Postdoctoral Associate University of Minnesota, United States
Abstract: In recent years, CRISPR-mediated gene editing has been achieved by harnessing the endogenous DNA repair pathways i.e. non-homologous end joining (NHEJ) and homology-directed repair (HDR), in various plant species. While targeted gene knockout can be obtained at high frequencies, the efficiency and precision of targeted insertion remain low. In this study, we sought to improve CRISPR-mediated targeted insertion through the classic Non-homologous end joining (NHEJ) pathway. Our results demonstrated that CRISPR-Cas9 frequently induces staggered cleavages with 5´ 1-nucleotide overhangs. This overhang structure can be harnessed for directional target insertion with improved precision by using double-stranded DNA donors with 5´ 1-nt complementary overhangs. We successfully applied this improved method to achieve efficient endogenous gene tagging and cis-regulatory element engineering in plants.
