In vivo genome editing rescues photoreceptor degeneration via a Cas9/RecA-mediated homology-directed repair pathway

Abstract

Although Cas9-mediated genome editing has been widely used to engineer alleles in animal models of human inherited diseases, very few homology-directed repair (HDR)-based genetic editing systems have been established in postnatal mouse models for effective and lasting phenotypic rescue. Here, we developed an HDR-based Cas9/RecA system to precisely correct Pde6b mutation with increased HDR efficiency in postnatal rodless (rd1) mice, a retinitis pigmentosa (RP) mutant model characterized by photoreceptor degeneration and loss of vision. The Cas9/RecA system incorporated Cas9 endonuclease enzyme to generate double-strand breaks (DSBs) and bacterial recombinase A (RecA) to increase homologous recombination. Our data revealed that Cas9/RecA treatment significantly promoted the survival of both rod and cone photoreceptors, restored the expression of PDE6B in rod photoreceptors, and enhanced the visual functions of rd1 mice. Thus, this study provides a precise therapeutic strategy for RP and other genetic diseases.

Fig. 1. Cas9/RecA-mediated in vitro genome editing.

(A) Schematic of Cas9- or Cas9/RecA-mediated HDR gene editing. Cas9-mediated HDR required sgRNA, spCas9, and ssDNA donors (dot line frame). Cas9/RecA-mediated HDR required sgRNA, spCas9, RecA, and ssDNA donors (solid line frame). RecA was enriched near sgRNA via the MS2-coated protein. (B) Experimental scheme for gene editing by NHEJ or HDR in the BFP HEK293FT line. (C) Representative FACS analysis of HEK293FT cells from the control (Cas9 without sgRNA group). (D) Representative FACS analysis of HEK293FT cells treated in the Cas9 group, with Cas9, sgRNA, and ssDNA donor. (E) Representative FACS analysis of HEK293FT cells treated in the Cas9/RecA group, with Cas9, sgRNA, ssDNA donor, and RecA. (F) Quantification results of Cas9-mediated HDR and NHEJ efficiencies and Cas9/RecA-mediated HDR and NHEJ efficiencies. n = 7. *P < 0.05 and **P < 0.01, paired Student’s t test. n.s., no significance.

Fig. 2. Cas9/RecA-mediated in vivo gene correction of Pde6b.

(A) Schematic of Pde6b gene correction by Cas9/RecA in rd1 (top) and wild-type (bottom) mice. The Pde6b point mutation (C to A) is marked in red, and the premature stop codon of the Pde6b rd1 mutation is labeled by a red arrowhead. The truncated rd1 protein is in red, and the wild-type sequence loss in the rd1 mutant is in blue. (B) Experimental design for Cas9/RecA-mediated HDR gene correction of Pde6b rd1 mutant mice. Plasmids required for Cas9/RecA were introduced into mouse retinae by retinal electroporation. (C) Schematic of genomic DNA and cDNA sequencing of Cas9/RecA-treated cells. EGFP⁺ cells from treated rd1 mice were collected using glass capillaries, with DNA extracted and enriched by Dde I restricted enzyme digestion for genome sequencing or cDNA collected through Smart-seq2 methods. (D) Representative direct genomic sequencing results from Cas9/RecA-mediated HDR gene correction. The Cas9-treated group (top) did not detect any gene correction. The Cas9/RecA-treated group (bottom) showed A to C conversion (red dashed frame). (E) Representative sequencing result of Pde6b cDNA from the Cas9/RecA-treated group. A was converted to C by Cas9/RecA-mediated gene correction. (F) Representative Western blot results from Cas9/RecA-mediated gene correction, and quantification of levels of PDE6B protein. Western blot for PDE6B shows the recovery of PDE6B expression. Actin was used as a loading control. Two retinae were mixed together for each Western blot sample, and three independent experiments were done. 1/10 wild type, loading ¹/₁₀ volume wild type and ⁹/₁₀ sample buffer to make up the volume; Cas9, rd1 mice treated with Cas9, sgRNA, and donor; Cas9/RecA, rd1 mice treated with Cas9, sgRNA, donor, and RecA.

Fig. 3. Cas9/RecA system rescued photoreceptor degeneration in rd1 mice in vivo.

(A) Representative immunofluorescence images of photoreceptor markers stained in mouse retinae from wild-type, Cas9, and Cas9/RecA HDR-treated rd1 mice electroporated on P0. Green, EGFP cells from Nrl-EGFP–labeled cells; red, cone markers of swOPN and mwOPN; blue, cell nucleus shown by 4′,6-diamidino-2-phenylindole (DAPI). OS, outer segment; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Arrowheads point to positive cells. Scale bars, 10 μm. (B) Quantification of rod photoreceptors from wild-type (n = 3), Cas9/RecA (n = 14), and Cas9 (n = 7) mice electroporated on P0. (C) Quantification of cone photoreceptors from wild-type (n = 3), Cas9/RecA (n = 10), and Cas9 (n = 5) mice electroporated on P0. ***P < 0.0001, unpaired Student’s t-test.

Fig. 4. Partial rescue of photoreceptor degeneration in postmitotic photoreceptors by Cas9/RecA-mediated repair.

(A) Ki67 staining in P0 and P3 mouse central retinae. Green, EGFP cells from Nrl-EGFP–labeled cells; red, Ki67 staining. (B) Representative immunofluorescence images of rod and cone photoreceptor markers stained in mouse retinae from wild-type, Cas9/RecA, and Cas9 mice electroporated on P3. Green, EGFP cells from Nrl-EGFP–labeled cells; red, cone markers of swOPN and mwOPN. Scale bars, 10 μm. (C) Quantification results of rod photoreceptors from wild-type (n = 3), Cas9/RecA (n = 17), and Cas9 (n = 8) mice electroporated on P3, and the mouse retinae analyzed on P31. (D) Quantification of double cone photoreceptors from wild-type (n = 3), Cas9/RecA (n = 10), and Cas9 (n = 7) mice electroporated on P3. ***P < 0.001, unpaired Student’s t-test.

Fig. 5. Visual function of wild-type mice, Cas9/RecA-treated rd1 mice, Cas9-treated rd1 mice, and Gnat1^−/−mice evaluated by ERG with STBs at P14.

(A) Representative flash response images of a-wave formed at low light intensities. (B) Representative flash response images of a-wave formed at medium light intensities. (C) Representative flash response images of a-wave formed at high light intensities. (D) Quantification of ERG a-wave amplitude results for P0 electroporation experiment from Cas9/RecA (n = 7) and Cas9 (n = 4) mice. (E) Quantification assay of ERG a-wave amplitude results for the P3 electroporation group from Cas9/RecA (n = 4) and Cas9 (n = 5) mice. *P < 0.05 and **P < 0.01, unpaired Student’s t-test.

Fig. 6. Retinal function of wild-type, Cas9/RecA-treated, and Cas9-treated mice evaluated by PLR.

(A) Representative images of eye constriction from wild-type, Cas9/RecA, and Cas9 mice. (B) Mice were treated with a light intensity of 1.7 × 10⁶ photons/μm²·s. Wild-type (n = 4), Cas9/RecA (n = 12), and Cas9 (n = 9) mice. *P < 0.05, unpaired Student’s t-test.