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

被引:18
|
作者
Kramarzova, Karolina Skvarova [1 ,2 ]
Osborn, Mark J. [1 ,3 ,4 ,5 ]
Webber, Beau R. [1 ]
Defeo, Anthony P. [1 ]
McElroy, Amber N. [1 ]
Kim, Chong Jai [6 ]
Tolar, Jakub [1 ,3 ,5 ]
机构
[1] Univ Minnesota, Dept Pediat, Div Blood & Marrow Transplantat, Minneapolis, MN 55455 USA
[2] Charles Univ Prague, Fac Med 2, Dept Pediat Hematol & Oncol, CLIP, Prague 15006, Czech Republic
[3] Univ Minnesota, Stem Cell Inst, Minneapolis, MN 55455 USA
[4] Univ Minnesota, Ctr Genome Engn, Minneapolis, MN 55455 USA
[5] Univ Minnesota, Asan Minnesota Inst Innovating Transplantat, Minneapolis, MN 55455 USA
[6] Asan Minnesota Inst Innovating Transplantat, Asan Med Ctr, Asan Inst Life Sci, Seoul 138736, South Korea
基金
新加坡国家研究基金会; 美国国家卫生研究院;
关键词
gene editing; CRISPR/Cas9; Fanconi anemia; fibroblasts; Fanconi anemia D1; poly adenosine diphosphate-ribose polymerase inhibitors; FANCONI-ANEMIA PATIENTS; ZINC-FINGER NUCLEASES; STRAND BREAK REPAIR; DNA-REPAIR; CRISPR-CAS9; NUCLEASES; HEMATOPOIETIC STEM; SOMATIC MOSAICISM; PARP INHIBITORS; THERAPY; INTEGRATION;
D O I
10.3390/ijms18061269
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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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页数:15
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