Construction of HEK293/K562 Cell Strain with G6PD Gene c.1388G>A Mutation Using CRISPR/Cas9
ZHANG Hongyang, SHU Yi, ZHU Dan, ZHANG Jia, ZOU Lin, ZHANG Penghui*
The aim of this study was to construct HEK293/K562 cell strain with G6PD gene c.1388G>A mutation using CRISPR/Cas9, and provide cell models for G6PD deficiency and its gene therapy research. Two sgRNAs (single-strand guide RNAs) and a mutant homology arm were designed for G6PD gene c.1388G>A site, the HEK293/K562 cell strain with G6PD gene c.1388G>A mutation were construced through CRISPR/Cas9 combined with HDR (homologous recombination repairing) pathway. qRT-PCR and Western blot were used to detect G6PD gene expression.CCK8 was used to detect cell proliferation. G6PD/6PGD ratio method was used to detect G6PD activity. Crystal violet staining and Annexin V-APC/7-AAD were used to verify the tolerance of the mutant cell strain to the oxidative active drug vitamin K3 and primaquine. The results showed that the CRISPR/Cas9 double plasmid vector system was successfully constructed and HEK293/K562 cell strain with G6PD gene c.1388G>A mutation were successfully constructed after monoclonal cell isolation and DNA sequencing, with no off-target effect. It was further found that the G6PD gene c.1388G>A mutation did not affect the mRNA transcription and protein translation of G6PD gene in HEK293 and K562 cells while the cell proliferation were inhibited and G6PD activity decreased in mutant cells. The tolerance to the oxidative active drug primaquine were weaker in both mutant HEK293 and K562 cells, and mutant HEK293 cell was less resistant to vitamin K3. This study successfully constructed HEK293 and K562 cell strain with G6PD gene c.1388G>A mutation, and provided cell models for G6PD deficiency and gene repair research.
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