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.
Services
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