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Lennart Trimborn, Mert Yanik, Annabella Janise, Knut Stieger; Gene Correction in Photoreceptors based on MMEJ. Invest. Ophthalmol. Vis. Sci. 2016;57(12):1153.
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© ARVO (1962-2015); The Authors (2016-present)
Retinitis pigmentosa comprises a heterogeneous group of retinal dystrophies with a prevalence of 1 in 3500 people. Mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene cause X-linked retinitis pigmentosa (XLRP). More than 80% of the mutations are located in the terminal exon ORF15 of the RPGR gene. Currently, no treatment option exists. The most common gene editing strategy is based on homologous recombination (HR) in combination with highly specific nucleases in mitotic cells. The aim of this study is to develop a strategy to increase the efficacy of micro homology mediated end joining (MMEJ) and HR in cell culture as the basis for a treatment option for XLRP.
An episomal fluorescence based reporter system for MMEJ was generated and tested in HEK293 T cells. It contains two plasmids with 20 bp homologous sequences (HS) defining the homology arms: one vector containing only a promotor and the HS, and the second plasmid with the HS and the cDNA for the blue fluorescence protein (BFP). The recombination is induced by CRISPR/ Cas9 cleavage. In a genome based reporter system, exon ORF15 flanked by HS and under the control of the CMV promotor was stably cloned into HEK293T cells by random integration (ie. the number of integration events is unknown). A plasmid containing renilla luciferase flanked by HS of varying length is used as template. Target sites for CRISPR/cas9 cleavage around the ORF15 sequence have been identified previously. Upon DNA double strand break (DSB) induction, the ORF15 exon is replaced by the luciferase, the luminescence can be measured using a plate reader (Tecan). Different homologous regions in length were generated to distinguish between the HR and MMEJ mediated DNA repair.
The episomal fluorescence based reporter system showed significant repair efficiency (3-5%) with 20 bp HS in the presence of a DSB, which is comparable to HR mediated repair. Efficacy of MMEJ was dependent on the HS sequence. Following a DSB in the HEK293T-ORF15 cell line, luciferase based luminescence was observed, which was correlated to the number of integration events.
The fluorescence / luciferase reporter systems to study HR and MMEJ efficacy will help to find the ideal strategy to repair disease causing mutations within the ORF15 exon. Further studies include the detailed analysis of HR and MMEJ activity in photoreceptors of retinal explants.
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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