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New Study Finds Three More CRISPR Proteins With Genome Editing Potential

NEW YORK (GenomeWeb) – The world of CRISPR continues to get wider. Scientists parsing metagenomic databases for sequences that look like the CRISPR/Cas9 system and the newly discovered CRISPR/Cpf1 system have found three new proteins that could have applications in genome editing, including one that potentially cleaves RNA.

The new proteins C2c1, C2c2, and C2c3 —named under the formula "Class 2 candidate x"— are distantly related to the known CRISPR systems used in human genome editing. The discovery and characterization of these proteins confirms the existence of several entirely new types of CRISPR systems. Researchers from the National Center for Biotechnology Information (NCBI), the Massachusetts Institute of Technology, and Rutgers University published the results of their study today in Molecular Cell, describing the unique and interesting characteristics of the proteins.

"This work shows a path to discovery of novel CRISPR/Cas systems with diverse properties, which are demonstrated here in direct experiments," Eugene Koonin, senior investigator at the NCBI, said. "The most remarkable aspect of the story is how evolution has achieved a broad repertoire of biological activities, a feat we can take advantage of for new genome manipulation tools."

Last month, scientists led by Feng Zhang of MIT and the Broad Institute, who is also a senior author of the new study, described CRISPR/Cpf1, a new single-RNA-guided CRISPR system that can edit DNA in human cells. It joined CRISPR/Cas9 in what is being called Class 2 of all the known CRISPR systems so far discovered in microbiology.

Class 2 CRISPR systems are characterized by one large CRISPR-associated (Cas) protein (Class 1 systems feature several smaller Cas proteins). The scientists found the three new Class 2 proteins by looking in metagenomic databases for genomic loci that appeared to contain a single, large protein as well as the gene for Cas1 , a protein essential to Cas9 and Cpf1. Using these criteria, they found 53 genomic loci to which they applied the C2c1, C2c2, and C2c3 labels.

C2c1 proteins are found in 18 bacterial species in Bacilli, Verrucomicrobia, alpha-proteobacteria, and delta-proteobacteria. Experiments showed that C2c1, like Cas9, could be repurposed into a dual-RNA guided DNA endonuclease active in human cells. Much like Cpf1, C2c1 requires a 5' protospacer adjacent motif that would work well in thymine-rich areas of the genome. The scientists were even able to combine the two RNAs into a single guide RNA, similar to those used with CRISPR/Cas9 genome editing applications.

C2c3 proteins are distantly related to C2c1 proteins and could not be assigned to specific taxa. The C2c1 and C2c3 proteins both contain RuvC-like nuclease domains, similar to Cpf1.

C2c2, however, is totally different from all other Class 2 proteins and shows no sequence similarity to Cas9, Cpf1, or the two other new proteins described in the paper. The researchers found C2c2 proteins in 21 bacterial species from alpha-proteobacteria, Bacilli, Clostridia, Fusobacteria, and Bacteroidetes. Although the researchers predicted C2c2 to be associated with two crRNAs, they only found one.

Closer inspection of the C2c2 sequences suggested that they contain higher eukaryotes and prokaryotes nucleotide-binding (HEPN) domains that could indicate RNase, or RNA-cleaving activity. "This is only a prediction or you may even say a speculation," Koonin told GenomeWeb. "But so far, all these [HEPN] domains that have ever been characterized indeed contain RNase activity." Interestingly, C2c2 contains two copies of this domain, while other proteins usually contain just one. "I would not be shocked if it turned out to be the first DNase in the HEPN family," he said.

While other types of CRISPR systems that haven't been used for genome editing have been known to target and cleave RNA, C2c2 could be the first Class 2 CRISPR system to offer that functionality.

Soon, scientists could have their hands full discovering, characterizing, and evaluating CRISPR systems.

"There are multiple ways to modify the search algorithm," Konstantin Severinov, a professor at Rutgers and an author of the study, said. "So more exciting and distinct CRISPR/Cas mechanisms should be expected soon."

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