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CRISPR enzymes might also diagnose disease
New CRISPR enzymes are variations of the CRISPR protein, Cas13a, unveiled last September in Nature by UC Berkeley. Turns out these enzymes can detect specific sequences of RNA, like those in a virus. Once CRISPR-Cas13a binds to a target RNA, it indiscriminately cuts up all RNA, easily severing RNA links to a molecule. This process makes the molecule fluoresce, scientists can then detect signals sent out by other infected cells.
Two teams of researchers at the Broad Institute paired CRISPR-Cas13a with amplified RNA, to show how this new process named SHERLOCK can detect viral RNA at extremely low concentrations. Detecting the early presence of viral RNA like that of dengue and Zika.
While the original Cas13a enzyme used by UC Berkeley and Broad Institute teams cut RNA at uracil, one of the four bases in the nucleic acid of RNA represented as letters: adenine (A), cytosine (C), and guanine (G), three of these new Cas13a variants cut RNA at adenine. This allows for simultaneous detection of two different RNA molecules, such as found in two viruses.
East-Seletsky PhD, and Jennifer A. Doudna PhD, and their UC Berkeley colleagues reported their findings May 4 in the journal Molecular Cell.
The CRISPR-Cas13a family (formerly called CRISPR-C2c2) is related to CRISPR-Cas9, which is revolutionizing biomedical research with its ease in targeting unique DNA sequences to be cut or edited as needed.
This RNA killing spree can also kill the cell. In their September Nature paper, the researchers argue this CRISPR-Cas13a, Pac-Man like activity in bacteria kills infectious viruses — its main role. As part of the immune system in some bacteria, it allows infected cells to commit suicide and save their sister microbes from infection. Similar non-CRISPR suicide systems exist in other bacteria.
The researchers searched databases of bacterial genomes to find 10 other Cas13a-like proteins. Of those, seven act like Cas13a, while three were different in where they cut RNA. RNA serves many functions inside a cell, as messenger RNA - working copies of DNA, consists of four different nucleotides: adenine, cytosine, guanine and uracil.
Co-authors with East-Seletsky and Doudna are former UC Berkeley postdoctoral fellow Mitchell O'Connell, now an assistant professor at the University of Rochester, and UC Berkeley postdocs David Burstein and Gavin Knott. The work was supported in part by a Frontiers Science award from the Paul Allen Institute and by the National Science Foundation (MCB-1244557).
Researchers at the Broad Institute paired CRISPR-Cas13a with amplified RNA, showing how
this new combination, called SHERLOCK, can detect viral RNA at extremely low concentrations.
Such a mix allows SHERLOCK to detect the presence of viral RNA, like dengue and Zika.
Dr. Doudna notes that detection of infectious RNA may or may not require amplification
which involves a complicated step.
Image Credit: Gregory Urquiaga/UC Davis