fig3

Figure 3. Double-guide treatment causes large deletions that prevent RD in WT/Rho.LΔ11Δ1 animals. (A) Agarose gel electrophoresis analysis of PCR products. Top row shows PCR products amplified using primers 2 and 4 [Supplementary Material 1] that are 1174 bases apart. The presence of the 1174 bp product indicates genomic DNA that does not contain large deletions. Bottom row contains PCR products amplified using the same genomic DNA samples with primers 1 and 4 [Supplementary Material 1], which are 1971 bases apart. Our standard PCR conditions do not efficiently amplify the full-length PCR product. Bands at 737 bp in this row indicate the presence of large deletions. Darker 737 bp bands in the bottom row in lanes 1 and 5 as well as the respective lighter 1174 bp bands in the top row indicate that the large deletion likely occurred to a greater extent in samples 1 and 5. Sample 4 is an example of a WT/Rho.LΔ11Δ1 animal treated with Sg2+Sg5, in which a large deletion was not introduced successfully. Markers are 1kb ladder (New England Biolabs). (B) Example sequence of a large deletion induced by editing with Sg2 and Sg5. The upper sequence shows the sequences of the Rho.LΔ11Δ1 allele that flank the Sg2 and Sg5 predicted cut sites (illustrated by vertical red lines and scissors icons), with 1234bp of intervening sequence between the cut sites (not shown). Sequences recovered from PCR products (the lower sequence is an example) are consistent with large deletions caused by editing with Sg2 and Sg5. In the example shown, the intervening 1234 bp is missing, as well as one additional guanine nucleotide. This sequence was recovered from the sample shown in lane 1 above. (C) Representative confocal micrograph from a WT/Rho.LΔ11Δ1 animal treated simultaneously with Sg2 and Sg5, in which a large inactivating deletion was detected. Green: mabB630N (rod opsin); red: WGA; blue: Hoechst dye. (D) Dot blot analysis for rod opsin of WT/WT animals treated with the Sg2 guide. Rod opsin was detected with mabB630N (n = 15 per group; P value is from Mann-Whitney U test). (E) Confocal micrographs of retinal sections from WT Sg2-treated animals are indistinguishable from confocal micrographs of retinal sections from untreated WT animals. Green: mabB630N (rod opsin); red: WGA; blue: Hoechst dye.