NEW YORK — Researchers have described a new CRISPR-associated nuclease that behaves differently from other Cas12a nucleases and could potentially be used as a molecular diagnostic.
"We were exploring CRISPR nucleases that were originally clumped with Cas12a. … Once we identified more of them, we realized that they were different enough from Cas12a to warrant a deeper dive," Oleg Dmytrenko, the first author of one of the studies and a postdoctoral fellow at the Würzburg Helmholtz Institute for RNA-based Infection Research (HIRI) in Germany, said in a statement. "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."
In a pair of papers appearing in Nature on Wednesday, researchers from HIRI and their US-based colleagues reported that the nucleases they dubbed Cas12a2 lead to an abortive infection response that triggers indiscriminate destruction of nucleic acids and affects cell growth — which, in the original bacterial system, was likely a last-ditch response to infection. In their structural analysis, the researchers found this action is mediated by the exposure of a cleft within Cas12a2. Such a tool targeting nucleic acids, they added, could be harnessed for a range of applications, including diagnostics.
"Considering how well different nucleases have been translated into new and improved technologies, any discovery in this field could bring new benefits to society," study author Chase Beisel, a group leader at HIRI, added in a statement.
Cas12a2 is a type V single-effector Cas nuclease, but as Dmytrenko and colleagues described in their paper, it has a protein sequence and architecture that is distinct from that of Cas12a. While Cas12a2 harbors conserved RuvC-like domains and N-termini, it contains a large domain of unknown function rather than the Cas12a bridge helix and has a zinc-finger domain instead of the Cas12a Nuc domain.
Through a series of analyses, they found that Cas12a2 relies on a different defense mechanism than that of Cas12a. It is activated by a protospacer-flanking sequence and recognizes target RNAs that are complementary to its guide RNA. After that binding, the active site is exposed and it degrades any single-stranded RNA, single-stranded DNA, and double-stranded DNA.
This activation triggers a sort of cellular SOS response, according to the researchers, which impedes cellular growth and leads to an abortive infection response.
In an accompanying paper, the researchers additionally used cryo electron microscopy to examine the structure of Cas12a2 while bound to a CRISPR RNA (crRNA), while bound to the crRNA and an RNA target, and while in a quaternary complex that also included a dsDNA collateral substrate mimetic. Cas12a2, they found, is auto-inhibited until it binds a cognate RNA target, which leads the RuvC active site to be exposed. That site then binds to different nucleic acids and destroys them.
"Cas12a2 basically grabs the two ends of the DNA double helix and bends it really tightly," co-first author Jack Bravo, a postdoctoral fellow at the University of Texas-Austin, said in a statement. "And so, the helix in the middle pops open, and then this allows this active site to destroy the bits of DNA that become single-stranded. This is what makes Cas12a2 different from all the other DNA-targeting systems."
Cas12a2 could be used in numerous applications, the researchers said. In the first paper, they showed that Cas12a2 could be harnessed to directly detect RNA. They further envisioned other applications for Cas12a2-fueled tools, including programmed killing of prokaryotic and eukaryotic cells or as a diagnostic that detects viral genetic material.
"If some new virus comes out tomorrow, all you have to do is figure out its genome and then change the guide RNA in your test, and you'd have a test against it," co-corresponding author David Taylor from UT-Austin added in a statement.