The Evolutionary Arms Race
Scientists have uncovered the mechanisms by which viruses can inactivate the bacterial immune system CRISPR to enable their survival and proliferation. The research, published in Cell, demonstrates how high-resolution images were used to identify the structure of the viral and bacterial protein complexes and how the virus inactivated the nuclease-activity of CRISPR.
The CRISPR system has become a very important part of recent genomic studies, largely because of its incredible accuracy and efficiency as a gene editing tool. The system involves a protein with nuclease activity to cleave DNA and a guide RNA which leads the protein to a specific locus within the genome. By manipulating the guide RNA, researchers have been able to cleave DNA at a range of desired location with great accuracy at will.
However, one of the problems that researchers have found when trying to utilise the CRISPR system is activating and deactivating the system as needed. To maintain the high accuracy of the tool, it is necessary to ‘switch off’ the nuclease activity when the desired edit has been made so that the CRISPR protein cannot continue to damage the DNA. With this in mind, several groups have started to investigate how viruses have adapted to cope with the CRISPR system.
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As DNA constructs, viruses are particularly susceptible to defence mechanisms like the CRISPR system, and so infecting bacteria has meant that they have needed to develop a range of proteins to protect themselves. To this end, viruses have developed ‘anti-CRISPR’ proteins which immobilise the bacterial defences.
This new research was focused on understanding the underlying mechanism of these anti-CRISPR proteins. While they have been identified in the past, their mode of action was relatively unknown, limiting researchers’ ability to utilise them in their own CRISPR experiments. However, using cryo-electron microscopy, a high resolution imaging technique, the team, lead by researchers from the Scripps Research Institute (TSRI), were able to observe how anti-CRISPR proteins and the bacterial CRISPR complex interacted with each other and how the bonding disabled the CRISPR system.
“It’s amazing what these systems do to one-up each other,” said Gabriel C. Lander, Ph.D., co-leader of the study. “It all comes back to this evolutionary arms race.”
The first thing the team investigated was how the CRISPR system analysed the viral genome. They were able to observe how the CRISPR proteins wrapped around the guide RNA, leaving specific regions exposed. It was these exposed sections which were then matched to viral sequences and therefore determined the specificity of the technique.
With this understanding, they were then able to examine the viral defence mechanisms. They were able to identify a range of different anti-CRISPR proteins that worked in a multitude of ways. One protein wrapped itself around the CRISPR complex, burying the exposed RNA regions and effectively blinding the system so that it could no longer target DNA.
Lander noted that these proteins were “exceptionally clever” in their approach to the problem. By targeting a vital component of the system that could not be removed, the viruses were preventing the bacteria from adapting to avoid the mechanism. “These anti-CRISPR proteins keep the bacteria from recognizing the viral DNA,” he said. “CRISPR systems cannot escape from these anti-CRISPR proteins without completely changing the mechanism they use to recognize DNA.”
Another protein appeared to be able to mimic DNA (based on observations of its location and negative charge), fooling the CRISPR complex into binding with it and then immobilising the entire structure.
The team behind this work hope that these discoveries will be able to improve future gene editing experiments that utilise the CRISPR system. Being able to activate and deactivate CRISPR at will is a very important part in developing a gene editing system that is both effective and safe.
“These findings are important because we knew that anti-CRISPR proteins were blocking bacterial defences, but we had no idea how,” said Lander. “That might work as an on-off switch for CRISPR.”