Fig. 2

The Anc80L65–SpCas9–Htra2 gRNA therapeutic system protected hair cells from neomycin-induced injury in vitro. a Experimental procedures for the in vitro studies with application of the Anc80L65–SpCas9 therapeutic system. b The indel frequency of the gRNAs was measured in the cultured basilar membrane at 8 days post-transduction with the Anc80L65–SpCas9 therapeutic system using high-throughput sequencing. Dots represent individual values, and bars represent the mean ± SD, n = 3. c Indels causing in-frame versus frameshift mutations in the coding sequence after co-transduction of Anc80L65–SpCas9 and Anc80L65–gRNA. d Representative confocal images of neomycin-treated cochlear explants without Anc80L65–SpCas9–Htra2 gRNA treatment. e Representative confocal images of Myo7a and caspase-3 immunofluorescence in neomycin-treated cochlear explants treated with Anc80L65–SpCas9–Htra2 gRNA for 8 days prior to neomycin exposure. IHC inner hair cell, OHC outer hair cell. Both scale bars (in d and e) represent 20 μm long and apply to all images. f–h Comparison of the numbers of Myo7a⁺ hair cells (f), caspase-3⁺ cells (g), and Myo7a⁺/caspase-3⁺ hair cells (h) per 100-μm sensory epithelium region between the neomycin-treated groups with or without Anc80L65–SpCas9–Htra2 gRNA treatment in the apical, middle, and basal turns of the cochlea. i Comparison of the ratio of Myo7a⁺/caspase-3⁺ cells to Myo7a⁺ cells between the neomycin-treated groups with or without Anc80L65–SpCas9–Htra2 gRNA treatment. Dots represent individual values, and bars represent the mean ± SD in f–i. For statistical analysis in f–i, Mann–Whitney U-tests, unpaired two-tailed Student’s t-tests, or unpaired t-tests with Welch’s correction were used as applicable. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. n = 5