CRISPR-Cas9 technology has emerged as a rapid and precise method
to generate genome edited mouse models. We offer full service mouse
projects from genome editing strategy design through validation of
the heterozygous N1 generation. We also offer injection services,
CRISPR reagent sourcing, and design review for investigators
wishing to design their own models using CRISPR-Cas9
Basic steps for VGER mouse genome editing
- Contact firstname.lastname@example.org
to schedule an advisory meeting. As of March 23, 2020, all advisory
meetings will be held by phone or ZOOM. Following the meeting, the
investigator completes the service request through iLab:
- A genome editing strategy is devised and reviewed with the
investigator. We have four classes of CRISPR-Cas9 genome editing
projects based on the donor DNA format, length of the desired gene
edit, and whether the edit is an insertion or
- Guide RNAs are sourced in a crRNA + tracrRNA format from
MilliporeSigma and tested for activity in a test tube assay. Guides
that pass the test tube activity assay are usually determined to be
sufficiently active in vivo, but more complex projects that require
mutliple guides may benefit from pre-screening in vivo.
- CRISPR editing reagents and a donor DNA, if required, are
delivered into the pronuclei of one-cell fertilized mouse zygotes.
Our standard strain options are C57BL/6J and C57BL/6N. Other
strains may also be used for genome editing. Inquire for more
- Resulting pups are biopsied and screened by PCR and Sanger
- Predicted founders are transferred to the investigator for
breeding and validation of the desired genome edit in the N1
generation. Alternatively, breeding and screening of the N1
generation can be performed by VGER.
- Investigators are provided with a validated genotyping protocol
and a genome editing report that includes a description of what the
model is, how it was designed and produced, and additional critical
information such as correct nomenclature for the new allele.
Vanderbilt Genome Editing Resource – Genome Editing
As of March 2019, VGER has performed embryo microinjections for
a total of 82 successful CRISPR gene editing projects.
100% of the last 26 VGER-designed and executed projects have
been technical successes (as defined by the desired modification
being introduced into a mouse). However, one project caused
embryonic lethality. Thus, live mice have been delivered for
Our projects fall into four categories:
End Joining (NHEJ). We recommend the use of this
high-efficiency editing strategy to create large deletions when
precise breakpoints are not required.
Directed Repair (HDR) with Single-Stranded DNAs (≤180
nucleotides). We recommend the use of this strategy
to introduce small edits, such as point mutations or small protein
tags (e.g., HA or Flag), and for the creation of precise DNA
- HDR with
Single-Stranded DNAs (181-5,000 nucleotides). This
approach enables the modification of longer DNA segments up to
approximately 5 kb. It is currently being used to insert loxP sites
around one or more exons, to introduce multiple point mutations,
and to insert exogenous coding sequences encoding fluorescent
proteins or Cre.
- HDR with
Double-Stranded DNAs (generally > 5 kb). We
recommend using double-stranded (ds) when the desired genome edit
exceeds 5 kb, or when commercial projection of a long ssDNA is not
feasible. We have used this approach to insert two cre-inducible
transgenes into Rosa26, and are currently working to improve the
efficiency of this approach by using 2-cell homologous
We guarantee mouse model delivery for Type I and II projects in
approved mouse strains with an appropriate number of microinjection
days for projects designed by the VGER.
here to contact the core
- Limited Genome Editing Guarantee: The
efficiency gene editing projects depends on design, reagent
quality, the genetic locus, and the type of edit desired. VGER
guarantees delivery of viable (see disclaimer #2) genome edits for
all Type 1 and 2 full-service projects in approved mouse strains
with the required number of injection days. Projects that do not
utilize a VGER designed or that utilize reagents from other sources
are not guaranteed.
- Non-Viable Genome Edits: Genome editing may
cause embryonic or perinatal lethality or infertility, resulting in
the inability to establish a viable line. This is suggested by any
the following observations: small F0 litter size with animals
containing only WT, non-frameshift, or heterozygous frameshift
mutant alleles. We will notify you if we suspect that your
gene-editing project is causing lethality or infertility.
- Undesired Mutations: Genome edited mice are
usually mosaic and will often contain small insertions or deletions
where cleavage occurred and was repaired by non-homologous
end-joining. Random DNA integrations and/or mutations in the DNA
sequence, particularly for longer insertions, may occur. Off-target
editing can occur. VGER chooses guide RNAs with low off-target
prediction scores to minimize the risk of edits at unwanted sites.
In mice, off-target mutations not in linkage with the desired edit
may be segregated over several generations of backcrossing to a WT
strain. VGER is not liable for models containing off-target
mutations, random insertions, or mutations in the DNA sequence
introduced during commercial synthesis or during integration into
- Mouse Husbandry: Mice are sensitive to their
physical environment, with noise and vibration being known to
affect reproductive success and pup survival. Control of these
variables lies with the Division of Animal Care (DAC) and is not
the responsibility of VGER.