Improving the genome editing efficiency of CRISPR/Cas9 in Arabidopsis and Medicago truncatula

<jats:title>Abstract</jats:title><jats:sec> <jats:title>Main conclusion</jats:title> <jats:p><jats:bold>An improved CRISPR/Cas9 system with the Arabidopsis UBQ10 promoter-driven Cas9 exhibits consistently high mutation efficiency in Arabidopsis and M. truncatula.</jats:bold></jats:p> </jats:sec><jats:sec> <jats:title>Abstract</jats:title> <jats:p>CRISPR/Cas9 is a powerful genome editing technology that has been applied in several crop species for trait improvement due to its simplicity, versatility, and specificity. However, the mutation efficiency of CRISPR/Cas9 in Arabidopsis and <jats:italic>M. truncatula</jats:italic> (Mt) is still challenging and inconsistent. To analyze the functionality of the CRISPR/Cas9 system in two model dicot species, four different promoter-driven Cas9 systems to target <jats:italic>phytoene desaturase</jats:italic> (<jats:italic>PDS</jats:italic>) genes were designed. <jats:italic>Agrobacterium</jats:italic>-mediated transformation was used for the delivery of constructed vectors to host plants. Phenotypic and genotypic analyses revealed that the Arabidopsis UBQ10 promoter-driven Cas9 significantly improves the mutation efficiency to 95% in Arabidopsis and 70% in <jats:italic>M. truncatula.</jats:italic> Moreover, the UBQ10-Cas9 system yielded 11% homozygous mutants in the T1 generation in Arabidopsis. Sequencing analyses of mutation events indicated that single-nucleotide insertions are the most frequent events in Arabidopsis, whereas multi-nucleotide deletions are dominant in bi-allelic and mono-allelic homozygous mutants in <jats:italic>M. truncatula.</jats:italic> Taken together, the UBQ10 promoter facilitates the best improvement in the CRISPR/Cas9 efficiency in <jats:italic>PDS</jats:italic> gene editing, followed by the EC1.2 promoter. Consistently, the improved UBQ10-Cas9 vector highly enhanced the mutation efficiency by four-fold over the commonly used 35S promoter in both dicot species.</jats:p> </jats:sec>