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。     

CRISPR-Cas生物传感器研究进展

成簇规律间隔短回文序列(clustered regularly interspaced short palindromic repeats,CRISPR)系统是广泛存在于细菌中的一种特有的免疫防御机制,与特殊的Cas蛋白结合后能够有效的对外源的核酸分子进行特异性片段化,并进一步促进其降解。CRISPR-Cas系统具有独特的靶向性,为开发针对于核酸为底物的生物传感器提供了新的概念。越来越多的研究人员根据不同Cas蛋白的性质,建立了独特的逻辑系统对靶标物质进行准确识别,基于CRISPR技术的生物传感器也开拓了该技术在基因编辑以外领域的应用。介绍了CRISPR-Cas系统的起源、作用机制和科学分类,根据生物传感器的作用方式以及识别底物进行了分类,并对基于CRISPR-Cas系统的高效生物传感器的应用前景进行了展望。     

CRISPR-Cas9 Circular Permutants as Programmable Scaffolds for Genome Modification

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Structure of the miniature type V-F CRISPR-Cas effector enzyme

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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.     

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.     

Genome editing methods in animal models

ABSTRACT

Genetically engineered animal models that reproduce human diseases are very important for the pathological study of various conditions. The development of the clustered regularly interspaced short palindromic repeats (CRISPR) system has enabled a faster and cheaper production of animal models compared with traditional gene-targeting methods using embryonic stem cells. Genome editing tools based on the CRISPR-Cas9 system are a breakthrough technology that allows the precise introduction of mutations at the target DNA sequences. In particular, this accelerated the creation of animal models, and greatly contributed to the research that utilized them. In this review, we introduce various strategies based on the CRISPR-Cas9 system for building animal models of human diseases and describe various in vivo delivery methods of CRISPR-Cas9 that are applied to disease models for therapeutic purposes. In addition, we summarize the currently available animal models of human diseases that were generated using the CRISPR-Cas9 system and discuss future directions.     

Efficient Genome Editing in Caenorhabditis elegans with a Toolkit of Dual-Marker Selection Cassettes

Use of the CRISPR/Cas9 RNA-guided endonuclease complex has recently enabled the generation of double-strand breaks virtually anywhere in the C. elegans genome. Here, we present an improved strategy that makes all steps in the genome editing process more efficient. We have created a toolkit of template-mediated repair cassettes that contain an antibiotic resistance gene to select for worms carrying the repair template and a fluorescent visual marker that facilitates identification of bona fide recombinant animals. Homozygous animals can be identified as early as 4–5 days post-injection, and minimal genotyping by PCR is required. We demonstrate that our toolkit of dual-marker vectors can generate targeted disruptions, deletions, and endogenous tagging with fluorescent proteins and epitopes. This strategy should be useful for a wide variety of additional applications and will provide researchers with increased flexibility when designing genome editing experiments.     

Temperature-Responsive Competitive Inhibition of CRISPR-Cas9

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CRISPR-Cas基因编辑系统升级:聚焦Cas蛋白和PAM

以CRISPR-Cas(clustered regularly interspaced short palindromic repeats and CRISPR associated proteins)系统为代表的基因编辑技术的出现极大地促进了人类改造自然界物种的能力.在医疗、工业、农业等多个研究领域,基因编辑技术正在...     

Multigene Editing in the Escherichia coli Genome via the CRISPR-Cas9 System

An efficient genome-scale editing tool is required for construction of industrially useful microbes. We describe a targeted, continual multigene editing strategy that was applied to the Escherichia coli genome by using the Streptococcus pyogenes type II CRISPR-Cas9 system to realize a variety of precise genome modifications, including gene deletion and insertion, with a highest efficiency of 100%, which was able to achieve simultaneous multigene editing of up to three targets. The system also demonstrated successful targeted chromosomal deletions in Tatumella citrea, another species of the Enterobacteriaceae, with highest efficiency of 100%.     

Target-Specific Precision of CRISPR-Mediated Genome Editing

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新RNA编辑技术CRISPR-Cas13

CRISPR-Cas13系统(clustered regularly interspaced short palindromic repeats associated Cas system,CRISPR-Cas)是一种快速、高效、精准的新型RNA编辑工具,具有易于设计、结构简单、操作方便、特异性强的特点。综述了CRISPR-Cas13在CRISPR分类系统中的地位、CRISPR-Cas13的结构基础以及作用机制、与其他RNA水平调节方法的比较以及目前的应用前景,以期为相关研究提供参考。