A Robust CRISPR/Cas9 System for Convenient, High-Efficiency Multiplex Genome Editing in Monocot and Dicot Plants  

作　　者：Xingliang Ma Qunyu Zhang Qinlong Zhu Wei Liu Yan Chen Rong Qiu Bin Wang Zhongfang Yang Heying Li Yuru Lin Yongyao Xie Rongxin Shen Shuifu Chen Zhi Wang Yuanling Chen Jingxin Guo Letian Chen Xiucai Zhao Zhicheng Dong Yao-Guang Liu 

机构地区：[1]State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, Guangzhou 510642, China [2]Key Laboratory of Plant Functional Genomics and Biotechnology of Guangdong Provincial Higher Education Institutions, Guangzhou 510642, China [3]Guangdong Provincial Key Laboratory of Protein Function and Regulation in Agricultural Organisms, Guangzhou 510642, China [4]College of Life Sciences, South China Agricultural University, Guangzhou 510642, China [5]Key Laboratory of South China Agriculture Plant Molecular Analysis and Genetic Improvement, South China Botanical Garden, Chinese Academy of Sciences,Guangzhou 510650, China

出　　处：《Molecular Plant》2015年第8期1274-1284,共11页分子植物（英文版）

摘　　要：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.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.

关 键 词：sequence-specific nucleases genome editing CRISPR/Cas9 rice Arabidopsis 

分 类 号：Q949.4[生物学—植物学] Q782