CRISPR/Cas9 system: Knock-out and Knock-in models
CRISPR/Cas9 is the most widely used method to perform gene editing in cells and mice. A guide RNA is able to bind and form a complex with the Cas9 protein that is guided to the target DNA by the presence of a complementary sequence (targeting sequence). Here the Cas9 protein creates a double strand break that is then repaired via Non Homologous End Joining (NHEJ) pathway or via Homology Directed Repair (HDR) pathway if a donor DNA with homologous sequences is present. NHEJ is used to generate KO models, the HDR for Kin. The illustration below shows the Cas9 system.
The direct microinjection of zygotes using the CRISPR/Cas9 approach allows us to obtain gene-edited mice without the manipulation of ES cells. Thereby, reducing the time to generate KO or Kin models to three to four months.
CFCM staff microinject the Cas9 protein and the guide RNA provided by the customer directly into C57BL/6 or FVB zygotes. Embryos are transferred into foster mothers and pregnancies are monitored.
Tail biopsies are sent to the customer for genotyping.
Three different models are generated:
- KO mice via introduction of indel mutations or specific deletions.
- Kin models via microinjection of a guide RNA and a single strand donor oligo carrying the mutation of interest.
- Conditional models via microinjection of 2 guide RNAs and two single strand donor oligos carrying loxP sites.