Shouldn’t a better mousetrap have a better off switch? That’s the question posed by researchers at the University of California, San Francisco (UCSF), who are working to make molecular biology’s better mousetrap—the CRISPR-Cas9 gene-editing system—even better. Although CRISPR-Cas9 is a tremendously exciting tool, one that can target selected genomic sites, it isn’t exactly glitch-free. It can snip at genomic sites that should be left alone, and may be more apt to meddle where it shouldn’t, given its tendency to loiter, remaining active even after it should retire.

But the CRISPR-Cas9 system can be deactivated. We know this because it doesn’t always work in its natural element, bacteria. While CRISPR-Cas9 serves as an immune system for bacteria, it is sometimes circumvented by viruses it ought to eliminate.

What do viruses know that we don’t? If only we knew, we could turn off CRISPR-Cas9 at will, preventing off-target effects and hastening CRISPR-Cas9 applications in the clinic.

Hoping to learn from the evolutionary struggle between bacteria and viruses, UCSF scientists decided to take a close look at viruses that are capable of frustrating CRISPR-Cas9. The scientists reasoned that they should be able to identify bacteria with inactivated CRISPR systems by looking for evidence of so-called “self-targeting”—bacterial strains where some virus had successfully gotten through the Cas9 blockade and inserted its genes into the bacterial genome. That is, viral DNA appearing both in a bacteria’s CRISPR array as well as elsewhere in its genome could be a sign of self-targeting.

The UCSF team hypothesized that CRISPR-defeating viruses must encode some anti-CRISPR agent, or else Cas9 would kill the bacteria by cutting its own genome where the viral DNA had been inserted. To find such agents, the team searched cas9-containing bacterial genomes for the co-existence of a CRISPR spacer and its target, a potential indicator for CRISPR inhibition.

The results of this investigation appeared December 29 in the journal Cell, in an article entitled, “Inhibition of CRISPR-Cas9 with Bacteriophage Proteins.”

“This analysis led to the discovery of four unique type II-A CRISPR-Cas9 inhibitor proteins encoded by Listeria monocytogenes prophages,” wrote the study’s authors. “More than half of L. monocytogenes strains with cas9 contain at least one prophage-encoded inhibitor, suggesting widespread CRISPR-Cas9 inactivation. Two of these inhibitors also blocked the widely used Streptococcus pyogenes Cas9 when assayed in Escherichia coli and human cells.”

The two inhibitors capable of blocking Streptococcus pyogenes Cas9, or SpyCas9, are called AcrIIA2 and AcrIIA4. Because SpyCas9 is the Cas9 variant that is now used by most labs around the world, AcrIIA2 and AcrIIA4 may prove to be an especially attractive anti-CRISPR-Cas9 tool. Other anti-CRISPR-Cas9 tools have been introduced, but they work against less common Cas9 variants.

The current study was led by Benjamin Rauch, Ph.D., a post-doctoral researcher in the laboratory of Joseph Bondy-Denomy, Ph.D., who is a UCSF Sandler Faculty Fellow in the Department of Microbiology and Immunology.

“The next step is to show in human cells that using these inhibitors can actually improve the precision of gene editing by reducing off-target effects,” said Dr. Rauch. “We also want to understand exactly how the inhibitor proteins block Cas9's gene targeting abilities, and continue the search for more and better CRISPR inhibitors in other bacteria.”

Drs. Rauch and Bondy-Denomy believe the ability to deactivate SpyCas9 will make CRISPR-based gene editing much safer and more precise by resolving the ongoing problem of unintended “off-target” gene modifications, which become more likely the longer the CRISPR gene editing machinery remains active in target cells.

The discovery could also be a boon for scientists using newer CRISPR techniques—such as CRISPR interference and CRISPR activation—which use Cas9 not to modify gene sequences but to precisely tune their activity up and down. Using anti-CRISPR proteins, researchers could boost or block gene activity temporarily, potentially even synchronizing choreographed bursts of activity from sets of interconnected genes across the genome, which could be key to studying and treating complex, multi-gene diseases.

CRISPR inhibitors could also prove to be a valuable safeguard, the researchers say, enabling scientists to quickly halt any application of CRISPR gene editing outside the lab.

“Researchers and the public are reasonably concerned about CRISPR being so powerful that it potentially gets put to dangerous uses,” noted Dr. Bondy-Denomy. “These inhibitors provide a mechanism to block nefarious or out-of-control CRISPR applications, making it safer to explore all the ways this technology can be used to help people.”

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