Dual-AAV delivering split prime editor system for in vivo genome editing

Abstract

Prime editor (PE), a new genome editing tool, can generate all 12 possible base-to-base conversions, insertion, and deletion of short fragment DNA. PE has the potential to correct the majority of known human genetic disease-related mutations. Adeno-associated viruses (AAVs), the safe vector widely used in clinics, are not capable of delivering PE (∼6.3 kb) in a single vector because of the limited loading capacity (∼4.8 kb). To accommodate the loading capacity of AAVs, we constructed four split-PE (split-PE994, split-PE1005, split-PE1024, and split-PE1032) using Rma intein (Rhodothermus marinus). With the use of a GFP-mutated reporter system, PE reconstituting activities were screened, and two efficient split-PEs (split-PE1005 and split-PE1024) were identified. We then demonstrated that split-PEs delivered by dual-AAV1, especially split-PE1024, could mediate base transversion and insertion at four endogenous sites in human cells. To test the performance of split-PE in vivo, split-PE1024 was then delivered into the adult mouse retina by dual-AAV8. We demonstrated successful editing of Dnmt1 in adult mouse retina. Our study provides a new method to deliver PE to adult tissue, paving the way for in vivo gene-editing therapy using PE.

Keywords: dual-AAV; gene editing; in vivo; prime editor; split.

Graphical abstract

Figure 1

Efficient T-to-C editing by Split-PEs in the reporter cells (A) Schematic of split-PEs split at four different sites (994−995, 1,005−1,006, 1,024−1,025, and 1,032−1,033). RuvC, endonuclease domain; BH, bridge helix; REC, recognition domain; HNH, His-Asn-His endonuclease domain; PI, protospacer-adjacent motif (PAM)-interacting domain; M-MLV RT, engineered Moloney murine leukemia virus (M-MLV) reverse transcriptase (D200N, L603W, T306K, W313F, T330P). (B) Schematic of Rma intein-mediated PE reconstitution through protein trans-splicing. (C) Reporter cell harbors a mutated GFP (GFPm) coding sequencing, containing a premature stop codon in the GFP coding sequence, downstream of the EF1α promoter. The GFP signal would be detected by flow cytometer when the TAG codon was converted to the CAG codon by split-PE. (D) The percentage of GFP-positive cells after different split-PE and pegRNA treatment. (E) The mean fluorescence intensity after different split-PE and pegRNA treatment. (F) The percentage of GFP-positive cells after varying split-PE3 treatment in GFPm reporter cells. (G) The mean fluorescence intensity after different split-PE3 and pegRNA2 treatment. non-guide, reporter cells transfected with full-length PE2 but not pegRNA. PE2 FL, reporter cells transfected with full-length PE2 and pegRNA. Values and error bars represent the mean ± SD of three independent biological repeats. One-way ANOVA is used for calculating statistical significance (NS, not significant; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001).

Figure 2

Prime editing of endogenous sites in HEK293T cells by split-PEs (A) Example of deep-sequencing data analysis of G-to-T conversion at endogenous sites targeted by split-PEs. Single-nucleotide transversion editing efficiency was analyzed by MATLAB, whereas insertion editing efficiency and indel frequency were analyzed by CRISPResso2. (B) Targeted insertion of GTA at RNF2 in HEK293T. (C) Targeted transversion of G to T at VEGFA in HEK293T. (D) Targeted insertion of CTT at HEK3 in HEK293T. (E) Targeted transversion of G to T at PRNP in HEK293T. Editing efficiencies represent sequencing reads that contain the correct edit and do not contain indels among total sequencing reads. Indels were also plotted for comparison. non-guide, HEK293T cells transfected with full-length PE2 but not pegRNA. PE3 FL, HEK293T cells transfected with full-length PE2, pegRNA, and nick-gRNA. Values and error bars represent the mean ± SD of three independent biological repeats. One-way ANOVA was used for calculating statistical significance (∗∗p < 0.01; ∗∗∗p < 0.001).

Figure 3

Efficient editing of endogenous sites by dual-AAV split-PEs in human cells (A) Schematic of split-PE delivered by dual-AAV1. N-terminal half and C-terminal half of split-PEs were packaged into two AAV1 separately, and then the cells were infected with both N-terminal half virus and C-terminal half virus, of which the ratio was 1:1. 5 days post-infection, cells were collected for sequencing. (B) Example of Sanger sequencing to detect editing at endogenous sites targeted by split-PEs. The red arrow indicates the edited site. (C) Targeted insertion of CTT at HEK3 in HEK293T cells. (D) Targeted transversion of G to T at VEGFA in HEK293T cells. (E) Targeted insertion of GTA at RNF2 in HEK293T cells. (F) Targeted insertion of GTA at RNF2 in HeLa cells. Editing efficiencies represent sequencing reads that contain the correct edit and do not contain indels among total sequencing reads, which were analyzed by MATLAB and CRISPResso2. Indels were plotted for comparison. AAV1-GFP, negative control. Values and error bars represent the mean ± SD of three independent biological repeats. One-way ANOVA was used for calculating statistical significance (∗∗p < 0.01; ∗∗∗p < 0.001).

Figure 4

Off-target analysis of endogenous sites targeted by dual-AAV-delivered split-PEs in human cells (A) Editing frequency of two known off-target sites of RNF2 in HEK293T and HeLa cells. (B) Editing frequency of four known top off-target sites of HEK3 in HEK293T cells. (C) Editing frequency of four predicted top off-target sites of VEGFA in HEK293T cells. The edits include all types of base substitutions as well as indels, which were analyzed by CRISPResso2. AAV1-GFP, negative control. Values and error bars represent the mean ± SD of three independent biological repeats. One-way ANOVA was used for calculating statistical significance (∗∗p < 0.01).

Figure 5

Efficient editing of Dnmt1 in adult mouse eye by split-PE1024 delivered with dual AAVs (A) Schematic illustration of subretinal injection. 1.1 × 10¹⁰ vg AAV8 (5 × 10⁹ vg N-terminal half of split-PE1024 + 5 × 10⁹ vg C-terminal half of split-PE1024 + 1 × 10⁹ vg GFP) was injected into 6-week-old mouse retina. Mouse retina genomic DNA was isolated and extracted 6 weeks post-injection. (B) Representative retinal flat-mount showing variable GFP intensity and uneven distribution. 1 week post-injection of 10⁹ vg AAV8-GFP, the mouse retina was isolated. Scale bar, 2,000 μm. (C) Fluorescence image of mouse retina section. RPE, retinal pigment epithelium; OS, outer segment; IS, inner segment; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GC, ganglion cell layer. Scale bar, 200 μm. (D) PCR amplification to detect the AAV genome in mouse retina injected with non-targeting or Dnmt1-targeting dual-AAV split-PEs with the EF1α or CMV promoter. Mock, noninjected control mouse retina. (E) Targeted editing efficiencies at the Dnmt1 site in adult mouse retina. Editing efficiencies represent sequencing reads that contain the correct edit and do not contain indels among total sequencing reads, which were analyzed by MATLAB and CRISPResso2. Indels were plotted for comparison. Retina injected with split-PE1024 and non-targeting pegRNA as the negative control. Values and error bars represent the mean ± SD of more than three independent mice eyes. t test was used for calculating statistical significance (∗p < 0.05).