CRISPR/Cas9 (D10A) nickase-mediated Hb CS gene editing and genetically modified fibroblast identification.

Wei-Hao Wu, Xiao-Mei Ma, Jian-Qing Huang, Qin Lai, Fu-Neng Jiang, Cui-Yun Zou, Long-Tian Chen, Lian Yu

Bioengineered 2022 May

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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.

Citation

Wei-Hao Wu, Xiao-Mei Ma, Jian-Qing Huang, Qin Lai, Fu-Neng Jiang, Cui-Yun Zou, Long-Tian Chen, Lian Yu. CRISPR/Cas9 (D10A) nickase-mediated Hb CS gene editing and genetically modified fibroblast identification. Bioengineered. 2022 May;13(5):13398-13406