Comparison of CRISPR/Cas Endonucleases for in vivo Retinal Gene Editing

Abstract

CRISPR/Cas has opened the prospect of direct gene correction therapy for some inherited retinal diseases. Previous work has demonstrated the utility of adeno-associated virus (AAV) mediated delivery to retinal cells in vivo; however, with the expanding repertoire of CRISPR/Cas endonucleases, it is not clear which of these are most efficacious for retinal editing in vivo. We sought to compare CRISPR/Cas endonuclease activity using both single and dual AAV delivery strategies for gene editing in retinal cells. Plasmids of a dual vector system with SpCas9, SaCas9, Cas12a, CjCas9 and a sgRNA targeting YFP, as well as a single vector system with SaCas9/YFP sgRNA were generated and validated in YFP-expressing HEK293A cell by flow cytometry and the T7E1 assay. Paired CRISPR/Cas endonuclease and its best performing sgRNA was then packaged into an AAV2 capsid derivative, AAV7m8, and injected intravitreally into CMV-Cre:Rosa26-YFP mice. SpCas9 and Cas12a achieved better knockout efficiency than SaCas9 and CjCas9. Moreover, no significant difference in YFP gene editing was found between single and dual CRISPR/SaCas9 vector systems. With a marked reduction of YFP-positive retinal cells, AAV7m8 delivered SpCas9 was found to have the highest knockout efficacy among all investigated endonucleases. We demonstrate that the AAV7m8-mediated delivery of CRISPR/SpCas9 construct achieves the most efficient gene modification in neurosensory retinal cells in vivo.

Keywords: AAV (adeno-associated virus); CRISPR (clustered regularly interspaced short palindromic repeats); gene editing; retina; retinal dystrophy.

FIGURE 1

In vitro YFP sgRNA validation and selection. (A) YFP-targeting sequence for sgRNA design. YFP-targeting sgRNAs were designed (3 sgRNAs for SpCas9, 1 sgRNA for SaCas9, 2 sgRNAs for Cas12a, and 2 for CjCas9). (B) T7E1 assay to detect cleavage efficiency for YFP. Expected cleavage products by T7E1 were detected in 2% TAE gel. * Cleavage products around 590 and 260 bp. (C) Representative fluorescence microscopy images showing YFP expression in cells transfected with different CRISPR/Cas constructs. Scale bar: 100 μm. (D) Flow cytometry analysis for sgRNA selection. Data are represented as mean ± SEM for 4–7 independent replicates. Intergroup comparisons were performed using a one-way ANOVA and corrected for multiple comparisons. HEK293A cells without YFP expression were also included as negative control. No significant difference in YFP editing was observed between single and dual CRISPR/SaCas9 vector systems (p = 0.9608). Selected sgRNAs for in vivo testing were SpCas9 YFPsgRNA2, Cas12a YFP sgRNA20nt, and CjCas9 YFPsgRNA2. *p < 0.05, **p < 0.01.

FIGURE 2

AAV7m8 mediated delivery of CRISPR/Cas to the mouse retina in vivo. (A) Schematic diagram of in vivo experiment. Mice were sacrificed 5 months after intravitreal injection. (B) Representative cross section image from retina co-transduced with AAV7m8-CRISPR/Cas and its selected YFP sgRNA. Mouse ID 29, right eye Robust AAV7m8 transductions in the retina were found. Scale bar: 200 μm. Images were taken by a Zeiss spinning disk confocal microscope. (C) Representative retinal whole-mount images from a mouse eye receiving AAV7m8-CRISPR/Cas and its selected YFP sgRNA. Mouse ID 76, right eye. Scale bar: 500 μm. Images were taken using an Olympus Slide Scanner. Please see Supplementary Figure S3 for representative cross-sectional images with higher magnification.

FIGURE 3

Comparison of YFP disruption in retinal cells with different CRISPR/Cas systems delivered by AAV7m8. (A) Schematic of the dual and single vector systems. For dual vector plasmids, the Cas endonuclease was driven by miniCMV or CMV promoter, whilst the sgRNA was driven by U6 promoter and mCherry under the control of CMV promoter to confirm vector transfection. For the single vector systems, an all-in-one plasmid with SaCas9 was designed with the Cas endonuclease being driven by a miniCMV promoter and sgRNA by U6 promoter. For Cas12a, we used the Cas endonuclease from Acidaminococcus (originally designated AsCpf1). A hemagglutinin (HA) tag was fused to the C-terminus of Cas endonuclease in the vector. (B) Representative FACS plots of dissociated retinal cells receiving different AAV7m8-CRISPR/Cas/AAV7m8-YFP sgRNA. The histograms in the lower panels (lower panel) were based on mCherry gating. Dissociated cells from one retina were used in each group. (C) Comparison of AAV7m8 transduction in the retina indicated by mCherry expression by FACS. Data are presented as mean ± SEM for 9–20 independent samples in each group. The D’Agostino-Pearson normality test was performed, and all groups were found to have a Gaussian distribution. Statistical analysis between groups was performed using one-way ANOVA followed by multiple comparisons test. (D) Comparison of YFP disruption in mCherry positive cells by FACS. Data are presented as mean ± SEM for 9–20 independent samples in each group. Data in two groups (Cas12a and Dual SaCas9) were found to not pass the D’Agostino-Pearson normality test, and as such the Kruskal–Wallis test was used. *p < 0.05, **p < 0.01, ***p < 0.001.