Abstract
BackgroundNuclease-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 Cdh23ahl allele, present in several commonly used inbred mouse strains, which predisposes to age-related progressive hearing loss.ResultsWe have used targeted CRISPR/Cas9-mediated homology directed repair (HDR) to correct the Cdh23ahl 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.ConclusionsCRISPR/Cas9-mediated HDR has been successfully utilised to efficiently correct the Cdh23ahl 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s13073-016-0273-4) contains supplementary material, which is available to authorized users.
Highlights
Nuclease-based technologies have been developed that enable targeting of specific DNA sequences directly in the zygote
We describe the use of targeted Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9mediated homology directed repair (HDR) to correct the Cdh23ahl allele directly in C57BL/6NTac zygotes. Both employing offset-nicking Cas9 (D10A) nickase with paired RNA guides and a single-stranded oligonucleotide as donor template, we show that allele repair was successfully achieved
We report the use of offset-nicking CRISPR/Cas9-mediated HDR to efficiently and precisely correct the Cdh23ahl allele directly in C57BL/6NTac zygotes
Summary
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 Cdh23ahl allele, present in several commonly used inbred mouse strains, which predisposes to age-related progressive hearing loss. Conclusions: CRISPR/Cas9-mediated HDR has been successfully utilised to efficiently correct the Cdh23ahl 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. Utilisation of knockout mice generated by this programme would provide a relatively quick and costeffective way to obtain models for the validation of genes arising from the human ARHL studies, and to assess the role of genes in ARHL
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