Optimising glyphosate tolerance in rapeseed (Brassica napus L.) by CRISPR/Cas9-based geminiviral donor DNA replicon system with Csy4-based single-guide RNA processing.

Rapeseed (Brassica napus L.) is an important oil crop worldwide, and effective weed control can protect its yields and quality. Farmers can benefit from tolerant cultivars when using herbicides, such as glyphosate. Amino acid substitutions in enolpyruvylshikimate-3-phosphate synthase (EPSPS) render the plant less sensitive to glyphosate. Therefore, we aimed to optimise the glyphosate tolerance trait in rapeseed via endogenous EPSPS modification. To achieve effective gene replacement in B. napus L., we employed a CRISPR/Cas9 system expressing single-guide RNAs (sgRNAs) cleaved by the CRISPR-associated RNA endoribonuclease Csy4 from Pseudomonas aeruginosa for targeted induction of double-strand breaks. Both the donor template and a geminiviral replicon harbouring an sgRNA expression cassette were introduced into plant cells. Using sgRNAs targeting adjacent donor DNA template containing synonymous mutations in sgRNA sites, we achieved precise gene replacements in the endogenous B. napus EPSPS gene, BnaC04EPSPS, resulting in TIPS and LFGAAGMCRL amino acid substitutions at frequencies up to 20%. Rapeseed seedlings harbouring EPSPSmTIPS or EPSPSmLFGAAGMCRL were glyphosate tolerant. Furthermore, modifications in BnaC04EPSPS were precisely transmitted to the next generation. Our genome editing strategy enables highly efficient gene targeting and the induction of glyphosate tolerance in oilseed rape.