基于AAVS1位点构建稳定表达Cas9蛋白的非小细胞肺癌细胞系

Establishment of a Non-Small Cell Lung Cancer Cell Line Stably Expressing Cas9 Protein at AAVS1 Safe Harbor Locus

该研究旨在利用CRISPR/Cas9介导的同源重组技术将Cas9基因序列整合入非小细胞肺癌A549细胞基因组中的AAVS1位点,建立稳定表达Cas9蛋白的A549单克隆细胞系。该技术避免了Cas9基因随机整合进入基因组带来的潜在风险。通过PCR、Western blot、CCK-8、STR技术分别检测A549单克隆细胞系的插入位点、Cas9蛋白水平、细胞增殖能力、基因编辑能力以及细胞身份信息。上述结果显示,在单克隆细胞系A549 Cas9-copGFP-1中Cas9基因准确插入至AAVS1安全位点并高表达Cas9蛋白,细胞增殖能力未发生改变。此外,该细胞还具有良好的基因编辑能力,细胞身份信息准确无误。总之,A549 Cas9-copGFP-1细胞可用于进一步的基因编辑,为肺癌相关基因的高通量筛选和功能性研究提供一种有力工具。

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.