The experiment is designed, Promega's Technical Services team is on speed dial, and, importantly, the products have arrived. It's time to put your lab safety gear on (gloves, lab coat, safety goggles, a smile) and head into the lab.
You can find our recommended detailed protocol that we referenced in the video here. This protocol will walk you through how to prepare the ribonucleoprotein (RNP) complex and deliver it to cells. It also includes how to validate the editing event in your cells.
To begin, you will first need to follow a simple, standard protocol to anneal the crisprRNA and tracrRNA to form the guide RNA duplex (Basically, add 1:1, heat to 95C for 5 minutes, then cool to room temperature – easy!). Then add purified Cas9 to the guideRNA duplex to form the RNP complex (combine and incubate at room temp for 10-20 minutes – so easy!).
Now that the RNP complex is ready to go, let's add it to the cells. There are multiple ways you can do this. If you have access to a Nucleofector instrument, that would be ideal. Don't have access to a Nucleofector? You can use lipid-based reagents designed for RNP transfection (CRISPRMAX is an example). Whether you are using a Nucleofector or a lipid-based transfection method, you will simply follow the manufacturer's cell line-specific directions for working with your cell line of interest.
After 24-48 hours have elapsed (depending on your transfection method), it is time for some serious fun. The grand finale. Did your editing event happen? Do you now have an endogenously tagged protein with which you can do all sorts of amazing studies?
Let's run the supremely easy assay to see if you have an insertion. With the small and mighty HiBiT tag, you do not have to perform laborious PCR experiments or Western blots to simply see if it worked or not. Instead you need to add one reagent and see if you have light.
he most straightforward option is to use the Nano-Glo® HiBiT Lytic Detection System. First, create a cell suspension (if you have adherent cells, trypsinize them). Then create two plates: one 6-well plate that you will use to continue to propagate the cells, and one 96-well plate that you will use to run the assay. Add the HiBiT lytic detection reagent to the 96-well plate, incubate for a short time, and then read the luminescence on a plate reader. In our video, we used the GloMax Discover plate reader, which has great dynamic range and sensitivity.
When the numbers are in, compare the light output to your unedited control cells. Do you see light? Do you have certain guide RNAs that resulted in increased luminescence? If you see light above your unedited cell control, then you have success!
Other tips and tricks for validating the CRISPR editing event
As you prepare the plates for HiBiT detection, count cells to normalize cell numbers among samples. If you don’t want to perform cell counts, you can do a very easy multiplex with a cell viability assay to normalize signal. Learn more about our CellTiter-Fluor assay for an easy multiplex with the HiBiT lytic detection reagent.
Are you targeting a cell membrane protein or secreted protein? If so, you could use our non-lytic HiBiT detection reagent. This reagent will leave your cells intact for further analysis. In this case, CellTiter-Glo is another multiplexing option to help you to normalize cell number among samples.
Are you interested in confirming expression of your full-length protein? How about a much easier, faster blotting technique? You could use a sample of the edited cells, lyse with preferred buffer, run the sample on a gel, and transfer to a membrane for detection with our Nano-Glo HiBiT blotting system.
That's it! How easy is that?!