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Int. J. Mol. Sci. 2017, 18(6), 1269; doi:10.3390/ijms18061269

CRISPR/Cas9-Mediated Correction of the FANCD1 Gene in Primary Patient Cells

1
Department of Pediatrics, Division of Blood and Marrow Transplantation, University of Minnesota, Minneapolis, MN 55455, USA
2
Childhood Leukemia Investigation Prague (CLIP), Department of Pediatric Hematology and Oncology, Second Faculty of Medicine, Charles University, Prague 15006, Czech Republic
3
Stem Cell Institute, University of Minnesota, Minneapolis, MN 55455, USA
4
Center for Genome Engineering, University of Minnesota, Minneapolis, MN 55455, USA
5
Asan-Minnesota Institute for Innovating Transplantation, University of Minnesota, Minneapolis, MN 55455, USA
6
Asan Institute for Life Sciences, Asan Medical Center, Asan-Minnesota Institute for Innovating Transplantation, Seoul 138-736, Korea
*
Author to whom correspondence should be addressed.
Received: 12 May 2017 / Revised: 2 June 2017 / Accepted: 9 June 2017 / Published: 14 June 2017
(This article belongs to the Special Issue Genome Editing 2018)
View Full-Text   |   Download PDF [2786 KB, uploaded 14 June 2017]   |  

Abstract

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 a FANCD1 gene 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). FANCD1 function 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 primary FANCD1 cells in a precise manner. These proof of principle studies support expanded application of gene editing for FA. View Full-Text
Keywords: gene editing; CRISPR/Cas9; Fanconi anemia; fibroblasts; Fanconi anemia D1; poly adenosine diphosphate-ribose polymerase inhibitors gene editing; CRISPR/Cas9; Fanconi anemia; fibroblasts; Fanconi anemia D1; poly adenosine diphosphate-ribose polymerase inhibitors
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).

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Skvarova Kramarzova, K.; Osborn, M.J.; Webber, B.R.; DeFeo, A.P.; McElroy, A.N.; Kim, C.J.; Tolar, J. CRISPR/Cas9-Mediated Correction of the FANCD1 Gene in Primary Patient Cells. Int. J. Mol. Sci. 2017, 18, 1269.

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