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

Area of Science:

• * Molecular Biology
• * Genetics
• * Ophthalmology

Background:

• * CRISPR/Cas gene editing shows potential for treating rare genetic diseases like inherited retinal dystrophies.
• * Challenges remain in delivering CRISPR/Cas components and achieving efficient homology-directed repair (HDR) in differentiated cells.

Purpose of the Study:

• * To investigate CRISPR/Cas9 and Cas12 nuclease delivery via lipoplexes in patient-derived induced pluripotent stem cells (hiPSCs) for treating Stargardt and Best diseases.
• * To evaluate gene editing efficiency, DNA cleavage, and HDR-based correction in hiPSC-derived retinal pigment epithelium (RPE) and retinal organoids.

Main Methods:

• * Utilized hiPSCs from patients with Stargardt disease (ABCA4 variant) and Best disease (BEST1 variant).
• * Delivered CRISPR/Cas9 and Cas12 nucleases using lipoplexes and compared with electroporation in hiPSC-derived RPE and retinal organoids.
• * Assessed transfection efficiency, sgRNA-mediated DNA cleavage, and HDR correction rates.

Main Results:

• * Achieved precise repair of the pathogenic BEST1 variant in hiPSC-derived RPE cells, with Cas12 demonstrating over 10% HDR efficiency.
• * Edited RPE cells maintained normal morphology and expressed key maturity markers.
• * Retinal organoids showed limited transfection and no detectable DNA cleavage, indicating a need for optimization in complex tissues.

Conclusions:

• * Demonstrated precise, single-nucleotide mutation correction in patient-derived RPE using lipoplex-delivered CRISPR/Cas9 and Cas12.
• * Established a proof of concept for CRISPR/Cas-based therapeutic strategies for inherited retinal dystrophies.
• * Highlighted the potential for clinical translation of gene editing in treating retinal diseases.