Extended Data Fig. 5.

Effects of SaCas9-KKH+sgRNA4.2 on inner ear function in WT & Bth mice. (a) Representative sensory transduction currents recorded at P14-16 from inner hair cells (IHCs) and outer hair cells (OHC) of WT mice injected with AAV-SaCas9-KKH-sgRNA4.2 at P1-P2. (b) Mean ± SEM maximal transduction current amplitudes for P14-16 IHCs (left, n = 8) and OHCs (right, n = 6) from WT mice injected with AAV-SaCas9-KKH-sgRNA4.2 at P1-P2. (c) Families of ABR waveforms recorded at eight weeks from an uninjected Tmc1^WT/WT mouse (left) and a Tmc1^WT/WT mouse injected with AAV-SaCas9-KKH-gRNA-4.2 at P1 (right). Bolded traces indicate threshold. Scale bar applies to all traces. (d) Mean ± S.D. ABR thresholds plotted as a function of stimulus frequency for six Tmc1^WT/WT mice (black, n = 6) and three Tmc1^WT/WT mice injected with AAV-SaCas9-KKH-gRNA-4.2 (gray) at 24 weeks of age (p = 0.9). (e) Mean ± S.D. ABR thresholds plotted as a function of stimulus frequency for six Tmc1^WT/WT mice (black), nine Tmc1^Bth/WT, and five Tmc1^Bth/WT mice injected with AAV-SaCas9-KKH-gRNA-4.2 (green) at 24 weeks of age. 8-kHz thresholds for injected: 38 ± 11 dB (n = 9) and uninjected: 64 ± 19 dB (n = 8) were significantly different (p = 0.004, unpaired two-tailed t-test). ABR thresholds at higher frequencies (22kHz) for injected (84 ± 15 dB, n = 9) and uninjected Tmc1^Bth/WT mice (103 ± 5 dB, n = 8; p = 0.004, unpaired two-tailed t-test). (f) Representative confocal images of 100-μm cochlear sections harvested at 24 weeks from the 8, 16, and 32 kHz regions from three uninjected Tmc1^WT/WT mice (top), and three Tmc1^WT/WT mice injected with AAV-SaCas9-KKH-gRNA-4.2 (bottom). The tissue was stained for MYO7A (red) and actin (green). (g) Mean ± S.D. number of surviving IHCs (left) and OHCs (right) per 100-μm section (n = 3).