压力源和保护海洋系统及其提供的生态系统服务至关重要。分子工具已经成为海洋生物监测的一种很有前途的解决方案。最新的进展之一是利用CRISPR-Cas技术建立可编程、快速、超灵敏和特异性的诊断方法。基于crispr的诊断(CRISPR-Dx)有可能实现强大、可靠和经济的近实时生物监测。然而,在CRISPR-Dx成为海洋生物监测的主流工具之前,必须克服几个挑战。一个关键的未满足的挑战是需要设计、优化和实验验证CRISPR-Dx分析。人工智能最近被认为是解决这一挑战的一种潜在方法。这一观点综合了CRISPR-Dx和机器学习建模方法的最新进展,展示了CRISPR-Dx作为海洋生物监测应用的新兴分子工具的潜力。
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引用次数: 0
CRISPR and the Plant Pathologists' Holy Grail. CRISPR和植物病理学家的圣杯。
IF 3.7 4区 生物学 Q3 Biochemistry, Genetics and Molecular Biology Pub Date : 2023-08-01 DOI: 10.1089/crispr.2023.29165.mwi
Matthew R Willmann
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引用次数: 0
CRISPR Comparison Toolkit: Rapid Identification, Visualization, and Analysis of CRISPR Array Diversity. CRISPR比较工具箱:快速鉴定,可视化和分析CRISPR阵列多样性。
IF 3.7 4区 生物学 Q3 Biochemistry, Genetics and Molecular Biology Pub Date : 2023-08-01 DOI: 10.1089/crispr.2022.0080
Alan J Collins, Rachel J Whitaker

CRISPR-Cas systems provide immunity against mobile genetic elements (MGEs) through sequence-specific targeting by spacer sequences encoded in CRISPR arrays. Spacers are highly variable between microbial strains and can be acquired rapidly, making them well suited for use in strain typing of closely related organisms. However, no tools are currently available to automate the process of reconstructing strain histories using CRISPR spacers. We therefore developed the CRISPR Comparison Toolkit (CCTK) to enable analyses of array relationships. The CCTK includes tools to identify arrays, analyze relationships between arrays using CRISPRdiff and CRISPRtree, and predict targets of spacers. CRISPRdiff visualizes arrays and highlights the similarities between them. CRISPRtree infers a phylogenetic tree from array relationships and presents a hypothesis of the evolutionary history of the arrays. The CCTK unifies several CRISPR analysis tools into a single command line application, including the first tool to infer phylogenies from array relationships.

CRISPR- cas系统通过在CRISPR阵列中编码间隔序列的序列特异性靶向,提供对移动遗传元件(MGEs)的免疫。间隔物在微生物菌株之间是高度可变的,并且可以快速获得,这使得它们非常适合用于密切相关生物的菌株分型。然而,目前还没有工具可以自动化使用CRISPR间隔器重建菌株历史的过程。因此,我们开发了CRISPR比较工具包(CCTK)来分析阵列关系。CCTK包括识别阵列的工具,使用CRISPRdiff和CRISPRtree分析阵列之间的关系,并预测间隔器的目标。CRISPRdiff可视化数组并突出显示它们之间的相似性。CRISPRtree从阵列关系中推断出一个系统发育树,并提出了阵列进化史的假设。CCTK将几个CRISPR分析工具统一到一个命令行应用程序中,包括第一个从数组关系推断系统发育的工具。
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引用次数: 4
期刊
CRISPR Journal
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