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Morgan L Maeder, Rina Mepani, Sebastian Gloskowski, Maxwell Skor, McKensie Collins, Gregory Gotta, Eugenio Marco, Hari Jayaram, David Bumcrot; Therapeutic Correction of an LCA-Causing Splice Defect in the CEP290 Gene by CRISPR/Cas-Mediated Genome Editing. Invest. Ophthalmol. Vis. Sci. 2016;57(12):1160.
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© ARVO (1962-2015); The Authors (2016-present)
Leber congenital amaurosis (LCA) comprises a genetically heterogeneous group of early-onset retinal disorders characterized by severe loss of vision in the first years of life. Approximately 20% of LCA patients harbor mutations in the CEP290 gene, which exceeds the packaging limit of AAV and is therefore not amenable to traditional gene therapy. Here, we report a gene editing approach in which the CRISPR/Cas9 system is used to modify the endogenous CEP290 locus and restore normal function of the gene.
Using the S. aureus CRISPR/Cas9 system, we employed a dual-cut approach in which two guide RNAs (gRNAs) are used to induce a pair of double strand breaks and excise the region containing the common IVS26 c.2991+1655 A>G mutation. Following transfection of primary fibroblasts derived from LCA10 patients, we quantify deletion rates by droplet digital PCR (ddPCR) and use qRT-PCR and Western blotting to measure expression of wildtype and mutant CEP290. We performed genome-wide specificity profiling of candidate gRNAs by deep sequencing of in silico predicted off-target sites as well as by the minimally-biased GUIDE-Seq method.
Following transfection of DNA encoding gRNA pairs and the S. aureus Cas9 protein, ddPCR quantification showed targeted deletion rates ranging from 10-20% in primary patient fibroblasts. qRT-PCR on the same samples demonstrated increased expression of wildtype CEP290 and concomitant decrease in expression of the aberrantly spliced mutant RNA species. Increased wildtype CEP290 protein expression was also evident by Western blot. Finally, specificity profiling allowed us us to select gRNA pairs with minimal off-target cleavage.
This work supports the development of a gene-editing approach for therapeutic treatment of LCA10 caused by the IVS26 c.2991+1655 A>G mutation in CEP290. The use of the S. aureus CRISPR/Cas9 system enables efficient packaging of the Cas9 gene, as well as two gRNA genes, into a single AAV vector and provides a method for delivery into patient photoreceptors.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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