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Mol Ther. 2017 Sep 6;25(9):1999-2013. doi: 10.1016/j.ymthe.2017.05.015. Epub 2017 Jun 12.

Using CRISPR-Cas9 to Generate Gene-Corrected Autologous iPSCs for the Treatment of Inherited Retinal Degeneration.

Author information

1
Stephen A. Wynn Institute for Vision Research and Department of Ophthalmology and Visual Sciences, Carver College of Medicine, University of Iowa, Iowa City, IA 52241, USA.
2
Stem Cell Program and Division of Hematology/Oncology, Boston Children's Hospital and Dana-Farber Cancer Institute, Howard Hughes Medical Institute, Harvard Stem Cell Institute, Harvard Medical School, Boston, MA 01451, USA.
3
Department of Animal Sciences, Iowa State University, Ames, IA 50011, USA.
4
Stephen A. Wynn Institute for Vision Research and Department of Ophthalmology and Visual Sciences, Carver College of Medicine, University of Iowa, Iowa City, IA 52241, USA. Electronic address: budd-tucker@uiowa.edu.

Abstract

Patient-derived induced pluripotent stem cells (iPSCs) hold great promise for autologous cell replacement. However, for many inherited diseases, treatment will likely require genetic repair pre-transplantation. Genome editing technologies are useful for this application. The purpose of this study was to develop CRISPR-Cas9-mediated genome editing strategies to target and correct the three most common types of disease-causing variants in patient-derived iPSCs: (1) exonic, (2) deep intronic, and (3) dominant gain of function. We developed a homology-directed repair strategy targeting a homozygous Alu insertion in exon 9 of male germ cell-associated kinase (MAK) and demonstrated restoration of the retinal transcript and protein in patient cells. We generated a CRISPR-Cas9-mediated non-homologous end joining (NHEJ) approach to excise a major contributor to Leber congenital amaurosis, the IVS26 cryptic-splice mutation in CEP290, and demonstrated correction of the transcript and protein in patient iPSCs. Lastly, we designed allele-specific CRISPR guides that selectively target the mutant Pro23His rhodopsin (RHO) allele, which, following delivery to both patient iPSCs in vitro and pig retina in vivo, created a frameshift and premature stop that would prevent transcription of the disease-causing variant. The strategies developed in this study will prove useful for correcting a wide range of genetic variants in genes that cause inherited retinal degeneration.

KEYWORDS:

CRISPR; iPSCs; retinal degeneration

PMID:
28619647
PMCID:
PMC5589061
DOI:
10.1016/j.ymthe.2017.05.015
[Indexed for MEDLINE]
Free PMC Article

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