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.

Fig. 1.

Schematic representation of the precise rescue of the Pde6b-L659P point mutation using BE, PE, and CE. Red indicates the target base, and blue indicates the bystander base. Due to the presence of 4 consecutive Cs at this locus, using gene editing tools for repair is prone to undesired bystander editing, making it suitable for comparing editing precision. The schematic diagram was created by biorender.com.

Fig. 2.

In vitro screening of CBE systems for Pde6b-L659P mutation correction. (A) Schematic representation of repairing Pde6b-L659P mutation by the CBE system (created by biorender.com). Bystander1 to 3 represent the C bases surrounding the target C that are easily edited simultaneously, leading to bystander mutations. (B) The 7 sgRNA sequences used for CBE system. Target base, red; PAM sequence, green. (C to F) Editing efficiencies of 7 tested sgRNAs with rA1-nSpRY-CBE (C), AID-nSpRY-CBE (D), CDA1-nSpRY-CBE (E), and eA3G-nSpRY-CBE (F) in the N2a cell model (n = 3 biologically independent experiments).

Fig. 3.

In vitro screening of PE systems for Pde6b-L659P mutation correction. (A) Schematic representation of repairing Pde6b-L659P mutation by the PE system (created by biorender.com). Bystander1 to 3 represent the C bases surrounding the target C that are easily edited simultaneously, leading to bystander mutations. (B) Comparison of editing efficiencies of 5 PE vectors in the N2a cell model. (C) Comparison of editing efficiencies of 5 different epegRNAs in the N2a cell model. (D) Comparison of editing efficiencies of 5 different ngRNAs in the N2a cell model. The ngRNAs have different nick positions (n = 3 biologically independent experiments).

Fig. 4.

In vitro screening of CE systems for Pde6b-L659P mutation correction. (A) Schematic representation of repairing Pde6b-L659P mutation by the CE system (created by biorender.com). Bystander1 to 3 represent the C bases surrounding the target C that are easily edited simultaneously, leading to bystander mutations. (B) Schematic representation of CE-gRNA, clkDNAs, and ngRNAs designed for Pde6b-L659P mutation correction. A total of 4 clkDNAs with varied lengths of PTT or PBS and 2 ngRNAs with varied nicking locations were evaluated. (C) Comparison of editing efficiencies across different CE system combinations in the N2a cell model analyzed by Sanger sequencing. (D) Editing efficiencies of optimal CE system in the N2a cell model analyzed by deep sequencing. (n = 3 biologically independent experiments).

Fig. 5.

Parallel comparison of optimal CBE, PE, and CE systems for Pde6b-L659P mutation correction by deep sequencing. (A) Heat maps showing C-to-T conversion efficiency of CBE, PE, and CE systems for Pde6b-L659P mutation repair in the N2a cell model. Bystander1 to 3 represent the C bases surrounding the target C that are easily edited simultaneously, leading to bystander mutations. (B) Comparison of target editing, bystander editing, and indel frequencies among CBE, PE, and CE systems at the Pde6b on-target site. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns: nonsignificant difference. (C) Evaluation of off-target editing frequencies of CBE, PE, and CE systems at the top 5 predicted off-target sites. OT, off-target (n = 3 biologically independent experiments).

Fig. 6.

In vivo correction of the Pde6b-L659P mutation by electroporation of the PE system. (A) Workflow of subretinal PE plasmid injection and electroporation at P0 (postnatal day 0), followed by rescue effect assessment at P30 (created with BioRender.com). (B) Editing efficiency of untreated and PE-electroporated mouse retinas at the Pde6b-L659P site (n = 3 eyes). (C) Representative deep sequencing result of PE-edited mouse retina at the Pde6b-L659P site. Target base, red dotted box. (D) Western blotting of PDE6B protein expression in WT mice, untreated and PE-treated Pde6b-L659P mice at P30. WT, wild type (n = 3 eyes). (E) Quantification of the relative PDE6B protein level normalized to alpha tubulin. *P < 0.05, ****P < 0.0001.

Fig. 7.

Preservation of retinal photoreceptors in Pde6b-L659P mice after PE treatment. (A) Representative immunofluorescence images of retinal sections stained with anti-PDE6B antibodies in untreated and PE-treated Pde6b-L659P mice at P30. Nuclei are counterstained with DAPI (blue), and GFP signal (green) marks successful electroporation. ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. Scale bar: 50 μm. (B) Immunofluorescence analysis of rhodopsin and cone arrestin in untreated and PE-treated Pde6b-L659P mice at P30. Scale bar: 50 μm. (C) Quantification of the fluorescence intensities of PDE6B, rhodopsin, and cone arrestin in untreated and PE-treated Pde6b-L659P mice. *P < 0.05, **P < 0.01. (D) Quantification of ONL thickness in untreated and Pde6b-L659P mice (n = 3 eyes, 3 values per eye). ****P < 0.0001.