CRISPR/Cas9-mediated lgp2 knockout and cross-species rescue reveal the immunoregulatory role of LGP2 in zebrafish.

Abstract

LGP2 is a key regulator within the RIG-I-like receptor (RLR) pathway with dual roles in antiviral immunity, but its function under basal conditions and its evolutionary variation among fish species require further investigation. In this study, we constructed a zebrafish lgp2 knockout model using CRISPR/Cas9. Knockout lgp2 led to upregulation of key RLR signaling components (mda5, mavs), type I interferons (ifnphi1, ifnphi3, ifnphi4), and the interferon-stimulated gene mxa. Transcriptomic profiling of 3 dpf embryos identified 3186 differentially expressed genes (DEGs), including significantly elevated expression of five claudin-family genes. Protein-protein interaction (PPI) analysis of DEGs indicated that irf7 and claudin b exhibit an interaction at the protein level. Gene ontology analysis showed that LGP2 was enriched in terms such as helicase activity and ATPase activity, and was also significantly enriched in extracellular structure terms. Comparative sequence analysis revealed conserved DEXDc, HELICc and CTD domains in LGP2 from zebrafish, grass carp and barbel chub, with notable species-specific sequence variations. Functional rescue experiments demonstrated that all three LGP2 orthologs could restore lgp2 expression and modulate downstream interferon responses in lgp2embryos; however, induction of the transcription factor irf7 was specifically mediated only by zebrafish-derived LGP2. These results provide insights into the functional diversity of LGP2 in teleost immunity and inform strategies for selective breeding aimed at disease resistance.