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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We thank J.-K. Zhu for the LbCpf1 plasmid. We thank C.-Y. Wu, whose lab provided the rice transformation service. This work is partly funded by the Ministry of Agriculture and Rural Affairs of China (grant no. 2018ZX0801003B to L.X. and Y.H.), the Ministry of Science and Technology of China (grant no. 2016YFD0100500 to LX), the Ministry of Agriculture of China (grant no. 2016ZX08010003 to L.X.), and the Central Non-Profit Fundamental Research Funding supported by the Institute of Crop Sciences, Chinese Academy of Agricultural Sciences (S2018QY05 to L.X.).
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Li, S., Li, J., He, Y. et al. Precise gene replacement in rice by RNA transcript-templated homologous recombination. Nat Biotechnol 37, 445–450 (2019). https://doi.org/10.1038/s41587-019-0065-7
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