CRISPR/Cas9 Gene Editing
CRISPR/Cas9 Gene Editing can be used to create KO, KI, and conditional (floxed) genes in mice. The CRISPR/Cas9 system uses a 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).
The real advantage of the CRISPR-Cas9 system is the ease and speed of design (compared to targeting vectors) as well as the ability to skip the mouse ES cell gene targeting step required for classic approaches to KO and KI mouse production. Genetically modified mice can be created by injecting or electroporating gRNA + Cas9 protein + repair DNA directly into 1-cell stage mouse embryos via cytoplasmic or pronuclear microinjection.
- CRISPR/Cas9 gene edited mice are created using our Pronuclear Microinjection Service.
- purchase and housing of mice used to produce embryos for injection
- superovulation of embryo donors and mating with stud males
- creation of pseudopregnant foster mothers by mating outbred ICR females with vasectomized males and checking for vaginal plugs the morning of injection
- harvesting of embryos from euthanized donors
- microinjection or electroporation of investigator’s gRNA + Cas9 protein (+ donor DNA repair template) into 1-cell stage mouse embryos
- surgically transfer all embryos that survive the injection process into the oviducts of pseudopregnant foster mothers
- monitoring of foster mothers before and after birth of pups
- weaning, ear-tagging, and tail-tipping pups
- provide tail biopsies to the client for genotyping
- arrange and assist with the transfer or shipment of transgenic mice to the investigator’s mouse colony
- For this standard service, the investigator is responsible for designing the guide(s) and provides the gRNA + Cas9 protein + DNA for microinjection.
- The TRP also offers a full-service CRISPR/Cas9 option through our collaboration with the UW Mouse CRISPR Core. Please contact Dr. Julie Mathieu for further information.
For our standard service, investigators using our core for the production of KO and KI mice are responsible for designing and preparing their own CRISPR/Cas9 constructs for injection. The TRP will provide guidance and resources. For an additional fee, the TRP offers a full-service option through our collaboration with the UW Mouse CRISPR Core. The full-service option includes gRNA design, pronuclear microinjection, and genotyping.
A few on-line CRISPR design tools:
- If preparing RNA, the investigator should provide several frozen aliquots of gRNA + Cas9 protein (+ donor DNA) in filtered RNase-free microinjection buffer (5mM Tris, pH 7.4; 0.1 mM EDTA in ultra-pure water)—see the Protocols page for more information.
- Each frozen aliquot should contain 30ul of mix at a concentration of 100ng/ul of Cas9 + 50ng/ul of gRNA (+ 100ng/ul of oligos).
- If preparing plasmid DNA (pX330 plasmid from Addgene), please provide a 30ul aliquot of mix at a concentration of 5ng/ul of circular plasmid +10ng/ul of oligos) in filtered microinjection buffer.
- The cleanliness of the construct is of utmost importance. It should be absolutely free of any phenol, chloroform, ethidium bromide, high salt and ethanol. Even trace amounts will quickly lyse the injected embryos or interfere with the development of the implanted embryos. "Sticky" RNA preps will clog the injection needles and kill embryos. Great care should be taken when preparing the injection mix as the success of the procedure is highly dependent upon the quality of the DNA and/or RNA being injected into the embryos. See Protocols page for more information.
- We guarantee that at least 70 injected embryos will be surgically implanted into pseudopregnant recipients, or 2 founder pups will be produced, whichever comes first when injecting hybrid embryos. The construct gRNA/Cas9 protein/DNA provided by the investigator must be clean and non-toxic or the guarantee is void. We cannot guarantee that the gRNA designed by the investigator will result in the desired mutation. Therefore, we highly recommend that you test your gRNA in vitro before injecting it into mouse embryos.
- In the event that an alternate host embryo strain is required, the additional costs incurred from purchasing and housing a special strain of stud males, will be charged to the investigator. The investigator will also pay the difference in purchase price between the cost of standard hybrid strains, and the cost of the embryo host strain selected. No guarantee will be made as to the number of injected embryos transferred, due to strain variability in embryo production.
- We will inject pure C57BL/6 embryos at no extra cost, but without guarantee. This strain tends to be less efficient due to high mortality post-injection, therefore two or three injection sessions are typically required to achieve the same results as one session using hybrid embryos.
- The investigator pays per diem cage charges beginning on the day of embryo transfer into pseudopregnant females.
- Cancellation of injection within two weeks of the scheduled date will result in a $500 fee to cover the cost of embryo donors, hormones, and technician time.
- Click here for current rates.