CRISPR/Cas系统在新型冠状病毒肺炎快速诊断中的应用

近年来,基于规律成簇间隔的短回文重复序列（ clustered regularly interspaced short palindromic repeats, CRISPR）及其相关蛋白（ CRISPR-associated protein, Cas）系统的新型分子诊断工具,为病原体的诊断开辟了新的机遇。该文将关注现有和正在研究的 CRISPR/Cas系统用于新型冠状病毒肺炎（coronavirus disease 2019, COVID-19）快速诊断的潜在能力,并重点探讨其在临床中的应用和面临的挑战。     

基于CRISPR/Cas系统诊断平台的研究进展

目前核酸检测技术已被广泛应用于临床实验室诊断,常规检测技术如实时荧光定量PCR技术耗时长且依赖特定的仪器设备。成簇规律间隔短回文重复序列(CRISPR)/CRISPR相关蛋白(Cas)系统是细菌和古细菌在与病毒斗争过程中获得的适应性免疫防御机制,已被发展成强大的基因组编辑技术。最近CRISPR领域的先驱团队基于Cas13a、Cas12a和新发现的Cas14蛋白开发出SHERLOCK、DETECTR等新型核酸检测工具,在传染性疾病的快速诊断、癌症中基因突变的检测和基因分型等方面意义重大,其灵敏度高、特异性强且快速经济,在临床分子诊断领域具有巨大潜力。本文综述了CRISPR/Cas系统的作用机制及新型诊断平台的原理和应用进展,总结了新型检测技术的优缺点并对其发展前景进行展望。     

CRISPR/Cas系统在分子检测中的应用

近年来,成簇规律间隔短回文重复序列/成簇规律短回文重复序列相关蛋白(CRISPR/Cas)系统凭借其简单、高效的基因编辑能力,已被广泛应用于生物、医学等多个研究领域。随着CRISPR技术的快速发展,CRISPR/Cas系统已被开发为一种快速、便携、低成本、高灵敏度的分子检测工具,在病原体检测、耐药性分析、单核苷酸多态性(SNP)分型、肿瘤基因突变检测等方面取得重大突破。文章就不同Cas蛋白在分子检测中的最新研究进展进行综述,并对其应用前景进行展望,以期为从事相关领域的科研工作者提供参考与帮助。     

CRISPR/Cas系统在SARS-CoV-2检测中的应用

摘要:新型冠状病毒( SARS-CoV-2)全球流行,快速有效的检测方法对于疫情防控和患者救治极为重要。目前已有多种方法应.用于SARS-CoV-2的检测,但均存在一定的局限性。研究发现,采用CRISPR/Cas系统构建的SARS-CoV-2检测方法具有快速、便捷的优势。目前应用于SARS-CoV-2检测的CRISPR/Cas系统主要包括CRISPR/Cas9. CRISPR/Cas12和CRISPR/Cas13,多以SARS-CoV-2的核酸和蛋白质作为检测靶标。因此,该文对基于CRISPR/Cas系统的SARS-CoV-2检测方法分别从核酸和蛋白质2个角度进行综述,并就各自的优缺点及未来发展趋势进行分析，期望为CRISPR/Cas系统在传染病检测中的应用提供参考。     

CRISPR/Cas12系统在肿瘤检测中的应用

快速、灵敏、特异的检测方法对于提高肿瘤患者的生存率并改善预后至关重要。除了在基因编辑领域的贡献，近年来成簇规律间隔短回文重复序列(CRISPR)/CRISPR相关蛋白(Cas)系统以其特有的靶标核酸识别切割能力和反式切割活性为特点，已成为新一代核酸检测工具被广泛应用于病原体、肿瘤和转基因等检测领域。基于此，该文对CRISPR/Cas12系统原理及其在不同肿瘤标志物中的检测应用进展作一综述，并对其应用前景进行展望，以期为CRISPR/Cas系统更好地应用于肿瘤筛查和诊断提供参考及借鉴。     

基因魔剪CRISPR/Cas:核酸精准检测的新宠和利器

规则成簇间隔短回文重复序列及其相关蛋白(CRISPR/Cas)系统是存在于大多数细菌及古细菌中的一种获得性免疫系统。作为一种高效的基因定点编辑工具,CRISPR/Cas系统不仅在基因敲出、基因治疗及基因修饰等领域炙手可热,而且正在发展成为核酸精准检测领域的新利器。随着众多研究者的不断探索,便携、快速、低成本、灵敏度高、特异性强的CRISPR/Cas核酸精准检测技术不断涌现。本文就CRISPR/Cas技术在核酸快速精准检测领域的最新成果作一概述,并总结展望其面临的困难和挑战。     

CRISPR/Cas基因编辑技术及其在血液系统疾病中的应用

CRISPR/Cas基因编辑技术是近年来新兴的对特定位点进行基因编辑的技术,利用CRISPR/Cas系统在某一特定位点导入目的基因,可以使有基因缺陷的细胞恢复正常功能,也可以在正常细胞内导入疾病相关基因从而构建相应疾病模型。CRISPR/Cas技术具有精确的打靶作用,作为基因剪切和编辑工具而广泛应用于各种实验,能够帮助深入研究基因功能以及治疗遗传性疾病。目前,CRISPR/Cas技术正成为农业、畜牧业、生物技术及医学领域的研究热点,本文就CRISPR/Cas系统在血液系统疾病中的最新研究进展作一综述。     

核酸检测和基因编辑的新工具：CRISPR/Cas13系统

成簇的规律间隔的短回文重复序列(clustered regularly interspaced short palindromic repeats,CRISPR)/CRISPR相关蛋白(CRISPR-associated proteins, Cas)系统是目前基因编辑、基因表达研究的热点,其中,研究较成熟的为CRISPR/Cas9系统,其在单链RNA的引导下可特异地切割靶DNA的特定位点,实现DNA水平的操作。而靶向RNA的CRISPR/Cas13系统开启了在RNA水平研究、诊疗的新时代。活化的Cas13具有独特的核酸酶活性,可特异性地切割靶向RNA,同时非特异性地切割周围环境中的RNA,利用以上特性可实现体外核酸检测。通过活性位点突变可产生无核酸酶活性但可与RNA结合的dCas13(dead Cas13),将dCas13与其他功能性蛋白质进行融合可进一步扩大dCas13的应用范围。该文主要概括了CRISPR/Cas13系统在核酸检测以及在RNA水平作为基因编辑工具的新进展。     

CRISPR-Cas13a系统在基因编辑和分子诊断领域中的研究进展

成簇的规律间隔的短回文重复序列(CRISPR)-CRISPR相关蛋白(Cas)系统是一个强大的基因编辑工具。相对于Cas9,Cas13a可靶向多基因转录产物,从而调控基因功能表达,填补了Cas9仅限于DNA水平的编辑及脱靶效应等缺陷。不仅如此,利用Cas13a靶向RNA的特性,该系统被成功地改造成下一代核酸诊断工具。文章概述了CRISPR-Cas13系统在基因编辑及分子诊断领域的最新研究进展,并对该系统的应用前景进行了展望。     

基于CRISPR/Cas蛋白的副溶血弧菌检测方法的研究

目的利用规律间隔性成簇短回文重复序列（CRISPR）免疫原理及Cas12a酶的特点构建一种快速检测副溶血弧菌（VP）的方法,实现对病原菌准确快速的检测和识别。方法本研究通过制备纯化Cas12a蛋白,筛选构建VP的gRNA,建立CRISPR-VP荧光检测系统,根据最终荧光扩增曲线判定CRISPR-VP检测方法的有效性。结果在CRISPR-VP检测方法中只在VP的序列存在时才会产生明显的荧光信号。结论本实验初步建立了基于CRISPR/Cas蛋白的VP的检测方法,为后续简易检测试剂的研制提供理论依据。     

Development of clustered regularly interspaced short palindromic repeats/CRISPR-associated technology for potential clinical applications

The clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) proteins constitute the innate adaptive immune system in several bacteria and archaea. This immune system helps them in resisting the invasion of phages and foreign DNA by providing sequence-specific acquired immunity. Owing to the numerous advantages such as ease of use, low cost, high efficiency, good accuracy, and a diverse range of applications, the CRISPR-Cas system has become the most widely used genome editing technology. Hence, the advent of the CRISPR/Cas technology highlights a tremendous potential in clinical diagnosis and could become a powerful asset for modern medicine. This study reviews the recently reported application platforms for screening, diagnosis, and treatment of different diseases based on CRISPR/Cas systems. The limitations, current challenges, and future prospectus are summarized; this article would be a valuable reference for future genome-editing practices.     

Progress in the application of CRISPR: From gene to base editing

The system of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated endonucleases (Cas) has been utilized for genome editing with great accuracy and high efficiency in generating gene knockout, knockin, and point mutations in eukaryotic genomes. However, traditional CRISPR/Cas9 technology introduces double-stranded DNA breaks (DSBs) at a target locus as the first step to make gene corrections, which easily results in undesired mutations. Thus, it is necessary to develop new methods for correcting the unwanted mutations. In this review, we summarize the recent developments and a new approach to genome and base editing by using CRISPR/Cas9. This methodology renders a conversion of one target base into another, for example, C to T (or G to A), and A to G (or T to C) without producing DSBs, requiring a donor DNA template, or generating excessive insertions and deletions. Furthermore, CRISPR/Cas9-derived base editing also improves efficiency in repairing point mutations in the genome.     

The ABCs of CRISPR in Tephritidae: developing methods for inducing heritable mutations in the genera Anastrepha,Bactrocera and Ceratitis

Tephritid fruit flies are destructive agricultural pests that are the targets of expensive population eradication and suppression efforts. Genetic pest management is one of the strategies for reducing or eliminating tephritid populations, relying upon the genetic manipulation of insects to render them sterile or capable of transmitting deleterious traits through gene drive. Currently, radiation, chemical mutagenesis, and transgenic techniques are employed to generate agents for genetic pest management, but new methods must be explored and developed for all tephritid pest species. Targeted mutagenesis induced by nonhomologous end join repair of clustered regularly interspaced short palindromic repeats and the CRISPR associated protein 9 (Cas9) (commonly known as CRISPR/Cas9) has been demonstrated to be an efficient method for creating knock‐out mutants and can be utilized to create germline mutations in Tephritidae. In this paper, we describe detailed methods to knockout the white gene in three tephritid species in the genera Anastrepha, Bactrocera and Ceratitis, including the first demonstration of CRISPR/Cas9 induced mutations in the genus Anastrepha. Lastly, we discuss the variables in tephritid systems that directed method development as well as recommendations for performing injections in remote containment facilities with little molecular biology capabilities. These methods and recommendations combined can serve as a guide for others to use in pursuit of developing CRISPR/Cas9 methods in tephritid systems.