Correction of Fanconi Anemia Mutations Using Digital Genome Engineering

Christopher J. Sipe, Mitchell G. Kluesner, Samuel P. Bingea, Walker S. Lahr, Aneesha A. Andrew, Minjing Wang, Anthony P. DeFeo, Timothy L. Hinkel, Kanut Laoharawee, John E. Wagner, Margaret L. MacMillan, Gregory M. Vercellotti, Jakub Tolar, Mark J. Osborn, R. Scott McIvor, Beau R. Webber, Branden S. Moriarity

Research output: Contribution to journalArticlepeer-review

2 Scopus citations


Fanconi anemia (FA) is a rare genetic disease in which genes essential for DNA repair are mutated. Both the interstrand crosslink (ICL) and double-strand break (DSB) repair pathways are disrupted in FA, leading to patient bone marrow failure (BMF) and cancer predisposition. The only curative therapy for the hematological manifestations of FA is an allogeneic hematopoietic cell transplant (HCT); however, many (>70%) patients lack a suitable human leukocyte antigen (HLA)-matched donor, often resulting in increased rates of graft-versus-host disease (GvHD) and, potentially, the exacerbation of cancer risk. Successful engraftment of gene-corrected autologous hematopoietic stem cells (HSC) circumvents the need for an allogeneic HCT and has been achieved in other genetic diseases using targeted nucleases to induce site specific DSBs and the correction of mutated genes through homology-directed repair (HDR). However, this process is extremely inefficient in FA cells, as they are inherently deficient in DNA repair. Here, we demonstrate the correction of FANCA mutations in primary patient cells using ‘digital’ genome editing with the cytosine and adenine base editors (BEs). These Cas9-based tools allow for C:G > T:A or A:T > C:G base transitions without the induction of a toxic DSB or the need for a DNA donor molecule. These genetic corrections or conservative codon substitution strategies lead to phenotypic rescue as illustrated by a resistance to the alkylating crosslinking agent Mitomycin C (MMC). Further, FANCA protein expression was restored, and an intact FA pathway was demonstrated by downstream FANCD2 monoubiquitination induction. This BE digital correction strategy will enable the use of gene-corrected FA patient hematopoietic stem and progenitor cells (HSPCs) for autologous HCT, obviating the risks associated with allogeneic HCT and DSB induction during autologous HSC gene therapy.

Original languageEnglish (US)
Article number8416
JournalInternational journal of molecular sciences
Issue number15
StatePublished - Aug 2022

Bibliographical note

Funding Information:
This research was funded by the Fanconi Anemia Research Fund (FARF), the University of Minnesota-Fairview Academic Investment Research Program (AIRP), the Children’s Cancer Research Foundation (CCRF), and the University of Minnesota’s Department of Pediatrics ‘R’ Award. C.J.S was funded by the University of Minnesota’s Stem Cell Institute INFUSE predoctoral award. M.G.K. was funded by an NIH predoctoral fellowship (T32GM007266).

Publisher Copyright:
© 2022 by the authors.


  • CRISPR-Cas9
  • Fanconi anemia (FA)
  • Fanconi anemia repair pathway
  • adenine base editing (ABE)
  • base editing
  • base excision repair
  • bone marrow failure
  • cytosine base editing (CBE)
  • digital genome engineering
  • double strand breaks
  • gene therapy

PubMed: MeSH publication types

  • Journal Article


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