Skip to main content

CRISPR/Cas9 Gene Editing

We are now using CRISPR/Cas9 to create targeted mutations directly in embryos or in embryonic stem (ES) cells. We have successfully used this technology to create the following mutations directly in single-cell fertilized embryos:

  • KO mutations
  • Single point mutation KI
  • Simultaneous targeting/functional deletion of two genes
  • Insertion of epitope tags within first exon to create tagged fusion protein
  • New mutations in established mutant mouse lines
  • Conditional KO: simultaneous insertion of LoxP sites of 5’ and 3’ of critical exon(s)

Other targeting strategies we are working on include deletion of 170Kb from a single chromosome.


Full-Scale Mutagenesis

  • Consultation to discuss project goals
  • CRISPR Vector Construction: includes gene editing strategy design, gRNA selection and cloning into all-in-one CRISPR vector, surveyor assay to test guide activity and identification of off-target sites
  • Oligo design/synthesis
  • Preparation of RNA/DNA for microinjection
  • Microinjection
  • Founder genotyping (PCR/topo/sequencing)

Microinjection of Provided Material

  • PI can submit RNA/DNA for injection
  • TTML will perform QC on submitted material and prepare for injection
  • Due to the complexities of CRISPR generated mutations, we strongly recommend that the TTML perform genotyping analysis of these mice

Creating New Mutations in Established Mutant Lines

  • Microinjection of embryos isolated in PI lab

ES Cell Mutagenesis

Follow Center for Genetic Medicine on