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

摘要:

对基于rBE3 (Rice base editor) 和rBE4碱基编辑系统创制获得OsSERK1(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OsSERK1(D428N) 和pi-ta(S918F) sgRNA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，OsSERK1(D428N)和Os08g07774含有相同的sgRNA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。

Abstract:

To gain more insights into the rice base editor (rBE3 and rBE4), we evaluated the mutation efficiency, off-target and inheritance of OsSERK1(D428N) and pi-ta(S918F) genes modified with rBE endonucleases. We predicted and analyzed the putative off-target sites of the sgRNA designed for OsSERK1(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 T0 plants. Analysis of the DNA sequencing data of T0 plants of OsSERK1(D428N) revealed no nucleotide change at any of the four potential off-target sites. For OsSERK1(D428N) and Os08g07774 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 OsSERK1(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.