Fanconi anemia (FA) is an inherited condition characterized by impaired DNA repair, physical anomalies, bone marrow failure, and increased incidence of malignancy. Gene editing holds great potential to precisely correct the underlying genetic cause such that gene expression remains under the endogenous control mechanisms. This has been accomplished to date only in transformed cells or their reprogrammed induced pluripotent stem cell counterparts; however, it has not yet been reported in primary patient cells. Here we show the ability to correct a mutation in Fanconi anemia D1 (FANCD1) primary patient fibroblasts. The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system was employed to target and correct aFANCD1gene deletion. Homologous recombination using an oligonucleotide donor was achieved and a pure population of modified cells was obtained by using inhibitors of poly adenosine diphosphate-ribose polymerase (poly ADP-ribose polymerase).FANCD1function was restored and we did not observe any promiscuous cutting of the CRISPR/Cas9 at off target sites. This consideration is crucial in the context of the pre-malignant FA phenotype. Altogether we show the ability to correct a patient mutation in primaryFANCD1cells in a precise manner. These proof of principle studies support expanded application of gene editing for FA.

Asan Institute for Life Sciences Asan Medical Center Asan Minnesota Institute for Innovating Transplantation Seoul 138 736 Korea

Department of Pediatrics Division of Blood and Marrow Transplantation University of Minnesota Minneapolis MN 55455 USA

Department of Pediatrics Division of Blood and Marrow Transplantation University of Minnesota Minneapolis MN 55455 USA Childhood Leukemia Investigation Prague Department of Pediatric Hematology and Oncology 2nd Faculty of Medicine Charles University Prague 15006 Czech Republic

Department of Pediatrics Division of Blood and Marrow Transplantation University of Minnesota Minneapolis MN 55455 USA Stem Cell Institute University of Minnesota Minneapolis MN 55455 USA Asan Minnesota Institute for Innovating Transplantation University of Minnesota Minneapolis MN 55455 USA

Department of Pediatrics Division of Blood and Marrow Transplantation University of Minnesota Minneapolis MN 55455 USA Stem Cell Institute University of Minnesota Minneapolis MN 55455 USA Center for Genome Engineering University of Minnesota Minneapolis MN 55455 USA Asan Minnesota Institute for Innovating Transplantation University of Minnesota Minneapolis MN 55455 USA

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