Gene editing strategy for development of an inherited retinal dystrophy model based on iPSC‐derived photoreceptors with mutation in the PROM1 gene

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AbstractPurpose: The PROM1 gene encodes the protein Prominin‐1 which plays a critical role in the morphogenesis of photoreceptors outer segments. Mutations in the PROM1 gene are related to Inherited Retinal Dystrophy (IRD) phenotypes. The c.1354dupT mutation in the PROM1 gene causes a premature stop codon. The aim of the study was to define the more accurate gene editing strategy for development of an IRD model based on iPSC‐derived photoreceptors.Methods: Firstly, the most accurate CRISPR/Cas9 machinery was determined by the web tool for genome editing CRISPOR and the DNAStar Lasergene software. gRNAs were selected, accordingly to their proximity to the mutation position and their specificity and efficiency scores. Then, silent mutations were added to the repair oligonucleotides (Alt‐R HDR Donor Oligos), by using the web tool MolBiotools, to generate a restriction site for the Ssp1enzyme. Afterwards, to confirm the in silico information, in vitro studies were performed. The U2OS cell line and the control iPSC line ([FiPS] Ctrl1‐Ep6F‐5) were transfected with each selected gRNA, their respective repair oligonucleotide, and the endonuclease Cas9‐protein, by using the Neon Transfection System. Finally, a digestion with Ssp1 were performed to specifically detect the edited genomes.Results: Several gRNAs were selected in silico. In vitro studies showed higher genome editing efficiency in two of them (40RE and 47RE). Finally, the gRNA guide 40RE and its respective repair oligonucleotide were selected.Conclusions: In‐silico and in‐vitro studies concluded that the gRNA guide 40RE was the most accurate for development of an in vitro IRD model based on iPSC‐derived photoreceptors with a mutation in the PROM1 gene. Gene editing strategy designed in silico should be validated in vitro before transfecting the target cells.