Researchers expand the known defensive abilities of CRISPR–Cas systems

Viruses can infect bacteria and archaea, just as they can humans. These microbes have evolved their own immune defense mechanisms to combat their pathogens. Bacterial defenses, like CRISPR-Cas systems, contain a variety of proteins and functions that aid bacteria in protecting themselves from foreign intruders.

The defense is based on a similar mechanism: as a “guide RNA,” a CRISPR ribonucleic acid (crRNA) detects sections of a foreign genome, such as virus DNA, for targeted cleavage. The CRISPR-associated (Cas) nuclease, which is guided by a crRNA, can cut its target with a pair of scissors, a natural method that humans have employed in many technologies.

Considering how well different nucleases have been translated into new and improved technologies, any discovery in this field could bring new benefits to society.”

Chase Beisel, Helmholtz Institute for RNA-based Infection Research, University of Würzburg

The institute is a site of the Braunschweig Helmholtz Centre for Infection Research, in collaboration with the Julius-Maximilians-Universität (JMU) in Würzburg. Beisel began the current investigation on a specific set of CRISPR-Cas systems in collaboration with Matthew Begemann at Benson Hill, Inc. (Missouri) and Ryan Jackson at Utah State University in the United States.

The findings were published in Nature, along with a comprehensive structural analysis by a second group headed by Ryan Jackson and David Taylor at the University of Texas.

Unique from any other known CRISPR nuclease

We were exploring CRISPR nucleases that were originally clumped with Cas12a, nucleases that defend bacteria by recognizing and cleaving invasive DNA. Once we identified more of them, we realized that they were different enough from Cas12a to warrant a deeper dive. This exploration led us to discover that these nucleases, which we called Cas12a2, do something very different not only from Cas12a but also from any other known CRISPR nuclease.”

Oleg Dmytrenko, Study First Author, University of Würzburg

The key distinction is in the mechanism of their defense action. Cas12a2 identifies invading RNA and cleaves it, but it can also destroy other RNA and DNA inside the cell, hindering growth and restricting infection transmission.

In general, such defense strategies that abort the infection have been known in bacteria.“A few other CRISPR-Cas systems work in this way. However, a CRISPR-based defense mechanism that relies on a single nuclease to recognize the invader and degrade cellular DNA and RNA has not been observed before.”

Oleg Dmytrenko, Study First Author, University of Würzburg

The findings in detail

Cas12a2 is distinguished from Cas12a by its protein sequence and architecture. Cas12a2 detects target RNAs that are complementary to its guide RNA when activated by a protospacer-flanking sequence (PFS). When RNA is targeted, it causes collateral nucleic acid cleavage, which degrades RNA, single-stranded DNA, and double-stranded DNA.

This activity causes cell arrest, likely by disrupting the cell’s DNA and RNA, impairing proliferation. Proof-of-principle shows that Cas12a2 can be employed for molecular diagnostics and direct detection of RNA biomarkers.

A destructive cleft

Cas12a2 was found to undergo considerable structural changes after attaching to its RNA target at different phases of the immune response in a second team's structural investigation of the nuclease published in the same issue of Nature.

This, in turn, exposes a cleft in the nuclease, allowing it to shred whatever nucleic acid it comes across, be it RNA, single-stranded DNA, or double-stranded DNA. The study also found strategies to tweak Cas12a2 in order to change the nucleic acid that the nuclease destroys after detecting its RNA target. These particulars open the door to potentially wide technology uses in the future.