Project 08

Approach

For that, single-guide RNa for the mutated allele will be designed. Plasmids containing single-guide RNas and the high-fidelity cas9 under a cones and rods promoter derived from the human rhodopsin kinase gene will be used. First, skin fibroblast from mice Prph2 KI/WT will be obtained and maintained in in vitro cultures. After, they will be transfected with the CRISPR system using the lipofectamine method. Mutated allele will be recognized by single-guide RNa and induce a cut in this region. Non-homologous end joining system will re-join the strand adding small inserts and deletions (indels) and interrupting the mutated sequence. As the mutation of interest is dominant, its ablation by CRISPR would be enough for therapy (without need of inserting the correct sequence), maintaining the peripherin expression in the non-mutated allele. After that, the efficiency of the CRISPR-mediated therapy for the reestablishment of the wild type phenotype will be analyzed in retinal organotypic cultures from Prph2 KI/WT mice. Finally, once the efficiency of the CRISPR system has been tested in fibroblasts and retinal organotypic cultures, in vivo treatments will be done to assess the efficacy and safety of the treatment in a living organism. Functional state of the retina at different ages will be assessed by ERG and optomotor test, as described in “specific aim 2”. Retinal structure and morphology will be determined by OCT, immunohistochemistry, confocal microscopy and transmission electron microscopy.