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

Genome Medicine
Joffrey MiannéMichael R Bowl

Abstract

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. 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 ha...Continue Reading

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Citations

Jul 8, 2016·Human Genetics·Channabasavaiah B GurumurthyXue Zhong Liu
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Methods Mentioned

BETA
Knockout
PCR
electrophoresis
biopsies
genotyping
dissection
transgenic
biopsy
targeted mutations
scanning electron microscopy

Software Mentioned

WTSI Genome Editing ( WGE )
WTSI Genome Editing tool
Genome Analysis Toolkit ( GATK )
mem
Indel realigner
GATK
BWA
UnifiedGenotyper
TDT system
MouseBook

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