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...
Autores principales: | , , , , , , |
---|---|
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 |