Data-driven optimization yielded a highly-efficient CRISPR/Cas9 system for gene editing in Arabidopsis

The CRISPR/Cas9 technology is widely used for plant gene editing. In Arabidopsis, the early CRISPR/Cas9 systems using constitutive promoters to drive Cas9 expression usually have low heritable mutation efficiencies in the T1 generation. Germ cell-specific or cell division-specific promoters, like EC1.2en-EC1.1p fusion, YAO and CDC45, have been used as alternatives to direct Cas9 expression, but the efficiency of getting nonchimeric mutations is still not high. To further improve gene editing efficiency, we used gene co-expression network analysis to identify NUC1 as a candidate promoter for driving Cas9 expression. NUC1 has expression patterns similar to YAO, but expresses at a much higher level. We constructed a CRISPR/Cas9 system pHY07 that uses the NUC1 promoter to drive Cas9 and carries the mCherry protein as a fluorescent marker for selecting transgenic seeds. Using this system to edit the GL2 gene, we obtained apparently nonchimeric mutations in 55% of the T1 transgenic plants. Among the Cas9-free T2 plants 99% harbored mutations that are stably inherited from the previous generation. Therefore, our system exhibited extremely high editing efficiency, and through fluorescent screening of transgenic seeds, it become easy to obtain Cas9-free and stable genetic mutants in the T2 generation.