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Light up the future

using CRISPR to knock in Promega bioluminescent tags.

Take on undruggable proteins.
Avoid overexpression artifacts.
Remove the limitations of classic epitope tagging.
Go from editing to assaying biology in as little as 24 hours.

Accelerate your research and light up the future.


Part One: Designing the Experiment

Read Part One: Designing the Experiment

The very first step of this DIY project is to determine your target of interest.

Try to answer these questions before moving into planning mode:

  • What protein would you like to analyze?
  • What cool things can you monitor when you endogenously tag this protein?
  • Where should you insert the tag based on your protein's function and localization?

Common sites of insertion include the C-terminus, N-terminus or within the protein. Once you know your insertion site, you must find the genomic sequence. The complete genomic sequence should include 5' and 3' UTRs. Here are a couple of websites that you can use to track down your genomic sequence:

EnsemBI ›

Now that you have the full genomic sequence, it's time to select a region to search for guide RNAs. You will do this by using DNA analysis software to zero in on the region of your protein where you want to insert the HiBiT tag.

Don't have analysis software? Don't worry.
Technical Services to the rescue.

If you are targeting the C-terminus, insert the tag before the stop codon. If you are targeting the N-terminus, insert the tag after the start codon. Once you plug your sequence into your DNA analysis program, find the region 50 nucleotides (nt) downstream of the insertion site for C-terminal fusions, or 50 nt upstream of the insertion site for N-terminal fusions.

You will take this 50 nt sequence and use an online program to identify potential guide RNAs. Here are a couple of examples of websites that are available to do this:

ChopChop ›
Broad Institute ›

These programs will identify the protospacer adjacent motif sites (PAMs). We recommend ordering 3-5 guide RNAs to try – they will likely have different efficiencies of insertion. For the HiBiT insertion, target PAM sites downstream of the stop codon for C-terminal fusions or upstream of the start codon for N-terminal fusions.

Cas9 will cut 3 nt upstream of the PAM, so we recommend using guide RNAs based on PAM sites within 10-20 nt of the insertion point and order your crisprRNA without including the PAM sequence. Once you have these sequences decided, you are ready to add your guide RNA to your order. The guide RNA consists of the crisprRNA (we just designed it!) and tracrRNA (a standard sequence). Ordering this duplex guide is easy - check out the IDT Alt-R CRISPR-Cas9 system as an example.

Next, we need to design the HiBiT donor DNA template. Head back to your genomic sequence in your DNA analysis software and focus in on your desired insertion site. Find regions of identity for the donor DNA template by determining the sequence that is 50 nt upstream and 50 nt downstream of the insertion site. Then position the HiBiT sequence symmetrically in the middle between these homology arms. You can find the HiBiT sequence and synthesis rights here ›

Tools & Materials Needed:

  • Lab Bench
  • Safety Equipment
  • Standard lab equipment (pipets, tubes, heat block or thermocycler)
  • Cell culture materials
  • Nucleofector or lipid-based reagents for RNP transfection
  • HiBiT detection reagents
  • DNA analysis software? (don't really NEED since TechServ can step in here)
  • Cell counter and/or multiplexing assays for normalization
Tip(!) – introduce a silent mutation in the PAM sequence of the donor DNA template to avoid re-cutting.

Now you are ready to add the donor DNA template to your order. Because HiBiT is small and mighty (no big tags needed!), you can insert using a single-stranded oligodeoxynucleotide as opposed to a large plasmid. For example, you can use the IDT Ultramer DNA oligonucleotide.

Designing your CRISPR reagents should only take a matter of a few hours or less. Are you feeling overwhelmed? Have you hit a wall? Promega's Technical Service team is here to help. They will guide you through your entire experiment. They can help you with:

  • Finding your correct genomic sequence
  • Designing your guideRNA
  • Designing your donor DNA template
  • Helping you order the correct reagents
  • And their help continues into the lab (Part 2)

Part 2 of this DIY project encompasses lab work, so make sure that you follow proper lab safety before you begin. If you need any additional assistance, don’t hesitate to contact Promega's Technical Services for your research project questions.

Part Two: Lab Work

Read Part Two: Lab Work

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?!


CRISPR HiBIT fusion cell lines and clones

Promega R&D has developed and characterized HiBiT fusions for several targets and backgrounds.  Click Request next to the cell line of interest and we'll reach out to discuss purchase options.

View Cell-Lines and Clones
Locus Knock-In Orientation Parental Cell Line
ADRB2 HiBiT N-terminal HEK293 Request
ANKRD37 HiBiT C-terminal HeLa Request
AXL HiBiT C-terminal HeLa Request
BNIP3 HiBiT C-terminal HeLa Request
BRD2 HiBiT N-terminal HEK293 + LgBiT Request
BRD4 HiBiT N-terminal HEK293 + LgBiT Request
BRD4 NanoLuc N-terminal HEK293 Request
BRD7 HiBit C-terminal HEK293 LgBiT Request
BRD9 HiBit C-terminal HEK293 LgBiT Request
CASP3 HiBiT C-terminal HeLa Request
CDK1 HiBiT C-terminal HEK293 Request
CDK2 NanoLuc C-terminal HEK293 Request
CDK4 NanoLuc N-terminal K562 Request
CDK6 HiBiT C-terminal HEK293 Request
CDK6 NanoLuc N-terminal K562 Request
CDK12 HiBiT C-terminal HEK293 Request
CDK13 HiBiT C-terminal HEK293 Request
CDK16 HiBiT C-terminal HEK293 Request
CDK17 HiBiT C-terminal HEK293 Request
CFL1 HiBiT C-terminal HeLa Request
cMyc HiBiT C-terminal MV4;11 Request
CTNNB1 HiBiT C-terminal HeLa Request
EGFR HiBiT N-terminal HeLa Request
EZR HiBiT C-terminal HeLa Request
FOS HiBiT C-terminal K562 Request
GSK3B HiBiT C-terminal HeLa Request
GSK3B HiBiT C-terminal Jurkat Request
HDAC2 HiBiT C-terminal HeLa Request
HIF1A HiBiT C-terminal HeLa Request
HILPDA HiBiT C-terminal HeLa Request
IRAK3 NanoLuc N-terminal THP1 Request
IRAK4 HiBiT C-terminal HeLa Request
IRAK4 HiBiT C-terminal K562 Request
JAK2(V617F) Nluc C-terminal Hel92.1.7 Request
JUN HiBiT C-terminal HeLa Request
KLF10 HiBiT C-terminal HeLa Request
MAP4K1(HPK1) NanoLuc N-terminal Jurkat Request
MAPK8 HiBiT C-terminal K562 Request
MAPK14 NanoLuc C-terminal HEK293 Request
PLCG1 HiBiT C-terminal K562 Request
PRKCA HiBiT C-terminal HeLa Request
PTK2 HiBiT C-terminal K562 Request
RIPK1 HiBiT C-terminal K562 Request
RIPK1 HiBiT C-terminal HeLa Request

Don't see what you need?

Email R&D now

Check out the recent cell line updates!

Study Proteins Under Endogenous Regulation

Using CRISPR/Cas9 gene editing, HiBiT-tagged proteins can be expressed under endogenous regulatory conditions, reducing overexpression artifacts and maintaining proper stoichiometry with other endogenous binding partners or regulatory machinery.

The small 11 aa HiBiT tag can be added to the desired protein in one of two ways:

  • Traditional recombinant methods with your promoter of choice
  • Insertion into endogenous loci using a simple, clone-free CRISPR/Cas9 genome editing protocol

Read about Quantifying Protein Abundance at Endogenous Levels

Support from planning to results

Get help designing your project. Ask questions along the way. See what materials we've already created. Fill out the form and a scientist will get back to you within two business days.