Targeted, random mutagenesis of plant genes with dual cytosine and adenine base editors

First author: Chao Li; Affiliations: CAS Institute of Genetics and Developmental Biology (中科院遗传与发育生物学研究所): Beijing, China

Corresponding author: Caixia Gao

Targeted saturation mutagenesis of crop genes could be applied to produce genetic variants with improved agronomic performance. However, tools for directed evolution of plant genes, such as error-prone PCR or DNA shuffling, are limited. We engineered five saturated targeted endogenous mutagenesis editors (STEMEs) that can generate de novo mutations and facilitate directed evolution of plant genes. In rice protoplasts, STEME-1 edited cytosine and adenine at the same target site with C > T efficiency up to 61.61% and simultaneous C > T and A > G efficiency up to 15.10%. STEME-NG, which incorporates the nickase Cas9-NG protospacer-adjacent motif variant, was used with 20 individual single guide RNAs in rice protoplasts to produce near-saturated mutagenesis (73.21%) for a 56-amino-acid portion of the rice acetyl-coenzyme A carboxylase (OsACC). We also applied STEME-1 and STEME-NG for directed evolution of the OsACC gene in rice and obtained herbicide resistance mutations. This set of two STEMEs will accelerate trait development and should work in any plants amenable to CRISPR-based editing.

作物基因的靶向饱和诱变可应用于获取带有改良农艺性状的遗传变异植株。然而，用于基因定向演化的工具，比如易错PCR和DNA改组技术，在植物中的使用不太成熟。作者开发了5个饱和的靶向内源诱变编辑器STEMEs，可以产生de novo变异，加速植物基因的定向演化。在水稻原生质体中，STEME-1在同一个靶向位点编辑胞嘧啶和腺嘌呤，其中C > T的效率最高为61.61%，而C > T和A > G同时存在的效率最高为15.10%。融合了切口酶Cas9-NG PAM变体的STEME-NG系统，利用20个单个gRNA就可以在水稻原生质体中对水稻乙酰辅酶A羧化酶（OsACC）上56个氨基酸产生近饱和的诱变。另外，作者还利用了STEME-1和STEME-NG对水稻OsACC基因进行了定向演化，获得了对除草剂具有抗性的突变植株。这两个STEMEs系统将有利于加速作物新的农艺性状的开发，同时也可应用于任何可以适用CRISPR基因编辑的植物。

通讯：高彩霞 (http://sourcedb.genetics.cas.cn/zw/zjrck/201001/t20100112_2725447.html)

个人简介：1991年，甘肃农业大学，学士；1994年，甘肃农业大学，硕士；1997年，中国农业大学，博士。

研究方向：主要从事植物基因组编辑技术、生物安全新型育种技术以及基因组编辑定向设计分子育种等方面的研究，致力于推动基因组编辑在分子设计育种中的应用。

doi: https://doi.org/10.1038/s41587-019-0393-7

Journal: Nature Biotechnology

Published date: January 13, 2020