Improving the efficiency of CRISPR/Cas9-induced multiplex editing in Brassica napus L.

Abstract

CRISPR/Cas9 is currently the most powerful genome editing tool for crop improvement and gene function exploration. However, the mutation efficiency of CRISPR/Cas9 in Brassica napus, a globally important allotetraploid oil crop, so far achieved remains significant potential for further improvement compared to that observed in model species. In this study, we systematically evaluated three key factors influencing editing efficiency: sgRNA promoters, sgRNA structural modifications, and Cas9-driven promoters. First, we compared all sgRNA promoters currently used in B. napus side by side. The average editing efficiency of AtU6–26 was the highest (44.74%), followed by AtU3b (36.18%), AtU6–29 (28.49%), AtU3d (17.73%) and AtU6–1 (0.53%). Second, we demonstrated that enhanced sgRNA (esgRNA) enhanced CRISPR/Cas9 editing efficiency by 52.1% compared to native sgRNA. The combination of AtU6–26 with esgRNA achieved the highest editing efficiency (average 61.4%). Third, we identified three endogenous promoters (pCAB1, pLTP2, and pTCTP) with high expression levels in the hypocotyls and callus, to replace the 35S promoter of Cas9. The CAB1 promoter was the most efficient and significantly enhanced the mutation efficiency by 31.46% over the commonly used 35S promoter. Thus, these optimizations provide valuable strategies for improving CRISPR/Cas9 efficiency in Brassica crops.