CORRECT介导的人HBB-28基因定点突变细胞株的建立及其应用

Construction and Application of CORRECT-mediated Human HBB-28 Gene Site-directed Mutagenesis Cell Line

关键词:  β-地中海贫血  HBB  阻断突变  定点突变  基因修复治疗

Key words:  β-thalassemia  HBB  blocking mutation  direct mutagenesis  gene repair therapy

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。

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.