There’s a war going on in your gut, and it has nothing to do with what you just ate. Viruses and bacteria are engaged in an age-old, life-or-death game of rock-paper-scissors (or, in this case, rock vs scissors). Insights into this battle could help researchers improve gene therapy or potentially find new solutions to antibiotic resistance.

In this rivalry within the gut microbiome, bacteria wield the scissors—CRISPR. Better known as the precise genome-editing tool that’s reinvigorated gene therapy’s potential as a treatment for human disease, CRISPR is derived from a defense mechanism that bacteria use to chop up the DNA from infecting viruses. Viruses strike back with “rocks:” anti-CRISPRs that block CRISPR’s DNA-chewing action.

The CRISPR system “is its own really interesting story,” said study lead Dr. Kevin Forsberg, a postdoctoral fellow in the lab of Hutch evolutionary biologist Dr. Harmit Malik, who studies the evolutionary arms race between bacteria and viruses. Both are members of Fred Hutch’s Basic Sciences Division.

CRISPR, short for CRISPR-Cas, is not one thing. "It's one concept but it has evolved independently many times in bacterial history,” Forsberg explained. “A relatively understudied aspect of CRISPR-Cas is its natural role as an immune system for bacteria that help bacteria combat viruses but also other types of selfish genetic elements that infect them.”

Similarly, there’s no one anti-CRISPR strategy and even less is known about these: “In this arms race, we really only know about tools used by one of the combatants,” Malik said.

There is a universe of potential anti-CRISPRs waiting to be explored, Forsberg said. In new work published September 10 in eLife, he, Malik and their collaborators describe a new type of anti-CRISPR. Using a method to screen DNA extracted from human microbiomes for activity against CRISPR, the team discovered an anti-CRISPR that blocks CRISPR in an as-yet unknown way.

Results from research like this into the evolutionary arms race between bacteria and viruses may have wide applications. A deeper understanding of anti-CRISPRs could help researchers seeking to improve control of CRISPR-based genome editing or finding new ways to manipulate bacteria for therapeutic purposes.

Viruses that attack bacteria are known as bacteriophages—which literally means “bacteria-eating virus” in Greek—or just phages. These are essentially capsules of genetic material. At some point in a virus’ lifecycle, it produces DNA copies of itself. CRISPR attacks this DNA. CRISPR and the Cas proteins include enzymes that slice up viral DNA, while CRISPR are the molecules a bacterium uses to help identify DNA that needs slicing. Ironically, viral infections have given bacteria the information they need to defend against viruses; CRISPR guide molecules are derived from remnants of phage genomes.

There are currently six known types of CRISPR-Cas complexes, each with multiple subtypes. (Cas9 is the variety on which the genome-editing tool is based.) Phage anti-CRISPRs are less understood. When Forsberg and Malik began their project, no anti-CRISPRs against Cas9 had been discovered.

Because the war began raging long before the advent of multicelled organisms, they’ve had billions of years to evolve their own weapons. Anti-CRISPRs against Cas9 had to be out there.

“Phages are battling for their lives,” Malik explained.

The first anti-CRISPR discovery relied on serendipity: Scientists encountered a bacteriophage that could resist a CRISPR-containing bacterium. Then, laborious experimentation revealed its anti-CRISPR.

But that’s a lot of time and effort to find just one anti-CRISPR. And it requires that scientists have both the phage and the bacterium, and that the bacterium is able to grow under laboratory conditions. Many don’t.

Forsberg took a different approach by focusing on DNA, specifically sequences from five human microbiomes, known as metagenomes. These contain DNA from bacteria as well as the viruses that infect them. It allowed high-throughput discovery of anti-CRISPRs without prior knowledge of either phage or bacterium.

Using DNA from two oral metagenomes and three fecal metagenomes, he set up a screening process that allowed him to sift through millions of DNA fragments to see which encoded genes that could keep Cas9 from chewing them up: anti-CRISPRs.

As attention-grabbing as CRISPR has been, the range of bacterial defenses against viruses remains little studied. Forsberg’s strategy could be used to discover other bacterial defense systems or new phage counterattack strategies, Malik said.

Human microbiome reveals new anti-CRISPR

The problem with going anti-CRISPR hunting is that there’s very little to guide a researcher. Oftentimes when sorting through DNA sequences, scientists can guess at a gene’s function by comparing its sequence to other genes with known function.

“Unfortunately, with anti-CRISPRs there are no good ways of doing that because they typically don't look like anything we've seen before,” Forsberg explained. “They are these small little proteins that have no sequence or structural relationship with known things.”

So instead of searching by gene sequence, Forsberg searched by function. With the help of Malik Lab technician Danica Schmidtke and former lab technician Ishan Bhatt, Forsberg ran the screening process twice to further enrich for genes that protected DNA against CRISPR and confirm their anti-CRISPR activity. At least 10 DNA sequences showed up as potential anti-CRISPRs, and one from the fecal microbiome stood out. None had been discovered before.

Dubbed AcrIIA11 — Acr for anti-CRISPR, IIA to denote the Cas system it inhibited, and 11 for the 11th anti-CRISPR to fit these criteria—the standout anti-CRISPR comes from a virus that infects bacteria of the genus Clostridium. In part because Clostridium are oxygen-hating bacteria that are difficult to grow, AcrIIA11 would have been difficult to uncover using more conventional methods, Forsberg said.

AcrIIA11, Forsberg and Malik found, counters a wide range of Cas9 systems. And it can block Cas9 action in mammalian cells, not just bacterial cells, as was shown through a collaboration with Dr. Ilya Finkelstein at the University of Texas. This surprised the team a bit—the Cas9 they used, the same one used in genome editing, came from Streptococcus, bacteria that are common in the oral, but not fecal, microbiome. Forsberg suspects that he found such a broad-acting anti-CRISPR because Clostridium uses a Cas9 that is very different from the one Forsberg used to screen his metagenomes, allowing him to uncover one that acts against several Cas9 types.

AcrIIA11 also doesn’t use the usual anti-CRISPR strategies against CRISPR-Cas9. Along with Fred Hutch colleagues Drs. Barry Stoddard and former Stoddard Lab member Brett Kaiser, Forsberg showed that AcrIIA11 works in a new way, one that they are still untangling. The finding highlights that, just as bacteria have evolved many strategies to combat viruses, viruses have evolved perhaps just as many to outmaneuver bacteria.

Uncharted possibilities

“Every year biology is discovering new bacterial defense strategies,” Malik said. “We’re at the stage when most defensive strategies are untapped.”

So far, every new anti-CRISPR discovered is new to biology—suggesting that there’s a lot of uncharted territory out there, he added.

Just as CRISPR-Cas9’s relevance to genome editing and potential disease fighting took years to understand, it will take time to reveal the potential applications of anti-CRISPRs.

One of the most obvious arguments for deepening our understanding of the arms race between phages and bacteria is the increasing problem of antibiotic resistance. We need improved strategies for dealing with disease-causing bacteria. One that’s gaining more attention is phage therapy, a century-old approach in which bacteriophages are used to treat bacterial infection.

“It's like an ecosystem down there,” Forsberg said of the gut microbiome. “You don't want to napalm the rainforest to get rid of just the bad bugs that you're interested in.”

Malik agreed, and noted that phage therapy could someday be a strategy with more precision. Once we better understand bacteria’s defense systems and how phages overcome them, we could potentially engineer phages to overcome specific bacteria with specific CRISPR-Cas systems.

In the meantime, Forsberg plans to further characterize the other anti-CRISPRs that showed up in his screening process. He is also continuing to examine how, exactly, AcrIIA11 works against CRISPR.

AcrIIA11 “is different and it's new. That's exciting and we're continuing to work on crossing the t's and dotting the i's,” he said.

Malik was more excited about the approach than even the findings.

“We are still not able to culture most of the bacteria that we have encountered in ecological niches like the human microbiome,” he said. “Being able to screen the activity of bacterial genes opens up an entirely new horizon of discovery of bacterial–phage interactions. It’s like reporting from the front lines of this ancient war.”