Establishment of a Non-Small Cell Lung Cancer Cell Line Stably Expressing Cas9 Protein at AAVS1 Safe Harbor Locus
FANG Weiying, ZHANG Xiaoyu, GU Tingyu, CHEN Yuelei, SHAO Ting*
This study aimed to create an A549 monoclonal cell line with stable expression of Cas9 protein by knocking in a Cas9 expression cassette into the AAVS1 safe harbor locus of non-small cell lung cancer A549 cell line using CRISPR/Cas9-mediated homologous recombination technology. This technology avoided the potential risks of random Cas9 integration into the genome. The insertion site, Cas9 protein expression, cell proliferation, gene editing capability and cell identity of A549 monoclonal cell lines were validated by PCR, Western blot, CCK-8 and STR analyses, respectively. The above results showed that Cas9 cassette was successfully inserted into the AAVS1 safe harbor locus of the monoclonal cell line named A549 Cas9-copGFP-1, which highly expressed Cas9 protein without significant differences in cell viability compared to unmodified A549 cells. Moreover, this cell line had significant gene editing capability and correct STR profiles. Overall, A549 Cas9-copGFP-1 could be further used for gene editing, providing a powerful tool for high-throughput screening and functional studies of lung cancer-related genes.
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