Pac-Man like CRISPR enzymes discovered
9 May 2017 by Evoluted New Media
New CRISPR enzymes have been found that could be used as sensitive detectors for infectious viruses.
New CRISPR enzymes have been found that could be used as sensitive detectors for infectious viruses.
US scientists were able to show that once CRISPR-Cas13a binds to its target RNA, it indiscriminately cuts up all RNA, like Pac-Man eating dots. Jennifer Doudna, professor of molecular biology and of chemistry at the Broad Institute, in Massachusetts, said: “Our intention is to develop the Cas13a family of enzymes for point-of-care diagnostics that are robust and simple to deploy.”
Researchers at the Institute paired CRISPR-Cas13a with RNA amplification and showed the system, dubbed SHERLOCK, could detect viral RNA at extremely low concentrations. This included RNA linked to a reporter molecule that would fluoresce, allowing it to be detected. The system has been shown to detect the presence of dengue and Zika viral RNA and potentially could detect RNA of distinctive cancer cells.
The CRISPR-Cas13a family, formerly referred to as CRISPR-C2c2, is related to CRISPR-Cas9, which is already revolutionising 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 – a nucleic acid-cutting enzyme referred to as a nuclease – latches onto specific RNA sequences, and not only cuts that specific RNA, but runs amok to cut and destroy all RNA present.
Alexandra East-Seletsky, a UC Berkeley graduate student working in the laboratory of Jennifer Doudna, one of the inventors of the CRISPR-Cas9 gene-editing tool, said: "We have taken our foundational research a step further. We found other homologs of the Cas13a family that have different nucleotide preferences, enabling concurrent detection of different reporters with, say, a red and a green fluorescent signal, allowing a multiplexed enzymatic detection system.”
While the original Cas13a enzyme used by the University of California Berkeley and Broad teams cuts RNA at one specific nucleic acid, uracil, three of the new Cas13a variants cut RNA at adenine. This difference allows simultaneous detection of two different RNA molecules, which could be from two different viruses. A full report of their findings will appear in Molecular Cell.
East-Seletsky said: "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." This RNA killing spree can kill the cell.
UC Berkeley researchers in Nature last September argued the ‘Pac-Man’ activity of CRISPR-Cas13a is its main role in bacteria, aimed at killing infectious viruses or phages. As part of the immune system of some bacteria, it allows infected cells to commit suicide to save their sister microbes from infection. Similar non-CRISPR suicide systems exist in other bacteria.
The UC Berkeley researchers subsequently searched databases of bacterial genomes and found 10 other Cas13a-like proteins. These have been synthesised 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.
East-Seletsky said: "Building on our original work, we now show that it is possible to multiplex these enzymes together, extending the scope of the technology. There is so much diversity within the CRISPR-Cas13a family that can be utilised for many applications, including RNA detection."
By Dermot Martin
