Knockout of <i>Ku70</i> and <i>Ku80</i> elevates homology‐directed repair efficiency in <i>Plutella xylostella</i>

The suppression of Ku70 and Ku80 has been verified to increase homology-directed repair (HDR) efficiency in fruit fly, silkworm and mosquito, but not in other insects. In this study, PxKu70 and PxKu80 were identified from the Plutella xylostella genome. Domain analysis revealed that PxKu70 contained three conserved domains: Ku N, Ku and Ku C, while PxKu80 comprised the Ku N, Ku and Ku PK bind domains. Phylogenetic analysis and multiple-sequence alignment indicated strong conservation of both proteins among lepidopteran insects. RT-qPCR analysis showed that PxKu70 and PxKu80 were highly expressed in adult stages, particularly in reproductive tissues such as the ovary and testis, suggesting their role in maintaining genomic stability during gametogenesis. Two homozygous knockout lines (ΔPxKu70 and ΔPxKu80) were successfully generated through CRISPR/Cas9-mediated genome editing. These knockout lines remained viable and fertile without observable fitness effects. A donor construct carrying an EGFP cassette designed for insertion at the PxKmo locus was generated to assess HDR-mediated integration. The HDR insertion rate was significantly elevated in both knockout lines compared with the wild-type. These findings demonstrate that suppression of either PxKu70 or PxKu80 can enhance HDR in P. xylostella, offering an effective approach for precise genome editing in lepidopteran species.