Enhancing CRISPR-Cas-based gene targeting in tomato using a dominant-negative ku80

The CRISPR-Cas-based gene targeting (GT) method has enabled precise modifications of genomic DNA ranging from single base to several kilobase scales through homologous recombination (HR). In plant somatic cells, canonical nonhomologous end-joining (cNHEJ) is the predominant mechanism for repairing double-stranded breaks (DSBs), thus limiting the HR-mediated GT. In this study, we implemented an approach to shift the repair pathway preference toward HR by using a dominant-negative ku80 mutant protein (KUDN) to disrupt the initiation of cNHEJ. The employment of KUDN conferred a 1.71- to 3.55-fold improvement in GT efficiency at the callus stage. When we screened transformants, there was a more remarkable increase in GT efficiency, ranging from 1.62- to 9.84-fold, at two specific tomato loci, SlHKT1;2 and SlEPSPS1. With practical levels of efficiency, this enhanced KUDN-based GT tool successfully facilitated a 9-bp addition at an additional locus, SlCAB13. These findings provide another promising method for more efficient and precise plant breeding.