hfCas12Max-mediated targeted integration at accessible chromatin regions with a goat-derived UCOE enhances stable...

Stable transgene expression in the mammary gland is crucial for recombinant protein production in livestock, yet it is frequently hampered by transgene silencing and random integration. To address this, we profile chromatin accessibility in goat mammary epithelial cells (GMECs) using ATAC-seq and identify 15 highly accessible genomic regions. Three of these regions are confirmed to support stable transgene expression. Notably, we identify a goat-derived ubiquitous chromatin opening element (UCOE) in the SF3B1-COQ10B intergenic region, with a high GC content (65%) and CpG island enrichment. This UCOE improves hfCas12Max-mediated integration of large DNA fragments and maintains high-level expression of human lactoferrin (hLTF) in GMECs. Subsequently, we precisely integrate the UCOE-hLTF cassette into the highly accessible loci and generate a transgenic goat via somatic cell nuclear transfer, without detectable off-target effects. Our pipeline, which integrates chromatin accessibility profiling, UCOE discovery, and precision editing, demonstrates the role of CpG island-containing UCOEs in preventing transgene silencing. The study provides valuable tools for enhancing recombinant protein production, supports the breeding of dairy goats for milk with high lactoferrin content, and advances the understanding of the interactions between chromatin, regulatory elements, and transgenes in molecular breeding.