Enhancement of the CRISPR/Cas9-Based Genome Editing System in Lettuce ( L.) Using the Endogenous Promoter.

The CRISPR/Cas9 system has been widely applied as a precise gene-editing tool for studying gene functions as well as improving agricultural traits in various crop plants. Here, we optimized a gene-editing system in lettuce ( L.) using the endogenous promoter and proved that the gene is a versatile target gene. We isolated the promoter from 10 snRNA genes identified from the lettuce genome database for comparison with the promoter that has been used to drive sgRNAs in lettuce. Two CRISPR/Cas9 vectors were constructed using the and promoters to drive sgRNA361 to target the gene. The chloroplast avoidance response was defective in lettuces with biallelic mutations in the targeted gene, as in the mutant. The gene mutations were stably heritable from the R0 to R2 generations, and the high gene-editing efficiency enabled the selection of transgene-free lines in the R1 generation and the establishment of independent mutants in the R2 generation. Our results suggest that the promoter is more effective than the promoter in driving sgRNA for the CRISPR/Cas9 system in lettuce and that is a useful target gene to verify gene editing efficiency without any detrimental effects on plant growth, which is often a consideration in conventional target genes.