CRISPR/Cas9-mediated γ-globin expression upregulation in HEK293T cells
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 des...
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Editorial Office of Journal of Guangxi Medical University
2025-04-01
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| Series: | Guangxi Yike Daxue xuebao |
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| Online Access: | https://journal.gxmu.edu.cn/article/doi/10.16190/j.cnki.45-1211/r.2025.02.011 |
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| _version_ | 1850190274220785664 |
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| author | HUANG Xianjuan LAI Yongrong LI Jing |
| author_facet | HUANG Xianjuan LAI Yongrong LI Jing |
| author_sort | HUANG Xianjuan |
| collection | DOAJ |
| description | 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. |
| format | Article |
| id | doaj-art-e931ed1ad720437c8474dbe258da810f |
| institution | OA Journals |
| issn | 1005-930X |
| language | zho |
| publishDate | 2025-04-01 |
| publisher | Editorial Office of Journal of Guangxi Medical University |
| record_format | Article |
| series | Guangxi Yike Daxue xuebao |
| spelling | doaj-art-e931ed1ad720437c8474dbe258da810f2025-08-20T02:15:20ZzhoEditorial Office of Journal of Guangxi Medical UniversityGuangxi Yike Daxue xuebao1005-930X2025-04-0142224124610.16190/j.cnki.45-1211/r.2025.02.011gxykdxxb-42-2-241CRISPR/Cas9-mediated γ-globin expression upregulation in HEK293T cellsHUANG Xianjuan0LAI Yongrong1LI Jing2The First Affiliated Hospital of Guangxi Medical University, Nan‐ ning 530021, ChinaThe First Affiliated Hospital of Guangxi Medical University, Nan‐ ning 530021, ChinaThe First Affiliated Hospital of Guangxi Medical University, Nan‐ ning 530021, ChinaObjective 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.https://journal.gxmu.edu.cn/article/doi/10.16190/j.cnki.45-1211/r.2025.02.011crispr/cas9 editingγ-globinhek293t cellsgene editing |
| spellingShingle | HUANG Xianjuan LAI Yongrong LI Jing CRISPR/Cas9-mediated γ-globin expression upregulation in HEK293T cells Guangxi Yike Daxue xuebao crispr/cas9 editing γ-globin hek293t cells gene editing |
| title | CRISPR/Cas9-mediated γ-globin expression upregulation in HEK293T cells |
| title_full | CRISPR/Cas9-mediated γ-globin expression upregulation in HEK293T cells |
| title_fullStr | CRISPR/Cas9-mediated γ-globin expression upregulation in HEK293T cells |
| title_full_unstemmed | CRISPR/Cas9-mediated γ-globin expression upregulation in HEK293T cells |
| title_short | CRISPR/Cas9-mediated γ-globin expression upregulation in HEK293T cells |
| title_sort | crispr cas9 mediated γ globin expression upregulation in hek293t cells |
| topic | crispr/cas9 editing γ-globin hek293t cells gene editing |
| url | https://journal.gxmu.edu.cn/article/doi/10.16190/j.cnki.45-1211/r.2025.02.011 |
| work_keys_str_mv | AT huangxianjuan crisprcas9mediatedgglobinexpressionupregulationinhek293tcells AT laiyongrong crisprcas9mediatedgglobinexpressionupregulationinhek293tcells AT lijing crisprcas9mediatedgglobinexpressionupregulationinhek293tcells |