Genome Editing Using CRISPR/Cas Systems in Legumes

Legumes are protein-rich grains of Fabaceae family that enrich soil through symbiotic nitrogen fixation (SNF). Genome editing employing clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein (Cas) system allows a precise modification of genome using either endogenous non-homologous end-joining (NHEJ), homology-dependent repair (HDR) or microhomology-mediated end-joining (MMEJ) pathways. This facile RNA-guided tool is extensively used to characterize gene functions and traits of agricultural importance. Here, we discuss the reports of genome editing using CRISPR/Cas system in five important legume species, viz. birdsfoot trefoil (Lotus japonicus), barrel medic (Medicago truncatula), alfalfa (Medicago sativa), soya bean (Glycine max) and cowpea (Vigna unguiculata), and some common web-based tools for designing reagents for editing. In birdsfoot trefoil, an efficient editing of representative symbiotic nitrogen-fixing genes via. Agrobacterium-mediated stable or hairy root transformation has been reported. Notably, this tool was used to decipher the synergistic roles of leghaemoglobin genes in SNF. In barrel medic, the amenability of CRISPR/Cas system was demonstrated for genes/loci using phytoene desaturase (PDS); both homoallelic and biallelic mutants were reported with albino phenotype. Subsequently, editing genes for small RNA processing were also demonstrated to be heritable in nature. In polyploid alfalfa, successful CRISPR/Cas-mediated genome editing was reported for traits like hyperaccumulation of phosphate and regulators of plant developmental pathway. In cowpea, the disruption of symbiotic nitrogen fixation gene, symbiosis receptor-like kinase (SYMRK), in hairy root-transformed plant exhibits a complete blockage of nodule formation. The extensively worked-out legume is soya bean, where endogenous and exogenous genes/loci were amenable to genome editing via. hairy root composite plants, protoplast and whole-plant transformation employing genetic engineering and RNP delivery routes. Besides several endogenous loci, genes for traits like plant architecture, SNF, early flowering, oil composition etc. were reported to be edited. Possibilities of HDR, multiplexing and base editing were also demonstrated. Gene-edited high-oleic soya bean oil (Calyno™) developed by Calyxt, Inc is the first gene-edited food released for consumers in the USA. Current research efforts should be focused to translate the findings in model legumes to legumes of local importance, so that the benefit of the technology can be harnessed.