Knockout of Ligase4 gene enhances homology-directed repair efficiency in Plutella xylostella.

Authors

Faisal Munir, Uroosa Zaheer, Sheng Lin, Anam Noreen Abbas, Mubashir Tariq, Jianying Liao, Qingxuan Qiao, Yanpeng Chang, Yussuf Mohamed Salum, Ibrahim Adams, Hui Wei, Muhammad Asad, Guang Yang

Published in

Insect science. Oct 28, 2025. Epub Oct 28, 2025.

Abstract

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.

PMID:
41147058
Bibliographic data and abstract were imported from PubMed on 28 Oct 2025.

Read full publication at:
Please sign in to see all details.

Stats

1-terrible, 9-excellent. How would you rate this publication? Sign in in to submit your rating.

Post a comment

You need to be signed in to post comments. You can sign in here.

Comments

There are no comments yet.