CRISPR/Cas9 Gene Editing

The CRISPR/Cas9 system uses the guide RNA (gRNA) in conjunction with the Cas9 endonuclease to create a double stranded break in the DNA at the targeted locus. The break will be repaired by non- homologous end-joining (NHEJ), which tends to add or delete base pairs resulting in a frame shift mutation (KO mouse). Alternatively, if a repair DNA is included in the injection mix, the DNA break can be repaired by homology directed repair (HDR) allowing for gene-targeted insertions (KI mouse).

CRISPR reagents are injected into fertilized one-cell-stage embryos, and the microinjected embryos are transferred into pseudopregnant mothers that develop, deliver and nurse mutant offspring. It takes about two to four months to generate a gene-targeted founder mouse with CRISPR technology. We have successfully used this technology to create the following mutations:

  • KO mutations
  • Single point mutation KI
  • Simultaneous Epitope (2-3X) KI
  • Insertion of epitope tags (<2 kb) to create tagged fusion protein
  • Gene Deletion (<1 kb in size)
  • Conditional KO: simultaneous insertion of LoxP sites of 5’ and 3’ of critical exon(s)

Lin Gan, PhD
Director, Genome Editing Core

Services

Full-Scale Mutagenesis

  • Consultation to discuss project goals.
  • Bio-information analysis, targeting strategy and design of targeting construct and other reagents, design and testing of PCR genotyping methods.
  • Targeting by CRISPR/Cas9 microinjection to produce germline quality founder mice in C57BL/6J background.
  • Breeding founder mice with C57BL/6J mice for germline transmission (GLT) to generate the heterozygous mice and genotype GLT F1 pups.
  • Guaranteed delivery for each project: one pair or more of heterozygous mice will be transferred to investigator

Microinjection of Provided Material

  •  PI can submit DNA templates and gRNAs, the core will provide Cas9 mRNA or protein.
  • There is no guarantee that any founder mice will be produced or will contain the desired edited alleles as the end results depend on the targeting efficiency as well as the mutation effect of the gene locus and targeting designs, and the quality (purity and toxicity) of reagents provided by the users.
  • For each microinjection session, we will inject ≥150 C57BL/6J embryos, perform embryo transfers to pseudopregnant females.
  • Tail tip biopsies will be provided to the investigator's laboratory for genotyping. Mouse pups will be transferred to the investigator at weaning.

Synthego's chemically modified sgRNA

  • The core offers discount price for Synthego's chemically modified sgRNA orders placed though the core.

Customized CRISPR/Cas9 project design and reagents

  • The core will design targeting and genotyping strategy, and generate CRISPR/Cas9 reagents (one sgRNA and one DNA template) to target a specific location in the mouse genome.