Correcting HBB −28 (A>G) mutation in primary skin fibroblast cells of beta thalassemia patient. (A) Sanger sequencing to detect the genotype of the patient. Genomic DNA from the patient’s cells was extracted for PCR amplification of the target region. PCR amplicons were then sequenced by Sanger sequencing. HBB −28 (A>G) mutation were labelled with red arrowhead. (B) Primary skin fibroblast cells from the HBB −28 (A>G) mutant patient. (C) Schematic of base editing in HBB −28 (A>G) homozygous mutant skin fibroblast cells and single cell genotyping. Skin fibroblast cells were transfected with BE3 and gRNA-1. 48 h after transfection, single cell was isolated and whole genome amplified. The genomic DNA was then used as the template for PCR amplification of HBB site. The PCR product was sequenced by Sanger sequencing. (D) Representative sequencing chromatographs of homozygous mutant cells (G−28G−25/G−28G−25), heterozygous cells (A−28G−25/G−28G−25), and wild-type cells (A−28G−25/A−28G−25). (E) A summary of the base editing efficiency in homozygous skin fibroblast cells from the patient. A total of 30 single cells were whole-genome amplified. And 28/30 cells were successfully amplified by PCR. Both G−28 and G−25 were converted to A (A−28 and A−25 respectively). *PCR amplification failed in 2 cells