BACKGROUND: Nuclease-based technologies have been developed that enable 
targeting of specific DNA sequences directly in the zygote. These approaches 
provide an opportunity to modify the genomes of inbred mice, and allow the 
removal of strain-specific mutations that confound phenotypic assessment. One 
such mutation is the Cdh23 (ahl) allele, present in several commonly used inbred 
mouse strains, which predisposes to age-related progressive hearing loss.
RESULTS: We have used targeted CRISPR/Cas9-mediated homology directed repair 
(HDR) to correct the Cdh23 (ahl) allele directly in C57BL/6NTac zygotes. 
Employing offset-nicking Cas9 (D10A) nickase with paired RNA guides and a 
single-stranded oligonucleotide donor template we show that allele repair was 
successfully achieved. To investigate potential Cas9-mediated 'off-target' 
mutations in our corrected mouse, we undertook whole-genome sequencing and 
assessed the 'off-target' sites predicted for the guide RNAs (≤4 nucleotide 
mis-matches). No induced sequence changes were identified at any of these sites. 
Correction of the progressive hearing loss phenotype was demonstrated using 
auditory-evoked brainstem response testing of mice at 24 and 36 weeks of age, 
and rescue of the progressive loss of sensory hair cell stereocilia bundles was 
confirmed using scanning electron microscopy of dissected cochleae from 
36-week-old mice.
CONCLUSIONS: CRISPR/Cas9-mediated HDR has been successfully utilised to 
efficiently correct the Cdh23 (ahl) allele in C57BL/6NTac mice, and rescue the 
associated auditory phenotype. The corrected mice described in this report will 
allow age-related auditory phenotyping studies to be undertaken using 
C57BL/6NTac-derived models, such as those generated by the International Mouse 
Phenotyping Consortium (IMPC) programme.