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New CRISPR Protein Targets, Cleaves RNA


NEW YORK (GenomeWeb) – A newly characterized CRISPR protein called C2c2 can target and cleave RNA in bacterial cells, opening up the possibility of a host of applications that have been developed for DNA-targeting CRISPR systems.

Co-senior authors  Feng Zhang of the Massachusetts Institute of Technology and Broad Institute and Eugene Koonin of the National Center for Biotechnology Information, along with other scientists at those institutions and Rutgers University, published the results of a study describing C2c2 today in Science.

"It's a completely different enzyme than Cas9 or Cpf1," Zhang told GenomeWeb. But, as he's done with Cas9 and Cpf1, he plans to turn it into a broadly applicable tool for biological research. "There are many ways to use it," he said.

In their study, the scientists showed that C2c2 belongs to a Class 2 CRISPR system which uses a single CRISPR RNA (crRNA) to find its target, has a preference for targets flanked by bases other than guanine, and cleaves single-stranded regions featuring short stretches of uracils. It doesn't appear to cleave double-stranded RNA or any type of DNA.

While these characteristics make it a unique among known CRISPR proteins, Zhang said they are not enormous limitations. And because it opens up the other half of the nucleic acid world to CRISPR-based applications it could become a technology on par with RNAi.

The study is the latest result from a collaboration with NCBI scientists to systematically scan genomic databases for sequences that resembled known CRISPR systems. The group found Cpf1 this way and, as reported by GenomeWeb in October 2015, published a study in Molecular Cell describing three new CRISPR proteins — C2c1, C2c2, and C2c3, all named under the rubric "Class 2, Candidate X."

While C2c1 and C2c3 both more resembled familiar DNA-cleaving CRISPR systems, C2c2 appeared to have higher eukaryote and prokaryote nucleotide-binding (HEPN) domains, suggesting it was an RNase.

"This is another interesting example of RNA-guided nucleic acid targeting employed by bacterial adaptive immune systems," Jennifer Dounda of the University of California, Berkeley told GenomeWeb in an email. "Single-celled organisms have evolved a diverse set of pathways for viral defense, and it's notable that RNA-targeting is relatively rare, perhaps due to the rarity of RNA viruses that infect bacteria."

The HEPN domains made it an especially intriguing project to move forward with, although Zhang said his lab is also investigating C2c1 and C2c3.

He said they chose to focus on the C2c2 from Leptotrichia shahii because it was readily available. "There's a whole family of them," he said. "There are many others we're looking into exploring."

The Science study showed that engineering Escherichia coli with the C2c2 system was able to offer immunity to the MS2 phage, along with other basic information about how it worked.

"Just like with Cas9, you can use it as an RNA-guided nuclease," Zhang said. In the study, the researchers successfully programmed it to knock down RFP production in cells by targeting messenger RNA; C2c2 did not successfully knock down RFP production when targeting DNA.

They also showed that by mutating the protein, they could inhibit cleavage but not RNA-binding ability, similar to the nuclease-null dCas9. "As a binding protein, you can use it to recruit different markers to track RNA or alter splicing," Zhang said. "Or you could try to upregulate translation by recruiting ribosomes." More options might include creating an RNA sensor or coupling other enzymatic activities to specific sites in RNA.

Despite the new information about C2c2, Zhang said it still needs to be further characterized. "We need to see how specific it is, how it works in mammalian cells," he said.

"More work is needed to turn this into a tool but we're excited to be working on that," he said.