CRISPR-Cas9-induced targeted mutagenesis of feruloyl CoA 6-hydroxylase gene reduces postharvest physiological deterioration in cassava roots

Cassava is a starch-rich, multipurpose root crop grown by millions of smallholder farmers in tropical and subtropical countries. Cassava cultivation is seriously constrained by post-harvest physiological deterioration (PPD) of roots that occurs within 24-72 h after harvest, making them unpalatable and unmarketable. The inevitable wounding of roots during harvest triggers an oxidative burst throughout the root together with the accumulation of secondary metabolites with scopoletin being the most abundant. Blue-black discoloration and vascular streaking observed during PPD occur due to over-accumulation of scopoletin whose biosynthesis involves key enzymes including feruloyl CoA 6 '-hydroxylase.We applied the CRISPR-Cas9 system for targeted mutagenesis of feruloyl CoA 6 '-hydroxylase genes (MeF6'H1, MeF6'H12 MeF6'H2 and MeF6'H3) in cassava to delay PPD. To generate cassava plants with enhanced root shelf-life, CRISPR-Cas9 cassettes with Cas9 gene and sgRNA targeting three genes; MeF6'H1, MeF6'H2, and MeF6'H3 were introduced into cassava via Agrobacterium-mediated transformation. Integration of transgenes in putative knockout plants were confirmed by PCR while targeted sanger sequencing revealed CRISPR-Cas9 induced mutations with insertion, deletions and substitution being reported upstream and downstream of the PAM sequence of target genes. Targeted for edition of dual genes exhibited significant reduction of scopoletin below HPLC detectable levels and had improved shelf-life compared to one gene knockout events and wild-type plants. Evaluation of phenotypic variations between mutant and wild type cassava plants revealed mutation-event associated traits including stem and petiole depigmentation, auxiliary budding, claw-like leaf appearance, loss of apical dominance and leaf chlorosis. This study demonstrated an effective and feasible approach to extend cassava root shelf-life to stabilize yield production, prevent postharvest wastage, and improve farmers' income.