[Construction and characterization of a stable Cas9-expressing monoclonal WSL cell line]

The molecular mechanism underlying the cellular invasion of African swine fever virus (ASFV) remains incompletely understood, particularly with respect to its key cellular receptors, which constitutes a major bottleneck in the development of effective vaccines and targeted antiviral therapies. To establish a robust experimental platform that supports efficient ASFV replication and enables genome-wide CRISPR screening for systematic identification of host factors involved in viral entry, we employed the CRISPR/Cas9 system to generate stable monoclonal cell lines expressing Cas9 protein based on the wild boar lung (WSL) cell line. Recombinant lentiviruses co-expressing Cas9 and blasticidin resistance gene were packaged via a lentiviral vector system and transduced into WSL cells. Following blasticidin selection, a polyclonal population stably expressing Cas9 was obtained and subjected to fluorescence-activated cell sorting (FACS) to derive monoclonal cell lines. Cas9 expression was determined by Western blotting. To assess the functional gene editing activity of the established clones, we introduced the lentiviruses carrying an EGFP reporter gene along with its specific single-guide RNA (sgRNA) into the monoclonal cell lines, and quantitatively evaluated the editing efficiency via flow cytometry. Furthermore, sgRNAs specifically targeting the ASFV B646L gene and the host TMEM239 gene were designed and synthesized to validate the cell line's capacity for editing both viral and host genomic loci. The results demonstrated the successful establishment of seven stable WSL-Cas9 monoclonal cell lines expressing Cas9 protein, among which clone WSL-Cas9-3# exhibited the highest editing efficiency, enabling effective genetic modification of both ASFV and host genes, while maintaining favorable genetic stability and normal growth properties. This study reports the generation of a WSL-Cas9 monoclonal cell line with high CRISPR/Cas9 editing efficiency, stable proliferation, and permissiveness for robust ASFV replication. This engineered cell line provides a reliable platform for future genome-wide functional screening to systematically identify host factors governing ASFV entry and establishes a critical technical foundation for delving into virus-host interactions.