New CRISPR inhibitors found with help from U.S. Department of Defense funding

As scientists march closer to using CRISPR gene editing to treat diseases, the U.S. Department of Defense is prepping for the possibility of a more nefarious use of CRISPR: its weaponization to harm humans, animals, or crops.

In July 2017, the Defense Advanced Research Projects Agency (DARPA) announced a $65 million research program to prepare for these scenarios. Called Safe Genes, the DARPA program is funding projects to develop countermeasures and prophylactic treatments against unwanted gene editing. It also supports researchers who are designing systems to better control intentional gene editing.

Last week, two papers published in Science offered the first glimpse of the kind of research that DARPA is supporting. Two groups, one led by Jennifer Doudna from the University of California, Berkeley (Science 2018, DOI: 10.1126/science.aau5138), and another led by Joseph Bondy-Denomy at the University of California, San Francisco (Science 2018, DOI: 10.1126/science.aau5174), discovered several new CRISPR inhibitors, including one for CRISPR/Cas12a, an increasingly popular alternative to the standard CRISPR/Cas9 gene editor.

Bondy-Denomy is well versed in these CRISPR inhibitors. In 2013, he helped discover the first anti-CRISPR proteins, as they’re called, and has been on the hunt for more ever since. Those first anti-CRISPRs weren’t helpful for halting gene editing because they didn’t work on the Cas enzymes that cut and edit DNA in the CRISPR methods widely used in labs.

An obvious place to look for more anti-CRISPRs is in bacteriophages, the viruses that besiege and kill bacteria. In fact, CRISPR evolved in bacteria as a primitive immune system that the cells use to chop up their viral enemies. Cas enzymes recognize the microbial attackers with the help of a mugshot of the viruses’ DNA stored in an RNA code in the bacteria.

But there’s an evolutionary arms race between viruses and bacteria. Bacteriophages themselves have acquired genes that make anti-CRISPR proteins. “Cas inhibition is actually quite rampant,” Bondy-Denomy says. Even though many bacteria have CRISPR, it actually fails to protect a lot of them because of the viral countermeasures.

In 2016, multiple labs, including Bondy-Denomy’s, found the first Cas9 inhibitors, but Cas12a inhibitors have remained elusive. Scientists are interested in the therapeutic gene-editing applications of Cas12a because it is a more compact protein than Cas9 and therefore may be easier to get into humans. Cas12a is also better for inserting new DNA into genomes, whereas the traditional Cas9 is better suited to turning genes on or off.

To find Cas12a inhibitors, Bondy-Denomy’s and Doudna’s labs started with a similar strategy. They scanned a database of bacterial genomes to find species whose CRISPR/Cas12a systems over time became programmed to chop up their own DNA instead of bacteriophage DNA. The existence of such a system would be suicidal for the bacteria, unless something like an anti-CRISPR protein prevented Cas12a from doing its job.

That search led both teams to a cow pathogen called Moraxella bovoculi. They then used different methods to scan the bacteria’s genome for a lurking bacteriophage gene that halted Cas12a activity. Both groups converged on an anti-CRISPR called AcrVA1, which effectively inhibited Cas12a when the scientists tried to use it for gene editing in human cells in culture.

“It is always a good sign when two independent studies converge on the same picture,” says Erik Sontheimer, a CRISPR scientist at the University of Massachusetts Medical School whose lab helped discover Cas9 inhibitors. “Whether it will be useful therapeutically or not, I’m not sure. It is still pretty early days,” he adds.

One potential use would be to halt unwanted gene editing. “DARPA is clearly thinking about CRISPR being used as a biological weapon,” says Karen Maxwell, a scientist who studies anti-CRISPRs at the University of Toronto. Administering that defense could be difficult, however. Since anti-CRISPRs are proteins, they can’t be taken in a pill like many small-molecule drugs.

Maxwell says that even if the anti-CRISPRs are not used to deter an attack, they could still prove handy for making CRISPR therapies safer.

Scientists developing CRISPR gene editing systems to treat a disease don’t want Cas enzymes to hang around long after they’ve made the desired edits to DNA. The longer that Cas lingers, the more likely it is to make unintended and cell-damaging breaks in DNA. Preventing these off-target cuts is one of the biggest priorities for making CRISPR therapies safer. In fact, Doudna and Bondy-Denomy have already shown that administering the Cas9-specific anti-CRISPR proteins just hours after applying CRISPR/Cas9 can result in a 75% decrease in off-target cuts (Sci. Adv. 2017, DOI: 10.1126/sciadv.1701620).

Anti-CRISPRs may also help make CRISPR therapies more specific by limiting the kinds of tissues CRISPR will work in. DARPA also wants to develop more controllable versions of gene drive, a technology that uses CRISPR to propagate a gene throughout a population of animals or plants. Several groups are developing the technique to prevent the spread of malaria in mosquitoes. But since this technique would alter the genes of wild animals, scientists would like to design an emergency kill switch for the system in case something goes awry. Anti-CRISPRs could help.

“It could be a national security interest to ensure that CRISPR goes forward in a safe manner,” Bondy-Denomy says. The Cas12a inhibitors are just one small part of many ongoing projects funded by DARPA, he adds. “Weaponized gene editing might sound a little bit like science fiction, but in the short term it is very clear that having safe gene editing makes sense.”