Journal
NATURE BIOTECHNOLOGY
Volume 37, Issue 4, Pages 445-+Publisher
NATURE PORTFOLIO
DOI: 10.1038/s41587-019-0065-7
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Funding
- Ministry of Agriculture and Rural Affairs of China [2018ZX0801003B]
- Ministry of Science and Technology of China [2016YFD0100500]
- Ministry of Agriculture of China [2016ZX08010003]
- Institute of Crop Sciences, Chinese Academy of Agricultural Sciences [S2018QY05]
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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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