Cargando…

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

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 endogen...

Descripción completa

Detalles Bibliográficos
Autores principales: Skvarova Kramarzova, Karolina, Osborn, Mark J., Webber, Beau R., DeFeo, Anthony P., McElroy, Amber N., Kim, Chong Jai, Tolar, Jakub
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2017
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5486091/
https://www.ncbi.nlm.nih.gov/pubmed/28613254
http://dx.doi.org/10.3390/ijms18061269
_version_ 1783246199199891456
author Skvarova Kramarzova, Karolina
Osborn, Mark J.
Webber, Beau R.
DeFeo, Anthony P.
McElroy, Amber N.
Kim, Chong Jai
Tolar, Jakub
author_facet Skvarova Kramarzova, Karolina
Osborn, Mark J.
Webber, Beau R.
DeFeo, Anthony P.
McElroy, Amber N.
Kim, Chong Jai
Tolar, Jakub
author_sort Skvarova Kramarzova, Karolina
collection PubMed
description 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.
format Online
Article
Text
id pubmed-5486091
institution National Center for Biotechnology Information
language English
publishDate 2017
publisher MDPI
record_format MEDLINE/PubMed
spelling pubmed-54860912017-06-29 CRISPR/Cas9-Mediated Correction of the FANCD1 Gene in Primary Patient Cells Skvarova Kramarzova, Karolina Osborn, Mark J. Webber, Beau R. DeFeo, Anthony P. McElroy, Amber N. Kim, Chong Jai Tolar, Jakub Int J Mol Sci Article 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. MDPI 2017-06-14 /pmc/articles/PMC5486091/ /pubmed/28613254 http://dx.doi.org/10.3390/ijms18061269 Text en © 2017 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Skvarova Kramarzova, Karolina
Osborn, Mark J.
Webber, Beau R.
DeFeo, Anthony P.
McElroy, Amber N.
Kim, Chong Jai
Tolar, Jakub
CRISPR/Cas9-Mediated Correction of the FANCD1 Gene in Primary Patient Cells
title CRISPR/Cas9-Mediated Correction of the FANCD1 Gene in Primary Patient Cells
title_full CRISPR/Cas9-Mediated Correction of the FANCD1 Gene in Primary Patient Cells
title_fullStr CRISPR/Cas9-Mediated Correction of the FANCD1 Gene in Primary Patient Cells
title_full_unstemmed CRISPR/Cas9-Mediated Correction of the FANCD1 Gene in Primary Patient Cells
title_short CRISPR/Cas9-Mediated Correction of the FANCD1 Gene in Primary Patient Cells
title_sort crispr/cas9-mediated correction of the fancd1 gene in primary patient cells
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5486091/
https://www.ncbi.nlm.nih.gov/pubmed/28613254
http://dx.doi.org/10.3390/ijms18061269
work_keys_str_mv AT skvarovakramarzovakarolina crisprcas9mediatedcorrectionofthefancd1geneinprimarypatientcells
AT osbornmarkj crisprcas9mediatedcorrectionofthefancd1geneinprimarypatientcells
AT webberbeaur crisprcas9mediatedcorrectionofthefancd1geneinprimarypatientcells
AT defeoanthonyp crisprcas9mediatedcorrectionofthefancd1geneinprimarypatientcells
AT mcelroyambern crisprcas9mediatedcorrectionofthefancd1geneinprimarypatientcells
AT kimchongjai crisprcas9mediatedcorrectionofthefancd1geneinprimarypatientcells
AT tolarjakub crisprcas9mediatedcorrectionofthefancd1geneinprimarypatientcells