by Michael Vinyard
figures by Nicholas Lue

Most therapeutic drug candidates that are put through clinical trials fail. Given that most of these fail during early development, the cost of bringing a single drug to market is now over $2.5 billion. If we focus on cancer alone, this high cost of drug development, combined with the fact that cancer is one of the leading killers in the US, means that any acceleration in cancer drug discovery could save lives. CRISPR-Cas9, the recently-harnessed genome editing technology, might find itself as a driver of such an acceleration.

CRISPR-Cas9 technology

CRISPR-Cas9 has been making waves in scientific communities for the past several years. It is a bio-engineering tool that enables genetic editing. In other words, this tool can be used to make physical cuts in our genetic material, DNA, creating changes called mutations. DNA is composed of many units, called genes, that provide a set of instructions for making proteins, which are the molecules that execute the functions necessary for life. For instance, proteins do the work that allows our muscles to move and our hearts to pump, as well infinitely more minute yet vital actions inside of every cell in the body.

This direst relationship between DNA/genes, protein, and human health is what makes CRISPR-Cas9 technology so exciting. When CRISPR is used to create a mutation in the DNA sequence, this mutation will be propagated to the protein product, which can impact the health of the cell and the person as a whole (Figure 1). For example, to directly reverse certain genetic diseases, CRISPR-Cas9 can theoretically be used to cut disease-causing genes, altering that gene’s protein product and ameliorating disease symptoms. In addition to these therapeutic aims, researchers have also been exploring CRISPR-Cas9’s use as a tool in drug discovery.

Figure 1: CRISPR-Cas9 induces lesions in DNA that are propagated to proteins. Cas9 is used to cut DNA, generating lesions or scars (panel A). RNA, an intermediary between DNA and protein, retains these CRISPR-Cas9-mediated scars, which are indicated by colored segments (panel B). RNA is used as instructions to make protein by the cell. The colored portions indicate the mutated parts of the protein (panel C).

CRISPR-Cas9 as a drug discovery tool

Therapeutic drugs, such as those intended to treat cancer, work by altering the function of the body’s workforce: proteins. In order to create such a drug, the first step is to determine which proteins should be targeted. In other words, which proteins actually matter for, say, the formation of cancer? To find these disease-relevant genes and proteins, researchers can use CRISPR-Cas9 to systematically inactivate, or knock out, the ~20,000 protein-coding genes found in humans. This approach is commonly referred to as a “genome-wide screen.”

For example, one might imagine investigating tumor formation in mice. To better understand which genes are responsible for driving the growth of a specific cancer, researchers can use CRISPR to remove, one by one, nearly every gene in these cancer cells. This approach allows them to identify a subset of genes that, when removed using CRISPR, block tumor formation. This information then can then be used to inform drug development. Specifically, if a drug could be designed that inactivates these proteins, identified by CRISPR as being necessary for tumor formation, then this could represent a very effective anti-cancer therapy.

While CRISPR’s role in identifying disease-related proteins is very useful, this is only the first step. This is because proteins are complex molecules that contain various parts, also known as domains. This is similar to a car that contains many parts, some of which are more important to the critical function of driving than others. If you take the doors off of a car, chances are it will still run. However, if you take the wheels off, things might be a little more difficult. All the same with proteins; if you mess up an accessory portion, it might still work; if you mess up a critical portion, it might be fatal. When researchers are designing drugs, they often look for those that might target critical regions of a protein. In the process of identifying lead anti-cancer drug candidates, researchers often modify the target protein to better understand which parts of the target are most critical to the interaction with the drug molecule. To do this, researchers have again turned to the power of CRISPR-Cas9 technology.


CRISPR-scanning is an approach that uses the cutting ability of CRISPR to make lesions across a gene that encodes a particular protein of interest. Rather than try to make a precise genome edit, CRISPR-scanning cuts DNA and then uses the cells’ built-in DNA repair machinery to fuse the DNA back together, making an imperfect and somewhat random scar. These scars are used as part of the instructions to make proteins. So, when CRISPR is used to generate many mutant versions of a gene, this generates many mutant versions of the corresponding protein, which can be studied to determine which parts of the protein matter for its function. So, for example, by sequentially mutating every domain in a protein, scientist can figure out which parts are the metaphorical doors, and which parts are the wheels. This distinction will allow for the design of drugs that target the important parts of proteins, like those that contribute to tumor formation.  In other words, CRISPR-scanning can identify the exact region of a protein that a therapeutic drug might do well to attach itself to in order to inactivate the protein.

CRISPR-scanning can also be done in the presence of a drug (CRISPR-suppressor scanning). If a drug is any good, it kills cancer cells. In this context, researchers can observe the extent to which cells containing various genetic scars thrive despite the presence of a drug. If a mutation caused by a scar prevents a drug from binding, the growth of that cancer cell might be rescued and cells with that scar will multiply (Figure 2). This is useful because part of making a good drug (or set of drugs) is knowing what might defeat it – cancer is a notoriously adaptable disease and one of the pitfalls of several cancer drugs is the development of cancer-resistance. Generating and studying this drug-resistance in the lab, before a drug ever gets to humans, is thus extremely useful. This is different than simple CRISPR-scanning in that instead of the interesting cells being those that died, it is those that thrive in the presence of a drug that normally kills them, due to their new CRISPR-induced mutation. Information about which protein regions, when mutated, lead to cell growth or cell death can be overlaid onto the 3-D structure of the protein target and drug-interacting hotspots can be identified. As such, the ability of CRISPR to identify which parts of the protein are important for function or disrupt the drug’s ability to engage its target make this technology an indispensable tool for therapeutic development.

Figure 2: CRISPR-suppressor scanning. The blue object represents a drug. The white shapes represent a hypothetical target protein. The colored portions of the proteins indicate the mutated portions of the protein. The magenta mutation happens to be located in the drug binding site and prevents the drug from binding, whereas in the other two (red and orange) examples, the mutation does not interfere with drug binding. In the case of the magenta mutation, the drug is blocked and the cell lives and multiplies.

CRISPR will aid biological discovery and will likely help us find new drugs

CRISPR genome editing is relatively new technology and, while its use in research laboratories as a tool is now widespread, the use of this technology in a living human is still considerably risky, especially given its irreversible nature and the number of safety trials it has yet to undergo. CRISPR-scanning and CRISPR-suppressor scanning have the opportunity to leverage CRISPR genome editing by taking advantage of its strengths, such as its programmable ability to alter DNA. Both strategies hold promise as new players in the drug discovery pipeline and may play a key role in the development of many future therapies. As a whole, it is my opinion that CRISPR may be the catalyst that helps us find more drugs for diseases like cancer and may indirectly save many lives through the research it enables.

Michael Vinyard is a third-year PhD student at Harvard University in the Department of Chemistry and Chemical Biology. He has been studying the role of chromatin regulators in cancer biology through CRISPR screens and other functional genomics and molecular biology-based approaches.

Nicholas Lue is a graduate student in the Chemical Biology PhD program at Harvard University. You can find him on Twitter as @nicklue8.

For more information:

  • A recent open-access review of the “CRISPR tool kit”  available to researchers can be found here.
  • Here is a very brief writeup on the first instance of CRISPR-scanning being used in the lab of Professor Chris Vakoc at Cold Spring Harbor Laboratories.
  • A more practical explanation of a genome-wide screen can be found here.

This article is part of our SITN20 series, written to celebrate the 20th anniversary of SITN by commemorating the most notable scientific advances of the last two decades. Check out our other SITN20 pieces!

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