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 versus 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 Sept. 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.
Microbes at war
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.
A faster way to find anti-CRISPRs
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.
