Fig. 1

Strategy for correction of the thalassemia mutations and evaluation of the cleavage activity of gRNAs. A Schematic of the gRNAs for targeting the β-41/42 deletion mutant in the HBB gene. The oligos for β-sgRNA were designed in 600 bp downstream of the last exon of HBB. The primers used in this construct are AEXON-F/BEXON-R. B Schematic of the gRNAs for targeting the Westmead point mutation in the HBA2 gene. The oligos for α2-sgRNA were designed in 200 bp downstream of the last exon of HBA2. The primers used in this construct are HBA2 mut-F/R. C The complementary annealed sgRNA oligonucleotides were inserted into vector PX330. The sgRNA and PAM (NGG) sequence was inserted into the middle of the GFP gene and introduced into pTP53-GFP-reporter. After the targeting DNA was cut by Cas9, homologous recombination of the duplications occurred, resulting in the formation of a full-length GFP. D GFP signals were significantly increased, demonstrating the efficient cleavage activity of HBA2-sgRNA. E GFP signals were significantly increased, demonstrating the efficient cleavage activity of HBB-sgRNA