Fig. 2

Genetic correction of HbE mutation of the HBB gene. a T7E1 assay of gRNA target sites. At 5 days post transfection, genomic DNA of PX459-gRNA transfected cells was extracted and the region spanning the gRNA target sites was PCR amplified using on-target primer pairs (arrows), giving PCR products of 306 bp (uncut) and 150 and 156 bp (cut). M indicates marker. b Multiplex PCR screening for HbE mutation of isolated clones after genetic correction by CRISPR/Cas9 and the ssODN template. HbE-negative clone (clone 297) indicated in red. c Representative DNA sequences of PCR products of the region spanning the gRNA target site in HbE-negative clones with HDR in the corrected clones and indels in others. DSB indicates the double-stranded break site generated by gRNA1. d Chromatogram of the parental Eβ-iPSC2 cells and the corrected C46 cells at the mutation site. Red box indicates HbE mutation (G → A) at codon 26. Note the overlapped peaks in the Eβ-iPSC2 cells, which occurred as a result of G in one allele (normal) and A in another allele (HbE). After genetic correction, both alleles contained the right nucleotide “G”. e Representative karyotype of the corrected C46 cells, which exhibited a normal karyotype (46, XY). f Potential off-target sites for gRNA1 as identified by BLAST search. Mismatch nucleotides are indicated in red. Eβ-iPSC2 iPSC lines derived from a patient with HbE/β-thalassemia, HbE hemoglobin E, HBD delta hemoglobin