Validation of caprine H11 and Rosa26 safe harbor loci via CRISPR- based system: Investigations on stable transgene expression and genetic biosafety

CRISPR/Cas9 technology is an efficient tool for livestock gene editing. However, host genome function can be disrupted by the random integration of exogenous genes. To circumvent this issue, site-specific integration is required. This study established a multi-dimensional assessment system to evaluate the biological applicability of H11/Rosa26 safe harbor loci as targeted integration platforms for exogenous genes in goats. Donor cells carrying the enhanced green fluorescent protein (EGFP) reporter gene at the H11 and Rosa26 loci were generated via CRISPR/Cas9-mediated homology-directed repair; this was followed by somatic cell nuclear transfer to produce transgenic cloned embryos and healthy offspring. Multi-dimensional analyses revealed the following. At the cellular level, there was stable and efficient EGFP expression at integration sites, with donor cells maintaining normal cell cycle progression, proliferation capacity, and apoptosis levels, and with no alterations in the transcriptional integrity of adjacent genes. At the embryonic level, there was sustained EGFP expression across pre-implantation embryonic stages, with developmental metrics statistically indistinguishable from wild-type embryos. Finally, at the individual level, cloned offspring exhibited growth phenotypes consistent with wild-type counterparts, and EGFP showed broad-spectrum expression in eight tissues. This study provides the first demonstration of H11/Rosa26 loci as dual-functional sites that enable both high-efficiency integration and biosafety in goat models using a cross-scale (cellular-embryonic-individual) validation system, offering a precise and low-risk technical paradigm for livestock genetic improvement.