NEW ORLEANS (GenomeWeb) – At the annual meeting of the American Association for Cancer Research in New Orleans on Monday, a panel of researchers took the stage to discuss how they use CRISPR technology to study cancer and to find ways to treat it.
Whitehead Institute principal investigator David Sabatini presented his lab's method for using CRISPR to screen cancer cell lines in search of target genes and to identify components of oncogenic pathways.
The researchers use CRISPR to knock out genes in cancer cell lines, and assign each gene a CRISPR score depending on whether the knockout increases the number of cancer cells or decreases them. Sabatini's team is applying this screening method to several different cancer cell lines — some of the genes they've identified as essential apply to many cancers, he said, whereas others are very specific not just to a kind of cancer, but to a particular cell line.
The hope, Sabatini added, is to find genes that are essential to cancer, but not to normal cells, and in that way perhaps find druggable targets. The team is also hoping to identify oncogenic drivers for different cancer types and then identify robust synthetic lethal interactions.
They're also trying to find patterns of co-essentiality — genes that work together to drive cancer growth — as well as to possibly eliminate certain targets that have been identified in the literature and deemed possibly druggable. For example, Sabatini said, the CRISPR screen his lab developed has found that the likely relevant target for the FURIN gene is IGF1. Even though other possibilities have been suggested by the literature, narrowing down the possibilities like this gives others a place to start in developing therapies.
The team has also found new components of gene pathways using the screen, which have been confirmed by biochemical experiments. Identifying such components can help researchers fill in the gaps in their knowledge and perhaps further elucidate how a specific gene pathway contributes to the development or growth of cancer.
"It's a very interesting way to go about de-orphaning a large number of genes," Sabatini said.
Also presenting was University of California, San Francisco professor Jonathan Weissman, who detailed his lab's efforts with two CRISPR variants they've developed: CRISPRactivation and CRISPRinterference.
In cancer, the inappropriate expression or shutting off of genes are central. But it's one thing to know about these changes and watch them, and it's another to control them, Weissman said. While this has changed thanks to CRISPR, it's possible to take this one step further and fine-tune the technology to allow researchers to adjust gene expression, rather than merely shutting it off.
CRISPRi is used to turn genes off and CRISPRa is used to turn genes on, Weissman said, but both can also be used to tweak gene expression one way or the other. "What we've built is a volume button to turn up or down the expression of any gene," he added.
And the lab has recently made improvements to their CRISPR variants. Initially, they built and published a genome-wide library, and were able to take pre-selected genes and use CRISPRi and CRISPRa to pick out 80 percent of the essential genes before starting to see false positives. In the newest version of the library, the researchers have increased that rate to 95 percent.
"One of the reasons we're so sensitive at detecting genes is because we don't have any toxicity from a binding event, unlike a cutting event, which can confer toxicity solely due to cutting," Weissman said, adding that the method is even sensitive enough to pick up mild growth defects in genes.
Using these CRISPR variants, the team has been able to conduct genome-wide forward genetic screens for any transcript; over-expression screens important for finding mechanisms of drug resistance, redundant genes, and gain of function; and massively parallel combination synthetic lethal screens.
Importantly, CRISPRi can also be used to identify drug targets by finding molecular targets for small molecules that have an unknown mechanism, and CRISPRa to identify how certain compounds act to kill cells, Weissman said.
The CRISPR variants can even be used to guide the development of compounds called chaperones. Oncogenes are often unstable and depend on chaperones to maintain activity, and chaperones can be important for enabling cancer cells to explore mutation space and develop resistance. However, effectively targeting chaperones is hard because there are many of them and they tend to overlap in networks. Using CRISPRi or CRISPRa to modulate their activity rather than simply turning them on or off could help researchers find therapies aimed at chaperone activity, Weissman said.