Correction of the auditory phenotype in C57BL/6N mice via CRISPR/Cas9-mediated homology directed repair

The most common form of sensory disability in the human population is age-related hearing loss (ARHL), which not only causes communication difficulties, but also is associated with social isolation, depression and reduced physical and cognitive function [1]. ARHL is known to be a complex disorder with both genetic and environmental components. Given the high prevalence of the condition (60 % of people aged ?70 years), coupled with an ageing population, there is a drive to elucidate the genes and pathology associated with ARHL, thus enabling the development of potential therapeutic strategies. To date, several major genetic studies have investigated adult hearing function in humans, providing candidate gene sets for ARHL susceptibility factors [2–6]. However, the lack of genome-wide significance and absence of replication between studies means validation of these candidate ARHL genes in a model organism is required.

The International Mouse Phenotyping Consortium (IMPC) aims to produce knockout mice for every gene in the mouse genome and test each mutant line through a broad-based phenotyping pipeline, in order to elaborate upon the function of every mouse gene [7, 8]. IMPC uses mutant embryonic stem cells developed by the International Knockout Mouse Consortium (IKMC). The mice generated by the IMPC are preserved in repositories and are available to the scientific community. Utilisation of knockout mice generated by this programme would provide a relatively quick and cost-effective way to obtain models for the validation of genes arising from the human ARHL studies, and to assess the role of genes in ARHL.

A main strength of the IKMC and IMPC programmes is that the respective embryonic stem cell resource and knockout mice produced are generated in a single inbred strain background, namely C57BL/6NTac. However, a major genetic impact of the use of C57BL/6N and the related C57BL/6J strain is that they harbour a fixed hypomorphic allele in the Cadherin23 gene (Cdh23
^ahl
) that causes these mice to exhibit a high-frequency hearing loss by 3–6 months of age that progresses to a profound impairment by 15 months of age [9, 10]. This renders C57BL/6NTac an unsuitable background strain for investigating potential ARHL-causing genes.

Over recent years several technologies have been developed that allow targeting of specific DNA sequences directly in the zygote, e.g. zinc-finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), and clustered regularly interspaced short palindromic repeats (CRISPR/Cas9). These are nuclease-based approaches which generate DNA double-stranded breaks (DSBs) at user-defined genomic sequences. The presence of a DSB initiates a repair mechanism that typically leads to non-homologous end joining (NHEJ), resulting in insertion or deletion (indels) events at the targeted locus. However, if the nucleases are used in conjunction with a donor DNA sequence carrying the desired insert with flanking homology to the targeted region, integration by homology directed repair (HDR) can occur. Recently, Low et al. [11] successfully corrected the Crb1
^rd8
mutation directly in C57BL/6N zygotes using a TALEN-mediated HDR approach, showing recovery of a normal retinal phenotype in heterozygous repaired animals.

Here, we describe the use of targeted CRISPR/Cas9-mediated HDR to correct the Cdh23
^ahl
allele directly in C57BL/6NTac zygotes. Using two different designs, both employing offset-nicking Cas9 (D10A) nickase with paired RNA guides and a single-stranded oligonucleotide (ssODN) as donor template, we show that allele repair was successfully achieved.

Importantly, we demonstrate that unlike inbred C57BL/6NTac mice (Cdh23
^ahl/ahl
), the heterozygous Cdh23 repair mice (Cdh23
^ahl/753AG
) have normal hearing thresholds and a full complement of cochlear sensory hair cell stereocilia bundles at 36 weeks of age. Thus, the repaired C57BL/6NTac mice described here provide an enhanced defined genetic background in which IMPC knockout mouse models can be generated, which are suitable for both assessment of age-related auditory function and age-related behavioural studies that utilize acoustic stimuli as part of the test paradigm.