Figure 2.

Correction of the mutation in TGFBI R124H mutant keratocytes using CRISPR-mediated HDR. (a) Result of an RFLP analysis of edited R124H cells. TGFBI exon 4 was amplified by PCR, and the products were treated with the BsiWI restriction enzyme. The lane with three bands was edited heterozygously and the lane with two bands was edited homozygously. (b) DNA sequences of PCR products amplified from the TGFBI gene of wild-type cells, a heterogeneous R124H mutant, and a repaired allele by HDR after transfection of Cas9 guide RNA and ssDNA. Two peaks were observed in the sequence of the R124H heterogeneous mutant, while the base of HDR-repaired cells was corrected to T. (c) Editing efficiency of CRISPR/Cas9-mediated HDR of an R124H mutation. RFLP, restriction fragment length polymorphism TGFBI, transforming growth factor β-induced; HDR, homology-directed repair.