Improved SpCas9 and LbCas12a genome editing systems in Brassica oleracea and Brassica napus

We report highly efficient genome editing in Brassica species. We compare the efficiency of targeted mutagenesis using four Streptococcus pyogenes Cas9 ( Sp Cas9) systems in Brassica oleracea and Brassica napus over 3 target genes and five guides to identify two which show a striking improvement to our first published system (Lawrenson et al., 2015). Targeted mutagenesis occurred in up to 100% of T0 plants with the improved systems, compared to 20% in the original system. This is the only reported comparison of Sp Cas9 systems we are aware of in Brassica species. Secondly, we report the first successful use of Lachnospiraceae bacterium Cas12a ( Lb Cas12a) in Brassica oleracea . We test three Lb Cas12a coding sequences and two guide architectures against one target gene using four guides. From this we identify the best performing combination of our novel, multi-intron coding sequence and a ribozyme flanked guide expression cassette. In this case 68% of T0 plants contained targeted mutations. Heritability of Lb Cas12a mutations is shown. We show that our two useful and novel Lb Cas12a coding sequences have utility in Brassica species.