Genome-Editing Custom Project General
CRISPR-Cas9 technology has emerged as a rapid, highly-precise
method to generate knock-out and knock-in mouse models. We can
perform your genome editing project for you from strategy design
through founder genotyping. We successfully created 34 new mouse
models at a 100% technical success rate in 2018. These included
seven gene deletions, fourteen point mutations, six epitope tags or
STOP codon insertions, four conditional alleles (floxed), one
fluorescent reporter knock-in, and two site-directed transgenes
inserted into the Rosa26 locus.
Basic steps for VGER mouse genome editing
- Contact firstname.lastname@example.org
to schedule an advisory meeting. Following the meeting, the
investigator completes the service request through iLab:
- A genome editing strategy is devised and reviewed with the
investigator. We now have four classes of CRISPR/Cas9 editing
strategies based on the donor DNA format and length of the desired
gene edit. See the attachment below for efficiency data fon each
type of project.
- ctRNAs are ordered and tested in vitro in a
ribonucleoprotein format with tracrRNA and Cas9 protein for
on-target efficiency at the desired genetic locus. See
the "MilliporeSigma 2018 Pricing" attachment below for
current reagent pricing.
- A donor ssDNA is designed for homology-directed repair projects
and reviewed with the investigator.
- Typically, Cas9 protein, ctRNA(s), and a ssDNA oligo or dsDNA
are injected into mouse zygotes at the one-cell stage.
- F0 pups are biopsied for genotyping and screened by a
VGER-developed PCR based assay.
- F0 Founder(s) are analyzed by Sanger Sequencing to identify
those containing the predicted desired gene edit.
- F0 Founder(s) is transferred to the investigator for
breeding to WT and validation of the desired genome edit in the N1
- Breeding and screening of the N1 generation is also available
by estimate upon request.
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.