Alfalfa ( Medicago sativa ) is an outcrossing tetraploid legume species widely cultivated in the world. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9) system has been successfully used for genome editing in many plant species. However, the use of CRISPR/Cas9 for gene knockout in alfalfa is still very challenging. Our initial single gRNA-CRISPR/Cas9 system had very low mutagenesis efficiency in alfalfa with no mutant phenotype. In order to develop an optimized genome editing system in alfalfa, we constructed multiplex gRNA-CRISPR/Cas9 vectors by a polycistronic tRNA-gRNA approach targeting the Medicago sativa stay-green ( MsSGR ) gene. The replacement of CaMV35S promoter by the Arabidopsis ubiquitin promoter (AtUBQ10) to drive Cas9 expression in the multiplex gRNA system led to a significant improvement in genome editing efficiency, whereas modification of the gRNA scaffold resulted in lower editing efficiency. The most effective multiplex system exhibited 75% genotypic mutagenesis efficiency, which is 30-fold more efficient than the single gRNA vector. Importantly, phenotypic change was easily observed in the mutants, and the phenotypic mutation efficiency reached 68%. This highly efficient multiplex gRNA-CRISPR/Cas9 genome editing system allowed the generation of homozygous mutants with a complete knockout of the four allelic copies in the T0 generation. This optimized system offers an effective way of testing gene functions and overcomes a major barrier in the utilization of genome editing for alfalfa improvement.

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Genome editing is an advanced genetic modification tool that facilitates the removal, insertion, or substitution of nucleotides in the genome of an organism for mutation induction. CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR‐associated protein 9) is the most widely used genome editing tool that can precisely modify the specific sequence of a genome. Significant progress has been made in the use of CRISPR/Cas9 for the genetic improvement of food crops. However, in forage crops, the use of CRISPR/Cas9 is still in the infant stage with limited understanding and application. This article reviews current advances in the establishment and application of the CRISPR/Cas9 system in alfalfa, summarizes the application of CRISPR/Cas9 in several other forage legume and grass species, and discusses the prospects of CRISPR/Cas9 in the genetic improvement of forage crops.

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