Mianne, J;
Chessum, L;
Kumar, S;
Aguilar, C;
Codner, G;
Hutchison, M;
Parker, A;
... Bowl, MR; + view all
(2016)
Correction of the auditory phenotype in C57BL/6N mice via CRISPR/Cas9-mediated homology directed repair.
Genome Medicine
, 8
, Article 16. 10.1186/s13073-016-0273-4.
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Correction of the auditory phenotype in C57BL6N mice via CRISPRCas9-mediated homology directed repair.pdf - Published Version Download (2MB) | Preview |
Abstract
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.
| Type: | Article |
|---|---|
| Title: | Correction of the auditory phenotype in C57BL/6N mice via CRISPR/Cas9-mediated homology directed repair |
| Open access status: | An open access version is available from UCL Discovery |
| DOI: | 10.1186/s13073-016-0273-4 |
| Publisher version: | https://doi.org/10.1186/s13073-016-0273-4 |
| Language: | English |
| Additional information: | This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ |
| Keywords: | Science & Technology, Life Sciences & Biomedicine, Genetics & Heredity, GENOME-WIDE ASSOCIATION, HEARING IMPAIRMENT, MOUSE, GENERATION, VARIANTS, SPECIFICITY, CONSORTIUM, NUCLEASES, DATABASE, SYSTEM |
| UCL classification: | UCL UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Brain Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Brain Sciences > The Ear Institute |
| URI: | https://discovery.ucl.ac.uk/id/eprint/10112371 |
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