Fig. 3

CRISPR-Cas9 mediated repair of the Cdh23 ^ahl allele in C57BL/6NTac mice preserves age-related high-frequency hearing and sensory hair cell stereocilia bundles. a ABR measurements from 24-week-old C57BL/6NTac mice (Cdh23 ^ahl/ahl) and their heterozygous ‘repaired’ C57BL/6NTac littermates (Cdh23 ^ahl/753A>G). As previously reported, by 24 weeks of age the WT Cdh23 ^ahl/ahl (n = 17) mice show elevated hearing thresholds (>60 dB SPL) for the 32 kHz stimulus, the highest frequency tested. However, at 24 weeks of age the Cdh23 ^ahl/753A>G (n = 13) littermates do not have elevated thresholds at 32 kHz, but instead display thresholds similar to those measured for the 8 and 16 kHz stimuli (~20–35 dB SPL). b By 36 weeks of age the Cdh23 ^ahl/ahlmice (n = 12) have very elevated hearing thresholds (≥80 dB SPL) for the 32 kHz stimulus, showing progression of the high-frequency hearing impairment. However, at 36 weeks of age the Cdh23 ^ahl/753A>G littermate mice (n = 9) still exhibit hearing thresholds within the normal range (~20–35 dB SPL) for all frequencies tested (8, 16 and 32 kHz). These results indicate that the CRISPR/Cas9 repaired Cdh23 allele is sufficient to preserve high-frequency hearing in C57BL/6NTac mice. ABR data analysed using an unpaired t test with Welch’s correction, and shown as mean ± standard deviation. c Scanning electron micrographs of the sensory epithelia in the apex, mid and base regions of the cochlea in WT C57BL/6NTac (Cdh23 ^ahl/ahl) and heterozygous repaired C57BL/6NTac (Cdh23 ^ahl/753A>G) mice at 36 weeks of age. Loss of outer hair cell (OHC) bundles is evident at the cochlear base of Cdh23 ^ahl/ahl mice. No loss of OHC bundles is evident in the age-matched Cdh23 ^ahl/753A>G littermate mice. d, e Cochleograms showing the number of inner hair cell (IHC) and OHC bundles present in the apex, mid and base regions of the cochlea in WT C57BL/6NTac (Cdh23 ^ahl/ahl) (n = 4) and heterozygous repaired C57BL/6NTac (Cdh23 ^ahl/753A>G) (n = 4) mice at 36 weeks of age. By 36 weeks of age, no significant loss of IHC bundles in any cochlear region of Cdh23 ^ahl/ahl or Cdh23 ^ahl/753A>G mice is observed. Significant loss of OHC bundles is found at the cochlear base of Cdh23 ^ahl/ahl mice, whereas no loss is found in the cochleae of Cdh23 ^ahl/753A>G mice. Hair cell count data analysed using an unpaired t test with Welch’s correction, and shown as mean ± standard error of the mean. *p < 0.05, **p < 0.01, ****p < 0.0001, ns not significant