• This study establishes a CRISPR/Cas9-mediated site-specific integration strategy to precisely target the nanobody PD-L1-Fc gene into the C12orf35 locus in CHO cells, overcoming limitations of random integration.
• The C12orf35 locus is identified as a 'hotspot' for stable transgene expression, with disruption of this gene potentially enhancing productivity and reducing recovery times.
• The use of EGFP as a reporter enables real-time monitoring of exogenous gene expression, facilitating efficient screening of stable cell lines.
• This approach lays the foundation for developing industrial recombinant CHO cell lines with improved genetic stability and consistent productivity.