Enhanced Bombyx genome editing via Cas9 ribonucleoprotein injection

Dear Editor, The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) system has become a popular and powerful method for manipulating genomes with considerably little effort and high efficiency. It allows for the generation of targeted indels, genomic structure variations, and insertion of foreign fragments. However, increasing concerns have been raised about possible off-target effects.     

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在丝状真菌基因组编辑中的应用

丝状真菌(filamentous fungi)通常指那些菌丝体较发达且不产生大型肉质子实体结构的真核微生物。丝状真菌不仅在自然界物质循环中发挥着重要作用,还与人类健康和工农业生产有着紧密的联系。然而,对丝状真菌进行遗传操作相对困难,极大地妨碍了丝状真菌的遗传学研究。成簇的规律间隔的短回文重复序列及其相关系统(clustered regulatory interspaced short palindromic repeats/CRISPR-associated protein 9, CRISPR/Cas9)是近年来发现的一种存在于细菌和古菌中保守的获得性免疫防御机制。最近,CRISPR/Cas9被开发成为了一种方便灵活的基因组编辑技术。目前,该技术已经广泛应用在不同物种的基因组编辑中。本文概述了CRISPR/Cas9在丝状真菌基因组编辑中的应用进展,旨在为开展该领域的研究工作提供参考。     

CRISPR/Cas9介导的基因组定点编辑技术在丝状真菌中的研究进展

CRISPR/Cas9技术是在特定的RNA引导下,利用特异的核酸酶实现对基因组进行编辑的新技术。自2013年该技术体系建立起来已成功应用于动物、植物及真菌中。本文简述了3种基于核酸酶的基因编辑技术及其应用,概述了CRISPR/Cas9系统的组成及其作用机理,总结了CRISPR/Cas9在模式真菌酿酒酵母及丝状真菌中的应用,并就在丝状真菌中应用该技术时sg RNA表达盒的设计、Cas9表达盒的优化、抗性标记的筛选、受体的选择等方面提出具体的研究方法。另外,针对该技术应用过程中出现的脱靶效应、Cas9核定位信号的添加、启动子的选择及多个靶基因的编辑等问题提出了建议与展望,希望能够为初次涉足该领域的科研人员提供理论参考和技术支持。     

Robust genome editing of CRISPR-Cas9 at NAG PAMs in rice

正Dear Editor,The CRISPR-Cas9(clustered regularly interspaced short palindromic repeats/Cas9)system has been widely used for a variety of applications,including targeted gene knockout,gene insertion,gene replacement and base editing.Despite its wide use,the genome editing using CRISPR-Cas9 is performed almost exclusively at sites containing canonical NGG protospacer adjacent motifs(PAMs).To overcome the PAM constraint of the CRISPR-Cas9 system,many attempts have been made to develop various Cas9 orthologs and     

Rapid and efficient generation of GFP-knocked-in Drosophila by the CRISPR-Cas9-mediated genome editing

The CRISPR-Cas9 technology has been a powerful means to manipulate the genome in a wide range of organisms. A series of GFP knocked-in (GFP^KI) Drosophila strains have been generated through CRISPR-Cas9-induced double strand breaks coupled with homology-directed repairs in the presence of donor plasmids. They visualized specific cell types or intracellular structures in both fixed and live specimen. We provide a rapid and efficient strategy to identify KI lines. This method requires neither co-integration of a selection marker nor prior establishment of sgRNA-expressing transgenic lines. The injection of the mixture of a sgRNA/Cas9 expression plasmid and a donor plasmid into cleavage stage embryos efficiently generated multiple independent KI lines. A PCR-based selection allows to identify KI fly lines at the F1 generation (approximately 4 weeks after injection). These GFP^KI strains have been deposited in the Kyoto Drosophila stock center, and made freely available to researchers at non-profit organizations. Thus, they will be useful resources for Drosophila research.     

CRISPR/Cas9-based genome editing of the filamentous fungi: the state of the art

Applied Microbiology and Biotechnology - In recent years, a variety of genetic tools have been developed and applied to various filamentous fungi, which are widely applied in agriculture and the...     

Efficient Genome Editing in Clostridium cellulolyticum via CRISPR-Cas9 Nickase

The CRISPR-Cas9 system is a powerful and revolutionary genome-editing tool for eukaryotic genomes, but its use in bacterial genomes is very limited. Here, we investigated the use of the Streptococcus pyogenes CRISPR-Cas9 system in editing the genome of Clostridium cellulolyticum, a model microorganism for bioenergy research. Wild-type Cas9-induced double-strand breaks were lethal to C. cellulolyticum due to the minimal expression of nonhomologous end joining (NHEJ) components in this strain. To circumvent this lethality, Cas9 nickase was applied to develop a single-nick-triggered homologous recombination strategy, which allows precise one-step editing at intended genomic loci by transforming a single vector. This strategy has a high editing efficiency (>95%) even using short homologous arms (0.2 kb), is able to deliver foreign genes into the genome in a single step without a marker, enables precise editing even at two very similar target sites differing by two bases preceding the seed region, and has a very high target site density (median interval distance of 9 bp and 95.7% gene coverage in C. cellulolyticum). Together, these results establish a simple and robust methodology for genome editing in NHEJ-ineffective prokaryotes.     

Homology-based repair induced by CRISPR-Cas nucleases in mammalian embryo genome editing

Recent advances in genome editing, especially CRISPR-Cas nucleases, have revolutionized both laboratory research and clinical therapeutics. CRISPR-Cas nucleases, together with the DNA damage repair pathway in cells, enable both genetic diversification by classical non-homologous end joining (c-NHEJ) and precise genome modification by homology-based repair (HBR). Genome editing in zygotes is a convenient way to edit the germline, paving the way for animal disease model generation, as well as human embryo genome editing therapy for some life-threatening and incurable diseases. HBR efficiency is highly dependent on the DNA donor that is utilized as a repair template. Here, we review recent progress in improving CRISPR-Cas nuclease-induced HBR in mammalian embryos by designing a suitable DNA donor. Moreover, we want to provide a guide for producing animal disease models and correcting genetic mutations through CRISPR-Cas nuclease-induced HBR in mammalian embryos. Finally, we discuss recent developments in precise genome-modification technology based on the CRISPR-Cas system.Supplementary InformationThe online version of this article (10.1007/s13238-021-00838-7) contains supplementary material, which is available to authorized users.     

A modified pCas/pTargetF system for CRISPR-Cas9-assisted genome editing in Escherichia coli

The clustered regularly interspaced short palindromic repeats(CRISPR)-associated nuclease 9(Cas9)-based genome editing tool pCas/pTargetF system that we establi...     

Efficient amplification of genes involved in microbial secondary metabolism by an improved genome walking method

Genome walking is a commonly used technique for the identification of DNA sequences adjacent to known regions. Despite the development of various genome walking methods, nonspecific products are often produced in certain circumstances, especially when GC-rich DNA sequences are dealt with. To effectively resolve such technical issues, a simple nested polymerase chain reaction-based genome walking method has been developed by implementing a progressively decreased annealing temperature from 70°C to 47.5°C in the first round of amplification and a high annealing temperature of 65°C in the second round of amplification. During the entire process, a lower ramp rate of 1.5°C s⁻¹ and cooling rate of 2.5°C s⁻¹ are performed to reach the annealing temperature. Using this method, we successfully obtained the upstream and downstream sequences of three GC-rich genes involved in the biosynthetic pathways of secondary metabolites from two bacterial genomes. The efficient amplification of DNA target longer than 1.5 Kb with GC content up to 75.0% indicates that the present technique could be a valuable tool for the investigation of biosynthetic pathways of various secondary metabolites.     

Enhancing plasmid transformation efficiency and enabling CRISPR-Cas9/Cpf1-based genome editing in Clostridium tyrobutyricum

Clostridium tyrobutyricum ATCC 25755 is known as a natural hyper-butyrate producer with great potentials as an excellent platform to be engineered for valuable biochemical production from renewable resources. However, limited transformation efficiency and the lack of genetic manipulation tools have hampered the broader applications of this micro-organism. In this study, the effects of Type I restriction-modification system and native plasmid on conjugation efficiency of C. tyrobutyricum were investigated through gene deletion. The deletion of Type I restriction endonuclease resulted in a 3.7-fold increase in conjugation efficiency, while the additional elimination of the native plasmid further enhanced conjugation efficiency to 6.05 ± 0.75 × 10³ CFU/ml-donor, which was 15.3-fold higher than the wild-type strain. Fermentation results indicated that the deletion of those two genetic elements did not significantly influence the end-products production in the resultant mutant ΔRMIΔNP. Thanks to the increased conjugation efficiency, the CRISPR-Cas9/Cpf1 systems, which previously could not be implemented in C. tyrobutyricum, were successfully employed for genome editing in ΔRMIΔNP with an efficiency of 12.5–25%. Altogether, approaches we developed herein offer valuable guidance for establishing efficient DNA transformation methods in nonmodel micro-organisms. The ΔRMIΔNP mutant can serve as a great chassis to be engineered for diverse valuable biofuel and biochemical production.     

利用CRISPR/Cas9技术对人多能干细胞进行高效基因组编辑

CRISPR/Cas9技术提供了一个全新的基因组编辑体系。本文利用CRISPR/Cas9平台,在人胚胎干细胞株中对选取的一段特定基因组区域进行了多种基因组编辑：通过在基因编码框中引入移码突变进行基因敲除;通过单链DNA提供外源模板经由同源重组定点敲入FLAG序列;通过同时靶向多个位点诱导基因组大片段删除。研究结果表明CRISPR/Cas9可以对多能干细胞进行高效基因编辑,获得的突变干细胞株有助于对基因和基因组区域的功能进行分析和干细胞疾病模型的建立。     

Efficient genome editing in Caenorhabditis elegans by CRISPR-targeted homologous recombination

Cas9 is an RNA-guided double-stranded DNA nuclease that participates in clustered regularly interspaced short palindromic repeats (CRISPR)-mediated adaptive immunity in prokaryotes. CRISPR–Cas9 has recently been used to generate insertion and deletion mutations in Caenorhabditis elegans, but not to create tailored changes (knock-ins). We show that the CRISPR–CRISPR-associated (Cas) system can be adapted for efficient and precise editing of the C. elegans genome. The targeted double-strand breaks generated by CRISPR are substrates for transgene-instructed gene conversion. This allows customized changes in the C. elegans genome by homologous recombination: sequences contained in the repair template (the transgene) are copied by gene conversion into the genome. The possibility to edit the C. elegans genome at selected locations will facilitate the systematic study of gene function in this widely used model organism.     

Modulating mutational outcomes and improving precise gene editing at CRISPR-Cas9-induced breaks by chemical inhibition of end-joining pathways

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