Fig. 2: In vitro optimization of the ABE AAV vectors.
From: High-efficiency base editing in the retina in primates and human tissues
a, Schematic of the dual AAV split-intein ABE8.5m(N) and ABE8.5m(C) vectors. b, Base-editing efficiencies in ABCA41961E/E human retinal organoids with split-Cfa intein versus split-Npu intein and 2× bpNLS versus 4× bpNLS sequences. Results were obtained from three biological replicates and are presented as the mean ± s.d. ***P = 1.70 × 10−4, by three-way mixed-effects ANOVA with Dunnett’s correction, compared to split-Cfa intein and 2× bpNLS. The dashed lines indicate editing rates using a construct with Cfa intein and 2× bpNLS. c, Base-editing efficiencies using different 3′ UTRs in ABCA41961E/E human retinal organoids. Results were obtained from three biological replicates and presented as the mean ± s.d. **P < 0.01, ***P < 0.001, by three-way mixed-effects ANOVA with Dunnett’s correction, compared to rabbit β-globin polyA (rbGlob). The dashed lines indicate editing rates using a construct with rbGlob polyA. d, Base-editing efficiencies using different 3′ UTRs in human retinal explants, and representative immunofluorescence images of ABE(N) and ABE(C) expression in the photoreceptor layer in whole-mount retinas (scale bars, 25 µm). Results were obtained from two biological replicates and are presented as means. The dashed lines indicate editing rates using a construct with rbGlob polyA. Cyan, ABE(N); yellow, ABE(C); gamma correction has been applied to obtain an optimal dynamic range for visualization. e, Base-editing efficiencies using different 3′ UTRs in human RPE/choroid explants. Results were obtained from two biological replicates and are presented as means. The dashed lines indicate editing rates using a construct with rbGlob polyA. Illustrations in b–e and illustrations of the AAV ITRs and AAV capsid in a created with BioRender.com. f, Representative immunofluorescence images of human retinal explants transduced with the AAV9-PHP.eB or AAV5 capsid encoding CMV-eGFP. Scale bars, 500 µm (left) and 25 µm (middle); right, quantification of eGFP-expressing cones and rods. Results were obtained from two to three biological replicates and are presented as the mean ± s.d. Gray, Hoechst; green, eGFP; magenta, arrestin3. g, Base-editing efficiencies with AAV5-SABE1 in human retinal explants and RPE/choroid explants, human iPS cell-RPE cells and sorted cones and rods. Results were obtained from three to four biological replicates and are presented as the mean ± s.d.
