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:
Lin Gan, PhD
Director, Genome Editing Core
Microinjection of Provided Material
Synthego's chemically modified sgRNA
Customized CRISPR/Cas9 project design and reagents