Gene Editing of Caffeic-O-methyltransferase (COMT1) in the model grass Setaria viridis Improves Biomass Saccharification Without Compromising Plant Growth or Abiotic Stress Tolerance

Second-generation bioethanol production is limited by the recalcitrance of lignocellulosic biomass, largely driven by lignin content and composition. Genetic strategies targeting lignin biosynthesis offer a promising alternative to improve biomass digestibility without relying on energy-intensive pretreatments. Here, we used CRISPR/Cas9 editing to disrupt the caffeic-O-methyltransferase gene (COMT1) in Setaria viridis, a C monocot model related to major bioenergy crops. COMT1 was selected based on its strong expression in lignifying tissues and its central role in the biosynthesis of syringyl (S) units, guaiacyl (G) units, and the flavone tricin, a non-canonical lignin monomer in grasses. Edited plants showed no visible growth defects, maintaining normal development and salt-stress tolerance. Chemical analyses revealed a drastic reduction in S units and tricin, accompanied by moderate but significant decreases in G and p-hydroxyphenyl (H) monomers. Despite these compositional changes, total soluble lignin content was only slightly reduced, suggesting compensatory incorporation of non-canonical monomers. Metabolome analysis of edited plants suggested a redirection of carbon flux towards phenylpropanoid and flavonoid pathways. These modifications resulted in a substantial increase in saccharification efficiency, demonstrating that COMT1 disruption can enhance biomass digestibility without compromising plant viability. Our findings highlight COMT1 as a key target for engineering improved feedstocks for bioenergy production. HighlightsCRISPR knockout of COMT1 in Setaria viridis markedly enhances biomass digestibility by reducing lignin monomers, without compromising plant growth or salt-stress tolerance. Genome editing of COMT1 in Setaria viridis reveals unexpected lignin plasticity, reducing S, G, H and tricin units and improving saccharification efficiency without developmental penalties. Combined thiacidolysis/GC-MS and metabolomics suggests a compensatory mechanism in the lignin portion of the S. viridis cell wall.