Optimization of CRISPR/LbCas12a-mediated gene editing in Arabidopsis.

CRISPR/LbCas12a system (LbCpf1) has been widely used for genome modification including plant species. However, the efficiency of CRISPR/LbCas12a varied considerably in different plant species and tissues, and the editing efficiency needs to be further improved. In this study, we tried to improve the editing efficiency of CRISPR/LbCas12a in Arabidopsis by optimizing the crRNA expression strategies and Pol II promoters. Notably, the combination of tRNA-crRNA fusion strategy and RPS5A promoter in CRISPR/LbCas12a system has highest editing efficiency, while CRISPR/LbCas12a driven by EC1f-in(crR)p had the highest ratio of homozygous & bi-allelic mutants. In addition, all homozygous & bi-allelic mutants can be stably inherited to the next generation and have no phenotypic separation. In this study, the editing efficiency of the CRISPR/LbCas12a system was improved by selecting the optimal crRNA expression strategies and promoter of LbCas12a in Arabidopsis, which will prove useful for optimization of CRISPR/LbCas12a methods in other plants.