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UCSF Team Describes Anti-CRISPR/Cas9 Proteins

NEW YORK (GenomeWeb) – Researchers from the University of California, San Francisco have discovered a way to block CRISPR/Cas9 gene editing using newly identified anti-CRISPR proteins found in bacteriophages. Theoretically, the technique could improve the safety and accuracy of CRISPR in certain applications.

Led by first author Benjamin Rauch and senior author Joseph Bondy-Denomy, both of UCSF, the researchers published the results of their study today in Cell. Looking in Listeria monocytogenes, they found four candidate anti-CRISPR proteins, collectively dubbed AcrIIA proteins, numbered one through four. They're not the first anti-CRISPR proteins discovered, but they're the first to work against the CRISPR/Cas9 system from Streptococcus pyogenes (SpCas9), the most widely used and best-characterized system used in research. In validation experiments, acrII1 and acrII4 showed an ability to inhibit SpCas9 in both Escherichia coli and human cells.

"Just as CRISPR technology was developed from the natural anti-viral defense systems in bacteria, we can also take advantage of the anti-CRISPR proteins that viruses have sculpted to get around those bacterial defenses," Rauch said in a statement.

Their findings also could lead to the discovery of more SpCas9 inhibitors, which the authors said could be used to address the problem of off-target editing with CRISPR by limiting the window of opportunity, as well as provide a "fail-safe" to address intentional or accidental harmful applications. 

In the study, the authors used a bioinformatics-driven approach to find strains of Listeria — which has a CRISPR system similar to S. pyogenes — that exhibited phage DNA integrated into the bacterial genome, even though the host CRISPR system should have excised it.

The team hypothesized that these phages 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.

"Cas9 isn't very smart," Bondy-Denomy said. "It's not able to avoid cutting the bacterium's own DNA if it is programmed to do so. So, we looked for strains of bacteria where the CRISPR/Cas9 system ought to be targeting its own genome. The fact that the cells do not self-destruct was a clue that the whole CRISPR system was inactivated."

The study looked at more than 300 strains of Listeria and found that three percent of strains exhibited self-targeting. From these, the researchers identified the previously unknown acrIIA proteins.

"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," Rauch said. "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."

The authors said that the ability to deactivate SpCas9 will make CRISPR-based gene editing safer and more precise by helping address the problem of unintended "off-target" gene modifications, which become more likely the longer the CRISPR gene editing machinery remains active in target cells.

CRISPR inhibitors could also prove to be a valuable safeguard, the researchers said, 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," Bondy-Denomy said. "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."