Redirecting DNA repair for efficient CRISPR-Cas-based gene targeting in tomato

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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 various approaches to shift the repair pathway preference toward HR by using a dominant-negative KU80 mutant protein (KUDN) to disrupt the initiation of cNHEJ and enhance DSB end resection through nucleases. Our results show from 1.71- to 3.55-fold improvement of the GT efficiency at the callus stage and a more remarkable, up to 9.84-fold, increase in GT efficiency at two specific tomato loci, SlHKT1;2 and SlEPSPS1 , when we screened transformants obtained from the KUDN-mediated cNHEJ suppression approach. With practical levels of efficiency, this enhanced KUDN-based GT tool successfully facilitated GT at an additional locus, SlCAB13 . These findings provide a promising method for more efficient and precise plant breeding in the future.

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