Knockout of <i>Ligase4</i> gene enhances homology‐directed repair efficiency in <i>Plutella xylostella</i>

Knock-in efficiency via CRISPR/Cas9-based homology-directed repair (HDR) is low in lepidopteran insects due to predominance of knockout through non-homologous end joining (NHEJ) pathway. DNA ligase 4 (LIG4), a core protein of NHEJ complex and double-strand break (DSB) binding repair, is a promising target for enhancing HDR efficiency in diverse species. However, the role of Lig4 in modulating HDR efficiency in Plutella xylostella, a highly destructive pest of vegetable crops, remains unclear. The PxLig4 gene was identified from the P. xylostella genome with 4 conserved domains: ATP-dependent DNA ligase core-adenylation middle domain, ATP-dependent DNA ligase core‑adenylation N-terminal domain, ATP-dependent DNA ligase core‑adenylation C‑terminal domain, and BRCA1 C terminus domain. Multiple sequence alignment and phylogenetic analysis confirmed strong conservation of PxLig4 among Lepidoptera. Real-time quantitative polymerase chain reaction (RT-qPCR) analysis showed that PxLig4 was highly expressed in eggs, female adults, and male adults, particularly in the ovary, followed by the testis, fat body, and midgut tissues. A homozygous PxLig4 knockout line (ΔPxLig4) with a 2-bp deletion was successfully generated by using the CRISPR/Cas9 technique. ΔPxLig4 exhibited significant developmental and reproductive defects. A donor vector to knock in the enhanced green fluorescent protein (EGFP) gene at the eye color gene PxKmo was developed to assess knock-in efficiency. The knock-in efficiency in ΔPxLig4 was 2.86%, significantly higher than the 0.26% observed in the wild-type P. xylostella. These results implicate Lig4 as a supporting factor for NHEJ and demonstrate that suppression of PxLig4 enhances HDR efficiency in P. xylostella, providing a potential strategy to improve the efficiency of gene knock-in in insect pests.