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刘永祥,麦庆云,黎允诗,梅珺琰,梁爱军,彭新良,周瑞鸿,周少虎.CORRECT介导的人HBB-28基因定点突变细胞株的建立及其应用[J].广西科学,2022,29(6):1125-1133. [点击复制]
- LIU Yongxiang,MAI Qingyun,LI Yunshi,MEI Junyan,LIANG Aijun,PENG Xinliang,ZHOU Ruihong,ZHOU Shaohu.Construction and Application of CORRECT-mediated Human HBB-28 Gene Site-directed Mutagenesis Cell Line[J].Guangxi Sciences,2022,29(6):1125-1133. [点击复制]
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| CORRECT介导的人HBB-28基因定点突变细胞株的建立及其应用 |
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刘永祥1, 麦庆云2, 黎允诗1, 梅珺琰1, 梁爱军1, 彭新良1, 周瑞鸿3, 周少虎1
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| (1.广州中医药大学第一附属医院生殖医学科, 广东广州 510405;2.中山大学附属第一医院生殖医学中心, 广东广州 510080;3.广州医药研究总院有限公司, 广东广州 510220) |
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| 摘要: |
| 为利用CRISPR/Cas9系统建立一种新型、高效的定点突变和基因修复的新方法——引入阻断突变碱基的CORRECT(Consecutive re-Guide or re-Cas steps to Erase CRISPR/Cas blocked Targets)方法，本研究使用规律间隔成簇短回文重复序列(Clustered Regularly Interspaced Short Palindromic Repeats,CRISPR)在线设计工具，针对人β-珠蛋白(HBB)基因设计小向导RNA (small guide RNA，sgRNA)，构建PX459-sgRNA-Cas9共表达质粒，然后以含阻断突变碱基(G>T)的单链寡核苷酸(single-stranded Oligo DNA Nucleotides,ssODNs)为同源模板，通过脂质体转染HEK293T细胞，并通过Sanger测序和酶切(T7EⅠ和AatⅡ)检测编辑效率，最后通过Sanger 测序分析敲除HBB单克隆细胞的基因型。结果表明，成功构建β-地中海贫血(简称β-地贫)HBB-28(A>G)基因纯合定点突变的HEK293T细胞株。阻断突变碱基优化的ssODNs减少Cas9蛋白对靶点的再次编辑，提高了整合效率。本研究建立的新型点突变技术可以高效获得纯合定点突变的HEK293T细胞株，既为单碱基突变疾病模型的建立提供实验依据，又为基因修复治疗提供高效的方法。 |
| 关键词: β-地中海贫血 HBB 阻断突变 定点突变 基因修复治疗 |
| DOI：10.13656/j.cnki.gxkx.20230110.012 |
| 投稿时间：2022-06-17修订日期：2022-09-29 |
| 基金项目:广州中医药大学“双一流”与高水平大学学科协同创新团队培育基金项目(2021xk64)资助。 |
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| Construction and Application of CORRECT-mediated Human HBB-28 Gene Site-directed Mutagenesis Cell Line |
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LIU Yongxiang1, MAI Qingyun2, LI Yunshi1, MEI Junyan1, LIANG Aijun1, PENG Xinliang1, ZHOU Ruihong3, ZHOU Shaohu1
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| (1.Department of Reproductive Medicine, The First Affiliated Hospital of Guangzhou University of Chinese Medicine, Guangzhou, Guangdong, 510405, China;2.Reproductive Medicine Center, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, Guangdong, 510080, China;3.Guangzhou General Pharmaceutical Research Institute Company Limited, Guangzhou, Guangdong, 510220, China) |
| Abstract: |
| In order to establish a new and efficient method named CORRECT (Consecutive re-Guide or re-Cas steps to Erase CRISPR/Cas blocked Targets) for site-directed mutagenesis and gene repair using CRISPR/Cas9 system,Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) online design tool was used in this study to design small guide RNA (sgRNA) for human β-globin (HBB) gene. PX459-sgRNA-Cas9 co-expression plasmid was constructed,and then single-stranded Oligo DNA Nucleotides (ssODNs) containing blocking mutation bases (G>T) were used as homologous templates. HEK293T cells were transfected with liposomes and the editing efficiency was examined by Sanger sequencing and enzyme digestion (T7E Ⅰ and Aat Ⅱ). Finally,the genotype of HBB knockout monoclonal cells was analyzed by Sanger sequencing. The results showed that β-thalassemia HBB-28(A>G) homozygous mutant HEK293T cell line was successfully constructed. Blocking mutant base-optimized ssODNs reduced the re-editing of Cas9 protein to the target and improved the integration efficiency. The HEK293T cell strain with homozygous point mutation can be efficiently obtained by the novel point mutation technology,which not only provides experimental basis for the establishment of single base mutation disease model,but also provides an efficient method for gene repair therapy. |
| Key words: β-thalassemia HBB blocking mutation direct mutagenesis gene repair therapy |
