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A Multiplexed Cas13-Based Assay with Point-of-Care Attributes for Simultaneous COVID-19 Diagnosis and Variant Surveillance. 一种基于多点cas13的检测方法,可同时用于COVID-19诊断和变异监测。
IF 3.7 4区 生物学 Q3 Biochemistry, Genetics and Molecular Biology Pub Date : 2023-04-01 DOI: 10.1089/crispr.2022.0048
Maturada Patchsung, Aimorn Homchan, Kanokpol Aphicho, Surased Suraritdechachai, Thanyapat Wanitchanon, Archiraya Pattama, Khomkrit Sappakhaw, Piyachat Meesawat, Thanakrit Wongsatit, Artittaya Athipanyasilp, Krittapas Jantarug, Niracha Athipanyasilp, Juthamas Buahom, Supapat Visanpattanasin, Nootaree Niljianskul, Pimchai Chaiyen, Ruchanok Tinikul, Nuanjun Wichukchinda, Surakameth Mahasirimongkol, Rujipas Sirijatuphat, Nasikarn Angkasekwinai, Michael A Crone, Paul S Freemont, Julia Joung, Alim Ladha, Omar Abudayyeh, Jonathan Gootenberg, Feng Zhang, Claire Chewapreecha, Sittinan Chanarat, Navin Horthongkham, Danaya Pakotiprapha, Chayasith Uttamapinant

Point-of-care (POC) nucleic acid detection technologies are poised to aid gold-standard technologies in controlling the COVID-19 pandemic, yet shortcomings in the capability to perform critically needed complex detection-such as multiplexed detection for viral variant surveillance-may limit their widespread adoption. Herein, we developed a robust multiplexed clustered regularly interspaced short palindromic repeats (CRISPR)-based detection using LwaCas13a and PsmCas13b to simultaneously diagnose severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and pinpoint the causative SARS-CoV-2 variant of concern (VOC)-including globally dominant VOCs Delta (B.1.617.2) and Omicron (B.1.1.529)-all the while maintaining high levels of accuracy upon the detection of multiple SARS-CoV-2 gene targets. The platform has several attributes suitable for POC use: premixed, freeze-dried reagents for easy use and storage; convenient direct-to-eye or smartphone-based readouts; and a one-pot variant of the multiplexed detection. To reduce reliance on proprietary reagents and enable sustainable use of such a technology in low- and middle-income countries, we locally produced and formulated our own recombinase polymerase amplification reaction and demonstrated its equivalent efficiency to commercial counterparts. Our tool-CRISPR-based detection for simultaneous COVID-19 diagnosis and variant surveillance that can be locally manufactured-may enable sustainable use of CRISPR diagnostics technologies for COVID-19 and other diseases in POC settings.

即时护理(POC)核酸检测技术有望帮助金标准技术控制COVID-19大流行,但在执行急需的复杂检测(如病毒变异监测的多路检测)能力方面的缺陷可能会限制其广泛采用。在此,我们开发了一种基于LwaCas13a和PsmCas13b的强大的多路聚类定期间隔短重复序列(CRISPR)的检测方法,可以同时诊断严重急性呼吸综合征冠状病毒2 (SARS-CoV-2)感染,并确定引起SARS-CoV-2的致病变体(VOC),包括全球主要的VOCs Delta (B.1.617.2)和Omicron (B.1.1.529),同时在检测多个SARS-CoV-2基因靶点时保持高水平的准确性。该平台具有适合POC使用的几个属性:预混,冷冻干燥试剂,便于使用和储存;方便直接看眼或智能手机读数;以及多路检测的一锅变体。为了减少对专有试剂的依赖,并使这种技术能够在低收入和中等收入国家持续使用,我们在当地生产和配制了我们自己的重组酶聚合酶扩增反应,并证明了其与商业对应物的同等效率。我们的工具——基于CRISPR的同时诊断COVID-19和变异监测的检测,可以在当地生产——可以在POC环境中可持续地使用CRISPR诊断技术来诊断COVID-19和其他疾病。
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引用次数: 0
Multiplex gRNAs Synergically Enhance Detection of SARS-CoV-2 by CRISPR-Cas12a. 多重gRNAs协同增强CRISPR-Cas12a对SARS-CoV-2的检测
IF 3.7 4区 生物学 Q3 Biochemistry, Genetics and Molecular Biology Pub Date : 2023-04-01 Epub Date: 2023-03-21 DOI: 10.1089/crispr.2022.0074
Melissa D Morales-Moreno, Erick G Valdés-Galindo, Mariana M Reza, Tatiana Fiordelisio, Jorge Peon, Armando Hernandez-Garcia

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) diagnostic methods have a large potential to effectively detect SARS-CoV-2 with sensitivity and specificity nearing 100%, comparable to quantitative polymerase chain reaction. Yet, there is room for improvement. Commonly, one guide CRISPR RNA (gRNA) is used to detect the virus DNA and activate Cas collateral activity, which cleaves a reporter probe. In this study, we demonstrated that using 2-3 gRNAs in parallel can create a synergistic effect, resulting in a 4.5 × faster cleaving rate of the probe and increased sensitivity compared to using individual gRNAs. The synergy is due to the simultaneous activation of CRISPR-Cas12a and the improved performance of each gRNA. This approach was able to detect as few as 10 viral copies of the N-gene of SARS-CoV-2 RNA after a preamplification step using reverse transcription loop-mediated isothermal amplification. The method was able to accurately detect 100% of positive and negative clinical samples in ∼25 min using a fluorescence plate reader and ∼45 min with lateral flow strips.

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)诊断方法在有效检测SARS-CoV-2方面具有很大的潜力,其灵敏度和特异性接近100%,与定量聚合酶链反应相当。然而,仍有改进的余地。通常,使用一个引导CRISPR RNA (gRNA)来检测病毒DNA并激活Cas附带活性,从而切割报告蛋白探针。在这项研究中,我们证明了平行使用2-3个grna可以产生协同效应,与使用单个grna相比,导致探针的切割速度提高4.5倍,灵敏度提高。这种协同作用是由于CRISPR-Cas12a的同时激活和每个gRNA的性能提高。该方法能够在使用逆转录环介导的等温扩增的预扩增步骤后检测到SARS-CoV-2 RNA n基因的10个病毒拷贝。该方法能够在~ 25分钟内使用荧光板阅读器准确检测100%的阳性和阴性临床样品,并在~ 45分钟内使用横向流动条。
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引用次数: 3
Special Issue: Manipulating the Microbiome with CRISPR. 特刊:用CRISPR操纵微生物组。
IF 3.7 4区 生物学 Q3 Biochemistry, Genetics and Molecular Biology Pub Date : 2023-04-01 DOI: 10.1089/crispr.2023.0005.cfp
Brady Cress, Rodolphe Barrangou
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引用次数: 0
Specific High-Sensitivity Enzymatic Reporter UnLOCKing-Mediated Detection of Oncogenic BCR::ABL1 and EGFR Rearrangements. 特异性高灵敏度酶报告基因解锁介导的致癌BCR::ABL1和EGFR重排检测。
IF 3.7 4区 生物学 Q3 Biochemistry, Genetics and Molecular Biology Pub Date : 2023-04-01 DOI: 10.1089/crispr.2022.0070
Grégoire Cullot, Samuel Amintas, Laura Karembé, Valérie Prouzet-Mauléon, Julie Rébillard, Lisa Boureau, David Cappellen, Aurélie Bedel, François Moreau-Gaudry, Stéphanie Dulucq, Sandrine Dabernat, Béatrice Turcq

Advances in molecular medicine have placed nucleic acid detection methods at the center of an increasing number of clinical applications. Polymerase chain reaction (PCR)-based diagnostics have been widely adopted for their versatility, specificity, and sensitivity. However, recently reported clustered regularly interspaced short palindromic repeats-based methods have demonstrated equivalent to superior performance, with increased portability and reduced processing time and cost. In this study, we applied Specific High-Sensitivity Enzymatic Reporter UnLOCKing (SHERLOCK) technology to the detection of oncogenic rearrangements. We implemented SHERLOCK for the detection of BCR::ABL1 mRNA, a hallmark of chronic myeloid leukemia (CML), and EGFR DNA oncogenic alleles, frequently detected in glioblastoma and non-small cell lung cancer (NSCLC). SHERLOCK enabled rapid, sensitive, and variant-specific detection of BCR::ABL1 and EGFR alterations. Compared with the gold-standard PCR-based methods currently used in clinic, SHERLOCK achieved equivalent to greater sensitivity, suggesting it could be a new tool in CML and NSCLC, to detect low level of molecular residual disease.

分子医学的进步使核酸检测方法在越来越多的临床应用中处于中心地位。基于聚合酶链反应(PCR)的诊断因其通用性、特异性和敏感性而被广泛采用。然而,最近报道的基于集群的规则间隔短回文重复的方法已经证明具有同等的优越性能,具有更高的可移植性和更少的处理时间和成本。在这项研究中,我们应用了特异性高灵敏度酶报告解锁(SHERLOCK)技术来检测致癌重排。我们使用SHERLOCK检测BCR::ABL1 mRNA(慢性髓性白血病(CML)的标志)和EGFR DNA致癌等位基因(常在胶质母细胞瘤和非小细胞肺癌(NSCLC)中检测到)。SHERLOCK能够快速、敏感和变异特异性地检测BCR::ABL1和EGFR的改变。与目前临床使用的基于金标准pcr的方法相比,SHERLOCK达到了相当高的灵敏度,提示它可能成为CML和NSCLC中检测低水平分子残留疾病的新工具。
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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 : 2023-02-20 DOI: 10.1101/2022.08.26.505498
Yen-Ho Chen, Shakuntala Sharma, W. 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. Here, we used CRISPR-Cas9 to mutagenize a tandem array of seven Nucleoredoxin1 (NRX1) genes spanning ~100 kb in Populus tremula × alba. We demonstrated efficient multiplex editing with one single gRNA in 42 transgenic lines. The mutation profiles ranged from small indels 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 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。在这里,我们使用CRISPR-Cas9诱变了白杨(Populus tremula × alba)中长度约100 kb的7个NRX1基因串联阵列。我们在42个转基因品系中展示了一个单一gRNA的高效多重编辑。突变谱范围从单个基因的小缺失和局部缺失到大的基因组缺失和跨串联基因的重排。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,以帮助功能表征。
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引用次数: 6
CRISPR Crops and Sustainable Agriculture. CRISPR作物与可持续农业。
IF 3.7 4区 生物学 Q3 Biochemistry, Genetics and Molecular Biology Pub Date : 2023-02-01 DOI: 10.1089/crispr.2023.0002.editorial
Rodolphe Barrangou
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引用次数: 0
Automated Good Manufacturing Practice-Compatible CRISPR-Cas9 Editing of Hematopoietic Stem and Progenitor Cells for Clinical Treatment of β-Hemoglobinopathies. 自动化良好生产规范兼容的CRISPR-Cas9编辑造血干细胞和祖细胞用于β-血红蛋白病的临床治疗
IF 3.7 4区 生物学 Q3 Biochemistry, Genetics and Molecular Biology Pub Date : 2023-02-01 DOI: 10.1089/crispr.2022.0086
Guillermo Ureña-Bailén, Milena Block, Tommaso Grandi, Faidra Aivazidou, Jona Quednau, Dariusz Krenz, Alberto Daniel-Moreno, Andrés Lamsfus-Calle, Thomas Epting, Rupert Handgretinger, Stefan Wild, Markus Mezger

Cellular therapies hold enormous potential for the cure of severe hematological and oncological disorders. The forefront of innovative gene therapy approaches including therapeutic gene editing and hematopoietic stem cell transplantation needs to be processed by good manufacturing practice to ensure safe application in patients. In the present study, an effective transfection protocol for automated clinical-scale production of genetically modified hematopoietic stem and progenitor cells (HSPCs) using the CliniMACS Prodigy® system including the CliniMACS Electroporator (Miltenyi Biotec) was established. As a proof-of-concept, the enhancer of the BCL11A gene, clustered regularly interspaced short palindromic repeat (CRISPR) target in ongoing clinical trials for β-thalassemia and sickle-cell disease treatment, was disrupted by the CRISPR-Cas9 system simulating a large-scale clinical scenario, yielding 100 million HSPCs with high editing efficiency. In vitro erythroid differentiation and high-performance liquid chromatography analyses corroborated fetal hemoglobin resurgence in edited samples, supporting the feasibility of running the complete process of HSPC gene editing in an automated closed system.

细胞疗法在治疗严重的血液和肿瘤疾病方面具有巨大的潜力。包括治疗性基因编辑和造血干细胞移植在内的创新基因治疗方法的前沿需要通过良好的生产规范进行处理,以确保患者的安全应用。在本研究中,使用CliniMACS Prodigy®系统(包括CliniMACS Electroporator (Miltenyi Biotec))建立了一种用于自动化临床规模生产转基因造血干细胞和祖细胞(HSPCs)的有效转染方案。作为一项概念验证,BCL11A基因的增强子是正在进行的用于治疗β-地中海贫血和镰状细胞病的临床试验中聚集的规则间隔短回弹重复(CRISPR)靶点,通过模拟大规模临床场景的CRISPR- cas9系统被破坏,产生了1亿个具有高编辑效率的HSPCs。体外红细胞分化和高效液相色谱分析证实了编辑样本中胎儿血红蛋白的复活,支持了在自动化封闭系统中运行HSPC基因编辑完整过程的可行性。
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引用次数: 1
A CRISPR-dCas9 System for Assaying and Selecting for RNase III Activity In Vivo in Escherichia coli. 基于CRISPR-dCas9的大肠杆菌RNase III活性测定与筛选系统
IF 3.7 4区 生物学 Q3 Biochemistry, Genetics and Molecular Biology Pub Date : 2023-02-01 DOI: 10.1089/crispr.2022.0041
Pricila Hauk, Ryan Weeks, Marc Ostermeier

Ribonuclease III (RNase III) and RNase III-like ribonucleases have a wide range of important functions and are found in all organisms, yet a simple and high-throughput in vivo method for measuring RNase III activity does not exist. Typical methods for measuring RNase III activity rely on in vitro RNA analysis or in vivo methods that are not suitable for high-throughput analysis. In this study, we describe our development of a deactivated Cas9 (dCas9)-based in vivo assay for RNase III activity that utilizes RNase III's cleavage of the 5'-untranslated region (UTR) of its own messenger RNA. The key molecule in the system is a hybrid guide RNA (gRNA) between the 5'-UTR of RNase III and gGFP, a gRNA that works with dCas9 to repress GFP expression. This fusion must be cleaved by RNase III for full GFP repression. Our system uses GFP fluorescence to report on Escherichia coli RNase III activity in culture and on an individual cell basis, making it effective for selecting individual cells through fluorescence-activated cell sorting. Homology between enzymes within the RNase III family suggests this assay might be adapted to measure the activity of other enzymes in the RNase III family such as human Dicer or Drosha.

核糖核酸酶III (RNase III)和RNase III样核糖核酸酶具有广泛的重要功能,存在于所有生物体中,但尚不存在一种简单、高通量的体内测定RNase III活性的方法。测量RNase III活性的典型方法依赖于体外RNA分析或不适合高通量分析的体内方法。在这项研究中,我们描述了我们开发的一种基于失活Cas9 (dCas9)的RNase III活性体内检测方法,该方法利用RNase III对其自身信使RNA的5'-未翻译区(UTR)的切割。该系统中的关键分子是RNase III的5'-UTR和gGFP之间的混合引导RNA (gRNA),该gRNA与dCas9一起抑制GFP的表达。为了充分抑制GFP,这种融合必须被RNase III切割。我们的系统使用GFP荧光报告大肠杆菌RNase III在培养和单个细胞基础上的活性,使其有效地通过荧光激活细胞分选来选择单个细胞。RNase III家族内酶之间的同源性表明,该方法可能适用于测量RNase III家族中其他酶的活性,如人类Dicer或Drosha。
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引用次数: 1
PASTE: The Way Forward for Large DNA Insertions. 大DNA插入的前进之路。
IF 3.7 4区 生物学 Q3 Biochemistry, Genetics and Molecular Biology Pub Date : 2023-02-01 DOI: 10.1089/crispr.2023.0001
Muhammad Arslan Mahmood, Shahid Mansoor
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引用次数: 2
Replication Protein Rep Provides Selective Advantage to Viruses in the Presence of CRISPR-Cas Immunity. 复制蛋白Rep在CRISPR-Cas免疫存在下对病毒提供选择性优势。
IF 3.7 4区 生物学 Q3 Biochemistry, Genetics and Molecular Biology Pub Date : 2023-02-01 DOI: 10.1089/crispr.2022.0037
Weijia Zhang, Yuvaraj Bhoobalan-Chitty, Xichuan Zhai, Yan Hui, Lars Hestbjerg Hansen, Ling Deng, Xu Peng

Anti-Clustered regularly interspaced small palindromic repeat (CRISPR) (Acr) phages cooperate to establish a successful infection in CRISPR-containing host. We report here the selective advantage provided by a replication initiator, Rep, toward cooperative host immunosuppression by viruses encoding Acrs. A rep knockout mutant (Δgp16) of Sulfolobus islandicus rod-shaped virus 2 produced around fourfold less virus in a CRISPR-null host, suggesting that Rep is the major replication initiator. In addition to Rep-dependent replication initiation from the viral genomic termini, we detected Rep-independent replication initiation from nonterminal sites. Intriguingly, despite the presence of Acrs, lack of Rep showed a profound effect on virus propagation in a host carrying CRISPR-Cas immunity. Accordingly, the co-infecting parental virus (rep-containing) outcompeted the Δgp16 mutant much more quickly in the CRISPR-containing host than in CRISPR-null host. Despite the nonessentiality, rep is carried by all known members of Rudiviridae, which is likely an evolutionary outcome driven by the ubiquitous presence of CRISPR-Cas in Sulfolobales.

抗聚集规则间隔小回文重复(CRISPR) (Acr)噬菌体合作在含有CRISPR的宿主中成功建立感染。我们在这里报告了复制启动器Rep对编码Acrs的病毒的协同宿主免疫抑制所提供的选择性优势。岛硫虫棒状病毒2的rep敲除突变体(Δgp16)在crispr无效的宿主中产生的病毒数量减少了约四倍,这表明rep是主要的复制启动器。除了来自病毒基因组末端的rep依赖性复制起始外,我们还检测到了来自非末端位点的rep非依赖性复制起始。有趣的是,尽管存在Acrs,但缺乏Rep对携带CRISPR-Cas免疫的宿主中的病毒传播有深远的影响。因此,在含有crispr的宿主中,共感染的亲本病毒(含rep)比在没有crispr的宿主中更快地战胜了Δgp16突变体。尽管没有必要,但所有已知的Rudiviridae成员都携带rep,这可能是由Sulfolobales中普遍存在的CRISPR-Cas驱动的进化结果。
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引用次数: 0
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CRISPR Journal
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