Precise gene replacement in rice by RNA transcript-templated homologous recombination

文献类型： 外文期刊

第一作者： Li, Shaoya

作者： Li, Shaoya;Li, Jingying;Zhang, Jiahui;Du, Wenming;Xia, Lanqin;He, Yubing;Xu, Meilian;Zhao, Yunde;He, Yubing;Xu, Meilian;Zhao, Yunde;Zhao, Yunde

作者机构：

期刊名称：NATURE BIOTECHNOLOGY （ 影响因子：54.908； 五年影响因子：50.516 ）

ISSN： 1087-0156

年卷期： 2019 年 37 卷 4 期

页码：

收录情况： SCI

摘要： One of the main obstacles to gene replacement in plants is efficient delivery of a donor repair template (DRT) into the nucleus for homology-directed DNA repair (HDR) of double stranded DNA breaks. Production of RNA templates in vivo for transcript-templated HDR (TT-HDR) could overcome this problem, but primary transcripts are often processed and transported to the cytosol, rendering them unavailable for HDR. We show that coupling CRISPR-Cpf1 (CRISPR from Prevotella and Francisella 1) to a CRISPR RNA (crRNA) array flanked with ribozymes, along with a DRT flanked with either ribozymes or crRNA targets, produces primary transcripts that self-process to release the crRNAs and DRT inside the nucleus. We replaced the rice acetolactate synthase gene (ALS) with a mutated version using a DNA-free ribonucleoprotein complex that contains the recombinant Cpf1, crRNAs, and DRT transcripts. We also produced stable lines with two desired mutations in the ALS gene using TT-HDR.

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