Improving gene editing of CRISPR/Cas9 using the callus-specific promoter pYCE1 in cassava (Manihot esculenta Crantz)

Previous studies have demonstrated that an appropriate promoter can drive <i>Cas9</i> transcription in the CRISPR/Cas9 system, which improves the efficiency of gene editing. Here, we identified and characterized callus-specific promoters to enhance gene editing efficiency in cassava. From the transcriptome data of 11 cassava tissues, the gene named <i>YCE1</i> was identified to exhibit callus-specific expression. Its promoter (<i>pYCE1</i>) could efficiently and specifically drive <i>EGFP</i> transcription in callus tissues. Given that friable embryogenic callus (FECs) is the recipient for genetic transformation in cassava, we replaced the original <i>35S</i> promoter with <i>pYCE1</i> to drive <i>Cas9</i> transcription for improving the CRISPR/Cas9 gene editing system. In single-gene editing, the mutation rate was significantly increased, which reached an overall mutation rate of 95.24% and a homozygous mutation rate of 52.38%, compared with 62.07% and 37.93% with the <i>35S</i> promoter, respectively. Furthermore, achieving a dual-gene homozygous mutation rate of 64.71% in dual-gene editing demonstrated the high efficiency of <i>pYCE1</i> in the gene editing application for cassava. These results underscore the potential of <i>pYCE1</i> to enhance gene editing efficiency in the CRISPR/Cas9 system of cassava. This approach paves the way for advanced gene function research and genetic breeding in cassava.