Construction Of ATRX Gene Knockout HeLa Cell Line Based On CRISPR / Cas9 Technique

Type â…¡ CRISPR/Cas9 system recognizes target genome sequence through its pair with sgRNA, which guides Cas9 to cleave DNA, leading to DNA double-strand breaks (DSBs). One way to repair DSBs is Non-homologous end joining (NHEJ), which frequently results in gene mutation and therefore realizes gene knockout. Cancer cells need mechanisms to maintain the length of telomeres in order to gain unlimited proliferation. It has been shown that about 85% of tumors rely on the activation of telomerase to keep the length of telomeres, whereas 15% of tumors do so through alternative lengthening of telomeres (ALT) by homologous recombination. The inactivation mutation of ATRX gene is found to have close relationship with ALT mechanism in pancreatic endocrine tumors and gliomas. For the purpose of revealing the role of ATRX gene in the activation of ALT mechanism and tumor development, our research established HeLa cell lines with ATRX gene knocked out by the usage of CRISPR/Cas9 technique. The results of identifications of ATRX expression level by western-blot and ATRX gene mutations by Sanger sequencing showed that we successfully established HeLa cell line with ATRX gene knocked out.