CRISPR-Cas系统的研究进展及应用

CRISPR-Cas系统是一种靶向基因编辑工具,操作简单,逐渐取代人工锌指核酸酶(ZFN)和类转录激活因子效应物核酸酶(TALEN)而成为近年的研究热点。Cas9蛋白能在一段小的RNA引导下特异性结合并切割DNA,在基因组结构和功能学研究中发挥重要作用。后来发现的Cas13等蛋白能特异性结合和编辑RNA,开启了转录组研究的新篇章。我们对CRISPR-Cas系统近年的研究发现做了简要概述,并对该系统作为一种工具在基础研究、生物工程、疾病治疗上的应用进行了总结。     

CRISPR/Cas系统的研究进展及前沿应用

CRISPR/Cas系统作为一种高效的基因组编辑工具,已经被广泛地研究和应用于各个领域。CRISPR/Cas系统已从最初的CRISPR/Cas9发展到现在的CRISPR/Cas12a、CRISPR/Cas13a、CRISPR/dCas等十多种基因编辑系统;从原来的靶向作用于DNA到现在的除了靶向作用于DNA和RNA外,还能应用于转录调控、DNA循环等无需基因编辑的领域。CRISPR/Cas系统以往存在的诸多局限性正在被一个一个突破,该系统的应用已经进入了一个新的时代。本文对CRISPR/Cas系统近些年的发展情况以及新发现的各种CRISPR/Cas系统做了一个总结,并列举了各个系统最新的应用情况。     

CRISPR/Cas:可应用于病毒检测和抗病毒治疗的前沿技术（英文）

近年来,病毒感染疫情频发,凸显了高效便利的病毒检测技术以及抗病毒药物研制的迫切性。基于成簇的规则间隔短回文重复序列（CRISPR）和CRISPR相关蛋白（Cas）的工程系统在靶向和切割核酸方面具有较高的特异性和效率,是目前使用最为广泛的基因编辑工具。该系统目前也广泛应用于病毒学研究和相关医疗实践。本文重点介绍了Cas9、Cas12和Cas13这三种最常用的CRISPR/Cas系统在病毒检测和抗病毒治疗中的应用。在病毒检测方面,Cas9通过与荧光传感器、电化学传感器和侧流层析试纸等生物传感器相结合,提高了生物传感器检测的灵敏度和准确性。Cas12和Cas13则基于其反式切割活性,目前已经开发了多种技术来检测DNA和RNA病毒,如SHERLOCK和DETECTER。在抗病毒治疗方面,Cas9已被用于靶向切割病毒DNA,从而抑制病毒的复制,其靶标包括DNA病毒的基因组和逆转录病毒的中间产物DNA;而Cas13则被用于靶向病毒RNA,其靶标包括RNA病毒的基因组和病毒mRNA。尽管CRISPR/Cas系统在灵敏度、效率和便利度等方面具有多种优势,但在一些方面仍不可避免地存在局限性,如脱靶效应、...     

CRISPR-Cas9介导的基因组编辑技术的研究进展

CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated proteins)系统为细菌与古生菌中抵御外源病毒或质粒DNA入侵的获得性免疫系统。该系统在crRNA的指导下,使核酸酶Cas识别并降解外源DNA。其中,Ⅱ型CRISPR-Cas系统最为简单,仅包括一个核酸酶Cas9与tracrRNA：crRNA二聚体便可完成其生物功能。基于CRISPR-Cas9的基因组编辑技术的核心为将tracrRNA:crRNA设计为引导RNA,在引导RNA的指导下Cas9定位于特定DNA序列上,进行DNA双链切割,实现基因组的定向编辑。CRISPR-Cas9系统以设计操纵简便、编辑高效与通用性广等优势成为新一代基因组编辑技术,为基因组定向改造调控与应用等带来突破性革命。从CRISPR-Cas9介导的基因组编辑技术的发展与应用等方面综述其最新研究进展,并着重介绍该技术的关键影响因素,为相关研究者提供参考。     

CRISPR/Cas9:基因编辑的新时代

基因编辑技术是通过核酸内切酶对基因组DNA进行定向改造的技术,可以实现对特定DNA碱基的缺失、替换等,常用的四种基因编辑工具分别是:巨型核酸酶、锌指核酸酶、转录激活因子样效应物核酸酶以及CRISPR/Cas9系统。其中CRISPR/Cas9系统作为一种新型的基因组编辑技术具有组成简单、特异性好、切割效率高的优点。该文对CRISPR/Cas9系统的结构组成和功能机制,动植物基因靶向编辑和人类在遗传性疾病、病毒感染性疾病以及肿瘤方面进行综述,旨在对CRISPR/Cas9系统的现状和发展进行总结和展望。     

基于CRISPR/Cas系统的生物传感策略研究进展

CRISPR/Cas9系统是继锌指核酸内切酶、类转录激活因子效应物核酸酶之后的第三代基因组定点编辑工具,因其具有特异性切割双链DNA的能力,被广泛应用于基因编辑、生物传感等领域。Cas12a(Cpf1)、Cas13a(C2c2)等蛋白"附属切割"活性的发现,拓展了CRISPR/Cas系统在生物传感中的应用。近年来,研究人员开发出一系列快速、超敏、高特异性的生物传感系统用于分子检测,如SHERLOCK,DETECTR等。本文主要综述了基于CRISPR/Cas系统的生物传感策略的研究进展,并展望了其未来发展的方向。     

CRISPR-Cas9基因编辑技术在病毒感染疾病治疗中的应用

CRISPR-Cas9基因编辑技术是基于细菌或古细菌CRISPR介导的获得性免疫系统衍生而来,由一段RNA通过碱基互补配对识别DNA,指导Cas9核酸酶切割识别的双链DNA,诱发同源重组或非同源末端链接,进而实现在目的DNA上进行编辑。病毒通过特异的受体侵染细胞,其基因组在细胞内发生复制、转录、翻译等过程完成其生活周期,某些DNA病毒或逆转录病毒基因组会整合到宿主基因组中。基因治疗是病毒感染疾病治疗的新趋势。因此,基因编辑技术在持续感染的病毒或潜伏感染病毒疾病治疗中具有重大的潜在意义。文章主要从CRISPR-Cas9作用机制以及在病毒感染疾病治疗中的应用等方面进行了综述。     

基于CRISPR-Cas13家族的RNA编辑系统及其最新进展

存在于细菌和古菌中的获得性免疫系统CRISPR-Cas目前已被广泛应用到生物技术领域,尤其是靶向DNA的CRISPR-Cas9技术。然而CRISPR-Cas系统靶向RNA的技术还处于初步应用阶段。Ⅵ型CRISPR-Cas系统(CRISPR-Cas13)的发现,揭示了RNA引导的RNA靶向性。CRISPR-Cas13是目前CRISPR-Cas家族中唯一只靶向ssRNA的系统,为RNA靶向和RNA编辑奠定了基础。根据Cas13系统发育已证明将Ⅵ型CRISPR-Cas系统分为4种亚型(A-D)。主要对目前最新的靶向RNA技术的CRISPR-Cas13家族的分类以及防御机制进行了综述,介绍了CRISPR-Cas13技术的应用以及基于CRISPR-Cas13家族的RNA编辑系统的最新研究进展。最后,对目前CRISPR-Cas13 RNA编辑技术体系存在的问题进行了分析和对未来的发展进行展望。     

基于CRISPR-Cas9的筛选文库的类型与应用

文库筛选技术广泛应用于生命科学研究各领域,加速了生物医药基础科研和临床实践的进展。本文对基于CRISPR-Cas9的文库类型和应用进行综述。CRISPR-Cas9文库包括敲除、活化和抑制文库。敲除文库通过Cas9/sgRNA靶向切割DNA序列,产生移码突变进行基因敲除。活化文库包括两种:一种是dCas9/sgRNA与转录活化蛋白质融合,例如dCas9-SAM,dCas9-SunTag和dCas9-VPR系统;另一种是dCas9与表观遗传修饰酶融合,例如d Cas9-Tet1和d Cas9-p300系统。CRISPR-Cas9抑制文库通过dCas9与表观遗传修饰蛋白质融合,抑制转录,例如d Cas9-KRAB和d Cas9-Dnmt3a系统。目前,CRISPR-Cas9文库广泛用于功能基因筛选、药物靶点和耐药靶点筛选、病毒靶点筛选和揭示信号通路,并在基因互作筛选及揭示顺式调节元件功能等方面初步展现其优势。CRISPR-Cas9文库优势在于其设计灵活、操作便捷、筛选高效。伴随基因编辑系统的研发,新的筛选文库靶向性和突变将更加精准,应用将更加拓展和深化。基于CRISPR-Cas9筛选文库不仅可以筛选病理和生理过程中的关键基因和非编码DNA,还可以揭示其发挥功能的分子机制,是剖析生命复杂调控网络的手术刀。     

Advances in CRISPR-Cas systems for RNA targeting,tracking and editing

Clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) systems, especially type II (Cas9) systems, have been widely used in gene/genome targeting. Modifications of Cas9 enable these systems to become platforms for precise DNA manipulations. However, the utilization of CRISPR-Cas systems in RNA targeting remains preliminary. The discovery of type VI CRISPR-Cas systems (Cas13) shed light on RNA-guided RNA targeting. Cas13d, the smallest Cas13 protein, with a length of only ~930 amino acids, is a promising platform for RNA targeting compatible with viral delivery systems. Much effort has also been made to develop Cas9, Cas13a and Cas13b applications for RNA-guided RNA targeting. The discovery of new RNA-targeting CRISPR-Cas systems as well as the development of RNA-targeting platforms with Cas9 and Cas13 will promote RNA-targeting technology substantially. Here, we review new advances in RNA-targeting CRISPR-Cas systems as well as advances in applications of these systems in RNA targeting, tracking and editing. We also compare these Cas protein-based technologies with traditional technologies for RNA targeting, tracking and editing. Finally, we discuss remaining questions and prospects for the future.     

Genome Editing with CRISPR-Cas9:Can It Get Any Better?

The CRISPR-Cas revolution is taking place in virtually all fields of life sciences.Harnessing DNA cleavage with the CRISPR-Cas9 system of Streptococcus pyogenes has proven to be extraordinarily simple and efficient,relying only on the design of a synthetic single guide RNA(sgRNA) and its co-expression with Cas9.Here,we review the progress in the design of sgRNA from the original dual RNA guide for S.pyogenes and Staphylococcus aureus Cas9(SpCas9 and SaCas9).New assays for genome-wide identification of offtargets have provided important insights into the issue of cleavage specificity in vivo.At the same time,the on-target activity of thousands of guides has been determined.These data have led to numerous online tools that facilitate the selection of guide RNAs in target sequences.It appears that for most basic research applications,cleavage activity can be maximized and off-targets minimized by carefully choosing guide RNAs based on computational predictions.Moreover,recent studies of Cas proteins have further improved the flexibility and precision of the CRISPR-Cas toolkit for genome editing.Inspired by the crystal structure of the complex of sgRNA-SpCas9 bound to target DNA,several variants of SpCas9 have recently been engineered,either with novel protospacer adjacent motifs(PAMs) or with drastically reduced off-targets.Novel Cas9 and Cas9-like proteins called Cpf 1 have also been characterized from other bacteria and will benefit from die insights obtained from SpCas9.Genome editing with CRISPR-Cas9 may also progress with better understanding and control of cellular DNA repair pathways activated after Cas9-induced DNA cleavage.     

CRISPR-Cas9研究进展及在基因治疗上的应用

CRISPR-Cas9[Clustered regularly interspaced short palindromic repeats（CRISPR）/CRISPR-associated （Cas）9]是近年兴起的一种高特异性和高效的基因编辑新技术,由向导RNA（single guide RNA,sgRNA）和cas9（CRISPR-associated 9）蛋白组成,引起DNA位点特异性双链断裂（double-strand breaks,DSBs）,引发同源重组修复（homology-directed repair,HDR）或非同源末端连接修复（non-homologous end joining,NHEJ）,达到靶基因修饰的作用。CRISPR-Cas9技术自发现以来,因其便于操作、花费较低、高特异性、可同时打靶任意数量基因等优点而被应用。近年研究显示,对于一些遗传性疾病,可通过CRISPR-Cas9精确的基因编辑破坏致病的内源基因、改正引起疾病的突变体或插入新的保护性基因进行治疗,该技术为基因治疗开启了一个新方向。主要从CRISPR-Cas9结构、作用机制及在疾病基因治疗上的应用等方面进行了综述。     

Non-viral and viral delivery systems for CRISPR-Cas9 technology in the biomedical field

The clustered regularly interspaced short palindromic repeats(CRISPR)-associated protein 9(CRISPR-Cas9) system provides a novel genome editing technology that can precisely target a genomic site to disrupt or repair a specific gene. Some CRISPR-Cas9 systems from different bacteria or artificial variants have been discovered or constructed by biologists, and Cas9 nucleases and single guide RNAs(sgRNA) are the major components of the CRISPR-Cas9 system. These Cas9 systems have been extensively applied for identifying therapeutic targets, identifying gene functions, generating animal models, and developing gene therapies.Moreover, CRISPR-Cas9 systems have been used to partially or completely alleviate disease symptoms by mutating or correcting related genes. However, the efficient transfer of CRISPR-Cas9 system into cells and target organs remains a challenge that affects the robust and precise genome editing activity. The current review focuses on delivery systems for Cas9 mRNA, Cas9 protein, or vectors encoding the Cas9 gene and corresponding sgRNA. Non-viral delivery of Cas9 appears to help Cas9 maintain its on-target effect and reduce off-target effects, and viral vectors for sgRNA and donor template can improve the efficacy of genome editing and homology-directed repair. Safe, efficient, and producible delivery systems will promote the application of CRISPR-Cas9 technology in human gene therapy.     

A simple,flexible and high‐throughput cloning system for plant genome editing via CRISPR‐Cas system

CRISPR-Cas9 system is now widely used to edit a target genome in animals and plants. Cas9 protein derived from Streptococcus pyogenes(Sp Cas9) cleaves double-stranded DNA targeted by a chimeric single-guide RNA(sg RNA). For plant genome editing, Agrobacterium-mediated T-DNA transformation has been broadly used to express Cas9 proteins and sg RNAs under the control of Ca MV 35 S and U6/U3 promoter, respectively. We here developed a simple and high-throughput binary vector system to clone a 19 20 bp of sg RNA, which binds to the reverse complement of a target locus, in a large T-DNA binary vector containing an Sp Cas9 expressing cassette. Twostep cloning procedures:(1) annealing two target-specific oligonucleotides with overhangs specific to the Aar I restriction enzyme site of the binary vector; and(2) ligating the annealed oligonucleotides into the two Aar I sites of the vector, facilitate the high-throughput production of the positive clones. In addition, Cas9-coding sequence and U6/U3 promoter can be easily exchanged via the GatewayTMsystem and unique Eco RI/Xho I sites on the vector, respectively. We examined the mutation ratio and patterns when we transformed these constructs into Arabidopsis thaliana and a wild tobacco, Nicotiana attenuata. Our vector system will be useful to generate targeted large-scale knock-out lines of model as well as non-model plant.     

Cas9HF1 enhanced specificity in Ustilago maydis

The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system is widely used as a tool to precisely manipulate genomic sequence targeted by sgRNA (single guide RNA) and is adapted in different species for genome editing. One of the major concerns of CRISPR-Cas9 is the possibility of off-target effects, which can be remedied by the deployment of high fidelity Cas9 variants. Ustilago maydis is a maize fungal pathogen, which has served as a model organism for biotrophic pathogens for decades. The successful adaption of CRISPR-Cas9 in U. maydis greatly facilitated effector biology studies. Here, we constructed an U. maydis reporter strain that allows in vivo quantification of efficiency and target specificity of three high fidelity Cas9 variants, Cas9HF1, Cas9esp1.1 and Cas9hypa. This approach identified Cas9HF1 as most specific Cas9 variant in U. maydis. Furthermore, whole genome sequencing showed absence of off-target effects in U. maydis by CRISPR-Cas9 editing.     

Detailed Phenotypic and Molecular Analyses of Genetically Modified Mice Generated by CRISPR-Cas9-Mediated Editing

The bacterial CRISPR-Cas9 system has been adapted for use as a genome editing tool. While several recent reports have indicated that successful genome editing of mice can be achieved, detailed phenotypic and molecular analyses of the mutant animals are limited. Following pronuclear micro-injection of fertilized eggs with either wild-type Cas9 or the nickase mutant (D10A) and single or paired guide RNA (sgRNA) for targeting of the tyrosinase (Tyr) gene, we assessed genome editing in mice using rapid phenotypic readouts (eye and coat color). Mutant mice with insertions or deletions (indels) in Tyr were efficiently generated without detectable off-target cleavage events. Gene correction of a single nucleotide by homologous recombination (HR) could only occur when the sgRNA recognition sites in the donor DNA were modified. Gene repair did not occur if the donor DNA was not modified because Cas9 catalytic activity was completely inhibited. Our results indicate that allelic mosaicism can occur following -Cas9-mediated editing in mice and appears to correlate with sgRNA cleavage efficiency at the single-cell stage. We also show that larger than expected deletions may be overlooked based on the screening strategy employed. An unbiased analysis of all the deleted nucleotides in our experiments revealed that the highest frequencies of nucleotide deletions were clustered around the predicted Cas9 cleavage sites, with slightly broader distributions than expected. Finally, additional analysis of founder mice and their offspring indicate that their general health, fertility, and the transmission of genetic changes were not compromised. These results provide the foundation to interpret and predict the diverse outcomes following CRISPR-Cas9-mediated genome editing experiments in mice.