Genome Editing

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 technology.

Basic steps for VGER mouse genome editing projects
  1. Contact to schedule an advisory meeting. Following the meeting, the investigator completes the service request through iLab:  iLab Organizer (
  2. 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 replacement. 
  3. Guide RNAs are sourced in a crRNA + tracrRNA format 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.
  4. CRISPR editing reagents and a donor DNA, if required, are delivered into the pronuclei of one-cell fertilized mouse zygotes. Our standard strain options is C57BL/6J. Other strains may also be used for genome editing. Inquire for more information.
  5. Resulting pups are biopsied and screened by PCR and Sanger sequencing.
  6. 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.
  7. 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 Efficiencies

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 25/26 projects.

Our projects fall into four categories: 

  1. Non-Homologous End Joining (NHEJ). We recommend the use of this high-efficiency editing strategy to create large deletions when precise breakpoints are not required.
  2. Homology 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 deletions. 
  3. 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.
  4. 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 recombination.

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.  


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VGER Disclaimers

  1. 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.
  2. 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.
  3. 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 the genome.
  4. 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.

An outline of CRISPR-Cas9 mouse model generation services available through the VGER. Contact for more information.