Development of a single transcript CRISPR/Cas9 toolkit for efficient genome editing in autotetraploid alfalfa

Alfalfa (Medicago sativa. L.) is a globally significant autotetraploid legume forage crop. However, despite its importance, establishing efficient gene editing systems for cultivated alfalfa remains a formidable challenge. In this study, we pioneered the development of a highly effective ultrasonic-assisted leaf disc transformation system for Gongnong 1 alfalfa, a variety widely cultivated in Northeast China. Subsequently, we created a single transcript CRISPR/Cas9 (CRISPR_2.0) toolkit, incorporating multiplex gRNAs, designed for gene editing in Gongnong 1. Both Cas9 and gRNA scaffolds were under the control of the Arabidopsis ubiquitin-10 promoter, a widely employed polymerase II constitutive promoter known for strong transgene expression in dicots. To assess the toolkit's efficiency, we targeted PALM1, a gene associated with a recognizable multifoliate phenotype. Utilizing the CRISPR_2.0 toolkit, we directed PALM1 editing at two sites in the wild-type Gongnong 1. Results indicated a 35.1% occurrence of editing events all in target 2 alleles, while no mutations were detected at target 1 in the transgenic-positive lines. To explore more efficient sgRNAs, we developed a rapid, reliable screening system based on Agrobacterium rhizogenes-mediated hairy root transformation, incorporating the visible reporter MtLAP1. This screening system demonstrated that most purple visible hairy roots underwent gene editing. Notably, sgRNA3, with an 83.0% editing efficiency, was selected using the visible hairy root system. As anticipated, tetra-allelic homozygous palm1 mutations exhibited a clear multifoliate phenotype. These palm1 lines demonstrated an average crude protein yield increase of 21.5% compared to trifoliolate alfalfa. Our findings highlight the modified CRISPR_2.0 system as a highly efficient and robust gene editing tool for autotetraploid alfalfa.