Correction of the auditory phenotype in C57BL/6N mice via CRISPR/Cas9-mediated homology directed repair
Mianné et al. Genome Medicine
Correction of the auditory phenotype in C57BL/6N mice via CRISPR/Cas9-mediated homology directed repair
Michelle M. Simon
Steve D. M. Brown
Michael R. Bowl
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 Cdh23ahl 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 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. 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.
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 [
]. 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 [
]. However, the lack of
genomewide 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 [
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
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.
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 (Cdh23ahl)
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 [
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. [
] successfully corrected the Crb1rd8
mutation directly in C57BL/6N zygotes using a
TALENmediated HDR approach, showing recovery of a normal
retinal phenotype in heterozygous repaired animals.
Here, we describe the use of targeted
CRISPR/Cas9mediated HDR to correct the Cdh23ahl 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 (Cdh23ahl/ahl), the heterozygous
Cdh23 repair mice (Cdh23ahl/753A>G) 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.
All animals were housed and maintained in the Mary
Lyon Centre, MRC Harwell under specific opportunistic
pathogen-free (SOPF) conditions, in individually
ventilated cages adhering to environmental conditions as
outlined in the Home Office Code of Practice. All animal
studies were licensed by the Home Office under the
Animals (Scientific Procedures) Act 1986 Amendment
Regulations 2012 (SI 4 2012/3039), UK, and additionally
approved by the Institutional Ethical Review Committee.
Mice were euthanized by Home Office Schedule 1
CRISPR/Cas plasmid generation
Oligonucleotide sequences and quality control of constructs
The sequences of all single-stranded oligonucleotides are
presented in Additional file 1: Table S1. All constructs
were checked using Sanger sequencing (SourceBioscience).
Construction of p_1.1 plasmid backbone A pair of
ssODNs containing two unique restriction sites, StuI
and AflII (T7GibsonF and T7GibsonR, Integrated DNA
Technologies) was annealed and cloned into linearised
gRNA_Cloning Vector (Addgene, 41824; George Church,
Harvard) digested with PvuI and AflII, using Gibson
Assembly Master Mix (New England BioLabs (NEB)). As a
result, the new restriction sites have been integrated
between the T7 promoter and the single guide RNA
(sgRNA) backbone sequence, and can be used to linearise
the plasmid for further cloning of chosen protospacer
Construction of p_1.3_D10A plasmid Cas9 mutant
D10A nickase sequence was PCR amplified from pX335
plasmid (Addgene, 42335; Feng Zhang, MIT) using high
fidelity Expand Long Range dNTPack (Roche) and
oligonucleotides Cas9f4gibson/Cas9r4gibson (Sigma-Aldrich).
The PCR product was cloned using Gibson Assembly
Master Mix (NEB) into linearised p_1.1 plasmid digested
with StuI and AflII in order to express Cas9 mutant
D10A under the T7 promoter.
Construction of p_1.1_sgRNA plasmids For each
sgRNA (Cdh23_U1, U2 and D1), a pair of ssODNs
(Sigma-Aldrich; forward 5′-TAATACGACTCACTAT
AGG-protospacer-3′; reverse 5′-GACTAGCCTTATTT
antisense-3′) was hybridised and cloned using Gibson
Assembly Master Mix (NEB) into linearised p_1.1 plasmid
digested with StuI and AflII in order to express sgRNAs
under the T7 promoter.
In vitro RNA transcription
Cas9 mutant D10A nickase mRNA and sgRNAs were in
vitro transcribed from linear forms of p_1.3_D10A and
p_1.1_sgRNA plasmids, respectively. Plasmids were
linearised with XbaI and phenol-chloroform purified.
mRNA was synthesised using Message Max T7 Arca
Capped Message Transcription Kit (Cellscript) and
polyadenylated using poly(A) polymerase Tailing Kit
(Epicentre). Single-stranded guide RNAs were synthesised
using MEGAshortscript (Ambion).
RNAs were purified using MEGAclear kit (Ambion).
RNA quality was assessed using a NanoDrop (Thermo
Scientific) and by electrophoresis on 2 % agarose gel
containing Ethidium Bromide (Fisher Scientific).
In vitro evaluation of sgRNA efficacy
In vitro efficacy of each in vitro transcribed sgRNA (U1,
U2 and D1) was assessed using the Guide-itTM sgRNA
Screening kit (Clontech) following the manufacturer’s
instructions. Analysis of the enzymatic digestions were
analysed by electrophoresis on 1.5 % agarose gel
containing ethidium bromide (Fisher Scientific).
Pronuclear microinjections of zygotes
Pronuclear microinjection was performed as per
Gardiner and Teboul [
], employing a FemtojJet (Eppendorf )
and C57BL/6NTac embryos. Specifically, injection
pressure (Pi) was set between 100 and 700 hPa, depending
on needle opening; injection time (Ti) was set at 0.5
seconds and the compensation pressure (PC) was set at
Microinjection buffer (MIB; 10 mM Tris–HCl, 0.1 mM
EDTA, 100 mM NaCl, pH7.5) was prepared and filtered
through a 2 nm filter and autoclaved. Cas9 mutant D10A
nickase mRNA, sgRNAs and ssODNs were diluted and
mixed in MIB to the working concentrations of 200 or
100 ng/μl, 100 or 50 ng/μl each and 40 or 20 ng/μl,
respectively. Injected embryos were re-implanted in CD1
pseudo-pregnant females. Host females were allowed to
litter and rear F0 progeny.
Genomic DNA from F0 and F1 animals was extracted from
ear clip biopsies using a DNA Extract All Reagents Kit
(Applied Biosystems). The targeted region was PCR
amplified using high fidelity Expand Long Range dNTPack
(Roche) and genotyping primers Geno_Cdh23_F1/R1 or
F2/R2. PCR products were further purified using a gel
extraction kit (Qiagen) and analysed by Sanger sequencing.
PCR products amplified from DNA obtained from F0
animals that showed mixed sequencing traces were
subcloned using a Zero-Blunt PCR cloning Kit (Invitrogen)
and 12–24 clones per founder were analysed by Sanger
Detection of ‘off-target’ sequence variations
Genomic DNA was prepared from the spleen of the F0
mouse that gave rise to the ‘repaired’ line that
underwent auditory phenotyping. Phenol-chloroform extracted
DNA was assessed using a NanoDrop (Thermo
Scientific), Epoch Microplate Spectrophotometer (Bio-Tek)
and by electrophoresis on 1.5 % agarose gel containing
ethidium bromide (Fisher Scientific). Whole genome
sequencing (WGS) was performed using an Illumina
HiSeq 2000 Sequencer. The sequence was mapped using:
the BWA-mem aligner with default parameters, v.0.7.10;
mouse genome reference mm10 from UCSC (original
GRCm38 from NCBI, January 2012); base Phred quality
cutoff, NA; keep duplicate reads, no; variable read length
support, yes; and, realign gaps, no. Sequence variants
were called by The Genome Analysis Toolkit (GATK)
]. The BAM file was realigned for indel calling by
Indel realigner. Indels were called by GATK’s
HaplotypeCaller and single nucleotide variants (SNVs) were called
by GATK’s UnifiedGenotyper, both using dbSNP version
137 as the background single nucleotide polymorphism
(SNP) set. SNV annotations were done using NGS-SNP
]. Indels were annotated using The Variant Effect
SNVs and indels were then compared against the
precompiled list found in 18 inbred strains from the Mouse
Genome Project [
]. Further filtering was done by
comparing the novel sequence variations with a wild-type
(WT) C57BL/6NTac mouse genome and other in-house
mouse sequences. Only high confidence sequence
variations were considered in this study. High confidence
SNVs and indels refer to those with Phred base quality
>150 and read depth >3.
Coding SNVs were investigated by PCR amplification
(primers are listed in Additional file 1: Table S1) and
Sanger sequencing of DNA from the F0 and four
C57BL/6NTac WT animals, including that of a stud
male employed to produce the microinjected embryos.
Analysis of the sgRNA predicted off-target sites
Genomic DNA from F1 animals was extracted from ear
clips using a DNA Extract All Reagents Kit (Applied
Biosystems). Potential off-target sites predicted by the
WTSI Genome Editing (WGE) webtool for sgRNA_U1
and sgRNA_D1 (design 1), and containing ≤3
mismatches (Additional file 1: Table S2) were PCR amplified
using High fidelity Expand Long Range dNTPack
(Roche) and the corresponding genotyping primers
(Additional file 1: Table S3). PCR amplicons were
gelpurified (QIAGEN) and analysed by Sanger sequencing.
Copy counting for ssODN_U1 in the F0 founder and F1
Genomic DNA from F0 and F1 animals was extracted
from ear clips using DNA Extract All Reagents Kit
(Applied Biosystems). Reaction mixtures (20 μl) contained
1 μl crude DNA lysate, 1× ddPCR Supermix for probes
(Bio-Rad), 225 nM of each primer (two primers per
assay used; Additional file 1: Table S1) and 50 nM of
each probe (one VIC-labeled probe for the reference
gene assay and one FAM-labeled Taqman assay specific
to the ssODN_U1 sequence, designed by Biosearch
Technologies). These were loaded into the Bio-Rad
QX200 AutoDG and droplets generated as per the
manufacturer’s instructions. After droplet generation, the oil/
reagent emulsion was transferred to a 96-well plate
(Eppendorf ) and the samples were amplified (95 °C for
10 min, followed by 40 cycles of 94 °C for 30 s and 58 °
C for 60 s, with a final elongation step of 98 °C for
10 min). The plate containing the droplet amplicons was
analysed as a CNV2 experiment in a QX200 Droplet
Reader (Bio-Rad), with channel 1 as unknown and
channel 2 as the two-copy reference assay. Standard reagents
and consumables supplied by Bio-Rad were used,
including cartridges and gaskets, droplet generation oil and
droplet reader oil. Analysis was performed using
Quantasoft software (Bio-Rad) and copy number determined
using a minimum of 10,000 partitions for each sample.
Auditory-evoked brainstem response
Auditory-evoked brainstem response (ABR) testing was
performed as previously described by Hardisty-Hughes
et al. [
]. Briefly, mice were anaesthetised using a
mixture of ketamine and xylazine and placed on a heated
mat in an acoustic chamber (ETS-Lindgren). Broadband
click and tone-burst stimuli (8 kHz, 16 kHz, and
32 kHz) were presented free field to the right ear of the
mouse. TDT system III hardware and software (Tucker
Davis Technology) were used for stimulus presentation
and response averaging, starting at the highest level
(90 dB sound pressure level (SPL)) and reducing in 5 or
10 dB increments until no response trace could be
observed. Mice that displayed no response to a 90 dB SPL
stimulus were recorded as 100 dB SPL for subsequent
analysis. Recovery of anaesthetized mice was assisted by
Cochlear scanning electron microscopy
Animals were euthanized and excised inner ears were
fixed overnight in 2.5 % gluteraldehyde in 0.1 M
phosphate buffer (Sigma-Aldrich). Fixed ears were decalcified
for 48 hours in 4.3 % EDTA in 0.1 M phosphate buffer
(Sigma-Aldrich). Fine dissection was performed to reveal
the organ of Corti, before osmium tetroxide (Agar
Scientific)–thiocarbohydrazide (Fluka) processing (adapted
from Hunter-Duvar [
]) was carried out. The inner ears
were then dehydrated through increasing strength
ethanol solutions (Fisher Scientific) and critical point dried
using an Emitech K850 (EM Technologies Ltd). The
specimens were then mounted on stubs using silver
paint (Agar Scientific) and sputter coated with platinum
using a Quorum Q150R S sputter coater (Quorum
Technologies). Prepared cochlea were visualised with a
JEOL LSM-6010 (Jeol Ltd) scanning electron
microscope. Hair cell stereocilia bundle counts were
performed by counting the number of adjacent inner hair
cells (IHCs) and outer hair cells (OHCs) to ten pillar
cells. For the analysis the cochlea was divided into three
separate regions (turns), apical (<180° from apex), mid
(180–450° from apex), and basal (450–630° from apex).
Four ears (one ear per mouse) were analysed for each
genotype at each region.
Results and discussion
Strategy for Cdh23ahl allele correction
The Cdh23ahl allele is a synonymous SNP affecting the
last nucleotide of the seventh coding exon of the Cdh23
gene (c.753). The presence of an adenine (A) rather than
a guanine (G) at this position leads to an increased
frequency of exon 7 skipping, predisposing inbred mouse
strains carrying the A allele to age-related hearing loss
]. Our strategy for correcting this allele involved
directly injecting CRISPR reagents into one-cell stage
mouse embryos. This approach has previously been
shown to introduce subtle modifications into the
genome at high efficiency [
]. Given reported concerns
regarding the specificity of the Cas9 nuclease and
potential off-target effects, we opted to use paired offset
guides along with a nickase version (D10A) of the Cas9
protein to correct the B6N.Cdh23ahl allele [
Employing the Zhang Lab CRISPR design tool (http://
crispr.mit.edu/), three protospacers (sgRNA_U1, sgRNA_
U2 and sgRNA_D1) were selected allowing for two
different designs (design 1, U1/D1, +42 nucleotide offset; and
design 2, U2/D1, +13 nucleotide offset) (Fig. 1). The
criteria for protospacer sequence selection was based on the
following previously suggested recommendations: double
nicking resulting in 5′ overhang; low potential off-target
effects; as close as possible to the targeted nucleotide; and
a short offset [
]. In vitro assessment of these sgRNAs
revealed that sgRNA_U1 and sgRNA_D1 both have a high
efficacy for directing Cas9 nuclease activity (85 % and
88 % cleavage, respectively), whereas sgRNA_U2 has a
lower efficacy (30 %) (Additional file 1: Fig. S1). As such,
while design 1 employs two very efficient sgRNAs, the
large offset will likely reduce the overall efficiency of this
design. In comparison, for design 2 the offset distance is
within the optimum range (−4 to +20), but it employs a
less efficient sgRNA that will reduce the overall efficiency
of this second design . Each design required a specific
121 bp ssODN to be synthesised with the desired
correction (A > G) at their centre; these act as DNA donor
templates for HDR. In addition to the Cdh23ahl allele
correction, both ssODN templates also contain substitutions
in the region complementary to the protospacer
sequences (AG > TC in ssODN_U1 and C > T in
ssODN_U2). These additional substitutions were added to
prevent further modification by the CRISPR/Cas9 post
HDR repair. In design 1, the AG > TC substitution does
not change the encoded residue (AGT > TCT, Ser > Ser),
and in design 2 the C > T substitution is within the intron
Correction of the Cdh23ahl allele
For each experimental design, in vitro transcribed Cas9
(D10A) nickase mRNA, two sgRNAs and one ssODN
were co-injected into one-cell-stage mouse embryos. To
optimise the CRISPR/Cas9-mediated HDR, we varied
the concentration of these reagents such that we used a
‘low’ and a ‘high’ concentration for each of the two
designs (Table 1). For design 1, the percentage of survival
(born/injected) was lower when using the higher
concentration. However, this likely reflects the smaller
number of embryos injected with the lower concentration, as
no difference in survival was noted for design 2 when
altering concentration (Table 1). Using design 1, a total of
244 embryos were injected over three microinjection
sessions giving rise to 72 live founder (F0) pups (29.5 %).
Whereas, using design 2 a total of 212 embryos were
injected over two microinjection sessions, giving rise to
32 live F0 pups (15.1 %).
To identify CRISPR-mediated events within the 104 F0
pups, DNA extracted from ear biopsies was utilised for
PCR amplification and Sanger sequencing of the
targeted locus. Animals with a complex genotype (i.e.
mosaic with two or more mutated alleles) or potentially
ET embryos transferred, TG transgenic
Generation of mice carrying the Cdh23753A>G repair using pronuclear injection of CRISPR/Cas9 reagents. For designs 1 and 2, two sets of injections were performed using either 100, 50, 50 and 20 ng/μl or 200, 100, 100
and 40 ng/μl of Cas9 (D10A) nickase mRNA, sgRNAs_U, sgRNA_D and ssODN, respectively. Higher efficiency was obtained when using the higher concentrations. The percentages of mutation rate and legitimate repair
rate have both been calculated using the number of pups born as the denominator
harbouring the correctly repaired allele were further
characterised by sub-cloning and Sanger sequencing of
the targeted region. Of the 72 F0 pups generated using
design 1, 11 were found mutated on-target, including two
containing the repaired Cdh23753A>G allele. For design 2, 4
of the 32 F0 pups were found mutated on-target, including
two containing the repaired Cdh23753A>G allele.
Interestingly, in addition to the repaired Cdh23753A>G allele, these
animals were also carrying alleles incorrectly repaired
through HDR (which we called “illegitimate repair”)
(Fig. 2). Thus, from a total of 456 injected embryos we
recovered 104 pups (22.8 %), 15 of which are transgenic
(14.4 %), including four carrying the correct
Cdh23753A>Grepair (3.8 %) (Table 1). Interestingly, 14 of the 15
transgenic mice (including the four with the repaired
allele) were obtained from microinjections using the
higher concentration of CRISPR/Cas9 reagents, while
only one transgenic mouse was produced using the
The four F0 mice identified as having the correctly
repaired Cdh23753A>G allele were shown to be highly
mosaic at the target region. To further characterise
the alleles present in these mice, PCR amplification of
the targeted locus was undertaken, and the resulting
amplicons were sub-cloned and Sanger sequenced.
This confirmed that the four F0 mice had undergone
CRISPR/Cas9-mediated repair of the Cdh23ahl allele,
all showing legitimate repair sequences (Fig. 2). Going
forward, this experimental design can be employed to
repair the Cdh23ahl allele in other mouse strains, e.g.
To establish heritability of the repaired Cdh23753A>G
allele and to segregate the alleles detected in mosaic
founders, three of the Cdh23753A>G F0 mice were bred to
stock C57BL/6NTac mice.
Analysis of the F1 offspring obtained from each of
these three founders revealed transmission of the
repaired Cdh23753A>G allele (Fig. 2a).
However, analysis of the F1 generation also
demonstrated that alleles represented in F0 somatic cells (e.g.
an ear biopsy) can be absent, or under-represented, in F0
germ cells, as determined by their non-transmission to
the F1 progeny. In addition, the reciprocal can occur
where alleles not detected within the somatic cells of a
F0 mouse are identified in their F1 progeny. This latter
case was observed for both of our design 2 founder mice,
which transmitted an additional WT allele not previously
detected at the F0 stage (Fig. 2b). These data highlight
three important issues associated with CRISPR-aided
mutagenesis: firstly, while F0 mice may give initial insight into
phenotypic outcomes of CRISPR/Cas9 targeting, they are
genotypically unpredictable due to potential mosaicism —
therefore, more detailed studies should be undertaken
using ≥ F1 animals; secondly, analysis of F1 mice may
identify ‘hidden’ alleles not seen in the somatic cells of F0 mice;
and thirdly, it is important to sequence the flanking
regions when producing point mutations by CRISPR-aided
mutagenesis to confirm that targeted mutations are not
associated with unwanted indels.
Analysis of predicted off-target sites
A major concern within the research community
regarding CRISPR/Cas9 technology is the potential for
‘off-target’ events to occur, as these could cause deleterious/
confounding phenotypic traits [
]. In order to
increase the specificity of our system we decided to use
the Cas9 mutant (D10A) double nicking system, which
has been proposed to reduce the likelihood of ‘off-target’
mutations . Moreover, the sgRNAs used in this study
were specifically selected as they show very few potential
off-target sites, particularly on chromosome 10, which
could not be easily segregated out through breeding,
unlike off-target mutations on other chromosomes.
Potential off-target binding sites for the design 1 guides were
determined using the WTSI Genome Editing tool [
When allowing up to four nucleotide mismatches, 55
and 173 off-target sites were predicted for sgRNA_U1
and sgRNA_D1, respectively (Additional file 1: Table
S2). To assess the possibility of off-target Cas9-mediated
damage, WGS was performed for the F0 used to
establish the line that underwent phenotyping as part of
this study. From approximately 152 million paired-end
reads (150 bp), the alignment had a 9× average read
depth with 1.5 % assembly gaps. To enable the
identification of CRISPR/Cas9-induced variants we also
sequenced a WT C57BL/6NTac from our breeding colony.
Sequence variants found to be common between the
CRISPR/Cas9 F0, WT C57BL/6NTac, and those variants
present in public repositories (including the Mouse
Genomes Project and dbSNP [
]) were eliminated
from further analysis. Using a standard mutation
detection tool we searched for potential sequence variations
(SNVs and small indels) in the predicted off-target sites
and surrounding the on-target site. No putative SNVs or
indels were detected at any of the 228 predicted
offtarget sites examined (Table 2). Due to the potential of
mosaicism within the F0 mouse to confound the WGS
data, we also assessed the F1 progeny for the presence of
‘hidden’ off-target damage transmitted through the
germline. We amplified and Sanger sequenced the 14
most closely related off-target sites (three or fewer
mismatches) predicted for sgRNA_U1 and sgRNA_D1
(Additional file 1: Table S3). No sequence variants were
identified, confirming the high specificity of the double
nicking system. In addition to the Cdh23753A>G repair
edit, two additional genome edits were also added as
part of design 1 (Fig. 1 and Table 3). All three edits were
detected by WGS. To investigate the coding (missense,
stop gain/loss and splice) mutation frequency in the F0
repaired Cdh23ahl/753A>G genome, we first used an
automated SNV detection pipeline. This identified 42
potential coding SNVs. Subsequently we manually compared
these with the WT C57BL/6NTac sequence and other
mouse strains from our in-house sequence library. We
found the majority of these coding SNVs (41) were
present at low allele frequencies in more than two
sequences or in regions with misaligned reads, and so were
eliminated from further analysis. The one remaining
coding SNV and six predicted false positives (randomly
selected) were assessed using Sanger sequencing (Table 3).
Of these: four SNVs were found not to be present in the
repaired Cdh23ahl/753A>G sequence and therefore
confirmed as false positives; one SNV (Ubox5) was found to
be present in both the WT C57BL/6NTac sequence and
the repaired Cdh23ahl/753A>G sequence; one SNV remained
Summary of the predicted off-target sites with four or fewer mismatches for both sgRNA_U1 (total of 55 sites) and sgRNA_D1 (total of 173 sites) separated into
genic type. Whole genome sequence analysis of the founder F0 mouse, used to establish the line that was phenotyped in this study, demonstrated no modifications to
be present at these 228 predicted off-target sites
Chr chromosome, B6J Ref C57BL/6J reference genome sequence, F0SNV founder-identified single nucleotide variant, AA amino acid, UD undetermined due to the
repetitive nature of the sequence encompassing the SNV
aPresent in the WT C57BL/6NTac strain
bThese two nucleotide changes were specifically introduced as part of correction design 1, and when both are present led to a synonymous change (p.S242S)
undetermined due to the repetitive nature of the genomic
locus (Vmn2r114); and one SNV (Fam184b) was found
only in the repaired Cdh23ahl/753A>G sequence. Sanger
sequencing of additional C57BL/6NTac mice from our WT
stock identified that the Fam184b SNV is heterogeneously
present, indicating that this mutation has recently arisen
spontaneously within the colony (less than ten
generations, as the WT C57BL/6NTac colony is restocked every
ten generations from the supplier, Taconic Biosciences).
This allele on chromosome 5 will be easily segregated from
the repaired Cdh23ahl/753A>G allele located on
chromosome 10. Importantly, no novel coding small indels were
predicted in the repaired Cdh23ahl/753A>G genome.
Another reported concern regarding genome editing is
the potential for the donor ssODN to randomly
incorporate at strand breaks in the genome. To address this
possibility we interrogated the F0 WGS data, which only
identified the ssODN_U1 sequence at the on-target site.
In addition, copy counting of the ssODN_U1 using
Droplet Digital™ PCR was undertaken for this F0 animal
and its F1 progeny. This confirmed that the ssODN has
only incorporated once within the genome of our
Correction of C57BL/6N auditory phenotype
The age-related hearing loss observed in C57BL/6 mice
(B6J and B6N) has been extensively characterized [
The Cdh23ahl hearing loss susceptibility allele (c.753A)
carried by these inbred strains cause the mice to develop
a high-frequency hearing loss by 3 to 6 months of age
that progresses to a profound impairment by 15 months
of age. Consistent with the exhibited auditory decline,
age-related histopathological changes occur within the
ageing cochleae of C57BL/6 mice. It has been shown
that loss of sensory hair cell stereocilia bundles begins in
the base of the cochlea (the region of the cochlea that
detects high-frequency sound) and gradually spreads
apically (the region that detects low-frequency sound)
with advancing age. While the loss includes both inner
and outer hair cell bundles, the loss of outer hair cell
bundles precedes and is more extensive than inner hair
cell bundle loss [
Hearing assessment by ABR threshold analysis was
undertaken for wild-type C57BL/6NTac (Cdh23ahl/
ahl) mice and heterozygous repaired C57BL/6NTac
(Cdh23ahl/753A>G) littermates (Cdh23ahl/753A>G) at 24
and 36 weeks of age. Figure 3 shows the ABR
threshold means for each auditory stimulus. By 24 weeks of
age the Cdh23ahl/ahl mice (n = 17) already have
significantly elevated hearing thresholds (>60 dB SPL) at
the highest frequency tested (32 kHz), whereas their
lower frequency (8 and 16 kHz) hearing thresholds
are within the normal range (15–35 dB SPL). However, at
24 weeks of age the Cdh23ahl/753A>G mice (n = 13) have
hearing thresholds within the normal range (15–35 dB
SPL) at all frequencies tested (8, 16 and 32 kHz) (Fig. 3a).
By 36 weeks of age the Cdh23ahl/ahl mice (n = 12)
show an expected increase in hearing threshold at
32 kHz (>80 dB SPL) compared with their 24-week
threshold, while their lower frequency hearing
thresholds are not greatly increased. At 36 weeks of age the
Cdh23ahl/753A>G mice (n = 9) still have hearing
thresholds within the normal range (15–35 dB SPL) at all
frequencies tested (8, 16 and 32 kHz) (Fig. 3b).
Sensory hair cells were assessed using scanning
electron microscopy for Cdh23ahl/ahl and Cdh23ahl/753A>G
mice at 36 weeks of age. Figure 3c shows micrographs
taken of the sensory epithelia from three regions of the
cochlea (apex, mid and base). This shows that by
36 weeks of age the Cdh23ahl/ahl mice have a normal
complement of hair cells (one row of IHCs and three
rows of OHCs) in the apex and mid regions of the
cochlea. However, as expected, they show loss of OHC
stereocilia bundles in the base. In contrast, at 36 weeks of
(See figure on previous page.)
Fig. 3 CRISPR-Cas9 mediated repair of the Cdh23ahl allele in C57BL/6NTac mice preserves age-related high-frequency hearing and sensory hair cell
stereocilia bundles. a ABR measurements from 24-week-old C57BL/6NTac mice (Cdh23ahl/ahl) and their heterozygous ‘repaired’ C57BL/6NTac littermates
(Cdh23ahl/753A>G). As previously reported, by 24 weeks of age the WT Cdh23ahl/ahl (n = 17) mice show elevated hearing thresholds (>60 dB SPL) for the
32 kHz stimulus, the highest frequency tested. However, at 24 weeks of age the Cdh23ahl/753A>G (n = 13) littermates do not have elevated thresholds at
32 kHz, but instead display thresholds similar to those measured for the 8 and 16 kHz stimuli (~20–35 dB SPL). b By 36 weeks of age the
Cdh23ahl/ahlmice (n = 12) have very elevated hearing thresholds (≥80 dB SPL) for the 32 kHz stimulus, showing progression of the high-frequency hearing impairment.
However, at 36 weeks of age the Cdh23ahl/753A>G littermate mice (n = 9) still exhibit hearing thresholds within the normal range (~20–35 dB SPL) for all
frequencies tested (8, 16 and 32 kHz). These results indicate that the CRISPR/Cas9 repaired Cdh23 allele is sufficient to preserve high-frequency hearing in
C57BL/6NTac mice. ABR data analysed using an unpaired t test with Welch’s correction, and shown as mean ± standard deviation. c Scanning electron
micrographs of the sensory epithelia in the apex, mid and base regions of the cochlea in WT C57BL/6NTac (Cdh23ahl/ahl) and heterozygous repaired
C57BL/6NTac (Cdh23ahl/753A>G) mice at 36 weeks of age. Loss of outer hair cell (OHC) bundles is evident at the cochlear base of Cdh23ahl/ahl mice. No loss
of OHC bundles is evident in the age-matched Cdh23ahl/753A>G littermate mice. d, e Cochleograms showing the number of inner hair cell (IHC) and OHC
bundles present in the apex, mid and base regions of the cochlea in WT C57BL/6NTac (Cdh23ahl/ahl) (n = 4) and heterozygous repaired C57BL/6NTac
(Cdh23ahl/753A>G) (n = 4) mice at 36 weeks of age. By 36 weeks of age, no significant loss of IHC bundles in any cochlear region of Cdh23ahl/ahl or Cdh23ahl/
753A>G mice is observed. Significant loss of OHC bundles is found at the cochlear base of Cdh23ahl/ahl mice, whereas no loss is found in the cochleae of
Cdh23ahl/753A>G mice. Hair cell count data analysed using an unpaired t test with Welch’s correction, and shown as mean ± standard error of the mean.
*p < 0.05, **p < 0.01, ****p < 0.0001, ns not significant
age the Cdh23ahl/753A>G mice do not show loss of IHC
or OHC bundles, in any region of the cochlea.
Hair cell bundle counts were undertaken to quantify
the number of IHC and OHC stereocilia bundles present
in the different cochlear regions of these mice. Figure 3d
shows there is no difference in the number of IHC
bundles found in Cdh23ahl/ahl and Cdh23ahl/753A>G mice at
36 weeks of age. Figure 3e shows that while there is no
difference in the number of OHC bundles found in the
apex and mid regions of Cdh23ahl/ahl and Cdh23ahl/
753A>G mice at 36 weeks of age, there is a significant
difference in the number of OHC bundles present in the
cochlear base. The Cdh23ahl/ahl mice show a loss of
>50 % of their OHC stereocilia bundles, whereas the
Cdh23ahl/753A>G mice have a full complement of OHC
bundles at the base of the cochlea.
Together, these data show that CRISPR/Cas9-mediated
HDR using ssODN genotypically corrected the Cdh23ahl
allele in C57BL/6NTac mice and phenotypically rescued
their age-related auditory function. The repaired allele is
being maintained on a C57BL/6NTac background and
available from the Frozen Embryo and Sperm Archive
(FESA) at MRC Harwell via MouseBook, an integrated
portal of mouse resources [
We report the use of offset-nicking
CRISPR/Cas9-mediated HDR to efficiently and precisely correct the
Cdh23ahl allele directly in C57BL/6NTac zygotes. Our
sequencing data suggest the approach is highly specific,
with no lesions identified at any of the predicted
offtarget sites. Critically, mice heterozygous for the repaired
allele maintain normal hearing function, with complete
abrogation of both the progressive hearing loss and
sensory cell degeneration phenotypes common to the WT
It has previously been reported that nuclease-mediated
HDR can cause mosaicism in founder animals, and this
was found in our F0 mice [
]. The genotypic complexity
present in the founder animals precludes definitive
phenotypic assessment of these mice and confirms that
detailed phenotype data should be acquired from
subsequent generations (F1 onwards), once the alleles have
been segregated and a precise genotype for each animal
The study of mouse mutants, whether induced,
engineered, or spontaneous, has been invaluable for the
elucidation of genes required for mammalian audition, and
remain the model organism of choice. Importantly,
characterisation of these models has provided mechanistic
insight into the biology of congenital and early-onset
hearing loss by elucidating gene function [
less progress has been made regarding understanding the
genetics and pathological processes associated with
agerelated hearing loss . By crossing to the C57BL/
6NTac.Cdh23753A>G mice generated in this study, mouse
models derived from IKMC embryonic stem cells and
IMPC-produced knockout mutants can be maintained on
an enhanced C57BL/6N background that will permit
investigation of auditory function in aged animals, thus
providing insight into genes required for age-related hearing.
In addition, models maintained on the repaired
background can be employed for age-related behavioural
studies that utilize acoustic stimuli as part of the test paradigm,
such as acoustic startle and pre-pulse inhibition.
Additional file 1: The following additional data are available with
the online version of this paper. Figure S1. In vitro assessment of
sgRNA efficacy. Table S1. The sequences of the oligonucleotides used in
this study. Table S2. The sequences and locations of the predicted off-target
sites for the two sgRNAs used in design 1. Table S3. Oligonucleotide
sequences for Sanger sequencing of sgRNA_U1 and sgRNA_D1 predicted
off-target sites (three or fewer mismatches). (DOCX 922 kb)
ABR: Auditory-evoked brainstem response; ARHL: Age-related hearing loss;
bp: Base pair; CRISPR: Clustered regularly interspaced short palindromic
repeats; DSB: DNA double-stranded break; GATK: Genome Analysis Toolkit;
HDR: Homology directed repair; IHC: Inner hair cell; IKMC: International
Knockout Mouse Consortium; IMPC: International Mouse Phenotyping
Consortium; indel: Insertion or deletion; MIB: Microinjection buffer; NEB: New
England BioLabs; NHEJ: Non-homologous end joining; OHC: Outer hair cell;
PCR: Polymerase chain reaction; sgRNA: Single guide RNA; SNP: Single
nucleotide polymorphism; SNV: Single nucleotide variant; SPL: Sound
pressure level; ssODN: Single-stranded oligonucleotide; TALEN: Transcription
activator-like effector nuclease; WGS: Whole genome sequencing; WT: wild
type; ZFN: Zinc-finger nuclease.
The authors declare that they have no competing interests.
J.M., G.C. and L.T. designed the oligonucleotides, genotyped progeny mice
and validated SNVs. L.C., C.A., A.P. and M.R.B. carried out the auditory
phenotyping and/or data analysis. S.K., M.M.S. and A.M. carried out the WGS
data analysis. M.H. and S.W. undertook colony management and cohort
breeding. S.W., S.D.M.B. and M.R.B. conceived the study. All authors contributed
to the writing of the manuscript. All authors read and approved the final
The authors would like to thank the staff of the Mary Lyon Centre for
providing excellent animal husbandry and microinjection services. We also
thank Dr Richard Talbot of Edinburgh Genomics (University of Edinburgh) for
library preparation and WGS. This work was supported by the Medical
Research Council (MC_U142684175 to SDMB) and Action on Hearing Loss
(PA05 to MRB). L.C. is a Medical Research Council PhD student.
1Mary Lyon Centre, MRC Harwell, Harwell, Oxford OX11 0RD, UK.
2Mammalian Genetics Unit, MRC Harwell, Harwell, Oxford OX11 0RD, UK.
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