Mutating alfalfa NAP1 and NAP2 transcription factors by multiplex CRISPR /Cas9 genome editing leads to delayed senescence with improved forage biomass, nutritional quality, and salinity tolerance

Alfalfa (Medicago sativa L.) is one of the most valuable forage crops due to its high biomass yield potential and nutritional values. However, forage biomass and quality are affected by different environmental and genetic factors, including developmental and stress-induced senescence, management, and harvesting stage. The CRISPR/Cas9 technology targeting stress-responsive transcription factors (TFs) offers great potential to enhance plant resilience against abiotic stresses and to develop high-quality and high-yielding forage crops. Here, we employed a multiplex CRISPR/Cas9-mediated gene-editing approach to simultaneously target two alfalfa homologous NAC TFs family genes (MsNAP1 and MsNAP2) in one construct to enhance vegetative growth and attain improved biomass by delaying leaf senescence. Two guide RNAs (gRNAs) were designed and clustered in a polycistronic tRNA-gRNA system and introduced into alfalfa by Agrobacterium-mediated transformation. Seventy-five putative loss-of-function alfalfa plants targeting both MsNAP1 and MsNAP2 were generated. Phenotypic and genotypic analyses revealed that Msnap1 and Msnap2 double mutants with delayed leaf senescence produced greater forage biomass, with increases of up to 83% in fresh weight and 42% in dry weight compared to the empty vector control. Furthermore, acid detergent fiber and neutral detergent fiber content were reduced with a concomitant increase in crude protein content, total digestible nutrients, and relative feed value in both leaves and stems, indicating significant improvement in forage quality. Our findings suggest that CRISPR/Cas9-edited Msnap1 and msnap2 double mutant lines could be used as valuable genetic resources to generate elite transgene-free alfalfa cultivars with delayed leaf senescence and improved forage biomass and quality.