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

Karolina Skvarova Kramarzova,Mark Osborn,Amber Mcelroy,Anthony Defeo,Chong Kim,Beau Webber,Jakub Tolar

doi:10.3390/ijms18061269

Karolina Skvarova Kramarzova, Mark Osborn + Show 5 more

Open Access

https://doi.org/10.3390/ijms18061269

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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.

Highlights

Fanconi anemia (FA) is an inherited genetic disorder characterized by chromosomal instability, bone marrow failure (BMF), congenital malformations, and early cancer onset [1,2,3]
To determine whether the FA phenotype could be corrected in true primary cells we undertook efforts to target the Fanconi anemia D1 (FANCD1) gene in fibroblasts derived from an FA patient
The 886delGT was favorable for designing reagents derived from the Streptococcus pyogenes clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system due to the proximity of the guide RNA to the mutation site in exon 8 (Figure 1A)

Summary

Introduction

Fanconi anemia (FA) is an inherited genetic disorder characterized by chromosomal instability, bone marrow failure (BMF), congenital malformations, and early cancer onset [1,2,3]. FANCD1, known as the breast cancer 2 gene (BRCA2), functions as a downstream effector of the DNA repair pathway where it binds and stabilizes RAD51-nucleoprotein filaments at the site(s) of DNA breaks [17]. This is a crucial prerequisite for DNA damage repair by homologous recombination [18,19]. We hypothesized that genome editing along with PARP inhibition (PARPi) would allow us to selectively recover gene modified cells To accomplish this we delivered CRISPR/Cas reagents and an oligonucleotide donor molecule to primary FANCD1 fibroblasts and, following PARPi treatment, obtained six genotypically and functionally corrected clones. This is the first report of a causal gene correction by gene editing in FA patient primary fibroblasts

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