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。     

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.     

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.     

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。     

Improving CRISPR/Cas9-mediated genome editing efficiency in Yarrowia lipolytica using direct tRNA-sgRNA fusions

Yarrowia lipolytica is an important oleaginous yeast currently used in the production of specialty chemicals and has a great potential for further applications in lipid biotechnology. Harnessing the full potential of Y. lipolytica is, however, limited by its inherent recalcitrance to genetic manipulation. In contrast to Saccharomyces cerevisiae, Y. lipolytica is poor in homology-mediated DNA repair and thus in homologous recombination, which limits site-specific gene editing in this yeast. Recently developed CRISPR/Cas9-based methods using tRNA-sgRNA fusions succeeded in editing some genomic loci in Y. lipolytica. Nonetheless, the majority of other tested loci either failed editing or editing was achieved but at very low efficiency using these methods. Using tools of secondary RNA structure prediction, we were able to improve the design of the tRNA-sgRNA fusions used for the expression of single guide RNA (sgRNA) in such methods. This resulted in high efficiency CRISPR/cas9 gene editing at chromosomal loci that failed gene editing or were edited at very low efficiencies with previous methods. In addition, we characterized the gene editing performance of our newly designed tRNA-sgRNA fusions for both chromosomal gene integration and deletion. As such, this study presents an efficient CRISPR/Cas9-mediated gene-editing tool for efficient genetic engineering of Yarrowia lipolytica.     

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...     

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。     

A sequential transformation method for validating soybean genome editing by CRISPR/Cas9 system

This study was performed to evaluate the sequential transformation for soybean genome editing using the CRISPR/Cas9 system as well as to show a strategy for examining the activity of CRISPR/Cas9 constructs, especially the designed guide RNAs (gRNAs). The gRNAs for targeted mutations of an exogenous gene and multiple endogenous genes were constructed and transferred into a stably-overexpressed-Cas9 soybean line using Agrobacterium rhizogenes-mediated hairy root induction system. The targeted mutations were identified and characterized by the poly-acrylamide gel electrophoresis (PAGE) heteroduplex method and by sequencing. Induced mutations of the exogenous gene (gus) were observed in 57% of tested transgenic hairy roots, while 100% of the transgenic root lines showed targeted mutations of the endogenous (SACPD-C) gene. Multiple gRNAs targeting two endogenous genes (SACPD-C and SMT) induced mutation rates of 75% and 67%, respectively. Various indels including small and large deletions as well as insertions were found in target sites of the tested genes. This sequential transformation method could present the targeting efficacy of different gRNAs of each tested gene. Additionally, in this study differences in gRNA ratings were found between bioinformatics predictions and actual experimental results. This is the first successful application of the sequential transformation method for genome editing in soybean using the hairy root system. This method could be potentially useful for validating CRISPR/Cas9 constructs, evaluating gRNA targeting efficiencies, and could be applied for other research directions.     

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-mediated genome editing: From basic research to translational medicine

The recent development of the CRISPR/Cas9 system as an efficient and accessible programmable genome-editing tool has revolutionized basic science research. CRISPR/Cas9 system-based technologies have armed researchers with new powerful tools to unveil the impact of genetics on disease development by enabling the creation of precise cellular and animal models of human diseases. The therapeutic potential of these technologies is tremendous, particularly in gene therapy, in which a patient-specific mutation is genetically corrected in order to treat human diseases that are untreatable with conventional therapies. However, the translation of CRISPR/Cas9 into the clinics will be challenging, since we still need to improve the efficiency, specificity and delivery of this technology. In this review, we focus on several in vitro, in vivo and ex vivo applications of the CRISPR/Cas9 system in human disease-focused research, explore the potential of this technology in translational medicine and discuss some of the major challenges for its future use in patients.     

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.     

Application and future perspective of CRISPR/Cas9 genome editing in fruit crops

Fruit crops, including apple, orange, grape,banana, strawberry, watermelon, kiwifruit and tomato, not only provide essential nutrients for human life but also contribute to the major agricultural output and economic growth of many countries and regions in the world. Recent advancements in genome editing provides an unprecedented opportunity for the genetic improvement of these agronomically important fruit crops. Here, we summarize recent reports of applying CRISPR/Cas9 to fruit crops,including efforts to reduce disease susceptibility, change plant architecture or flower morphology, improve fruit quality traits, and increase fruit yield. We discuss challenges facing fruit crops as well as new improvements and platforms that could be used to facilitate genome editing in fruit crops, including d Cas9-base-editing to introduce desirable alleles and heat treatment to increase editing efficiency. In addition, we highlight what we see as potentially revolutionary development ranging from transgene-free genome editing to de novo domestication of wild relatives. Without doubt, we now see only the beginning of what will eventually be possible with the use of the CRISPR/Cas9 toolkit. Efforts to communicate with the public and an emphasis on the manipulation of consumerfriendly traits will be critical to facilitate public acceptance of genetically engineered fruits with this new technology.     

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.