Prime editing for the investigation of aberrant splicing defect associated with a pathogenic PRPH2 variant

The human Peripherin 2 (<i>PRPH2</i>) gene, essential for the structure and function of photoreceptor outer segments, is implicated in a range of inherited retinal diseases (IRDs). This study focuses on the pathogenic c.828+1G>A <i>PRPH2</i> splice site variant. We employed prime editing (PE) technology to install and correct this variant in human induced pluripotent stem cells (hiPSCs). We developed an all-in-one PE construct, featuring a GFP reporter to facilitate the identification of successfully edited clones. The resulting heterozygous and homozygous hiPSC clones exhibited no detectable off-target mutations or karyotype abnormalities. Crucially, we found in hiPSCs and DD50/DD100 precursor hiPSC-derived retinal organoids that the c.828+1G>A <i>PRPH2</i> mutation leads to activation of a cryptic splice site and intron retention, forming a mutant transcript. Importantly, correction of the c.828+1G>A <i>PRPH2</i> mutation in the homozygous hiPSC clone resulted in the restoration of the canonical <i>PRPH2</i> transcript and a reduction of the mutant transcript. Our findings highlight the potential of PE as a precise and safe method for installing and correcting pathogenic <i>PRPH2</i> mutations in hiPSCs, paving the way for future genotype-phenotype studies and therapy development for <i>PRPH2</i>-mediated IRDs.