利用rBE3和rBE4系统进行水稻基因单碱基编辑的特异性和遗传稳定性分析  

Specificity and inheritance of rBE3 and rBE4 endonuclease-induced gene modifications in rice

作　　者：任斌 严芳 旷永洁 李娜[1] 张大伟[2] 林宏辉[2] 周焕斌 

机构地区：[1]中国农业科学院植物保护研究所植物病虫害生物学国家重点实验室,北京100193 [2]四川大学生命科学学院生物资源与生态环境教育部重点实验室,四川成都610065

出　　处：《生物工程学报》2017年第10期1776-1785,共10页Chinese Journal of Biotechnology

基　　金：中国农业科学院科技创新工程和植物病虫害国家重点实验室开放基金资助~~

摘　　要：对基于rBE3(Rice base editor)和rBE4碱基编辑系统创制获得Os SERK1(D428N)和pi-ta(S918F)等基因的单碱基编辑突变体材料进行编辑特异性和遗传稳定性分析,旨在全面了解和更好地利用该碱基编辑系统。首先对Os SERK1(D428N)和pi-ta(S918F)sg RNA的潜在脱靶位点进行预测,并对T0代材料中的各脱靶位点进行PCR扩增和Sanger测序检测;同时对该两个基因的突变体材料自交获得的T1代植株的靶位点序列和外源T-DNA分离进行检测。结果显示各T0代材料均未检测到潜在脱靶位点发生单碱基编辑;此外,Os SERK1(D428N)和Os08g07774含有相同的sg RNA位点,且两个位点均能发生单碱基编辑;rBE3或rBE4系统介导产生的碱基编辑可稳定遗传至T1代,并在T1代可获得无外源T-DNA的纯合突变体。上述结果表明由rBE3或rBE4介导的碱基编辑具有较高的特异性,可进行多位点编辑,引入的碱基替换可稳定遗传至后代。To gain more insights into the rice base editor （rBE3 and rBE4）, we evaluated the mutation efficiency, off-target and inheritance of OsSERKl（D428N） and pi-ta（S918F） genes modified with rBE endonucleases. We predicted and analyzed the putative off-target sites of the sgRNA designed for OsSERKl（D428N） and pi-ta（S918F） by PCR amplification and Sanger sequencing. Then we further characterized the inheritance and stability of targeted base mutations and T-DNA segregation in the progeny of the self-fertilized TO plants. Analysis of the DNA sequencing data of TO plants of OsSERKl（D428N） revealed no nucleotide change at any of the four potential off-target sites. For OsSERKI（D428N） and OsOSg07774 carry the same sgRNA targeting sites, base substitution at both two loci were detected at a frequency of 41.67%. The targeted base mutations could be transmitted readily to T1 progeny. Furthermore, genetic segregation caused the loss of T-DNA at a frequency between 25.0% and 40.9% in the T1 transgenic plants of OsSERKl（D428N） and pi-ta（S918F）. These results demonstrated that the rBE3 and rBE4 systems could mediate specifically targeted base editing in one- or multi-site, and the targeted base editing could be stably inherited to next generation.

关 键 词：CRISPR/Cas9n 大鼠胞苷脱氨酶 脱靶效率 T-DNA分离 水稻 

分 类 号：Q943.2[生物学—植物学]