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 "VGER Gene Editing Service Form" (see
attachment below) and is provided with an estimate.
- 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 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 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 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 appropriate number of microinjection
days for projects designed by the VGER.
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