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

  作 者:Shaoya Li, Jingying Li, Yubing He, Meilian Xu, Jiahui Zhang, Wenming Du, Yunde Zhao & Lanqin Xia 


  刊物名称:Nature Biotechnology

  出版年份:18 March 2019


  文章摘要: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.