UC Berkeley researchers find Pac-Man-like CRISPR enzymes

Updated 2017-05-05 11:30:30 Xinhua

Researchers at the University of California, Berkeley, have described 10 new CRISPR enzymes that, once activated, behave like Pac-Man in the classic video game to chew up ribonucleic acid (RNA), a nucleic acid present in all living cells.

The researchers, who published a paper Thursday in the journal Molecular Cell, believe their new findings could lead to sensitive detectors of infectious viruses.

CRISPR, short for clustered regularly interspaced short palindromic repeats, are segments of prokaryotic DNA containing short, repetitive base sequences. It has been a contested research area in recent years, as it holds the promise to be a gene-editing tool.

The new CRISPR enzymes are variants of a CRISPR protein, Cas13a. Last September, the UC Berkeley researchers reported in Nature that once CRISPR-Cas13a binds to its target RNA, it begins to indiscriminately cut up all RNA, easily cutting RNA linked to a reporter molecule, making it fluoresce to allow signal detection.

Two teams of researchers at the Broad Institute later paired CRISPR-Cas13a with RNA amplification, and showed that the system could detect viral RNA at extremely low concentrations, detecting the presence of dengue and Zika viral RNA, for example.

Such a system could be used to detect any type of RNA, including RNA distinctive of cancer cells.

While the original Cas13a enzyme used by the UC Berkeley and Broad teams cuts RNA at one specific nucleic acid, uracil, according to the researchers, three of the new Cas13a variants cut RNA at adenine. This difference allows simultaneous detection of two different RNA molecules, such as from two different viruses.

RNA consists of four different nucleotides: adenine, cytosine, guanine and uracil.

"Think of binding between Cas13a and its RNA target as an on-off switch -- target binding turns on the enzyme to go be a Pac-Man in the cell, chewing up all RNA nearby," said first author Alexandra East-Seletsky, a UC Berkeley graduate student in the laboratory of Jennifer Doudna, one of the inventors of the CRISPR-Cas9 gene-editing tool.

The CRISPR-Cas13a family is related to CRISPR-Cas9, which is revolutionizing biomedical research and treatment because of the ease of targeting it to unique DNA sequences to cut or edit. While the Cas9 protein cuts double-stranded DNA at specific sequences, the Cas13a protein latches onto specific RNA sequences, and not only cuts that specific RNA, but runs amok to cut and destroy all RNA present.

After publishing their September Nature paper, the UC Berkeley researchers searched databases of bacterial genomes and found 10 other Cas13a-like proteins, which they synthesized and studied to assess their ability to find and cut RNA. Of those, seven resembled the original Cas13a, while three differed in where they cut RNA.

Doudna, a professor of molecular biology, noted that detection of infectious RNA may or may not require amplification, which is a complicated step. "Our intention is to develop the Cas13a family of enzymes for point-of-care diagnostics that are robust and simple to deploy," she was quoted as saying in a news release.

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