Direct delivery of Cas9 or base editor protein and guide RNA complex enables genome editing in the retina

Abstract

Genome editing by CRISPR-Cas holds promise for the treatment of retinal dystrophies. For therapeutic gene editing, transient delivery of CRISPR-Cas9 is preferable to viral delivery which leads to long-term expression with potential adverse consequences. Cas9 protein and its guide RNA, delivered as ribonucleoprotein (RNP) complexes, have been successfully delivered into the retinal pigment epithelium in vivo. However, the delivery into photoreceptors, the primary focus in retinal dystrophies, has not been achieved. Here, we investigate the feasibility of direct RNP delivery into photoreceptors and retinal pigment epithelium cells. We demonstrate that Cas9 or adenine-base editors complexed with guide RNA, can enter retinal cells without the addition of any carrier compounds. Once in the retinal cells, editing rates vary based on the efficacy of the guide RNA and the specific location edited within the genes. Cas9 RNP delivery at high concentrations, however, leads to outer retinal toxicity. This underscores the importance of improving delivery efficiency for potential therapeutic applications in the future.

Keywords: CRISPR/Cas9; MT: Delivery Strategies; RPE; base editor; gene editing; gene therapy; inherited retinal dystrophy; photoreceptor; retina.

Graphical abstract

Figure 1

Direct Cas9 RNP delivery induces indels in vivo in the RPE and the neural retina (A) Transmission electron microscopy (TEM) analysis of 60 μM Cas9 protein (10-nm-diameter circles are shown in yellow) and Cas9 RNP (17-nm-diameter circles are shown in yellow) imaged using 1% aqueous uranyl acetate as a negative stain. (B) Size (nm) of 30 μM Cas9 protein alone or Cas9 RNPs determined by dynamic light scattering (DLS). Three different measures (in blue, orange, and green but overlapping) were performed at 5-min intervals. Means of the peak by volume (nm) ± standard deviation. d.nm = diameter in nm. (C) Schematic representation of the subretinal injections performed in the eyes of adult wild-type mice. RPE and neural retinas are separated during dissections and analyzed independently. (D) Indels in the RPE and neural retina after subretinal injection of Cas9 RNPs at different concentrations in vivo. NGS analysis was performed 7 days after injection. Each dot represents RPE/neural retina isolated from a single mouse eye. Mean ± SEM. Ordinary one-way ANOVA test, Dunnett’s multiple comparisons test. (E) OCT images 7 days post-injection of buffer solution, or different Cas9 RNP concentration in the injection area (temporal images) in adult wild-type mice.

Figure 2

Localization of Cas9 protein inside the neural retina Eyes were collected 3 days after 2 μL subretinal injection of buffer (control) or 30 μM Cas9 RNPs. (A) Top view of the entire neural retina after segmentation from mouse whole eye after clearing and 3D imaging. Injection zone area is circled in blue. Staining of the photoreceptors (Recoverin, red) and Cas9 protein (green). Scale bar, 200 μm. (B and C) Neural retina cross-sections were stained for nuclei (DAPI, blue), photoreceptors (Recoverin, red), and Cas9 protein (green). OS: outer segment, ONL: outer nuclear layer, OPL: outer plexiform layer, INL: inner nuclear layer, IPL: inner plexiform layer, GCL: ganglion cell layer. (C) Zoom on the photoreceptors outer segment and nuclei.

Figure 3

RNP delivery to the retina using cationic lipids: heterogeneous assemblies of complexes and no efficacy improvement (A–H) TEM analysis of (A and B) Lipofectamine 2000 undiluted and (C–H) different complexes observed when mixing Cas9 RNP and Lipofectamine 2000 (diluted 100×). Monomers of RNP (D and F) are highlighted with black arrows. (I) Size of 30 μM Cas9 RNP complexed with Lipofectamine 2000 determined by DLS. Three different measures (in blue, orange, and green) were performed at 5-min intervals. Means of the peak by intensity (nm) ± standard deviation. d.nm = diameter in nm. (J) Indels induced in the whole RPE and neural retina after 2 μL of subretinal injection of 30 μM Cas9 RNPs complexed with cationic lipids: Lipofectamine 2000 or RNAiMAX in vivo in wild-type (WT) mice. NGS analysis was performed 7 days post-injection. Each dot represents RPE/neural retina isolated from a single mouse eye. Mean ± SEM. Ordinary one-way ANOVA test, Dunnett’s multiple comparisons test. (K) Neural retina cross-sections were stained for nuclei (DAPI, blue), photoreceptors (Recoverin, red) and Cas9 protein (green). OS: outer segment, ONL: outer nuclear layer.

Figure 4

Functional consequences of the RNP delivery and toxicity (A) OCT images 1-month post-injection of buffer solution in the bleb area (dorso-temporal injections), 30 μM Cas9 RNPs or Cas9 RNPs complexed with Lipofectamine 2000 in adult wild-type (WT) mice. (B) ERG analysis of control buffer vs. 30 μM Cas9 RNPs naked in adult WT mice. Only amplitudes for the highest light stimulations are represented (20 cd s/m² candela per square meter) for scotopic and 50 cd s/m² for photopic).

Figure 5

Efficiency of different sgRNAs targeting genes highly expressed in the retina (A) Validation of sgRNAs targeting genes highly expressed in photoreceptors (Sag, Rho, and Pde6b), compared with the sgRNA targeting Vegfa gene by TIDE assay. Transfection of Cas9 RNPs complexed with Lipofectamine 2000 in 661W cell line. Mean ± SEM. Ordinary one-way ANOVA test, Dunnett’s multiple comparisons test. sgRNAs that were selected for further in vivo studies are highlighted in colors. (B) Frequencies of indels induced in the whole RPE and whole neural retina of wild-type (WT) mice after subretinal injection of 30 μM Cas9 RNPs with Sag sgRNA 3; Rho sgRNA1 or Pde6b sgRNA1 and compared with the Vegfa sgRNA. NGS analysis was performed 7 days post-injection. Each dot represents RPE/neural retina isolated from a single mouse eye. Mean ± SEM. Ordinary one-way ANOVA test, Dunnett’s multiple comparisons test. (C) Frequencies of indels induced in the whole neural retina of WT mice after subretinal injection of 30 μM Cas9 RNPs with Sag sgRNA1; Sag sgRNA2 or Sag sgRNA 3. NGS analysis was performed 7 days post-injection. Each dot represents neural retina isolated from a single mouse eye. Mean ± SEM. (D) Heatmap representation of the log2 VST DESeq2 values for the main retinal cell types in three samples in which the whole neural retina was extracted and the three samples in which the photoreceptors were extracted using vibratome. (E) Frequencies of indels induced in the photoreceptors of WT mice after subretinal injection of 30 μM Cas9 RNPs with Sag sgRNA 3. NGS analysis was performed 7 days post-injection. Each dot represents photoreceptors isolated from a single mouse eye. Mean ± SEM.

Figure 6

AAV.Cas9 delivery: efficacy and genomic safety (A and B) Relative expression of SpCas9 (A) and sgRNA 3 (B) 21 days after subretinal injection of AAV.SpCas9 and AAV.sgRNA 3. Results of qPCR after 2ˆ(-ddCT) analysis using β-actin as a reference gene. Mean ± SEM. (C) Indels in the neural retina after subretinal injection of three different doses of AAV.Cas9 and AAV.sgRNA 3. NGS analysis was performed 21 days after injection. Each dot represents a neural retina isolated from a single mouse eye. Mean ± SEM. (D) Table of the five top off-targets of sgRNA 3 according to COSMID (https://crispr.bme.gatech.edu/). MMS: number of mismatches. (E) Indels of the five top off-targets of sgRNA3 in the neural retina. For AAV delivery, the highest dose of 2 × 10¹¹ vg/mL (total) was used. For the RNP delivery, the optimized 30 μM Cas9 RNP was used. NGS analysis was performed 7 days after injection for the RNP and 21 days after injection for the PBS and the AAV. Each dot represents a neural retina isolated from a single mouse eye. Mean ± SEM.

Figure 7

ABE RNP delivery to the retina is able to generate targeted substitution (A) Transmission electron microscopy (TEM) analysis of 30 μM ABE protein and ABE RNP imaged using 1% aqueous uranyl acetate as a negative stain. For the picture of ABE RNP at 200 nm, the Gamma was manually adjusted in order to see details within the protein aggregate. (B) Size of 30 μM ABE protein alone or ABE RNP determined by DLS. Means of the peak by volume (nm) ± standard deviation. PI: polydispesity index. (C) Neural retina cross-sections were stained for nuclei (DAPI, blue), photoreceptors (Recoverin, red), and ABE (SpCas9 protein, green). OS: outer segment, ONL: outer nuclear layer, OPL: outer plexiform layer, INL: inner nuclear layer. (D) Schematic of the base editing strategy. In black bold, the PAM; in blue, the spacer; in red, the targeted nucleotide; and in orange, the bystanders. (E) Frequencies of on-site substitutions in the RPE and the whole neural retina of WT mice after subretinal injection of 30 μM ABE RNPs with Sag sgRNA 3. NGS analysis was performed 7 days post-injection. Each dot represents RPE/neural retina isolated from a single mouse eye. Mean ± SEM. Student’s t test. (F) Correlation between the targeted substitution found at the DNA level and at the cDNA level by NGS sequencing performed 7 days post-injection.