Currently, a major challenge exists in CRISPR-mediated genome editing research in sheep: the low efficiency of exogenous large DNA fragment targeted integration without drug selection or fluorescence enrichment. This restriction significantly impedes the use of precise genome editing in sheep for agricultural, biological, and biomedical purposes. In this study, we employed the strategy of increasing the local concentration of the homologous repair template at the site of the DNA double-strand break (DSB). We achieved this by fusing the DNA binding domain (BD) of the Gal4 protein (Gal4-BD) to the N-terminal end of the SpCas9 protein using a 32-amino acid (aa) flexible linker. Additionally, we incorporated a 17 bp UAS at the 3′ end of the donor template, which can be specifically recognized and bound by Gal-BD. As a result, we observed a significant improvement in the knock-in efficiency of the exogenous large DNA fragment (2997 bp) in sheep fetal fibroblasts (SFFs), increasing it from 5.30% (8/151) to 16.67% (32/192), providing a comparatively efficient and user-friendly method to promote CRISPR-mediated gene knock-in in sheep.

Introduction: Defects in RAG1/RAG2 -mediated V(D)J recombination cause severe combined immunodeficiency (SCID), a disorder characterized by the absence of mature T and B lymphocytes resulting from impaired antigen receptor gene rearrangement. Although evolutionary analyses demonstrate strong conservation of the catalytic core domains of RAG1/RAG2 across jawed vertebrates, structural divergence among species highlights the need for physiologically relevant large-animal models to evaluate human disease mechanisms. Pigs represent an advantageous model for human pathologies because of their close immunological and physiological similarities to humans. Methods: In this study, we performed evolutionary profiling, conservation analysis, tertiary structural modeling, and protein docking to characterize pig RAG1 and RAG2 and predict their functional roles in V(D)J recombination. These predictions were experimentally validated through the generation of RAG1 and RAG2 knockout pigs using CRISPR/Cas9-mediated genome editing. Results: Our findings define key structural features of pig RAG1 and RAG2 that are essential for V(D)J recombination and establish an integrated framework that combines comprehensive computational analyses with in vivo experimental validation. Discussion: This approach supports the development of pigs as physiologically relevant models of RAG deficiency and provides a foundation for future studies in pig immunology, genome editing, and translational research. Furthermore, these immunodeficient pigs permit long-term engraftment of human tissue xenografts and stem cell-derived teratomas, thereby enabling investigations of human immune reconstitution, cancer biology, and regenerative medicine. Collectively, this model offers a robust platform for immunotherapy development and for advancing studies in evolutionary and comparative immunology research.