Tracking proteins in cells depends on tagging the molecules for identification, which can be done with the gene-editing tool CRISPR. With this technology, proteins can be tagged in live cells or the molecules can be captured for analysis. Still, scientists continue to assess and improve this approach.
“The biggest challenge is potential effects on protein activity or localization by the tag,” said Peter Kaiser—professor and chair of the department of biological chemistry at the University of California, Irvine. “This is less of a problem when tagging essential genes, because problems with activity will be either seen by getting exclusively heterozygous tag insertion or reduced proliferation rates.” For proteins from genes that are considered nonessential, though, scientists face a bigger issue. Here, “it is challenging because biological output assays to monitor activity of a particular protein are not always available,” Kaiser explained.
Modifying the methods
No one-size-fits-all method exists for applying CRISPR to protein tagging. “The ease or challenges of CRISPR insertion of fusion tags into cellular protein targets is highly dependent upon the choice of fusion tags one wishes to insert and/or the choice of cell line to modify,” said Danette Daniels, R&D group leader at Promega. “In principle, the design of CRISPR guide RNAs and donor DNA sequences for insertions has been significantly streamlined and aided with the availability of CRISPR design computational programs from several academic and commercial sources.”
Some rough guidelines do apply for the sorts of fusion tags and cells that are likely to improve the results. “In general, smaller fusion tags have higher efficiency of CRISPR insertion relative to larger ones, and cells which are more amenable to transfection or nucleofection also have higher efficiency,” Daniels explained. “Difficult to work with cell lines—oftentimes those which are specialized for the study of particular diseases—present the greatest challenge for initial CRISPR insertions and downstream clonal selection of the insertions, either heterozygous or homozygous allelic insertions.”
One CRISPR-based approach uses an HiBiT tag, which is an 11 amino-acid peptide that can be detected. Daniels noted that scientists at Promega have “generated hundreds of HiBiT CRISPR insertions into either the N- or C-terminal genomic loci of key disease proteins in various physiologically relevant cancer cell lines.” Then, binding between HiBiT and Promega’s LgBiT protein produces luminescence.
Now, even larger fusion tags—such as Promega’s NanoLuc luciferase and HaloTag protein, which are 19 and 34 kilodaltons, respectively—can be used. “Despite having lower efficiency than the HiBiT CRISPR insertions, we have developed protocols for enrichment,” Daniels said. With HaloTag CRISPR insertions and fluorescently activated cell (FAC) sorting, Daniels pointed out that scientists can take “advantage of our high-resolution Janelia Fluor HaloTag dyes to enrich CRISPR-modified cells from unmodified ones.”
Scientists also develop their own CRISPR-based methods of tagging points. As an example, Kaiser and his colleagues described a “versatile toolset for rapid tagging of endogenous proteins.” They added that their approach “utilizes CRISPR/Cas9 and microhomology-mediated end joining repair for efficient tagging.” As Kaiser and his colleagues concluded: “This approach and the developed tools should greatly facilitate functional analysis of proteins in their native environment.”
Exploring the efficiency
As already noted, protein tagging varies by cell type and other parameters. According to Thorsten Müller—group leader for cell signaling at Ruhr-University Bochum, “The transfection efficiencies as well as knock-in efficiencies in some cells are really low, especially in stem cells, and efficiency depends on the knock-in strategy you apply,” he explained. An often-used method is the homology directed repair (HDR), which uses DNA sequence homologies.
Müller added, “For HDR, you might need to invest a lot of time in molecular cloning.” Instead of using up the time, he said that the “best approach seems to be to order gBlocks, but it’s of course more expensive.”
By looking at several methods, Harvard Medical School research fellow Hassan Bukhari and Müller studied different protein-tagging methods. “We are currently comparing different methods in points of best efficiency,” Müller said. “The point is that many published papers just used the fluorescence readout to quantify efficiency, but this is not a good readout.” For example, when using a GFP knock-in with human embryonic kidney (HEK) cells, Müller said, “you will often have a lot of positive GFP cells, but most of them are false positives, as HEK cells are somehow able to activate the GFP independent of a knock-in.” Consequently, he noted, “You need to sequence the genomic DNA, but this is not regularly done in many publications.”
Expanding applications
As scientists explore the benefits and challenges of tagging proteins with CRISPR-based methods, more uses will be developed. In fact, interesting applications are already underway. For instance, Müller said, “Our goal is mostly to tag proteins in the context of neurodegeneration.” In this work, he said, “For example, we are tagging the amyloid precursor protein.”
With Promega’s tools, other protein-tagging tasks can also be approached with CRISPR. “Our collection of HiBiT CRISPR protein targets and cell lines have significantly enabled screening efforts and mechanistic understanding for targeted degradation compounds and internalization of extracellular receptors,” Daniels said. “Targeted degradation is a rapidly emerging therapeutic modality encompassing PROTACs—a proteolysis targeting chimera—and molecular glues, small molecules which target proteins for destruction via the ubiquitin-proteasomal pathway.”
From a therapeutic perspective, Daniels added that these tools can be used for “rapid triaging and rank-ordering of active compounds” and “monitoring the dynamics of receptor internalization after compound treatments,” which is useful in characterizing a compound’s mechanism of action.
There’s a lot that scientists can learn from tagging a protein—especially if that can be done easily and without affecting the protein’s function. CRISPR-based approaches improve some aspects—making it easier to use in some cases, for example—but there’s still work to do. In the end, biology might find that it too has a physics-like observer effect: Looking at a system changes it. Maybe that’s just how it will be, and we need to make the most of a limited situation. In the meantime, it never hurts to play a game of tag and see what can be seen.