Targeted knockout of CYP79A1 reduces cyanogenic potential in grain sorghum

Sorghum bicolor is a climate-resilient C4 crop used for food, forage, and bioenergy, but broader adoption is constrained by accumulation of the cyanogenic glucoside dhurrin, which can release toxic hydrogen cyanide (HCN) upon tissue damage. Dhurrin levels are especially high in juvenile tissues, creating risk for grazing animals and limiting use in mixed crop-livestock systems. Here, we establish a CRISPR-Cas9 genome-editing strategy targeting CYP79A1, which catalyzes the first committed step in dhurrin biosynthesis, in the elite grain sorghum inbred RTx430, yielding transgene-free lines with stable, heritable reduction in cyanogenic potential across early vegetative development. Homozygous cyp79a1 knockouts were negligibly cyanogenic, whereas heterozygous plants exhibited approximately half the cyanogenic potential of unedited nulls. Consistent with established livestock grazing guidelines, only homozygous knockouts fell below thresholds considered hazardous for incidental grazing. This work establishes CYP79A1 as a practical and heritable genome-editing target for reducing sorghum cyanogenesis and provides a clear path for deployment of low-cyanogenic alleles in elite breeding backgrounds.