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CRISPR/C2c2 Enzyme Possesses Two RNA-Cleaving Mechanisms

NEW YORK (GenomeWeb) – The exotic CRISPR enzyme C2c2 has two different RNA cleaving functions — one for cutting its target and another for processing its guiding CRISPR RNA. The distinct RNase activities of the RNA-guided, RNA-targeting C2c2 enzyme were parsed in a new Nature study led by University of California, Berkeley scientists Jennifer Doudna and Robert Tijan.

To pin down specifics about how C2c2 interacts with RNAs, the scientists looked at enzymes from three bacterial species: Leptotrichia buccalis, Leptotrichia shahii, and Listeria seeligeri. The study also detailed how each of the C2c2 enzymes process pre-crRNAs. "These dual RNase functions are chemically and mechanistically different from each other and form the crRNA-processing behavior of the evolutionarily unrelated CRISPR enzyme Cpf1," the authors wrote.

In a statement, Doudna said the study suggests C2c2 could be used in molecular diagnostics. "C2c2 is essentially a self-arming sentinel that attacks all RNAs upon detecting its target. This activity can be harnessed as an auto-amplifying detector that may be useful as a low-cost diagnostic," she noted.

It's one of the first proposed applications for the nascent C2c2 enzyme — it was reported less than a year ago by scientists led by Feng Zhang of the Massachusetts Institute of Technology and Eugene Koonin of the National Center for Biotechnology Information, and is one of three proteins found by looking through bacterial genome databases for sequences resembling CRISPR systems. C2c2 was uniquely different from previously discovered CRISPR proteins, including Cas9 and Cpf1, and was predicted to have RNA-cleaving abilities. This was confirmed by another study led by Zhang and Koonin, published in June.

In April, scientists led by Emmanuelle Charpentier of the Max Planck Institute for Infection Biology in Berlin — a senior co-author with Doudna on the seminal paper establishing CRISPR/Cas9 as a programmable nuclease — found that CRISPR/Cpf1 could both cleave target DNA and process crRNAs.

"We wondered whether C2c2 itself might possess pre-crRNA processing activity, and if so, whether the mechanism would be distinct from Cpf1, an unrelated class 2 CRISPR effector that can process pre-crRNAs," the Berkeley team wrote in its study.

Using RNA-sequencing and cleavage assays, the team mapped cleavage sites and determined that C2c2 enzyme activity resembled crRNA processing. Cleaving target RNA was thought to be enabled by HEPN domains in the C2c2 protein, but it was unclear which parts enabled crRNA processing. By systematically mutating amino acid residues in L. buccalis, the scientists found a mutated protein where crRNA processing was diminished while crRNA binding and targeted RNA cleavage were not affected. "These results show that distinct active sites within the C2c2 protein catalyze pre-crRNA processing and RNA-directed RNA cleavage," the authors wrote.

The researchers came up with strategy to use the dual-cutting nature of C2c2 to detect specific RNA transcripts in complex mixtures. "In bacteria, C2c2 probably operates as a sentinel for viral RNAs," they noted, adding that C2c2 seems to show indiscriminate RNA cleavage when activated, cutting non-target RNA as well. "We propose that when invasive transcripts are detected within the host cell via base pairing with crRNAs, C2c2 is activated for promiscuous cleavage of RNA," they said.

The team surmised that an activated C2c2 ribonucleoprotein would also cleave a single-stranded RNA oligo attached to a quenched fluorescent reporter, releasing the reporter and generating a signal.

The researchers were successful in experiments to detect a signal by having C2c2 target both phage and human RNA, suggesting it could potentially act as an RNA detection platform that does not rely on nucleic acid amplification.