Fig. 1

Strategy to target HbE mutation in Eβ-iPSCs using CRISPR/Cas9 to induce a double-stranded break at the HBB locus and a single-stranded oligo donor (ssODN) template to repair the mutation. a Multiplex PCR analysis for HbE mutation. wt-HDFs and wt-iPSCs indicate wild-type human dermal fibroblasts and iPSCs from a healthy individual. Eβ-HDFs, Eβ-iPSC1 and Eβ-iPSC2 are human dermal fibroblasts and two iPSC lines derived from a patient with HbE/β-thalassemia. b Schematic of targeted region of the HBB locus of the patient with HbE/β-thalassemia. The patient has a 4-bp deletion (–TCTT) in one allele and a point mutation at codon 26 (G → A) resulting in a structural variant hemoglobin E (HbE) in the other. Seamless and efficient correction of HbE is achieved using a gRNA targeting the point mutation (A) and the ssODN template carrying the correct nucleotide (G) with the left and right homology arms of 90 bp. Orange boxes indicate exons; red lines indicate introns. Red arrowheads show mutation sites; black arrowhead shows cleavage site of Cas9 by gRNA1; purple and red arrows indicate primer pairs for T7E1 assay and multiplex PCR for HbE detection, respectively. c Sequence of seven gRNAs designed to target the HbE mutation. Red “A” indicates point mutation in the Eβ-iPSC2 cells and gRNAs at HBB locus. HBB beta hemoglobin