High-Frequency Generation of Homozygous/Biallelic Mutants via CRISPR/Cas9 Driven by AtKu70/80 Promoters

CRISPR/Cas9 gene editing technology is widely used in plant gene editing to verify gene function or improve agronomic traits. In the CRISPR/Cas9 system, <i>Cas9</i> expression hinges on promoter choice, and CRISPR/Cas9 driven by a strong promoter or cell division-specific promoter has a higher editing efficiency. The CRISPR/Cas9 mechanism involves the CAS9 enzyme, which, directed by guide RNA, cleaves target double-stranded DNA and subsequently induces insertions or deletions (InDels) through the non-homologous end joining (NHEJ) repair pathway. The Ku protein plays a central role in the NHEJ repair process. It remains unclear whether driving <i>Cas9</i> with promoters of <i>AtKu70</i> and <i>AtKu80</i>, which are subunits of the Ku protein, will enhance gene editing efficiency. In this study, the promoters of <i>AtKu70</i> and <i>AtKu80</i> were cloned and used to drive <i>Cas9</i> in the CRISPR/Cas9 system. Four different genes, <i>GmRj7</i>, <i>GmNNL1</i>, <i>AtPDS3</i>, and <i>AtBRI1</i>, were designed for soybean hairy root transformation and <i>Arabidopsis</i> transformation. The results showed that the CRISPR/Cas9 systems driven by the promoters of <i>AtKu70</i> and <i>AtKu80</i> achieved higher homozygous/biallelic mutation efficiencies than the CRISPR/Cas9 system driven by the 35S promoter in hairy root transformation by <i>Rhizobium rhizogenes</i> and stable genetic transformation with <i>Rhizobium tumefaciens</i>.