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。     

CRISPR-Cas9驱动的基因编辑新纪元

在自然界生物长期的进化过程中,细菌和古细菌演化出了一种适应性免疫系统用以抵御外源病毒与质粒的入侵,该系统由成簇规律间隔的短回文重复序列与相关基因组成,称之为CRISPR-Cas。近年来,这一领域突飞猛进,如今已经发展成为一种功能强大的基因编辑工具并在生物学及其相关领域得到广泛应用。本文重点综述了近年来CRISPR-Cas9系统在基因编辑、基因调节以及作为体外工具酶和特异性等方面的若干前沿进展。     

诺贝尔化学奖授予CRISPR-Cas9基因编辑研究

2020年,诺贝尔化学奖授予现就职于德国马普感染生物学研究所的法籍科学家Emmanuelle Charpentier和美国加州大学伯克利分校的Jennifer Doudna,表彰她们发明CRISPR基因编辑方法.她们揭示了Cas9具有RNA介导的DNA核酸内切酶活性,可以切断任意DNA双链,产生双链断裂.她们指出CRISPR具有在活细胞中修改基因的作用,利用CRISPR-Cas9编辑工具,可以精确改变细胞中的DNA.由于简单、高效、廉价等特征,CRISPR已经成为最为流行的基因编辑技术,被称为基因编辑"魔剪".本文介绍了两位诺贝尔化学奖得主的研究成果,概述了CRISPR系统的发现历程,以及CRISPR-Cas9的功能和应用.     

基于CRISPR-Cas9技术的基因治疗研究进展

CRISPR-Cas9系统是细菌在与噬菌体抗争的进化过程中产生的一种抵御外源DNA入侵的机制,能有效识别并剪切外源DNA。基于其识别切除外源DNA的原理,CRISPR-Cas9系统被开发成为新一代基因编辑工具。与ES打靶、ZFN、TALEN等技术途径相比,CRISPR-Cas9系统操作简便、效率高、成本低,有着极其广阔的应用前景。本文整理了近年内有关CRISPR-Cas9系统的最新文献报道,对该系统工作原理以及针对基因治疗的研究进展进行综述。     

大肠杆菌双质粒CRISPR-Cas9系统的优化及应用

近年来,CRISPR-Cas9(clustered regularly interspaced short palindromic repeats)基因编辑系统已经成功应用于多种微生物中.由于CRISPR-Cas9系统仅受PAM(protospacer adjacent motif)位点NGG的限制,因此理论上CRIS...     

CRISPR-Cas9技术在干细胞中的应用

成簇规律间隔短回文重复序列系统(Clustered regularly interspaced short palindromic repeats,associated RNA guided endonuclease Cas9(CRISPR-Cas9)是细菌或古细菌在长期演化过程中形成的抵御外来遗传物质的一种获得性免疫防御机制,其中II型CRISPR-Cas系统依赖Cas9核酸内切酶靶向剪切外源DNA。Cas9内切酶在向导RNA的指导下靶向性地剪切特定基因位点,已被广泛应用在不同种属的基因编辑研究中。利用CRISPR-Cas9基因编辑系统的优势,结合现有干细胞研究技术,在小鼠、大鼠,甚至灵长类动物的功能基因组研究中,可以大幅提高各种基因修饰动物的获得效率,缩短获得的时间,从而快捷有效地研究基因功能;同时,可以建立包括灵长类疾病模型在内的多种动物疾病模型,促进生物医学的发展,造福人类。     

CRISPR-Cas9技术在细菌中的研究进展及应用

CRISPR (Clustered regularly interspaced short palindromic repeats)最早发现于绝大多数细菌和古细菌中,帮助其防御和抵抗噬菌体和外来质粒的入侵。近年来,随着对该系统结构及功能的逐步深入认识和研究,CRISPR-Cas9技术已经发展成为一种简易高效的基因组定点编辑手段,但是在使用过程中仍存在一些问题。本文重点介绍了CRISPR-Cas9系统功能和分类,在细菌基因编辑中的应用和问题以及应对措施。     

CRISPR-Cas系统在微生物研究中的应用进展

来源于细菌防御系统的CRISPR-Cas系统因其功能强大、多样性受到广泛关注,也在动物、植物、微生物等各个领域得到了应用并迅速发展。概述了CRISPR-Cas系统的作用机理和分类,并对CRISPR-Cas系统在微生物基因编辑和病毒核酸检测方面的研究及应用进展进行了综述,以期为微生物研究工作提供参考。     

CRISPR-Cas9基因编辑技术在秀丽线虫中的应用

基于细菌基因组规律成蔟的间隔短回文重复(Clustered regularly interspaced short palindromic repeats)发展而来的新型基因编辑方法(CRISPR-Cas9)对生物医学研究是一场划时代的革命。它几乎可用于大多数生物体的基因编辑。秀丽线虫是一种非常经典的遗传学模式生物,CRISPR-Cas9基因编辑技术进一步加速了对其基因功能及各种生物学问题的研究。文中主要总结CRISPR-Cas9基因编辑系统在遗传学模式生物秀丽线虫中的发展和应用。     

CRISPR-Cas9 for the genome engineering of cyanobacteria and succinate production

Cyanobacteria hold promise as a cell factory for producing biofuels and bio-derived chemicals, but genome engineering of cyanobacteria such as Synechococcus elongatus PCC 7942 poses challenges because of their oligoploidy nature and long-term instability of the introduced gene. CRISPR-Cas9 is a newly developed RNA-guided genome editing system, yet its application for cyanobacteria engineering has yet to be reported. Here we demonstrated that CRISPR-Cas9 system can effectively trigger programmable double strand break (DSB) at the chromosome of PCC 7942 and provoke cell death. With the co-transformation of template plasmid harboring the gene cassette and flanking homology arms, CRISPR-Cas9-mediated DSB enabled precise gene integration, ameliorated the homologous recombination efficiency and allowed the use of lower amount of template DNA and shorter homology arms. The CRISPR-Cas9-induced cell death imposed selective pressure and enhanced the chance of concomitant integration of gene cassettes into all chromosomes of PCC 7942, hence accelerating the process of obtaining homogeneous and stable recombinant strains. We further explored the feasibility of engineering cyanobacteria by CRISPR-Cas9-assisted simultaneous glgc knock-out and gltA/ppc knock-in, which improved the succinate titer to 435.0±35.0 μg/L, an ≈11-fold increase when compared with that of the wild-type cells. These data altogether justify the use of CRISPR-Cas9 for genome engineering and manipulation of metabolic pathways in cyanobacteria.     

CRISPR-Cas system: Toward a more efficient technology for genome editing and beyond

Genome engineering technology is of great interest for biomedical research that enables scientists to make specific manipulation in the DNA sequence. Early methods for introducing double-stranded DNA breaks relies on protein-based systems. These platforms have enabled fascinating advances, but all are costly and time-consuming to engineer, preventing these from gaining high-throughput applications. The CRISPR-Cas9 system, co-opted from bacteria, has generated considerable excitement in gene targeting. In this review, we describe gene targeting techniques with an emphasis on recent strategies to improve the specificities of CRISPR-Cas systems for nuclease and non-nuclease applications.     

构建CRISPR-Cas9介导的耻垢分枝杆菌基因组高效删除系统

【背景】耻垢分枝杆菌具有生长迅速和非致病性的特点,可作为结核分枝杆菌致病机理研究替代菌株和类固醇激素生产的工程菌,但目前耻垢分枝杆菌中缺乏高效率的基因组敲除方法。【目的】基于CRISPR-Cas9介导的定点、高效的DNA切割能力,构建耻垢分枝杆菌染色体DNA片段无痕敲除系统。【方法】构建了包含四环素诱导型启动子驱动的密码子优化的cas9基础载体pCas9101,在双侧同源臂长度约为1 kb条件下选用合适的gRNA表达模块,分别测试了对耻垢分枝杆菌mc2155染色体上的3β-羟基类固醇脱氢酶基因(MSMEG_5228,1 071 bp)和胆固醇降解基因簇(MSMEG_5990-MSMEG_6043,约48kb)敲除效率,使用相同大小的同源臂以经典p2NIL-pGOAL方法进行对照,并计算效率。【结果】使用CRISPR-Cas9方法对耻垢分枝杆菌mc2155的3β-羟基类固醇脱氢酶基因敲除效率为22%,胆固醇降解基因簇敲除效率也达到18%,两者连续敲除效率为4%。但对照p2NIL-pGOAL方法未能获得目标DNA片段敲除的菌株。【结论】本文建立的基于CRISPR-Cas9的耻垢分枝杆菌基因组无痕敲除系统显示出较高的敲除效率,该方法可为耻垢分枝杆菌后续研究提供快速高效的基因组操作方法。     

CRISPR-Cas9应用于病毒性传染病防控的研究进展

病毒性传染病是威胁人类健康的重要因素,迫切需要新的治疗方法来降低由急性病毒感染如鼻病毒和登革热病毒以及慢性病毒感染如人类免疫缺陷病毒1和乙型肝炎病毒引起的发病率和死亡率.随着分子生物学技术的发展,靶向序列特异性的基因编辑技术成为传染病治疗的有力工具.其中规律成簇间隔短回文重复序列(clustered regularly interspaced short palindromic repeats,CRISPR)-CRISPR相关蛋白9(CRISPR associated protein 9,Cas9)凭借其高效、简便、高特异性等特点被广泛应用于细胞系和动物模型中的传染病治疗,从而成为有前景的新型传染病治疗模式.目前,利用病毒和非病毒载体将Cas9以DNA、m RNA或蛋白质的形式递送到细胞中的可行性研究和评估CRISPR-Cas9体内适用性的临床试验已经在进行中.本篇综述中,我们将对CRISPR-Cas9的原理,其应用于传染病治疗的最新研究进展以及该技术面临的挑战和可预测性的解决方法等加以概述,并进一步展望其未来的发展方向.     

A high-throughput screening strategy for detecting CRISPR-Cas9 induced mutations using next-generation sequencing

Background

CRISPR-Cas9 is a revolutionary genome editing technique that allows for efficient and directed alterations of the eukaryotic genome. This relatively new technology has already been used in a large number of ‘loss of function’ experiments in cultured cells. Despite its simplicity and efficiency, screening for mutated clones remains time-consuming, laborious and/or expensive.

Results

Here we report a high-throughput screening strategy that allows parallel screening of up to 96 clones, using next-generation sequencing. As a proof of principle, we used CRISPR-Cas9 to disrupt the coding sequence of the homeobox gene, Evx1 in mouse embryonic stem cells. We screened 67 CRISPR-Cas9 transfected clones simultaneously by next-generation sequencing on the Ion Torrent PGM. We were able to identify both homozygous and heterozygous Evx1 mutants, as well as mixed clones, which must be identified to maintain the integrity of subsequent experiments.

Conclusions

Our CRISPR-Cas9 screening strategy could be widely applied to screen for CRISPR-Cas9 mutants in a variety of contexts including the generation of mutant cell lines for in vitro research, the generation of transgenic organisms and for assessing the veracity of CRISPR-Cas9 homology directed repair. This technique is cost and time-effective, provides information on clonal heterogeneity and is adaptable for use on various sequencing platforms.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1002) contains supplementary material, which is available to authorized users.     

Harnessing the native type I-B CRISPR-Cas for genome editing in a polyploid archaeon

Research on CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR associated protein) systems has led to the revolutionary CRISPR/Cas9 genome editing technique. However, for most archaea and half of bacteria, exploitation of their native CRISPR-Cas machineries may be more straightforward and convenient. In this study, we harnessed the native type I-B CRISPR-Cas system for precise genome editing in the polyploid haloarchaeon Haloarcula hispanica. After testing different designs, the editing tool was optimized to be a single plasmid that carries both the self-targeting mini-CRISPR and a 600–800 bp donor. Significantly, chromosomal modifications, such as gene deletion, gene tagging or single nucleotide substitution, were precisely introduced into the vast majority of the transformants. Moreover, we showed that simultaneous editing of two genomic loci could also be readily achieved by one step. In summary, our data demonstrate that the haloarchaeal CRISPR-Cas system can be harnessed for genome editing in this polyploid archaeon, and highlight the convenience and efficiency of the native CRISPR-based genome editing strategy.