Production of gRNAs by in-vitro transcription (IVT)
This is the protocol we used in the past to produce gRNAs for injection into embryos. We no longer use in-vitro transcribed gRNAs, as we prefer plasmid injection or gRNA transgenes. The protocol takes about 20h from start to finish, including an overnight incubation.
Materials you will need:
- specific primers
- PCR kit
- gel extract kit
- Ambion T7 shortscript kit
- Ambion MEGAclear kit
Step1: Production of a T7 template for IVT
We use PCR products as template for the IVT. This way one can easily introduce the specific target sequence of the gRNA through the fwd primer. As a template we use a self-made plasmid that encodes the 80nt gRNA core. If you don’t have one already, you can order one of the gRNA plasmids that are available on Addgene or we can send you our template plasmid. The fwd primer also introduces the T7 promoter into the template.
Note that the last two Guanines in the T7 promoter are the first two bases that are transcribed. If you have a target site that does not have GG at the 5′ end you can add these bases to the 5′ of the target sequence. We use the Q5 hot-start PCR kit for our PCRs, but any high fidelity polymerase should be fine. After PCR we run the DNA on an 1.5% agarose gel, cut out the bands and purify the DNA with the Qiagen gel purification kit. Dissolving the gel piece at room temperature gives better DNA recovery rates. We usually run 50ul PCR reactions and end up with a DNA concentration of around 60ng/ul.
Step2: IVT
The gRNA template is then in-vitro transcribed using the Ambion T7 shortscript kit according to the manual. We use 8ul template and incubate at least 4h, but usually over night.
Step3: Purification
We use the Ambion MEGAclear kit to purify the RNA from the IVT. We follow the instructions in the manual and elute in 50ul for 10min at 70C. At the end you should have 50ul of injection grade gRNA at a concentration somewhere between 1-2ug/ul. Run 200ng of gRNA on a gel to confirm integrity before injection.