CFCM

CRISPR/Cas9 system: Knock-out and Knock-in models

team-item

Contact person

Lorenza Ronfani

MORE

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.

 

crisp-cas9

 

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.

crisp-cas9_2

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.