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Sci Rep. 2019 Jan 25;9(1):768. doi: 10.1038/s41598-018-36506-w.

Effective CRISPR/Cas9-mediated correction of a Fanconi anemia defect by error-prone end joining or templated repair.

Author information

1
Division of Tumor Biology and Immunology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX, Amsterdam, The Netherlands. h.vandevrugt@vumc.nl.
2
Section of Oncogenetics, Department of Clinical Genetics, Cancer Center Amsterdam, Amsterdam University Medical Centers, De Boelelaan 1118, 1081 HV, Amsterdam, The Netherlands. h.vandevrugt@vumc.nl.
3
Division of Tumor Biology and Immunology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX, Amsterdam, The Netherlands.
4
Genome Engineering Oxford, Sir William Dunn School of Pathology, University of Oxford South Parks Road, OX1 3RE, Oxford, UK.
5
Section of Oncogenetics, Department of Clinical Genetics, Cancer Center Amsterdam, Amsterdam University Medical Centers, De Boelelaan 1118, 1081 HV, Amsterdam, The Netherlands.
6
Mouse Clinic for Cancer and Aging research (MCCA) Transgenic Facility, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX, Amsterdam, The Netherlands.
7
Division of Tumor Biology and Immunology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX, Amsterdam, The Netherlands. h.t.riele@nki.nl.
8
Section of Oncogenetics, Department of Clinical Genetics, Cancer Center Amsterdam, Amsterdam University Medical Centers, De Boelelaan 1118, 1081 HV, Amsterdam, The Netherlands. h.t.riele@nki.nl.

Abstract

Fanconi anemia (FA) is a cancer predisposition syndrome characterized by congenital abnormalities, bone marrow failure, and hypersensitivity to aldehydes and crosslinking agents. For FA patients, gene editing holds promise for therapeutic applications aimed at functionally restoring mutated genes in hematopoietic stem cells. However, intrinsic FA DNA repair defects may obstruct gene editing feasibility. Here, we report on the CRISPR/Cas9-mediated correction of a disruptive mutation in Fancf. Our experiments revealed that gene editing could effectively restore Fancf function via error-prone end joining resulting in a 27% increased survival in the presence of mitomycin C. In addition, templated gene correction could be achieved after double strand or single strand break formation. Although templated gene editing efficiencies were low (≤6%), FA corrected embryonic stem cells acquired a strong proliferative advantage over non-corrected cells, even without imposing genotoxic stress. Notably, Cas9 nickase activity resulted in mono-allelic gene editing and avoidance of undesired mutagenesis. In conclusion: DNA repair defects associated with FANCF deficiency do not prohibit CRISPR/Cas9 gene correction. Our data provide a solid basis for the application of pre-clinical models to further explore the potential of gene editing against FA, with the eventual aim to obtain therapeutic strategies against bone marrow failure.

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