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Nat Biotechnol. 2019 Apr;37(4):445-450. doi: 10.1038/s41587-019-0065-7. Epub 2019 Mar 18.

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

Author information

1
Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing, China.
2
National Key Laboratory of Crop Genetic Improvement and National Center of Plant Gene Research (Wuhan), Huazhong Agricultural University, Wuhan, China.
3
National Key Laboratory of Crop Genetic Improvement and National Center of Plant Gene Research (Wuhan), Huazhong Agricultural University, Wuhan, China. yundezhao@ucsd.edu.
4
Section of Cell and Developmental Biology, University of California, San Diego, La Jolla, CA, USA. yundezhao@ucsd.edu.
5
Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing, China. xialanqin@caas.cn.

Abstract

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.

PMID:
30886437
DOI:
10.1038/s41587-019-0065-7

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