“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 coauthor Benjamin Rauch, a post-doc in Bondy-Denomy’s lab, in a press release. “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 researchers found these Cas9 blockers by searching bacterial genomes for both a CRISPR sequence and its target, under the assumption that the genome likely contained an inhibitor to prevent CRISPR from cutting that target in the bacterium’s own genome. Indeed, Rauch and colleagues uncovered several anti-CRISPRs in Listeria whose sequences had been left behind in the bacterial genome by prior phage infection. “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 the statement.
Two of the inhibitors blocked Cas9 from Streptococcus pyogenes, the form of the DNA-cutting enzyme frequently used in genome editing. In a study published earlier this month in Cell, a different team of researchers reported the discovery of several anti-CRISPRs that block Cas9, but none of them acted against the activity of the Cas9 from S. pyogenes.
“The expression of these inhibiting proteins could be triggered in response to a given condition, or at a particular time point in the development of an organism, which would put a stop to the activity of Cas9,” Philippe Horvath, a senior scientist at DuPont in France, told The Scientist in reference to the earlier study. “A clever way to store the molecular scalpel into its safety case.”