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Amplifying CRISPR: Next-Generation Diagnostics. 扩增CRISPR:新一代诊断。
IF 3.7 4区 生物学 Q3 Biochemistry, Genetics and Molecular Biology Pub Date : 2023-04-01 DOI: 10.1089/crispr.2023.0004.editorial
Rodolphe Barrangou
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引用次数: 1
Periodontal Disease Pathogens, Pathogenesis, and Therapeutics: The CRISPR-Cas Effect. 牙周病病原体、发病机制和治疗:CRISPR-Cas效应。
IF 3.7 4区 生物学 Q3 Biochemistry, Genetics and Molecular Biology Pub Date : 2023-04-01 DOI: 10.1089/crispr.2022.0094
Madhurya N Kedlaya, Lakshmi Puzhankara, Rohit Prasad, Akshatha Raj

Periodontal disease (PD) is an immune-inflammatory disease affecting the supporting structures of the teeth, which results in progressive destruction of the hard and soft tissues surrounding teeth, ultimately resulting in tooth loss. The primary etiological factor for this disease is the presence of pathogenic microorganisms. Pathogenic bacteria face antagonistic conditions and foreign DNA components during the infection stage and depend on defense mechanisms such as clustered regularly interspaced short palindromic repeats (CRISPR)-Cas to counter them. Virulence genes regulated by the CRISPR-Cas system are often expressed by bacteria as part of the stress response to the presence of stress conditions and foreign elements. There is ever-growing evidence regarding the role of CRISPR-Cas in virulence of periodontal pathogens. The same CRISPR-Cas system may also be targeted to reduce bacterial virulence and it may also be utilized to develop diagnostic and therapeutic strategies for prevention and control of PD progression. This review article describes the CRISPR-Cas systems in the periodontal dysbiotic microbial communities, their role in the virulence of periodontal pathogens, and their potential role in understanding the pathogenesis of periodontitis and treatment of PD.

牙周病(Periodontal disease, PD)是一种影响牙齿支撑结构的免疫炎症性疾病,其结果是牙齿周围的硬组织和软组织逐渐被破坏,最终导致牙齿脱落。本病的主要病因是病原微生物的存在。病原菌在感染阶段面临拮抗条件和外源DNA成分,依赖于聚集规律间隔短回文重复序列(CRISPR -Cas)等防御机制来对抗它们。由CRISPR-Cas系统调控的毒力基因通常作为细菌对应激条件和外来因素的应激反应的一部分而表达。有越来越多的证据表明CRISPR-Cas在牙周病原体的毒力中的作用。同样的CRISPR-Cas系统也可以用于降低细菌毒力,也可以用于制定预防和控制PD进展的诊断和治疗策略。这篇综述文章介绍了CRISPR-Cas系统在牙周微生物群落中的作用,它们在牙周病原体的毒力中的作用,以及它们在了解牙周炎的发病机制和PD治疗中的潜在作用。
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引用次数: 1
Functional and Phylogenetic Diversity of Cas10 Proteins. Cas10蛋白的功能和系统发育多样性。
IF 3.7 4区 生物学 Q2 GENETICS & HEREDITY Pub Date : 2023-04-01 Epub Date: 2023-03-13 DOI: 10.1089/crispr.2022.0085
Tanner Wiegand, Royce Wilkinson, Andrew Santiago-Frangos, Mackenzie Lynes, Roland Hatzenpichler, Blake Wiedenheft

Cas10 proteins are large subunits of type III CRISPR RNA (crRNA)-guided surveillance complexes, many of which have nuclease and cyclase activities. Here, we use computational and phylogenetic methods to identify and analyze 2014 Cas10 sequences from genomic and metagenomic databases. Cas10 proteins cluster into five distinct clades that mirror previously established CRISPR-Cas subtypes. Most Cas10 proteins (85.0%) have conserved polymerase active-site motifs, while HD-nuclease domains are less well conserved (36.0%). We identify Cas10 variants that are split over multiple genes or genetically fused to nucleases activated by cyclic nucleotides (i.e., NucC) or components of toxin-antitoxin systems (i.e., AbiEii). To clarify the functional diversification of Cas10 proteins, we cloned, expressed, and purified five representatives from three phylogenetically distinct clades. None of the Cas10s are functional cyclases in isolation, and activity assays performed with polymerase domain active site mutants indicate that previously reported Cas10 DNA-polymerase activity may be a result of contamination. Collectively, this work helps clarify the phylogenetic and functional diversity of Cas10 proteins in type III CRISPR systems.

Cas10蛋白是III型CRISPR RNA (crRNA)引导的监视复合物的大亚基,其中许多具有核酸酶和环化酶活性。在这里,我们使用计算和系统发育方法从基因组和宏基因组数据库中识别和分析2014 Cas10序列。Cas10蛋白聚集成五个不同的分支,反映了先前建立的CRISPR-Cas亚型。大多数Cas10蛋白(85.0%)具有保守的聚合酶活性位点基序,而hd -核酸酶结构域的保守性较差(36.0%)。我们发现Cas10变异在多个基因上分裂,或者在遗传上融合到由环核苷酸(即NucC)或毒素-抗毒素系统成分(即AbiEii)激活的核酸酶上。为了阐明Cas10蛋白的功能多样化,我们克隆、表达并纯化了来自3个不同进化枝的5个代表。没有一个Cas10是分离的功能性环化酶,对聚合酶结构域活性位点突变体进行的活性测定表明,先前报道的Cas10 dna聚合酶活性可能是污染的结果。总的来说,这项工作有助于阐明III型CRISPR系统中Cas10蛋白的系统发育和功能多样性。
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引用次数: 0
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
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CRISPR Journal
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