Prime Editing Strategy to Install the PRPH2 c.828+1G>A Mutation

Area of Science:

• Genetics
• Ophthalmology
• Stem Cell Biology

Background:

• Peripherin 2 (PRPH2) mutations are linked to inherited retinal diseases (IRDs) like retinitis pigmentosa (RP) and macular degeneration.
• PRPH2 is crucial for photoreceptor outer segment structure; mutations cause outer segment disorganization or absence.
• A patient with PRPH2-linked RP presented with widespread retinal pigment epithelium (RPE) atrophy, notably sparing the fovea.

Purpose of the Study:

• To establish an efficient method for introducing specific mutations into induced pluripotent stem cells (iPSCs) for modeling rare genetic eye diseases.
• To develop a prime editing strategy for accurately inserting the pathogenic PRPH2 c.828+1 G>A splice-site mutation into wild-type iPSCs.
• To facilitate the generation of well-laminated retinal organoids for studying the pathobiology of PRPH2-based RP.

Main Methods:

• Development of a prime editing system tailored for precise genetic modification.
• Application of the prime editing strategy to introduce the c.828+1 G>A splice-site mutation in the PRPH2 gene.
• Utilizing engineered iPSCs to generate retinal organoids for disease modeling.

Main Results:

• An efficient prime editing strategy was successfully developed and implemented.
• The pathogenic PRPH2 c.828+1 G>A splice-site mutation was accurately installed in iPSCs.
• The developed method enables the generation of iPSC-derived retinal organoids carrying specific disease-causing mutations.

Conclusions:

• Prime editing offers an effective approach for introducing specific mutations into iPSCs for modeling rare genetic disorders.
• This strategy is crucial for advancing the study of PRPH2-linked inherited retinal diseases using iPSC-derived retinal organoids.
• The developed method holds promise for creating accurate disease models to investigate the pathobiology and potential therapies for IRDs.