Objective To edit the γ-globin promoter of HEK293T cells using the CRISPR/Cas9 technology to upregulate γ-globin and explore the feasibility of using gene editing technology for the treatment of β-thalassemia.

Methods Using gene editing technology to treat β-thalassemia, two sgRNA sequences were designed at 420 bp upstream of the γ -globin transcription start site (TSS). CRISPR/Cas9 plasmids were transfected via lipofectamine to induce cleavage of the hemoglobin gamma (HBG) gene promoter in HEK293T cells. Positive clone screening, Sanger sequencing validation, TIDE analysis for editing efficiency, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blotting were used to detect the expression level of the γ-globin gene.

Results Both sgRNAs achieved efficient gene cutting on γ-globin, with cutting of 44.20% (sgRNA1) and 32.90% (sgRNA2), respectively. The results of RT-qPCR and western blotting showed that, compared to the negative control groups (NCs transfected with empty PX459 plasmid), the expression levels of the γ-globin gene in the sgRNA2 group were elevated (P < 0.05).

Conclusion The CRISPR/Cas9 gene editing technology can achieve effective editing on the γ-globin promoter, thereby upregulating γ-globin.