Design of Specific and Efficient sgRNA for CRISPR/Cas9 System to Knockout Superoxide Dismutase 2 in Breast Cancer Stem Cells

Abstract

This study aimed to design specific and efficient single guide RNA (sgRNA) for CRISPR/Cas9 system to knockout human superoxide dismutase 2 (SOD2) in human breast cancer stem cells (BCSCs). To achieve this, two sgRNA targets were selected, located within the Ala16Val polymorphism and the conserved region of human SOD2 variants. Design process was carried out using CRISPRdirect tool, considering the on/off target efficiency score. Subsequently, these sgRNAs were cloned into CRISPR/Cas9 expression plasmid and transfected into both the CD24-/CD44+ and ALDH1+ human BCSCs. To determine the most efficient sgRNA, a cleavage activity assay was conducted. The effectiveness of CRISPR/Cas9 system in knocking out the mRNA and protein expression of SOD2 in BCSCs was determined using quantitative reverse transcriptase polymerase chain reaction and western blot assays, respectively. The results showed that the sodex2.1 sgRNA targeting the Ala16Val region within exon 2 was ineffective in knocking out the SOD2 protein expression. However, among the four sgRNAs targeting the conserved region of nine SOD2 variants, the sodex2.4 sgRNA spanning from nucleotide 532-554 showed the highest efficiency based on cleavage activity assays. The sodex2.4 sgRNA significantly decreased both mRNA and protein expressions of SOD2 in human BCSCs. In conclusion, this study successfully designed specific and efficient sgRNA to knockout SOD2 expression in human BCSCs using CRISPR/Cas9 system. Moreover, further investigations are recommended to understand the impact of SOD2 knockout on the aggressiveness of breast cancer, particularly in BCSCs.

Keywords

• breast cancer
• crispr/cas9
• sgrna
• sod2
• stem cells