4.7 Article

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

Journal

Publisher

MDPI
DOI: 10.3390/ijms18061269

Keywords

gene editing; CRISPR/Cas9; Fanconi anemia; fibroblasts; Fanconi anemia D1; poly adenosine diphosphate-ribose polymerase inhibitors

Funding

  1. Czech Science Foundation [17-04941Y]
  2. NIH [T32-HL007062]
  3. Regenerative Medicine of Minnesota
  4. National Research Foundation of Korea [NRF-2015K1A4A3046807]
  5. National Center for Advancing Translational Sciences of the National Institutes of Health [UL1TR000114]
  6. [8UL1TR000114-02]
  7. [R01 AR063070]
  8. [P01 CA065493]

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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.

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