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), the increased efficiency in mouse ES cell targeting, as well as the ability to skip the mouse ES cell gene targeting step required for classic approaches to KO and KI mouse production. KO mice and small KI (using ssDNA donors) can be generated by electroporating fertilized mouse embryos with gRNA + Cas9 protein + repair DNA. Conditional KO's and large inserts using targeting vectors are electroporated into mouse ES cells along with gRNA + Cas9 protein to increase the targeting efficiency.
- We offer a Standard Service in which the investigator is responsible for designing the guide(s) and provides the gRNA + Cas9 protein + DNA for microinjection or electroporation.
- We also offer a Full-Service CRISPR/Cas9 option through our collaboration with the UW Mouse CRISPR Core which includes gRNA design, embryo microinjection or electroporation, and genotyping. Please contact Dr. Julie Mathieu for further information.
- Both the Standard Service as well as the Full-Service options include the following services:
- Purchase and house mice used to produce embryos for injection.
- Superovulate embryo donors and mate with stud males to generate fertilized embryos for injection.
- Create pseudopregnant foster mothers by mating outbred ICR females with vasectomized males and checking for vaginal plugs the morning of injection.
- Harvest fertilized embryos from euthanized donors.
- Microinject or electroporate gRNA + Cas9 protein (+ donor DNA repair template) into 1-cell stage mouse embryos or targeted ES cell clone into mouse blastocysts.
- Surgical transfer of injected/electroporated embryos into pseudopregnant foster mothers.
- Monitor implanted pregnant mothers before and after birth of pups.
- Wean, ear-tag, and tail-tip (or ear-punch) pups for genotyping.
- Provide tail or ear biopsies to the client for genotyping.
- Arrange and assist with the transfer or shipment of transgenic mice to the investigator’s mouse colony.
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, embryo microinjection or electroporation, and genotyping.
A few on-line CRISPR design tools:
- If preparing RNA for microinjection, 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/electroporated embryos will be surgically implanted into pseudopregnant recipients, or 2 founder pups will be produced, whichever comes first when injecting or electroporating 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.
- 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.