Precise CRISPR/Cas9 and Cas12 Correction Using Lipoplexes in Retinal Models Derived from Patients with Inherited Retinal Dystrophies

TL;DR

Researchers used CRISPR/Cas9 and Cas12 with lipoplexes to successfully correct a genetic mutation in retinal cells from patients with inherited eye diseases.

Contribution

This is the first demonstration of precise gene correction in patient-derived retinal pigment epithelium using CRISPR/Cas9 and Cas12 delivered via lipoplexes.

Findings

CRISPR/Cas12 achieved over 10% HDR correction efficiency in edited RPE cells.

Corrected RPE cells maintained normal morphology and maturity markers.

Retinal organoids showed low transfection efficiency and no detectable DNA cleavage.

Abstract

Gene editing, particularly CRISPR/Cas technology, represents a promising approach for the treatment of rare genetic diseases, including inherited retinal dystrophies, for which effective therapies are largely unavailable. Despite extensive research investigating gene editing across a wide range of cell types, transient delivery of CRISPR/Cas components and efficient homology-directed repair (HDR) in differentiated cells remain challenging. In this study, we employed hiPSCs derived from patients with Stargardt disease or Best disease, carrying pathogenic variants in ABCA4 or BEST1, respectively, to explore gene editing in human models. CRISPR/Cas9 and Cas12 nucleases were delivered into hiPS-derived retinal pigment epithelium (RPE) and retinal organoids using lipoplexes and compared with electroporation. We evaluated transfection efficiency, sgRNA-mediated DNA cleavage, and HDR-based correction. Precise repair of the pathogenic BEST1 variant was successfully achieved in hiPS-derived RPE cells using both nucleases, with Cas12 yielding the highest efficiency, exceeding 10% of HDR correction. Edited RPE cells preserved normal morphology and expressed specific maturity markers. In contrast, retinal organoids exhibited moderate transfection efficiency but showed no detectable CRISPR/Cas-induced DNA cleavage, highlighting the need for further optimization of gene editing in more complex cellular tissues. This study demonstrates, for the first time, precise correction of a single-nucleotide mutation in patient-derived RPE using CRISPR/Cas9 and Cas12 delivered using lipoplexes. These findings underscore the therapeutic potential of CRISPR/Cas-based strategies for inherited retinal dystrophies and provide a proof of concept for future clinical approximations.

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