Fig. 3

CasRx selectively disrupts the Tmc1^Bth transcript in Bth mice. a Schematic of the AAV vector encoding CasRx and sgRNA3 (upper), and a control NT vector (lower). b Outline of the in vivo experiments. Mice were injected with AAV (~5 × 10⁹ vg) at P1–P2, and the organs of Corti were dissected and cultured at P5, and hair cell physiology was analyzed at P15–P16. Injected mice were sequenced after 2 weeks followed by hearing tests (ABR and DPOAE) after 4, 8, and 12 weeks, immunohistochemistry, and scanning electron microscopy at 10 weeks after injection. c The percentage of deep sequencing reads of Tmc1^Bth and Tmc1⁺. Pie charts indicate the mean composition of Tmc1^Bth and Tmc1⁺ transcripts in these samples, sequences show the single-nucleotide difference between the Tmc1^Bth and Tmc1⁺ transcripts (52.83 ± 5.33%, 53.39 ± 4.8%, and 14.88 ± 9.77% Tmc1^Bth transcript for non-injected, injected with AAV-CasRx + NT, and injected with AAV-CasRx + sgRNA3, respectively. n = 3 mice, data are shown as the mean ± SD). d Deep sequencing analysis of the ratios of transcripts between Tmc1^Bth and Tmc1⁺ for non-injected mice (n = 3 mice), mice injected with AAV-CasRx + NT (n = 3 mice), and mice injected with AAV-CasRx + sgRNA3 (n = 3 mice), respectively. Data are shown as the mean ± SD, **p < 0.01, P-values were determined by one-way ANOVA with Dunnett’s multiple comparisons test. e mRNA expressions in the cochlea at 2 weeks after injection as measured by RT-qPCR. The expression of CasRx mRNA (n = 11 mice), total Tmc1 mRNA (n = 5 mice), Tmc1^Bth mRNA (n = 4 mice), and Tmc1^Bth (n = 5 mice) between injected with AAV-CasRx + sgRNA3 and non-injected contralateral ears were showed in graphs. Relative mRNA expression levels were calculated with the ΔΔCt algorithm. Data are shown as the mean ± SD, *p < 0.05, ***p < 0.001, P-value was determined by unpaired two-tailed t-test. f Amplification of the Tmc1^Bth sequence. The amplicon was detected by a pair of specific targeting primers with heterozygous templates, and the primers cannot amplify with the wild-type template. g Representative MET recordings and maximal MET current amplitudes of apical IHCs at the equivalent of P15–P16. h The MET current amplitude was 461.134 ± 74.978 pA, 442.458 ± 82.805 pA, and 344.409 ± 114.591 pA in Tmc^+/+ mice (n = 16 OHCs), non-injected Tmc1^Bth/Bth mice (n = 18 OHCs), and Tmc1^Bth/Bth mice injected with AAV-CasRx + sgRNA3 (n = 16 OHCs), respectively. Data are shown as the mean ± SD, **p < 0.01, P-values were determined by one-way ANOVA with Sidak’s multiple comparisons test