Fig. 3.

Effects of Anc80L65–SpCas9 therapeutic system on hair cell survival and hearing function in neomycin-exposure mice. a Experimental overview. b The individual (dot) and mean ± SD (bar) indel frequency of the three gRNAs. n = 4. c Representative confocal images showing immunofluorescence staining with Myo7a (red) 2 weeks after the last dose of neomycin from an injected cochlea and the contralateral (non-injected) cochlea. IHC inner hair cell, OHC outer hair cell. Arrows indicate the missing IHCs. Scale bar represents 20 μm long and applies to all images. d Quantification of cochlear hair cells 2 weeks after the last dose of neomycin. Individual values are shown; bars represent the mean ± SD. Unpaired two-tailed Student’s t-tests or Mann–Whitney U-tests were used as applicable. e Comparison of the ABR thresholds between the non-injected and injected ears 2 weeks after the last dose of neomycin. f Families of ABR waveforms recorded at 2 weeks after the last dose of neomycin from the representative non-injected ear and the single best ear injected with Anc80L65–SpCas9 system. The blue trace indicates the threshold of the non-injected ear while the red trace indicates the threshold of the injected ear. The scale bar applies to all traces. g Comparison of the ABR thresholds between the non-injected and injected ears 4 weeks after the last dose of neomycin. The lighter red lines in e and g represent the ABR threshold of individual injected ears. For statistical analysis in e and g, two-tailed paired t-tests or Wilcoxon matched-pairs signed rank tests were used as applicable. h, i Peak amplitudes (h) and latencies (i) of ABR wave 1 evoked by 90 dB SPL in the Anc80L65–SpCas9–Htra2 gRNA-injected ears compared with the non-injected ears 4 weeks after the last dose of neomycin. Unpaired two-tailed Student’s t-tests or unpaired t-tests with Welch’s correction were used as applicable. Data are presented as the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001