Chul-Yong Park,1,6 Duk Hyoung Kim,2,3,6 Jeong Sang Son,4,6 Jin Jea Sung,1 Jaehun Lee,4 Sangsu Bae,5 Jong-Hoon Kim,4,7 Dong-Wook Kim,1,7,* and Jin-Soo Kim2,3,7,*
1Department of Physiology and Brain Korea 21 Plus Project for Medical Science, Yonsei University College of Medicine, Seoul 120-752, Korea
2Center for Genome Engineering, Institute for Basic Science, Seoul 151-742, Korea
3Department of Chemistry, Seoul National University, Seoul 151-742, Korea
4Laboratory of Stem Cell Biology, Division of Biotechnology, College of Life Science and Biotechnology, Korea University, Seoul 136-713, Korea
5Department of Chemistry, Hanyang University, Seoul 133-791, Korea
*Correspondence: Dong-Wook Kim, Jin-Soo Kim
Hemophilia A is an X-linked genetic disorder caused by mutations in the F8 gene, which encodes the blood coagulation factor VIII. Almost half of all severe hemophilia A cases result from two gross (140-kbp or 600-kbp) chromosomal inversions that involve introns 1 and 22 of the F8 gene, respectively. We derived induced pluripotent stem cells (iPSCs) from patients with these inversion genotypes and used CRISPR-Cas9 nucleases to revert these chromosomal segments back to the WT situation. We isolated inversion-corrected iPSCs with frequencies of up to 6.7% without detectable off-target mutations based on whole-genome sequencing or targeted deep sequencing. Endothelial cells differentiated from corrected iPSCs expressed the F8 gene and functionally rescued factor VIII deficiency in an otherwise lethal mouse model of hemophilia. Our results therefore provide a proof of principle for functional correction of large chromosomal rearrangements in patient-derived iPSCs and suggest potential therapeutic applications.