Precise Correction of the Pde6b-L659P Mutation Causing Retinal Degeneration with Minimum Bystander Editing by Advanced Genome Editing Tools.

Abstract

Recently developed base editing (BE), prime editing (PE), and click editing (CE) technologies enable precise and efficient genome editing with minimal risk of double-strand breaks and associated toxicity. However, their effectiveness in correcting real disease-causing mutations has not been systematically compared. Here, we aim to evaluate the potential of BE, PE, and CE technologies in rescuing the retinal degeneration-causing Pde6b (c.1976T>C, p.L659P) mutation. This site is prone to bystander effects, making it an ideal model for comparing the editing outcomes of these 3 novel technologies, particularly their editing precision. We optimized BE, PE, and CE systems in vitro using Pde6b-L659P cell models and compared their editing via deep sequencing. BE and PE had similar efficiency, but PE was the most precise, minimizing bystander edits. CE had lower efficiency and higher indel rates, needing further optimization. Using the optimal PE system for in vivo electroporation in Pde6b-L659P mice, we achieved 12.4% targeted repair with high precision, partially rescuing retinal degeneration. This study demonstrates proof of concept for the precise correction of the Pde6b-L659P mutation causing retinal degeneration using BE, PE, and CE tools. The findings offer valuable insights into the future optimization of precision gene editing techniques and their potential translational applications.