Mapping gigantic molecular scissors

Tech Science 30. aug 2020 3 min Research director, professor Guillermo Montoya Written by Kristian Sjøgren

Researchers have mapped the structure and function of a giant CRISPR-Cas complex. The mapping of these molecular scissors may advance researcher’s knowledge of how bacteria develop antimicrobial resistance and how this can be combatted.

Researchers from the University of Copenhagen have mapped the structure and function of the largest CRISPR-Cas system to date.

These molecular scissors contain 37 polypeptides and a crRNA molecule containing six active catalytic centres and play a key role in defending microorganisms against invading phages (viruses that attack bacteria).

The mapping of the CRISPR-Cas system known as Cmr-β gives researchers entirely new insight into the battle between microbes that leads to the development of antimicrobial resistance. The discovery could potentially lead to other biotechnological applications in the future.

“How this discovery can be exploited is difficult to say now. With CRISPR-Cas9, CRISPR technology has already revolutionized gene technology, and although this CRISPR-Cas system can probably not be used for the same purposes, it might be used in other contexts of which we are not yet aware, but this requires understanding the structure and function of Cmr-β,” explains Guillermo Montoya, Professor, Novo Nordisk Foundation Center for Protein Research, University of Copenhagen.

The research has been published in Molecular Cell.

CRISPR-Cas is the cornerstone in defending against phages

Cmr-β is an important part of the archaea microorganisms’ defence against invading phages.

When the phages settle on the surface of the cell, they fire their genetic material into the cell to get the host to copy the genetic material of the phages and make more phages at the expense of the cell’s own chances of survival.

One response from the archaeal cell is to mobilize Cmr-β, which identifies the invading RNA and cuts it into snippets.

The new research results show that Cmr-β has 4 catalytic sites that can bind and cleave invading RNA.

However, this is not the end of the story.

Over evolutionary time, many phages have evolved a response in which they fire anti-CRISPR molecules into the archaeal cell, knocking out complexes such as Cmr-β in the process.

However, the phages are not alone in evolving over time, and the new research suggests that some of the many protein moieties in Cmr-β actually form a protective sheath around the CRISPR-Cas system, so that the phages’ anti-CRISPR molecules cannot penetrate the catalytic parts of the complex and neutralize them.

In addition, Cmr-β also produces the cyclic oligoadenylate signalling molecule, which activates other molecular scissors and thus intensifies the antimicrobial defence.

“This is an incredibly extensive complex that has been very complicated to map because it has so many different features. Finding other examples of protein complexes that have multiple enzymatic activities in the same complex is difficult,” explains Guillermo Montoya.

Insight into how antimicrobial resistance develops

During the study, the researchers mapped the structure of Cmr-β using cryoelectron microscopy to capture thousands of blurred images of the protein complex and then compiled them into high-resolution 3D images.

This enabled them to determine what the protein complex looked like in its activated and inactivated forms.

Understanding the function of Cmr-β also means understanding how antimicrobial resistance develops.

In struggling against the phages, the archaea and the bacteria inhabit a soup of genetic material from the snippets of cleaved RNA.

The archaea or the bacteria can use this RNA soup in their own DNA if the snippets of RNA turn out to be able to protect them against various types of antibiotics that we may launch in their direction.

“By understanding how microorganisms’ defences against phages function, we can also better understand immunity in bacteria and how bacteria render our medicines useless. We can then develop methods to combat the development of antimicrobial resistance,” says Guillermo Montoya.

May become a biotechnology tool

In addition to advancing our understanding of the development of immunity in microorganisms, mapping Cmr-β may also have useful applications within biotechnology or in diagnosis.

The researchers still cannot be certain what the applications may be, but they are examining this.

CRISPR-Cas9 has revolutionized gene technology, and other CRISPR-Cas systems can also be used, for example, to remove all RNA and thus all proteins in cells to study the cells in a completely new way. Cmr-β may have similar potential.

“In the future, the complex may have applications that we cannot imagine right now, but that is how basic scientific discoveries work. Only a year ago, nobody took very much interest in coronaviruses in bats because nobody could see that this type of virus could generate a pandemics. Data that do not appear interesting today can suddenly become very topical or very useful,” says Guillermo Montoya.

"Structures of the Cmr-β complex reveal the regulation of the immunity mechanism of type III-B CRISPR-Cas" has been published in Molecular Cell. Several authors of the study are employed at the Novo Nordisk Foundation Center for Protein Research, University of Copenhagen. In 2019, the Novo Nordisk Foundation awarded a Distinguished Investigator grant to Guillermo Montoya entitled GENED_2.0: From Molecular Mechanisms to the Next Generation of Genome Editing Tools.

The Montoya Group works to illuminate the molecular details of cellular processes. This knowledge is the basis for the understanding of diseases and t...

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