The CRISPR/Cas9 gene-editing technique would seem a great way to identify cancer’s essential binding pockets. Use CRISPR/Cas9 to induce a mutation in a candidate gene, that is, a gene that codes for a protein known to serve as a binding pocket. The mutated gene becomes dysfunctional or produces dysfunctional variants. Cancer cells either take the loss in stride, or falter. In the latter case, the candidate gene may prove to be a druggable target. Develop a drug to fill the pocket. Kill the cancer.

Sounds simple. But CRISPR-induced mutations don’t necessarily disable binding proteins. If a mutation happens to alter a binding protein’s structure far from the binding site, the protein may retain sufficient functionality to mask just how essential it is to a cancer’s wellbeing.

To get around this problem, scientists at Cold Spring Harbor Laboratory used a modified version of the CRISPR/Cas9 screening technique. These scientists, led by Chris Vakoc, M.D., Ph.D., made sure to use a CRISPR/Cas9 method that would target functional protein domains.

These scientists found that their CRISPR/Cas9 approach could “generate a higher proportion of null mutations and substantially increase the potency of negative selection.” In addition, they found that “the magnitude of negative selection can be used to infer the functional importance of individual protein domains of interest.”

These findings appeared May 11 in Nature Biotechnology, in an article entitled, “Discovery of cancer drug targets by CRISPR-Cas9 screening of protein domains.” This article described how the scientists surveyed about 200 potential targets in leukemia cells and successfully identified six targets already known and validated by pharmaceutical scientists. The scientists also found an additional 19 targets never before recognized.

“This is just a single demonstration,” said Dr. Vakoc. “More broadly, what we provide is a way to comprehensively identify specific vulnerabilities in cancer cells, across cancer types.”

To confirm that their overall approach was working as planned, the scientists used SURVEYOR assays and deep-sequencing analysis to evaluate the differences in phenotypic severity between domain-targeting sgRNAs and 5′ exon sgRNAs. These differences, the scientists determined, could not be explained by variation in the overall efficiency of mutagenesis. Also, they could not be attributed to off-target cutting at exons encoding homologous protein domains. Instead, the scientists concluded that the CRISPR/Cas9-induced mutations within domains became depleted more rapidly from the cell population than mutations introduced outside of the domain.

Besides focusing on particular binding domains, Dr. Vacoc’s lab is interested in particular pockets. “My lab is focused on finding a small number of these binding pockets—ones whose function cancer cells are addicted to,” Dr. Vakoc points out. Implementing an extra degree of selectivity, the lab is starting with the kinds of binding pockets that drug makers like to target—considered “druggable” for various technical reasons.

“For entirely pragmatic reasons, our lab is prioritizing our search to the kinds of targets chemists like and are willing to design drugs against,” Dr. Vakoc explains. “We want to have an impact on cancers in the near-term. We want to provide pharmaceutical companies the kind of targets that they have extensive experience figuring out how to hit. And it all comes back to the question: can we identify specific things that cancer cells need—and then deprive them of those things. CRISPR should bring us to the end-game of finding all of the critical pockets of vulnerability in cancer.”

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