SA Genome Editing Facility (SAGE)

A one-stop-shop for custom Knock Out (KO) mice.

Are you interested in fast access to KO mice at a highly competitive price? The SAGE Facility uses cutting edge genome editing technology to generate mutant mice for a wide range of applications.

SAGE uses an innovative new approach to generate KO mice that does not rely on traditional ES cell-based methods. Instead, we use genome editing (CRISPR) technology to directly modify the genome of zygotic (1 cell) embryos. Recent publications have shown that this is a highly efficient approach and we have already generated over 15 KO mouse strains using this method. Genome editing in zygotes offers many advantages over traditional methods including much faster generation of KO animals (within 3-6 weeks of injection), lower cost and complete control over the genetic background.

As part of the University of Adelaide's Robinson Research Institute, the Centre of Molecular Pathology and SAHMRI, the SAGE Facility:

  • Functions as a one-stop-shop for KO mice production
  • Provides rapid generation of mouse models to support grant applications and generation of high quality in vivo data for high impact publications
  • Is the first Genome Editing Facility in Australia

SAGE can generate KO (frameshift/exon deletion) mutants and point mutations, as well as epitope tags, and knock-in reporter (eg. eGFP) alleles on a fee for service basis. We are also developing technology to generate conditional alleles and can offer to generate these on a collaborative basis.

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  • Generating knock out mice

    Strategy 1: Frameshift KO mice (fsKO)

    A Genome Editing Agent (GEA) based on CRISPR technology will be designed by SAGE to target the gene of interest. GEAs will target exonic sequences near the start of the open reading frame. Injection of the GEA into a mouse one cell embryo (zygote) will generate a double stranded break at the target sequence. Repair of the double stranded break by Non Homologous End Joining will result in small deletions (or insertions). A frameshift mutation occurs when the number of nucleotides that are deleted or inserted is not a multiple of three, which will likely result in a dysfunctional protein. Injected zygotes are transferred to psudopregnant females and the resulting pups are screened for indels using PCR and sequencing.

    The process for Strategy 1 includes:

    1. Design and generation of GEAs: SAGE will design a GEA to target the customer's gene of interest. Once the design has been approved by the customer, SAGE will prepare the custom GEA reagent for injection into mouse zygotes. GEAs will be designed to maximise the efficiency of GEA binding at the intended target site and to minimise off target binding.
    2. Injection: For most projects, one injection session will be sufficient to generate the fsKO mouse line. If necessary, two additional injection sessions will be performed. GEAs will be injected into C57Bl/6 mouse zygotes and the surviving embryos transferred into pseudopregnant females.
    3. Screening/genotyping: SAGE will perform the molecular screening assays to identify founders with GEA-induced mutations. This will involve extraction of genomic DNA from biopsy tissue, PCR across the putative mutation site, assaying for mutations by detection of heteroduplex formation, and identifying mutations by Sanger sequencing of PCR protects. Founders with frameshift mutations will then be transferred to the client's animal facility (cost not included).

    Strategy 2: Deletion KO mice (delKO)

    Two GEAs that are complementary to intronic sequences flanking a critical exon or exons of the gene of interest will be injected into mouse zygotes. Simultaneous cleavage of the DNA by both GEAs followed by NHEJ-repair will result in the deletion of the critical exon(s).

    Design and generation of the GEAs, injection and screening/genotyping for delKO projects will be performed as for fsKO mice (see above).

    It is anticipated KO mice will be generated in 3-4 months, although this make take longer during very busy periods.

  • Generation of point mutation and epitope tag alleles

    Using the method of Yang et al (2013) Cell 154, 1-10, SAGE has generated point mutations and epitope-tagged (HA-FLAG) alleles (unpublished data).

    All prices for SA Genome Editing projects includes: design and preparation of the genome editing agent(s) ie CRISPR gRNA, injection into mouse zygotes (3 sessions maximum), transfer of injected zygotes to pseudopregnant females, and screening and sequence verification of mutant alleles in founder mice.

    A deposit of 50% of the total fee is required prior to commencement of the project and is non-refundable. The remaining fee is due upon delivery of the mutant mice. Mouse transport costs are not included in the SAGE fee. A discount is available for Robinson Research Institute and University of Adelaide members

    Please contact SAGE for a more information and to discuss costs.

  • Generation of reporter alleles

    CRISPR-stimulated homologous recombination in zygotes has been used to generate fluorescent reporter alleles (Yang et al (2013) Cell 154, 1-10).

    All prices for SA Genome Editing projects includes: design and preparation of the genome editing agent(s) ie CRISPR gRNA, injection into mouse zygotes (3 sessions maximum), transfer of injected zygotes to pseudopregnant females, and screening and sequence verification of mutant alleles in founder mice.

    A deposit of 50% of the total fee is required prior to commencement of the project and is non-refundable. The remaining fee is due upon delivery of the mutant mice. Mouse transport costs are not included in the SAGE fee. A discount is available for Robinson Research Institute and University of Adelaide members

    Please contact SAGE for more information and to discuss cost.

  • Generation of conditional alleles

    An exciting development in the genome editing field was the recent demonstration that a conditionally targeted allele (containing LoxP sites flanking a critical exon) could be generated using CRISPR technology - Yang et al (2013) Cell 154, 1-10.

    SAGE is currently assessing the efficacy of this published approach and other strategies for conditional allele generation. Given the widespread use of conditional alleles, SAGE can offer to generate conditional alleles on a collaborative basis for a significant discount.

    Please contact SAGE for a quote.

  • Collaboration

    SAGE is willing to generate more technically challenging alleles (point mutations, reporter and conditional) on a collaborative basis. Collaborative projects will be available at a discounted fee and entitle SAGE to coauthorship of the initial publication describing the mutant strain.

  • Costs

    All prices for SA Genome Editing projects includes: design and preparation of the genome editing agent(s) ie CRISPR gRNA, injection into mouse zygotes (3 sessions maximum), transfer of injected zygotes to pseudopregnant females, and screening and sequence verification of mutant alleles in founder mice.

    A deposit of 50% of the total fee is required prior to commencement of the project and is non-refundable. The remaining amount is due upon delivery of the mutant mice. Mouse transport costs are not included in the SAGE fee. A discount is available for Robinson Research Institute and University of Adelaide members.

    Please contact SAGE for a quote or for more information.

For more information about the SAGE Facility and to discuss your mutation of interest please contact Ms Melissa White or the Facility Director, Prof Paul Thomas. SAGE is located at SAHMRI - North Terrace, Adelaide.