Universal Correction of Blood Coagulation Factor VIII in Patient-Derived Induced Pluripotent Stem Cells Using CRISPR/Cas9

Abstract

Hemophilia A (HA) is caused by genetic mutations in the blood coagulation factor VIII (FVIII) gene. Genome-editing approaches can be used to target the mutated site itself in patient-derived induced pluripotent stem cells (iPSCs). However, these approaches can be hampered by difficulty in preparing thousands of editing platforms for each corresponding variant found in HA patients. Here, we report a universal approach to correct the various mutations in HA patient iPSCs by the targeted insertion of the FVIII gene into the human H11 site via CRISPR/Cas9. We derived corrected clones from two types of patient iPSCs with frequencies of up to 64% and 66%, respectively, without detectable unwanted off-target mutations. Moreover, we demonstrated that endothelial cells differentiated from the corrected iPSCs successfully secreted functional protein. This strategy may provide a universal therapeutic method for correcting all genetic variants found in HA patients.