Secrets of the CRISPR Arms Race

Using cryo-electron microscopy, researchers based at The Scripps Research Institute (TSRI) and Montana State University (MSU) analyzed how viruses defeat CRISPR, the bacterial immune system. Viruses, these scientists learned, have at least a couple of anti-CRISPR strategies. One strategy is to simply counterattack. Viruses can deploy proteins that disable CRISPR machinery. Another strategy is to misdirect CRISPR. Evidently, viruses can fool the CRISPR system into binding an expendable viral protein instead of essential viral DNA.

While these strategies have evolved to preserve viruses, they could benefit people too. Adapted for use in our CRISPR genome-editing technologies, these strategies could effectively give us a CRISPR off switch.

This point was emphasized in a paper prepared by the TSRI and MSU scientists. The paper, entitled “Structure Reveals Mechanisms of Viral Suppressors that Intercept a CRISPR RNA-Guided Surveillance Complex,” appeared March 23 in the journal Cell, and focuses on viral anti-CRISPR proteins that subvert the CRISPR system in Pseudomonas aeruginosa. This system relies on a 350-kDa CRISPR RNA (crRNA)-guided surveillance complex (Csy complex) to bind foreign DNA and recruit a trans-acting nuclease for target degradation.

“Here, we report the cryo-electron microscopy (cryo-EM) structure of the Csy complex bound to two different Acr proteins, AcrF1 and AcrF2, at an average resolution of 3.4 Å,” wrote the article’s authors. “The structure explains the molecular mechanism for immune system suppression, and structure-guided mutations show that the Acr proteins bind to residues essential for crRNA-mediated detection of DNA.”

The study that yielded these results was led by TSRI’s Gabriel C. Lander, Ph.D., and MSU’s Blake Wiedenheft, Ph.D. “It’s amazing what these systems do to one-up each other,” said Dr. Lander. “It all comes back to this evolutionary arms race.”

“Although CRISPR/Cas9 is the ‘celebrity’ CRISPR system, there are 19 different types of CRISPR systems, each of which may have unique advantages for genetic engineering. They are a massive, untapped resource,” continued Dr. Lander. “The more we learn about the structures of these systems, the more we can take advantage of them as genome-editing tools.”

The researchers began by examining exactly how the CRISPR surveillance complex analyzes a virus’s genetic material to see where it should attack. The CRISPR surveillance complex consists of proteins that wrap around a guide RNA like a grasping hand, exposing specific sections of this RNA. These sections scan viral DNA, looking for genetic sequences they recognize.

“This system can quickly read through massive lengths of DNA and accurately hit its target,” explained Dr. Lander. “If the CRISPR complex identifies a viral DNA target, the surveillance machine recruits other molecules to destroy the virus’s genome.”

Next, the researchers analyzed how viral anti-CRISPR proteins paralyze the surveillance complex. They found that one type of anti-CRISPR protein covers up the exposed section of CRISPR RNA, thereby preventing the CRISPR system from scanning the viral DNA.

“These anti-CRISPR proteins keep the bacteria from recognizing the viral DNA,” noted Dr. Lander. He called these anti-CRISPR proteins “exceptionally clever” because they appear to have evolved to target a crucial piece of the CRISPR machinery. If bacteria were to mutate this machinery to avoid viral attacks, the CRISPR system would cease to function. “CRISPR systems cannot escape from these anti-CRISPR proteins without completely changing the mechanism they use to recognize DNA,” he observed.

Another anti-CRISPR protein uses a different trick. Based on its location and negative charge, the researchers believe this anti-CRISPR protein acts as a DNA mimic, fooling CRISPR into binding this immobilizing protein, rather than an invading viral DNA.

“These findings are important because we knew that anti-CRISPR proteins were blocking bacterial defenses, but we had no idea how,” Dr. Lander emphasized. Dr. Lander and the other authors of the current study look forward to further exploration of anti-CRISPR proteins—how they evolved, and how they may be exploited for practical gain.

“Genetic conflict between viruses and their hosts drives evolution and genetic innovation,” the authors of the Cell article noted. “Collectively, [our] data provide a snapshot of an ongoing molecular arms race between viral suppressors and the immune system they target.”

The researchers believe this new understanding of anti-CRISPR proteins may eventually lead to more sophisticated and efficient tools for genome editing. Perhaps anti-CRISPR proteins can be used in CRISPR systems to swoop in to block gene editing—or researchers could degrade anti-CRISPR proteins to trigger gene editing. “That might work as an on–off switch for CRISPR,” Dr. Lander said.