CRISPR/Cas9 genome targeting systems have been applied to a variety of species. However, most CRISPR/Cas9 systems reported for plants can only modify one or a few target sites. Here, we report a robust CRISPR/Cas9 vec...CRISPR/Cas9 genome targeting systems have been applied to a variety of species. However, most CRISPR/Cas9 systems reported for plants can only modify one or a few target sites. Here, we report a robust CRISPR/Cas9 vector system, utilizing a plant codon optimized Cas9 gene, for convenient and high- efficiency multiplex genome editing in monocot and dicot plants. We designed PCR-based procedures to rapidly generate multiple sgRNA expression cassettes, which can be assembled into the binary CRISPR/ Cas9 vectors in one round of cloning by Golden Gate ligation or Gibson Assembly. With this system, we edi- ted 46 target sites in rice with an average 85.4% rate of mutation, mostly in biallelic and homozygous status. We reasoned that about 16% of the homozygous mutations in rice were generated through the non-homol- ogous end-joining mechanism followed by homologous recombination-based repair. We also obtained uni- form biallelic, heterozygous, homozygous, and chimeric mutations in Arabidopsis T1 plants. The targeted mutations in both rice and Arabidopsis were heritable. We provide examples of loss-of-function gene mu- tations in To rice and T1Arabidopsis plants by simultaneous targeting of multiple （up to eight） members of a gene family, multiple genes in a biosynthetic pathway, or multiple sites in a single gene. This system has provided a versatile toolbox for studying functions of multiple genes and gene families in plants for basic research and genetic improvement.展开更多
Precise and straightforward methods to edit the plant genome are much needed for functional genomics and crop improvement. Recently, RNA-guided genome editing using bacterial Type II cluster regularly interspaced shor...Precise and straightforward methods to edit the plant genome are much needed for functional genomics and crop improvement. Recently, RNA-guided genome editing using bacterial Type II cluster regularly interspaced short palindromic repeats （CRISPR）-associated nuclease （Cas） is emerging as an efficient tool for genome editing in microbial and animal systems. Here, we report the genome editing and targeted gene mutation in plants via the CRISPR-Cas9 sys- tem. Three guide RNAs （gRNAs） with a 20-22-nt seed region were designed to pair with distinct rice genomic sites which are followed by the protospacer-adjacent motif （PAM）. The engineered gRNAs were shown to direct the Cas9 nuclease for precise cleavage at the desired sites and introduce mutation （insertion or deletion） by error-prone non-homologous end joining DNA repairing. By analyzing the RNA-guided genome-editing events, the mutation efficiency at these target sites was estimated to be 3-8%. In addition, the off-target effect of an engineered gRNA-Cas9 was found on an imper- fectly paired genomic site, but it had lower genome-editing efficiency than the perfectly matched site. Further analysis suggests that mismatch position between gRNA seed and target DNA is an important determinant of the gRNA-Cas9 tar- geting specificity, and specific gRNAs could be designed to target more than 90% of rice genes. Our results demonstrate that the CRISPR-Cas system can be exploited as a powerful tool for gene targeting and precise genome editing in plants.展开更多
The CRISPR/Cas system has been extensively applied to make precise genetic modifications in various organisms. Despite its importance and widespread use, large-scale mutation screening remains time-consuming, labour-i...The CRISPR/Cas system has been extensively applied to make precise genetic modifications in various organisms. Despite its importance and widespread use, large-scale mutation screening remains time-consuming, labour-intensive and costly. Here, we developed Hi-TOM(available at http://www.hi-tom.net/hi-tom/), an online tool to track the mutations with precise percentage for multiple samples and multiple target sites. We also described a corresponding next-generation sequencing(NGS) library construction strategy by fixing the bridge sequences and barcoding primers. Analysis of the samples from rice, hexaploid wheat and human cells reveals that the Hi-TOM tool has high reliability and sensitivity in tracking various mutations, especially complex chimeric mutations frequently induced by genome editing. Hi-TOM does not require special design of barcode primers,cumbersome parameter configuration or additional data analysis. Thus, the streamlined NGS library construction and comprehensive result output make Hi-TOM particularly suitable for high-throughput identification of all types of mutations induced by CRISPR/Cas systems.展开更多
The clustered regularly interspaced short palindromic repeat （CRISPR）-associated protein9 （Cas9） genome editing system （CRISPR/Casg） is adapted from the prokaryotic type II adaptive immunity system. The CRISPR/C...The clustered regularly interspaced short palindromic repeat （CRISPR）-associated protein9 （Cas9） genome editing system （CRISPR/Casg） is adapted from the prokaryotic type II adaptive immunity system. The CRISPR/Cas9 tool surpasses other programmable nucleases, such as ZFNs and TALENs, for its simplicity and high efficiency. Various plant-specific CRISPR/Cas9 vector systems have been established for adap- tion of this technology to many plant species. In this review, we present an overview of current advances on applications of this technology in plants, emphasizing general considerations for establishment of CRISPR/ Cas9 vector platforms, strategies for multiplex editing, methods for analyzing the induced mutations, fac- tors affecting editing efficiency and specificity, and features of the induced mutations and applications of the CRISPR/Cas9 system in plants. In addition, we provide a perspective on the challenges of CRISPR/Cas9 technology and its significance for basic plant research and crop genetic improvement.展开更多
概述了我国农作物田间杂草的主要类型、除草剂主要类型及其作用机制以及农作物除草剂抗性主要机制,综述了作物除草剂抗性育种的研究进展,比较了传统育种和转基因育种方式培育除草剂抗性作物的优缺点,总结了转基因育种存在的安全性问题...概述了我国农作物田间杂草的主要类型、除草剂主要类型及其作用机制以及农作物除草剂抗性主要机制,综述了作物除草剂抗性育种的研究进展,比较了传统育种和转基因育种方式培育除草剂抗性作物的优缺点,总结了转基因育种存在的安全性问题。同时介绍了转基因培育除草剂抗性作物中常用的抗性基因以及基因转入受体的方式,展望了CRISPR/Cas9(clustered regularly interspaced short palindromic repeats,CRISPRassociated protein 9)等基因编辑技术在农作物除草剂抗性育种研究中的应用与发展前景。展开更多
文摘CRISPR/Cas9 genome targeting systems have been applied to a variety of species. However, most CRISPR/Cas9 systems reported for plants can only modify one or a few target sites. Here, we report a robust CRISPR/Cas9 vector system, utilizing a plant codon optimized Cas9 gene, for convenient and high- efficiency multiplex genome editing in monocot and dicot plants. We designed PCR-based procedures to rapidly generate multiple sgRNA expression cassettes, which can be assembled into the binary CRISPR/ Cas9 vectors in one round of cloning by Golden Gate ligation or Gibson Assembly. With this system, we edi- ted 46 target sites in rice with an average 85.4% rate of mutation, mostly in biallelic and homozygous status. We reasoned that about 16% of the homozygous mutations in rice were generated through the non-homol- ogous end-joining mechanism followed by homologous recombination-based repair. We also obtained uni- form biallelic, heterozygous, homozygous, and chimeric mutations in Arabidopsis T1 plants. The targeted mutations in both rice and Arabidopsis were heritable. We provide examples of loss-of-function gene mu- tations in To rice and T1Arabidopsis plants by simultaneous targeting of multiple （up to eight） members of a gene family, multiple genes in a biosynthetic pathway, or multiple sites in a single gene. This system has provided a versatile toolbox for studying functions of multiple genes and gene families in plants for basic research and genetic improvement.
文摘Precise and straightforward methods to edit the plant genome are much needed for functional genomics and crop improvement. Recently, RNA-guided genome editing using bacterial Type II cluster regularly interspaced short palindromic repeats （CRISPR）-associated nuclease （Cas） is emerging as an efficient tool for genome editing in microbial and animal systems. Here, we report the genome editing and targeted gene mutation in plants via the CRISPR-Cas9 sys- tem. Three guide RNAs （gRNAs） with a 20-22-nt seed region were designed to pair with distinct rice genomic sites which are followed by the protospacer-adjacent motif （PAM）. The engineered gRNAs were shown to direct the Cas9 nuclease for precise cleavage at the desired sites and introduce mutation （insertion or deletion） by error-prone non-homologous end joining DNA repairing. By analyzing the RNA-guided genome-editing events, the mutation efficiency at these target sites was estimated to be 3-8%. In addition, the off-target effect of an engineered gRNA-Cas9 was found on an imper- fectly paired genomic site, but it had lower genome-editing efficiency than the perfectly matched site. Further analysis suggests that mismatch position between gRNA seed and target DNA is an important determinant of the gRNA-Cas9 tar- geting specificity, and specific gRNAs could be designed to target more than 90% of rice genes. Our results demonstrate that the CRISPR-Cas system can be exploited as a powerful tool for gene targeting and precise genome editing in plants.
基金supported by the National Key Research and Development Program of China (2017YFD0102002)the Agricultural Science and Technology Innovation Program of Chinese Academy of Agricultural Sciencesthe National Natural Science Foundation of China (31401363)
文摘The CRISPR/Cas system has been extensively applied to make precise genetic modifications in various organisms. Despite its importance and widespread use, large-scale mutation screening remains time-consuming, labour-intensive and costly. Here, we developed Hi-TOM(available at http://www.hi-tom.net/hi-tom/), an online tool to track the mutations with precise percentage for multiple samples and multiple target sites. We also described a corresponding next-generation sequencing(NGS) library construction strategy by fixing the bridge sequences and barcoding primers. Analysis of the samples from rice, hexaploid wheat and human cells reveals that the Hi-TOM tool has high reliability and sensitivity in tracking various mutations, especially complex chimeric mutations frequently induced by genome editing. Hi-TOM does not require special design of barcode primers,cumbersome parameter configuration or additional data analysis. Thus, the streamlined NGS library construction and comprehensive result output make Hi-TOM particularly suitable for high-throughput identification of all types of mutations induced by CRISPR/Cas systems.
文摘The clustered regularly interspaced short palindromic repeat （CRISPR）-associated protein9 （Cas9） genome editing system （CRISPR/Casg） is adapted from the prokaryotic type II adaptive immunity system. The CRISPR/Cas9 tool surpasses other programmable nucleases, such as ZFNs and TALENs, for its simplicity and high efficiency. Various plant-specific CRISPR/Cas9 vector systems have been established for adap- tion of this technology to many plant species. In this review, we present an overview of current advances on applications of this technology in plants, emphasizing general considerations for establishment of CRISPR/ Cas9 vector platforms, strategies for multiplex editing, methods for analyzing the induced mutations, fac- tors affecting editing efficiency and specificity, and features of the induced mutations and applications of the CRISPR/Cas9 system in plants. In addition, we provide a perspective on the challenges of CRISPR/Cas9 technology and its significance for basic plant research and crop genetic improvement.
文摘概述了我国农作物田间杂草的主要类型、除草剂主要类型及其作用机制以及农作物除草剂抗性主要机制,综述了作物除草剂抗性育种的研究进展,比较了传统育种和转基因育种方式培育除草剂抗性作物的优缺点,总结了转基因育种存在的安全性问题。同时介绍了转基因培育除草剂抗性作物中常用的抗性基因以及基因转入受体的方式,展望了CRISPR/Cas9(clustered regularly interspaced short palindromic repeats,CRISPRassociated protein 9)等基因编辑技术在农作物除草剂抗性育种研究中的应用与发展前景。
