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Herbicide resistance is one of the main crop traits that improve farming methods and crop productivity. CRISPR-Cas9 can be applied to the development of herbicide-resistant crops based on a target site resistance mechanism, by editing genes encoding herbicide binding proteins. The sgRNAs capable of editing the target genes of herbicides,  pds  (phytoene desaturase),  ALS  (acetolactate synthase), and  EPSPS  (5-Enolpyruvylshikimate-3-phosphate synthase), were designed to use with the CRISPR-Cas9 system in tomato ( Solanum lycopersicum  cv. Micro-Tom). The efficiency of the sgRNAs was tested using  Agrobacterium  mediated transient expression in the tomato cotyledons. One sgRNA designed for editing the target site of  PDS  had no significant editing efficiency. However, three different sgRNAs designed for editing the target site of  ALS  had significant efficiency, and one of them, ALS2-P sgRNA, showed over 0.8% average efficiency in the cotyledon genome. The maximum efficiency of ALS2-P sgRNA was around 1.3%. An sgRNA for editing the target site of  EPSPS  had around 0.4% editing efficiency on average. The sgRNA efficiency testing provided confidence that editing of the target sites could be achieved in the transformation process. We confirmed that 19 independent transgenic tomatoes were successfully edited by ALS2_P or ALS1_W sgRNAs and two of them had three base deletion mutations, which are expected to have altered herbicide resistance. In this study, we demonstrated the usefulness of performing an sgRNA efficiency test before crop transformation, and confirmed that the CRISPR-Cas9 system is a valuable tool for breeding herbicide-resistant crops.

Selection of the high efficient sgRNA for CRISPR-Cas9 to edit herbicide related genes, PDS, ALS, and EPSPS in tomato