Abstract

We report the first successful, high efficiency use of LbCas12a in barley and describe the development and application of two novel Cas12a variants. In total we compared five coding sequence (CDS) variants including the two novel ones and two guide architectures over 5 different target genes using twenty different guides. We found large differences in editing efficiencies between the different CDS versions (0-87%) and guide architectures (0-70%) and show our two novel CDS versions massively outperform the others on test in this species. We show heritability of mutations generated. Our findings highlight the importance of optimising CRISPR systems for individual species and are likely to aid the use of LbCas12a in other monocot species.