Highly efficient CRISPR-mediated homologous recombination via NHEJ deficiency rather than HDR factors overexpression in Populus
Efficient homology-directed DNA repair (HDR) is a vital difficulty confronting researchers to replace the target genome's desired fragment. In plants, scientists have performed meticulous investigations on herbal, crops, and citrus trees using HDR effector proteins, CtIP and MRE11, to obtain double-stranded breaks (DSBs) more precisely. Although HDR efficiency in plants previously has been reported, no record has been declared about HDR efficiency in woody perennial Populus. Here, we hypothesized that inhibition of non-homologous recombination cofactors XRCC4 and enhancing the HDR pathway activities enable us to improve the HDR efficiency. In this study, the BleoR gene was used to integrate into the interested site and generated transformants against Zeocin antibiotics. We designed plasmids, including different fusions of HDR proteins and, together with the XRCC4 target. Furthermore, we showed that TaqMan real-time PCR could be a powerful tool to verify HDR efficiency. We confirmed that both applying HDR proteins and XRCC4 deficiency simultaneously could improve HDR efficiency, which showed about fiftyfold more, and decremented polymorphisms about sixfold less than no affecting HDR and NHEJ pathways. We developed a new recombinant poplar genome to generate stable lines resistant to the Zeocin antibiotic.
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