基于优化sgRNA系统提高海岛棉CRISPR/Cas9基因组编辑功效的研究  

Enhancing CRISPR/Cas9 genomic editing efficiency based on optimization of sgRNA of Gossypium barbadense L.

作　　者：李继洋[1,2] 胡燕 姚瑞 代培红 刘晓东[1] LI Ji-Yang;HU Yan;YAO Rui;DAI Pei-Hong;LIU Xiao-Dong(Agricultural College,Xinjiang Agricultural University/Key Laboratory of Agricultural Biotechnology,Xinjiang Agricultural University,Urumqi 830052,Xinjiang,China;Life Sciences College,Shihezi University,Shihezi 832000,Xinjiang,China)

机构地区：[1]新疆农业大学农学院/新疆农业大学农业生物技术重点实验室,新疆乌鲁木齐830052 [2]石河子大学生命科学院,新疆石河子832000

出　　处：《作物学报》2019年第10期1522-1534,共13页Acta Agronomica Sinica

基　　金：南京农业大学-新疆农业大学联合基金项目(KYYJ201603);新疆农业大学作物学重点学科发展基金项目资助。

摘　　要：CRISPR/Cas9基因组编辑体系已经在多种作物中被建立,其最大的优势在于能简单高效定向创制突变体。然而,CRISPR/Cas9基因组编辑体系在实际操作过程中经常会出现没有编辑的情况,或者靶向编辑目的基因的同时也会引发不同程度的脱靶效应,这对CRISPR/Cas9基因组编辑技术的运用带来不利影响。本研究基于前期在海岛棉体细胞中建立的CRISPR/Cas9基因组编辑体系,通过对构建Cas9不同密码子优化方式、不同PAM位点个数和不同靶位点数量的编辑载体,来分析比较编辑效率和脱靶效应的差异。结果表明,2种Cas9密码子不同优化方式的编辑载体产生的编辑效率和脱靶效应无显著差异;优化后的双PAM位点的编辑效率显著高于单PAM位点,且脱靶效率显著较低;大部分双靶序列编辑效率均高于单靶序列且脱靶效率较低。上述研究结果为今后优化CRISPR/Cas9介导的海岛棉基因组编辑体系奠定了重要的理论依据。The CRISPR/Cas9 genome editing system has been established in many crops. The advantages of its directional creation of mutants are increasingly favored by researchers. However,the CRISPR/Cas9 genome editing technology targets the editing of the target gene and also triggers off-target effects at different frequencies,which determine the reliability of the genome editing system. This study was based on the previous CRISPR/Cas9 genome editing system established in the island-cotton somatic cells. Edit the vector by constructing different codon optimization methods for Cas9,different numbers of PAM sites and different target sites,and the difference in editing efficiency and off-target effect were analyzed and compared. There was no significant difference in editing effects and off-target effects caused by Cas9-edited vectors with different optimal codons. The partially double-sgRNA had significantly higher editing efficiency and significantly lower off-target efficiency than the single sgRNA;the editing efficiency of the transformed No shift sgRNA target sequence was significantly higher than that of the Shift sgRNA and the former had a significant decrease in off-target efficiency relative to the latter. Therefore,using No shift sgRNA type target sequence can effectively improve the editing efficiency and significantly reduce the off-target efficiency,thus laying a theoretical foundation for optimizing the CRISPR/Cas9 mediated island cotton genome editing system and accurately and efficiently creating island cotton functional gene mutants in the future.

关 键 词：棉花 CRISPR/Cas9 编辑效率 脱靶效率 sgRNA类型 

分 类 号：S562[农业科学—作物学] Q943.2[生物学—植物学]