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

Henri J. van de Vrugt, Tim Harmsen, Joey Riepsaame, Georgina Alexantya, Saskia E. van Mil, Yne de Vries, Rahmen Bin Ali, Ivo J. Huijbers, Josephine C. Dorsman, Rob M. F. Wolthuis, Hein te Riele

Research output: Contribution to journalArticleAcademicpeer-review

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.
Original languageEnglish
Article number768
JournalScientific Reports
Volume9
Issue number1
DOIs
Publication statusPublished - 1 Dec 2019

Cite this

van de Vrugt, Henri J. ; Harmsen, Tim ; Riepsaame, Joey ; Alexantya, Georgina ; van Mil, Saskia E. ; de Vries, Yne ; Bin Ali, Rahmen ; Huijbers, Ivo J. ; Dorsman, Josephine C. ; Wolthuis, Rob M. F. ; te Riele, Hein. / Effective CRISPR/Cas9-mediated correction of a Fanconi anemia defect by error-prone end joining or templated repair. In: Scientific Reports. 2019 ; Vol. 9, No. 1.
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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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Effective CRISPR/Cas9-mediated correction of a Fanconi anemia defect by error-prone end joining or templated repair. / van de Vrugt, Henri J.; Harmsen, Tim; Riepsaame, Joey; Alexantya, Georgina; van Mil, Saskia E.; de Vries, Yne; Bin Ali, Rahmen; Huijbers, Ivo J.; Dorsman, Josephine C.; Wolthuis, Rob M. F.; te Riele, Hein.

In: Scientific Reports, Vol. 9, No. 1, 768, 01.12.2019.

Research output: Contribution to journalArticleAcademicpeer-review

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AU - van Mil, Saskia E.

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