In the CRISPR toolkit, Cas12a2 is like a Swiss Army Knife—one tool that can do multiple things. The nuclease performs RNA-guided, sequence-nonspecific degradation of single-stranded RNA, single-stranded DNA, and double-stranded DNA. However, how the enzyme cuts so indiscriminately has remained unclear.
Now, researchers shed light on its nuclease activity by solving its structure. Using cryo-EM, the team reports structures of Cas12a2 in binary, ternary, and quaternary complexes to provide a structural basis for the system’s mechanism. This work could be leveraged to create rational mutants that degrade a spectrum of collateral substrates. In addition, the discovery may help the development of new, inexpensive, and highly sensitive at-home diagnostic tests for a wide range of infectious diseases, including COVID-19, influenza, Ebola, and Zika.
This work is published in Nature in the paper, “RNA targeting unleashes indiscriminate nuclease activity of CRISPR-Cas12a2.”
It was recently discovered that Cas12a2 from Sulfuricurvum sp. PC08-66 relies on abortive infection (dormancy or cell death in response to the presence of an invader) to achieve population-level immunity instead of the more typical system used by CRISPR–Cas systems to target and degrade foreign genetic elements, including phage and plasmids.
Using cryo-EM, researchers discovered that when Cas12a2 binds to a specific sequence of target RNA from a virus, a side portion of Cas12a2 swings out to reveal an active site which starts to indiscriminately cut any genetic material it comes into contact with.
More specifically, the authors write that their structures reveal “Cas12a2 is autoinhibited until binding a cognate RNA target, which exposes the RuvC active site within a large, positively charged cleft.” Double-stranded DNA substrates, they noted, are captured through duplex distortion and local melting, stabilized by pairs of “aromatic clamp” residues that are crucial for double-stranded DNA degradation and in vivo immune system function.
“Cas12a2 basically grabs the two ends of the DNA double helix and bends it really tightly,” said Jack Bravo, PhD, a postdoctoral fellow in the lab of David Taylor at the University of Texas at Austin. “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.”
With a single mutation to the Cas12a2 protein, the active site degrades only single-stranded DNA—a feature especially useful in developing new diagnostics tailored for any of a wide range of viruses.
A test based on this technology could theoretically combine the best features of PCR-based tests that detect genetic material from a virus (high sensitivity, high accuracy, and the ability to detect an active infection) with the best features of rapid at-home diagnostic tests (inexpensive to produce without requiring specialized lab equipment). It also would be easily adaptable to any new RNA virus.
“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,” said David Taylor, PhD, associate professor in the department of molecular biosciences at the University of Texas at Austin.
A companion paper in the same issue of Nature, titled “Cas12a2 elicits abortive infection through RNA-triggered destruction of dsDNA” describes the biological functions of Cas12a2.