Engineering herbicide-resistant oilseed rape by CRISPR/Cas9-mediated cytosine base-editing.

CRISPR/Cas9-based base editor (BE) technology that enables precise base-editing has been developed and applied in several plant species, including Arabidopsis, rice, wheat, maize, tomato and cotton. However, it is not clear whether base editing will work in allotetraploid oilseed rape (Brassica napus L.). In this study, we successfully established a base-editing system that can efficiently introduce C to T conversion in sgRNA targets in oilseed rape. BnALS (acetolactate synthase) gene, the target site of several important herbicides was closed edited at position P197 by the BE system, conferred tribenuron-methyl resistant to oilseed rape. The base editing efficiency for BnALS1 was about 1.8% (4/217). Homozygous mutants without exogenous T-DNA could be obtained in T1 generation. No off-target effect was detected in the mutated plants. In addition, in order to easily discriminate between the WT and the P197S mutant alleles, allele-specific PCR markers were developed. The BE would be a very useful tool for gene function study and precision molecular breeding in oilseed rape. The P197S substitution in BnALS1 generates a novel herbicide-resistant mutant in oilseed rape, and the transgene-free homozygous mutant with its allele-specific markers could be used for breeding herbicide resistance and thus might facilitate weed management in oilseed rape production.