Synthesis-dependent repair of Cpf1-induced double strand DNA breaks enables targeted gene replacement in rice

文献类型： 外文期刊

第一作者： Li, Shaoya

作者： Li, Shaoya;Li, Jingying;Zhang, Jiahui;Du, Wenming;Fu, Jindong;Sutar, Suhas;Xia, Lanqin;Zhao, Yunde;Zhao, Yunde;Zhao, Yunde

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关键词： Cpf1; gene replacement; homology-directed DNA repair (HDR); ribozyme; rice (Oryza sativa L); synthesis-dependent strand annealing (SDSA)

期刊名称：JOURNAL OF EXPERIMENTAL BOTANY （ 影响因子：6.992； 五年影响因子：7.86 ）

ISSN： 0022-0957

年卷期： 2018 年 69 卷 20 期

页码：

收录情况： SCI

摘要： The recently developed CRISPR (clustered regularly interspaced short palindromic repeats)/Cpf1 system expands the range of genome editing and is emerging as an alternative powerful tool for both plant functional genomics and crop improvement. Cpf1-CRISPR RNA (crRNA) produces double strand DNA breaks (DSBs) with long 5'-protruding ends, which may facilitate the pairing and insertion of repair templates through homology-directed repair (HDR) for targeted gene replacement and introduction of the desired DNA elements at specific gene loci for crop improvement. However, the potential mechanism underlying HDR of DSBs generated by Cpf1-crRNA remains to be investigated, and the inherent low efficiency of HDR and poor availability of exogenous donor DNA as repair templates strongly impede the use of HDR for precise genome editing in crop plants. Here, we provide evidence of synthesis-dependent repair of Cpf1-induced DSBs, which enables us precisely to replace the wild-type ALS gene with the intended mutant version that carries two discrete point mutations conferring herbicide resistance to rice plants. Our observation that the donor repair template (DRT) with only the left homologous arm is sufficient for precise targeted allele replacement offers a better understanding of the mechanism underlying HDR in plants, and greatly simplifies the design of DRTs for precision genome editing in crop improvement.

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