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

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

来源：Bioengineered, 2022, 13(5): 13398-13406.

DOI：10.1080/21655979.2022.2069940

• Fibroblast
• alpha-thalassemia
• CRISPR/Cas9 (D10A)
• genetic correction
• Hb constant spring

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 a-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 alpha-thalassemia. @@@ [GRAPHICS] @@@ .

• 单位
  1; Zhongkai University of Agriculture and Engineering