Multi-species Comparative Analysis Identifies Conserved Enhancers for Improving Casein Expression via Gene Editing

As the global population continues to grow, the demand for protein is correspondingly increasing. Milk serves as a significant source of high-quality protein, with casein being its primary nutrient. Consequently, the cultivation of dairy animals with elevated casein expression has become a critical objective in the field of livestock breeding. Compared with traditional breeding methods, gene editing offers a more efficient approach to enhancing casein expression in dairy animals. However, progress in this area is constrained by the lack of key editing targets. Enhancers, which are core cis-acting elements enriched with transcription factor and cofactor binding sites, play a crucial role in regulating milk protein expression and represent ideal targets for gene editing. At present, comprehensive research on enhancers at the casein gene locus in livestock remains limited. This study identified 8 ultra-conserved regions through a cross-species comparison of casein gene loci in humans, mice, cattles, zebu cattle, goats, sheep, and camels. Following functional validation in silico, cross-species conserved enhancer sequences regulated by STAT5a were validated, with the CSN2 conserved enhancer (CSN-EN3) demonstrating the most potent regulatory effect. Co-immunoprecipitation (Co-IP) and bimolecular fluorescence complementation assays have demonstrated that STAT5a interacts with cofactors such as MED1, GR, ELF5, and NFIB, thereby synergistically regulating gene expression. The findings suggest that transcription factors and cis-acting elements associated with lactation exhibit high interspecies conservation, elucidating the pivotal role of STAT5a in lactation regulation. By altering the conserved enhancer CSN-EN3, its cis-regulatory control over STAT5a-dependent transcription was changed, leading to a 3.57-fold increase in casein expression. In conclusion, this study developed an enhancer identification system that integrates multi-species genome alignment with model animal epigenetic marker analysis, successfully identifying cross-species conserved enhancers at the mammalian casein locus. This research introduces a novel strategy to augment casein expression in ruminants, providing a significant theoretical foundation and technical support for the precision breeding of high-casein dairy cows and goats.