CRISPR-Cas9 targeting

The Precision Mouse Engineering Core can develop mice with targeted mutations such as deletions, small insertions or point mutations directly in fertilized embryos. It takes about two months to generate a gene-targeted founder mouse with CRISPR technology. Typically, the positive founders are transferred to the investigator.

The core injects CRISPR reagents into fertilized one-cell-stage embryos. Then the core transfers microinjected embryos into pseudopregnant surrogate mothers who develop, deliver and nurse mutant offspring.

The service includes:

  • Design of guide RNAs (gRNAs) and single-stranded oligo donors.
  • Ordering and generating Cas9-gRNAs.
  • Microinjection of reagents into zygotes and transferal of microinjected embryos into surrogate mothers.
  • ID tagging and tail DNA isolation from pups.
  • Transfer of founder mice to investigator.

Each investigator is responsible for:

  • Confirming targeting design.
  • Screening founder pups.
  • Breeding founders.
  • Mutant genotyping design and establishment.

The core can provide all necessary assistance for procedures, such as screening, founder breeding planning and genotyping, upon request. Contact the core for assistance.

Some important notes about the CRISPR-Cas9 targeting service:

  • We have successfully generated mutants and offer mutagenesis in inbred C57BL/NHsd and FVB/NHsd (from Envigo). We also have modified existing mutants or other strains provided by investigators. Contact the core for gene editing in other strains.
  • Although gRNAs will be designed to have minimal off-targets in coding regions, off-target mutations can occur in founder mice. Backcrossing is highly recommended to eliminate or reduce off-target mutations.
  • Founder mice can be mosaic, meaning that they may have different mutations in different tissues or cells. They may not transmit the mutation found in screening. The core can provide assistance in screening and breeding strategies upon request.

Although the above services are set up to deliver the mutant mice to investigators as efficiently as possible, the core can perform screening, breeding or other services on a fee basis as our schedule allows. Contact us with any request or concern.