Ku80 gene knock out by clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeats-associated protein 9 enhances the sensitivity of HEK293T cells to doxorubicin

Abstract

Objective 293T cells with Ku80 knockout were constructed using clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeats-associated protein9 (CRISPR/Cas9) technique, and the effect of Ku80 deletion on DNA damage repair and doxorubicin resistance of 293T cells was investigated. Methods Firstly, a pair of DNA Oligos targeted Ku80 was designed and the single guide RNA (sgRNA) expression vectors were constructed using pSpCas9 (BB)-2A-Puro (PX459) vector. This vector was transfected into 293T cells. The effectiveness of the designed sgRNA was verified by site-directed Polymerase chain reaction (PCR) and T7E1 enzyme reaction. The 293T cell line was constructed using limited dilution method and puromycin screening. Single cell clones were selected and screened by Western blotting assay and sequencing. Furthermore, DNA damage was induced by doxorubicin treatment, and detected by DNA DSB marker phosphorylated Histone H2AX (γ-H2AX). Cell counting kit-8 (CCK-8) assay was carried out to detect the sensitivity of the cells to doxorubicin. Results The Ku80 gene knockout 293T cells were successfully constructed. After DNA damage induced by doxorubicin treatment and DNA repair, the positive rates of γ-H2AX staining in the control and Ku80 knockout groups were (30.67±2.49)% and (88.00±1.63)%, respectively (P=0.000), indicating that the DNA damage repair ability was significantly inhibited in Ku80 knockout 293T cells. Ku80 knockout increases the sensitivity of 293T cells to doxorubicin. The growth inhibition rates of Ku80 knockout group and control group were (53.99±1.29)% and (32.42±1.20)%, respectively (P=0.000). Conclusion The knockout of Ku80 in 293T cells inhibited the DNA damage repair ability and increased the sensitivity of cells to doxorubicin. Key words: Clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeats-associated protein 9 technique; 293T cell; Ku80 gene; Doxorubicin