Figure 2:

In vivo genome editing with SaCas9-KKH. (a) Schematic of the AAV vector used in the study. (b) Experimental overview for in vivo studies. (c) Targeted deep sequencing from noninjected or AAV-SaCas9-KKH-gRNA-4.2 injected whole cochleas (mean ± SD). In non-injected animals, background indel frequencies ranged between 0.05 - 0.06%. Every data point represents a unique sequencing reaction from pooled cochleas (Supplementary Table 6 includes the number of cochleas each data point, number of independent sequencing reactions for non-injected: 5 (P7), 2 (P14), 2 (P27), injected: 5 (P7), 2 (P14), 2 (P42), 2 (P55) and 2 (P196 WT)). ANOVA was performed to describe overall difference (F=445.8, p<0.0001) followed by Tukey’s post hoc test (for statistical results see Supplementary Table 1). (d) Indel profiles from AAV-SaCas9-KKH-gRNA-4.2 injected Tmc1^Bth/WT animals. Tmc1^Bth and Tmc1^WT reads are plotted separately. Minus numbers represent deletions, plus numbers represent insertions. Sequences without indels (value=0) are omitted from the chart. (e) The most abundant reads in the AAV-SaCas9-KKH + gRNA 4.2 injected Tmc1^Bth/WT animals, shown separately for Tmc1^Bth (top) and Tmc1^WT (bottom) reads. Representation is similar to Extended Data Figs. 1f. (f) AAV integration into the Tmc1^Bth and Tmc1^WT alleles in non-injected and injected Tmc1^Bth/WT animals at P14-P55. Mean ± SD, number of independent experiments and sequencing reactions: 2 for non-injected and 6 for injected). Supplementary Table 6 includes the age and number of cochleas for each data point. (g) Indel profiles and read abundance at the mRNA level from AAV-SaCas9-KKH-gRNA-4.2 injected Tmc1^Bth/WT animals. Data representation is similar to Extended Data Figs. 1f. (h) Relative read counts for Tmc1^Bth and Tmc1^WT representing mRNA in non-injected and injected animals (Supplementary Table 6 includes age and number of cochleas for each data point). The dashed line represents an equal ratio (ratio=1). (ANOVA, p=0.0001, Dunnett’s multiple comparisons: p=0.0005 for injected (P42) vs non-injected (P42) and p=0.0001 for injected (P55) vs non-injected (P42)).