hfCas12Max-Mediated Targeted Integration at Accessible Chromatin Regions with a Novel UCOE Enhances Stable Recombinant Lactoferrin Expression

Stable transgene expression in livestock mammary glands is crucial for recombinant protein production but is often disrupted by transgene silencing and random genomic integration. To overcome these issues, we used ATAC-seq to analyze chromatin accessibility in goat mammary epithelial cells (GMECs), identifying 15 highly accessible genomic regions. Three of these regions were confirmed to support stable transgene expression through dual-luciferase reporter assays and flow cytometry. Notably, we identified a new 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 improved CRISPR/hfCas12Max-mediated integration of large DNA fragments and maintained high-level expression of human lactoferrin (hLTF) in GMECs. Subsequently, we precisely integrated the UCOE-hLTF cassette into the ROSA26 safe harbor locus using hfCas12Max and successfully generated transgenic goats via somatic cell nuclear transfer, without detectable off-target effects. Our comprehensive pipeline, combining chromatin accessibility profiling, UCOE discovery, and precision editing, highlights the role of CpG island-containing UCOEs in preventing transgene silencing. This provides vital tools for improved recombinant protein production and aids in breeding dairy goats for lactoferrin-rich milk, while advancing understanding of chromatin-regulatory element-transgene interactions in molecular breeding.