Novel Anti-CRISPR Protein Targets Cas13b RNA Nuclease

There are numerous potential uses for the CRISPR-Cas gene scissors, ranging from antiviral treatments and diagnostics to the treatment of genetic disorders. Scientists are looking for ways to control or prevent the systems' activity, though, to safely use their abilities. Here comes the anti-CRISPR protein AcrVIB1, a promising inhibitor whose precise role has not yet been determined.

In cooperation with the Helmholtz Centre for Infection Research (HZI) in Braunschweig, a research team from the Helmholtz Institute for RNA-based Infection Research (HIRI) in Würzburg has discovered the exact mechanism by which AcrVIB1 functions, broadening the known mechanism by which Acrs can inhibit CRISPR. The findings were published in the journal Molecular Cell.

Phage viruses and bacteria are engaged in a long-standing arms race. Bacteria have developed complex defenses against phage attacks that enable them to identify and neutralize invasive viruses.

In response, phages have created creative ways to get past these defenses. The CRISPR-Cas defense system in bacteria is a prime example of this continuous conflict, as it is thwarted by anti-CRISPR proteins (Acrs) in phages, which selectively block these bacterial “gene scissors.”

In addition to their counter-defensive role, anti-CRISPR proteins have the potential to allow for more accurate control over CRISPR technologies. To realize their full potential, it is crucial to comprehend the underlying mechanisms.

In collaboration with the Julius-Maximilians-Universität Würzburg (JMU), researchers at the Braunschweig Helmholtz Centre for Infection Research (HZI) and the Helmholtz Institute for RNA-based Infection Research (HIRI) have now further clarified the role of a significant but as-yet-uncharacterized anti-CRISPR protein.

In a previous study, we used a deep learning algorithm to predict new Acrs. This led to the identification of AcrVIB1, the first anti-CRISPR protein targeting the Cas13b nuclease. The nuclease Cas13b can recognize and cut RNA. It is currently used to silence genes, whether to study their function, clear viruses or counteract genetic diseases linked to the gene.”

 Chase Beisel, Department Head and Professor, Helmholtz Institute for RNA-based Infection Research

Chase Beisel led the study together with the department of Professor Wulf Blankenfeldt at HZI.

However, how the protein AcrVIB1 inhibits Cas13b remained unknown until now. In this study, the research team presents this entirely new blocking mechanism.

An RNA Dead-End

The Cas13b nuclease functions by interacting with a CRISPR ribonucleic acid (crRNA), which acts as a guide to recognize and bind to complementary RNA sequences, such as those from phages. Once the target RNA is bound, Cas13b can cleave and degrade all nearby RNAs and these complementary RNA molecules.

AcrVIB1 takes a completely different approach from the majority of known anti-CRISPR proteins, which block steps along this pathway like target recognition or crRNA binding: AcrVIB1 actually enhances the binding of the crRNA to Cas13b rather than preventing it.

However, the generated pair is dysfunctional, which means that even in the presence of its target, the enzyme cannot start breaking down RNAs. Additionally, cellular ribonucleases, which degrade RNA molecules, can target the bound crRNA.

The tighter binding between nuclease and guide RNA was entirely unexpected. The simpler and therefore initially expected mechanism would have been to just prevent the guide RNA from binding in the first place. Nevertheless, the path taken by AcrVIB1 appears to be more effective: AcrVIB1 binds tightly to and thereby renders Cas13b inactive. At the same time, it increases the turnover of guide RNAs, making Cas13b a dead end for crRNAs.”

Dr. Katharina Wandera, Study First Author, Helmholtz Centre for Infection Research

Wandera has completed her doctorate in Chase Beisel's laboratory.

Wulf Blankenfeldt's lab at HZI and Chase Beisel's group at HIRI have teamed up to understand the structure of the inhibition mechanism better. Blankenfeldt's team demonstrated through cryo-electron microscopy that AcrVIB1 binds to Cas13b, releasing the crRNA-binding domain.

“Our finding provides a blueprint for the development of molecules that could mimic or modify the function of the anti-CRISPR protein,” says Blankenfeldt.

These are the first data from the HZI's new cryo-electron microscopy facility to be published.

A Vast Field

In the future, we could use molecules such as AcrVIB1 to regulate or temporarily deactivate CRISPR systems across a variety of applications.”

Wulf Blankenfeldt, Helmholtz Centre for Infection Research

The safety and accuracy of CRISPR-based technologies could be further improved by this discovery.

“Deciphering this mechanism also provides valuable insights into the co-evolution of bacteria and viruses, which are constantly trying to outsmart each other,” explained Wandera.

A better understanding of bacterial resistance may greatly benefit the creation of novel antibiotics and the advancement of synthetic biology.

In conclusion, this research not only advances the knowledge of anti-CRISPR tactics but also opens the door for novel medical treatments and diagnostic techniques.

“But this is just the beginning: There are certainly many more Acrs and novel inhibitory mechanisms waiting to be discovered,” said Beisel, giving an outlook on future research projects.