Locating CRISPRs Molecular On/Off Switch

Investigators have identified the structure of viral anti-CRISPR proteins attached to a bacterial CRISPR RNA-guided surveillance complex, and how they disable their defense system.

In a study published in Cell, investigators found that anti-CRISPR proteins block the ability of the gene-editing technology to identify and attack the viral genome. Furthermore, one of the anti-CRISPR proteins “mimics” DNA to evade detection.

“It’s amazing what these systems do to one-up each other,” said co-lead investigator Gabriel C. Lander. “It all comes back to this evolutionary arms race.” They described the process as “a molecular arms race between viral suppressors and the immune system they target.”

CRISPR technology has made waves among the research community, and scientists have discovered that they can use CRISPR’s natural ability to degrade sections of viral RNA and use CRISPR systems to remove unwanted genes from an organism.

“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,” Lander said. “They are a massive, untapped resource. The more we learn about the structures of these systems, the more we can take advantage of them as genome-editing tools.”

For the study, the investigators used cryo-electron microscopy—–a high-resolution imaging technique––to identify 3 key aspects of CRISPR and anti-CRISPR systems.

The method allowed the investigators to examine precisely how the CRISPR surveillance complex analyzes a virus’ genetic material to determine if it should attack, according to the study. The proteins within the complex wrap around the CRISPR RNA to expose specific sections of bacterial RNA. The sections of RNA then scan viral DNA to look for genetic sequences they recognize.

“This system can quickly read through massive lengths of DNA and accurately hit its target,” Lander said.

Once a CRIPSR complex identifies a viral DNA target, the surveillance machine will recruit other molecules to destroy the virus’ genome, according to the study.

For the next step, the investigators analyzed how viral anti-CRISPR proteins paralyze the surveillance complex. The results showed that 1 type of anti-CRISPR protein covers up the exposed section of CRISPR RNA, preventing the system from recognizing the viral DNA.

Lander referred to the anti-CRISPR proteins as “exceptionally clever,” because they appear to have evolved to target a key piece of the CRISPR machinery. If the bacteria were to mutate the machinery to avoid viral attacks, the CRISPR system would stop functioning.

“CRISPR systems cannot escape from these anti-CRISPR proteins without completely changing the mechanism they use to recognize DNA,” Lander said.

A different anti-CRISPR protein uses a different technique based on its location and negative charge. The investigators believe that the anti-CRISPR proteins act as a DNA mimic to trick CRISPR into binding the immobilizing proteins instead of 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,” Lander said.

The investigators hope that the new findings will eventually lead to more efficacious and sophisticated tools for gene editing.