Fig. 5: Intravitreal injection of Prpf31-KO vectors results in structural and functional changes primarily in the inner retina.

a Illustration of experimental approach. KO vector 7m8-U6-gRNA2-CAG-GFP (2 µl, 2.13E + 12 vg/ml) was delivered to SpCas9-expressing mice via intravitreal injection. 7m8-CAG-GFP (2 µl, 2.46E + 12 vg/ml) was delivered to the contralateral eye as control. b Deep sequencing of genomic DNA from whole retina of the KO eyes 5 weeks (p.i.) revealed 3.73 ± 0.19% editing of Prpf31 at the gRNA2 target region (n = 3). c Mutations found in modified Prpf31. The main editing event identified was the insertion of an adenosine at the predicted cut site (3 bp before PAM). d-h Retinal conditions at 15 weeks (p.i.) d Fundus images. Retina pallor was observed alongside blood vessels in KO eyes (arrows), suggesting retinal lesion (CON n = 19, KO n = 19). e OCT of retinas and quantification of total retinal thickness (neural retina and RPE) and IPL thickness (n = 8). Red arrows, IS/OS junction. ONH, optic nerve head; T, temporal; N, nasal. Scale bar vertical, 100 µm; Scale bar horizontal, 200 µm. f Retinal cross sections. GFP (green) was expressed in all retinal layers with the strongest expression in the inner retina and Müller cells. KO eyes had significant thinning of the IPL and INL, loss of the bipolar cell marker (PKCα in red) with shortened bipolar processes, as well as loss of the ganglion cell/amacrine cell marker (PAX6 in grey) compared to control eyes. Scale bar, 50 μm. g Quantification of ERG scotopic and photopic a- and b-waves (n = 9). The photopic and scotopic b-wave amplitudes in KO eyes decreased dramatically, while scotopic a-waves were moderately reduced. h Representative scotopic ERG recordings stimulated by a 10 cd·s/m² flash. Scale bar vertical, 50 µV; Scale bar horizontal, 50 ms. Data were shown as mean ± SEM. Significance of the overall treatment factor was analyzed using two-way ANOVA and stated as a P value in the plots. Significance of the treatment factor at each distance from ONH (for OCT) or each flash intensity (for ERG) was calculated by Sidak multiple comparisons test and marked as *P < .05; **P < .01; ***P < .001; ****P < .0001. ONH = optic nerve head. ONL = outer nuclear layer; INL = inner nuclear layer; IPL = inner plexiform layer. Source data are provided as a Source Data file.