Department of Hematology, Longyan First Hospital Affiliated Fujian Medical University, Longyan, China.
Guangdong Key Laboratory of Clinical Molecular Medicine and Diagnostics, Guangzhou First People's Hospital, School of Medicine, South China University of Technology, Guangzhou, China.
Bioengineered. 2022 May;13(5):13398-13406. doi: 10.1080/21655979.2022.2069940.
This study investigated whether CRISPR/Cas9 (D10A) nickase-mediated gene editing can correct the aberrant Hb Constant Spring mutation (Hb CS or HBA2: c.427 T > C) in fibroblasts. Vectors for repairing the α-globin-encoding gene, HBA2:c.427 T > C mutation, includingthe CRISPR/Cas9(D10A)-sg plasmid and donor with homology arms, were constructed and used to perform gene editing in patient-derived fibroblasts. We subsequently analyzed the genetic correction, the gene editing efficiency and off-target effect. Sequencing analysis and the BamHI assay showed that HB CS mutant cells were repaired with Hb CS point mutations, the editing efficiency was 4.18%~9.34% and no off-target effects were detected. The results indicate that the HB CS mutant gene is effectively repaired by the CRISPR/Cas9 (D10A)system, which may enable truly personalized therapy for precise repair of α-thalassemia.
本研究探讨了 CRISPR/Cas9（D10A）切口酶介导的基因编辑是否可以纠正成纤维细胞中异常的血红蛋白 Constant Spring 突变（Hb CS 或 HBA2:c.427T>C）。构建了用于修复α-珠蛋白编码基因 HBA2:c.427T>C 突变的载体，包括 CRISPR/Cas9（D10A）-sg 质粒和带有同源臂的供体，并用于对患者来源的成纤维细胞进行基因编辑。随后，我们分析了基因矫正、基因编辑效率和脱靶效应。测序分析和 BamHI 检测表明，HB CS 突变细胞的 Hb CS 点突变得到修复，编辑效率为 4.18%~9.34%，未检测到脱靶效应。结果表明，CRISPR/Cas9（D10A）系统可有效修复 HB CS 突变基因，可能为α-地中海贫血的精确修复提供真正的个体化治疗。
