Abstract
CRISPR/Cas is a simple, robust, versatile tool for plant biology studies and precision plant breeding. However, establishing a high-efficiency gene editing system for multiplex editing of the autotetraploid crop alfalfa (Medicago sativa L.), the most important forage legume worldwide, remains a formidable challenge. Here, we systematically identified endogenous U6 promoters in alfalfa through transient expression via Agrobacterium-mediated infiltration of alfalfa leaves. We further demonstrated the efficacy of the three most active promoters for genome editing using an optimized alfalfa hairy root system. Subsequently, we established an improved CRISPR/Cas9 multiplex system containing three or four tandemly arrayed MsU6-promoter-driven polycistronic tRNA-sgRNA (PTG) expression cassettes, each consisting of three tRNA-sgRNA units, to simultaneously edit three or four alfalfa genes, coupled with the visual reporter RH1 or RUBY. This toolkit showed efficient multiplex editing in the hairy root system with visual selection. We successfully obtained regenerated, red-colored shoots resulting from the stable transformation of alfalfa. These results highlight the potential application of the visual reporter system for the stable transformation of alfalfa. Our improved CRISPR/Cas9 multiplex system enables convenient, high-efficiency multiplex genome editing in alfalfa, providing a versatile toolset to facilitate functional studies of multiple genes and gene families for basic research and the genetic improvement of alfalfa. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s42994-025-00200-z.