Targeted correction of c.130_131insA restored HAX1 protein expression. (A) Schematic overview of the HAX1 protein and the corresponding genomic locus including the location of c.130_131insA insertional mutation, which leads to a premature stop codon. The c.130_131insA mutation and the location of the double-strand break are indicated in red and green, respectively. The ssODN provided the corrected sequence and a silent mutation in the serine codon upstream of the mutation site. (B-C) Upon successful correction, a restriction site for MwoI was gained in exon 2 that was used to distinguish corrected clones from uncorrected after agarose gel electrophoresis (C). In corrected and WT clones, the additional MwoI site would cut the 530-bp amplicon into 2 fragments of ∼190 bp and 1 fragment of 144 bp. Digestion of uncorrected clone amplicons lead to bands of 336 and 194 bp. (D) The corrected sequence and the silent mutation in the serine codon were detected via Sanger sequencing. (E) The 2 healthy isogenic cell lines and the positive control NB-4 cells expressed HAX1 protein analyzed by western blotting. HAX1^W44X-iPSCs showed no HAX1 expression. (F) Normal karyotype with no chromosomal aberrations could be detected in HAX1^corrected-iPSCs.