CRISPR/Cas9基因组编辑技术在农业动物中的应用

CRISPR(Clustered regularly interspaced short palindromic repeats)/Cas(CRISPR associated proteins)是在细菌和古细菌中发现的一种用来抵御病毒或质粒入侵的获得性免疫系统.目前已发现的CRISPR/Cas系统包括Ⅰ,Ⅱ和Ⅲ型,其中Ⅱ型系统的组成较简单,由其改造成的CRISPR/Cas9技术已成为一种高效的基因组编辑工具.自2013年CRISPR/Cas9技术成功用于哺乳动物基因组定点编辑以来,应用该技术进行基因组编辑的报道呈现出爆发式的增长.农业动物不仅是重要的经济动物,也是人类疾病和生物医药研究的重要模式动物.本文综述了CRISPR/Cas9技术在农业动物中的研究和应用进展,简述了该技术的脱靶效应及减少脱靶的主要方法,并展望了该技术的应用前景.     

Delivery of CRISPR/Cas9 for therapeutic genome editing

The clustered, regularly‐interspaced, short palindromic repeat (CRISPR)‐associated nuclease 9 (CRISPR/Cas9) is emerging as a promising genome‐editing tool for treating diseases in a precise way, and has been applied to a wide range of research in the areas of biology, genetics, and medicine. Delivery of therapeutic genome‐editing agents provides a promising platform for the treatment of genetic disorders. Although viral vectors are widely used to deliver CRISPR/Cas9 elements with high efficiency, they suffer from several drawbacks, such as mutagenesis, immunogenicity, and off‐target effects. Recently, non‐viral vectors have emerged as another class of delivery carriers in terms of their safety, simplicity, and flexibility. In this review, we discuss the modes of CRISPR/Cas9 delivery, the barriers to the delivery process and the application of CRISPR/Cas9 system for the treatment of genetic disorders. We also highlight several representative types of non‐viral vectors, including polymers, liposomes, cell‐penetrating peptides, and other synthetic vectors, for the therapeutic delivery of CRISPR/Cas9 system. The applications of CRISPR/Cas9 in treating genetic disorders mediated by the non‐viral vectors are also discussed.     

Mosaicism in CRISPR/Cas9-mediated genome editing

The CRISPR/Cas9 system is a rapid, simple, and often extremely efficient gene editing method. This method has been used in a variety of organisms and cell types over the past several years. However, using this technology for generating gene-edited animals involves a number of obstacles. One such obstacle is mosaicism, which is common in founder animals. This is especially the case when the CRISPR/Cas9 system is used in embryos. Here we review the pros and cons of mosaic mutations of gene-edited animals caused by using the CRISPR/Cas9 system in embryos. Furthermore, we will discuss the mechanisms underlying mosaic mutations resulting from the CRISPR/Cas9 system, as well as the possible strategies for reducing mosaicism. By developing ways to overcome mosaic mutations when using CRISPR/Cas9, genotyping for germline gene disruptions should become more reliable. This achievement will pave the way for using the CRISPR technology in the research and clinical applications where mosaicism is an issue.     

利用CRISPR/Cas9技术构建CTCF蛋白降解细胞系

CTCF是脊椎动物关键的绝缘子蛋白,在细胞生命过程中发挥重要作用,敲除CTCF基因会导致小鼠胚胎死亡。为进一步探讨CTCF的功能,本文利用CRISPR/Cas9介导的同源重组,在内源性CTCF表达框上游敲入一个有丝分裂期降解结构域(Mitosis-special degradation domain, MD),该结构域可以带动CTCF融合蛋白在M期降解。作为对照,将MD结构域的第42位的精氨酸突变为丙氨酸,形成无降解活性的MD^*,可使MD^*-CTCF融合蛋白始终稳定存在。将嘌呤霉素与融合蛋白同时表达,即可利用抗生素筛选,高效地筛选到纯合克隆。利用蛋白印迹技术和免疫荧光检测3种细胞在不同细胞周期的CTCF蛋白变化情况,发现MD-CTCF细胞系CTCF蛋白含量约为野生型细胞的10%,MD^*-CTCF细胞系的CTCF含量与野生型没有显著差别;通过流式细胞术观测降解CTCF对细胞的影响,发现MD-CTCF细胞系G1期明显延长。总之,利用CRISPR/Cas9技术在CTCF表达框上游高效地插入MD,首个CTCF特异性降解的人类细胞系获得成功构建。     

Insights into maize genome editing via CRISPR/Cas9

Maize is an important crop for billions of people as food, feed, and industrial raw material. It is a prime driver of the global agricultural economy as well as the livelihoods of millions of farmers. Genetic interventions, such as breeding, hybridization and transgenesis have led to increased productivity of this crop in the last 100 years. The technique of genome editing is the latest advancement in genetics. Genome editing can be used for targeted deletions, additions, and corrections in the genome, all aimed at genetic enhancement of crops. The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR associated protein 9 (CRISPR/Cas9) system is a recent genome editing technique that is considered simple, precise, robust and the most revolutionary. This review summarizes the current state of the art and predicts future directions in the use of the CRISPR/Cas9 tool in maize crop improvement.     

CRISPR/Cas9编辑大肠杆菌工程菌生产氨基葡萄糖

【背景】氨基葡萄糖(glucosamine, GlcN)及其衍生物N-乙酰氨基葡萄糖(N-acetylglucosamine,GlcNAc)是合成糖胺聚糖的重要前体物质,在医药、化妆品和保健品领域具有广泛的应用价值。传统的生产方式存在诸多弊端,如环境污染、原料限制、不适于海鲜易过敏人群等问题,因此利用微生物发酵法生产GlcN和GlcNAc越来越受到青睐。【目的】利用微生物发酵生产并提高N-乙酰氨基葡萄糖的产量,探索分子改造及发酵条件优化策略。【方法】以大肠杆菌MG1655为出发菌株,首先利用表达载体共表达大肠杆菌来源的glmS和酿酒酵母来源的gna1,构建GlcNAc的生物合成路径,然后利用CRISPR/Cas9技术敲除GlcNAc的分解代谢与转运途径,以提高GlcNAc的产量,最后结合发酵条件优化使GlcNAc的产量得到进一步提升。【结果】通过分子改造得到一株产GlcNAc菌株RY-5,发酵20 h后GlcNAc的产量达到了2.36 g/L,相较于初始构建的菌株RY-1提高了29倍,进一步对装液量和诱导剂IPTG的添加时间等条件进行发酵优化,GlcNAc产量达到了7.74g/L,与优...     

Functional characterization of SlitPBP3 in Spodoptera litura by CRISPR/Cas9 mediated genome editing

Functional gene analysis by using genome editing techniques is limited only in few model insects. Here, we reported an efficient and heritable gene mutagenesis analysis in an important lepidopteran pest, Spodoptera litura, using the CRISPR/Cas9 system. By using this system, we successfully obtained the homozygous S. litura strain by targeting the pheromone binding protein 3 gene (SlitPBP3), which allowed us to elucidate the role of this gene in the olfaction of the female sex pheromones. By co-injection of Cas9 mRNA and sgRNA into S. litura eggs, highly efficient chimera mutation in SlitPBP3 loci was detected both in injected eggs (39.1%) and in the resulting individual moths (87.5%). We used the mutant moths as parents to obtain the G1 offspring and the homozygous mutant strain in G2. The function of SlitPBP3 was explored by Electroantennogram (EAG) recordings with a homozygous mutant strain. The result showed that the EAG responses were significantly decreased in mutant males than in control males when treated with the major sex pheromone component (Z9,E11-14:Ac) and a minor component (Z9-14:Ac) at higher dosages. The results demonstrate that s SlitPBP3 gene plays a minor role in the perception of the female sex pheromones. Furthermore, our study provides a useful methodology with the CRISPR/Cas9 system for gene in vivo functional study, particular for lepidopteran species in which the RNAi approach is not efficient.     

A CRISPR/Cas9-based genome editing system for Rhodococcus ruber TH

Rhodococcus spp. are organic solvent-tolerant strains with strong adaptive abilities and diverse metabolic activities, and are therefore widely utilized in bioconversion, biosynthesis and bioremediation. However, due to the high GC-content of the genome (~70%), together with low transformation and recombination efficiency, the efficient genome editing of Rhodococcus remains challenging. In this study, we report for the first time the successful establishment of a CRISPR/Cas9-based genome editing system for R. ruber. With a bypass of the restriction-modification system, the transformation efficiency of R. ruber was enhanced by 89-fold, making it feasible to obtain enough colonies for screening of mutants. By introducing a pair of bacteriophage recombinases, Che9c60 and Che9c61, the editing efficiency was improved from 1% to 75%. A CRISPR/Cas9-mediated triple-plasmid recombineering system was developed with high efficiency of gene deletion, insertion and mutation. Finally, this new genome editing method was successfully applied to engineer R. ruber for the bio-production of acrylamide. By deletion of a byproduct-related gene and in-situ subsititution of the natural nitrile hydratase gene with a stable mutant, an engineered strain R. ruber THY was obtained with reduced byproduct formation and enhanced catalytic stability. Compared with the use of wild-type R. ruber TH, utilization of R. ruber THY as biocatalyst increased the acrylamide concentration from 405 g/L to 500 g/L, reduced the byproduct concentration from 2.54 g/L to 0.5 g/L, and enhanced the number of times that cells could be recycled from 1 batch to 4 batches.     

计算生物学分析在基因组编辑研究中的应用

基因组编辑是对基因组遗传信息进行定向改造的技术,其中CRISPR/Cas系统是目前应用最广泛的基因组编辑新技术。将先进的高通量测序以及相关计算生物分析应用于基因编辑研究,可进一步优化基因编辑效率和精度等检测流程,实现对全基因组功能基因筛选的监测。同时,利用基于生物信息及机器学习和深度学习等新方法,可对向导RNA(gRNA)的高效设计和实现对编辑效果的预测。本文将对计算生物学分析在CRISPR/Cas基因编辑系统的应用及研究进展等进行概述。     

DNA-free genome editing with preassembled CRISPR/Cas9 ribonucleoproteins in plants

Transgenic Research - Processes of traditional trait development in plants depend on genetic variations derived from spontaneous mutation or artificial random mutagenesis. Limited availability of...     

Efficient genetic transformation and CRISPR/Cas9‐mediated genome editing in Lemna aequinoctialis

The fast growth, ease of metabolic labelling and potential for feedstock and biofuels production make duckweeds not only an attractive model system for understanding plant biology, but also a potential future crop. However, current duckweed research is constrained by the lack of efficient genetic manipulation tools. Here, we report a case study on genome editing in a duckweed species, Lemna aequinoctialis, using a fast and efficient transformation and CRISPR/Cas9 tool. By optimizing currently available transformation protocols, we reduced the duration time of Agrobacterium‐mediated transformation to 5–6 weeks with a success rate of over 94%. Based on the optimized transformation protocol, we generated 15 (14.3% success rate) biallelic LaPDS mutants that showed albino phenotype using a CRISPR/Cas9 system. Investigations on CRISPR/Cas9‐mediated mutation spectrum among mutated L. aequinoctialis showed that most of mutations were short insertions and deletions. This study presents the first example of CRISPR/Cas9‐mediated genome editing in duckweeds, which will open new research avenues in using duckweeds for both basic and applied research.     

CRISPR/Cas9 for plant genome editing: accomplishments,problems and prospects

The increasing burden of the world population on agriculture requires the development of more robust crops. Dissecting the basic biology that underlies plant development and stress responses will inform the design of better crops. One powerful tool for studying plants at the molecular level is the RNA-programmed genome editing system composed of a clustered regularly interspaced short palindromic repeats (CRISPR)-encoded guide RNA and the nuclease Cas9. Here, some of the recent advances in CRISPR/Cas9 technology that have profound implications for improving the study of plant biology are described. These tools are also paving the way towards new horizons for biotechnologies and crop development.     

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基因组编辑技术在癌症研究中的应用

CRISPR/cas9基因组编辑技术因其设计简单以及操作容易,使其在基因编辑的研究中越来越受到欢迎。利用该技术,科研人员可以实现在碱基的水平对基因组进行定点修饰。CRISPR系统现已经被广泛地应用到多个物种的基因组编辑以及癌症的相关研究中。本文在最新研究进展的基础上,结合对癌症研究及基因组编辑技术的理解,对CRISPR/Cas9技术在癌症研究中的应用进行了综述。     

CRISPR/Cas9 genome editing demonstrates functionality of the autoimmunity-associated SNP rs12946510

Genome-wide association studies (GWAS) map genetic associations of complex traits with precision limited to a linkage disequilibrium group. To translate GWAS results into new understanding of disease mechanisms, individual causative polymorphisms and their target genes should be identified. CRISPR/Cas9 genome editing can be used to create isogenic cell lines bearing alternative genotypes of candidate single-nucleotide polymorphisms to test their causality and to reveal gene targets. An intergenic polymorphism rs12946510 is associated with multiple sclerosis, inflammatory bowel disease and asthma. We created sublines of the T-helper cell line bearing alternative genotypes of rs12946510 and showed that its risk (“T”) allele is associated with lower expression of IKZF3 and ORMDL3 genes and reduced cell activation. Our editing procedure can become an effective tool for discovering new genes involved in pathogenesis of complex diseases.