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CRISPR enzymes might also diagnose disease

University of California, Berkeley, research describes 10 new CRISPR enzymes that when activated, act like Pac-Man chewing up RNA and detecting infectious viruses.


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.


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


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.


"We have taken our foundational research a step further ... allowing for a multiplexed enzymatic detection system."

Alexandra East-Seletsky PhD, UC Berkeley graduate student in the laboratory of Jennifer A. Doudna PhD, one of the inventors of the CRISPR-Cas9 gene-editing tool and first author. East-Seletsky was also a co-first author of the September Nature paper.


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.


While Cas9 protein cuts double-stranded DNA at specific sequences, Cas13a protein is an enzyme to cut target RNA, but runs amok destroying all RNA.

"Think of Cas13a and its RNA target as an on-off switch binding to a target turns the enzyme into Pac-Man in the cell, chewing up all nearby RNA."

Alexandra East-Seletsky PhD


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.


"Building on our original work, we now show it is possible to multiplex enzymes together, and extend the scope of the technology. The diversity within the CRISPR-Cas13a family can be used for many applications, including detecting RNA."

Alexandra East-Seletsky PhD

"Our intention is to develop the Cas13a family of enzymes for point-of-care diagnostics that are robust and simple to deploy."

Jennifer A. Doudna PhD, Professor, Molecular Biology and Chemistry, Howard Hughes Medical Institute investigator.


Highlights
• Cas13a family contains two distinct subfamilies
• Cas13a subfamilies possess distinct nuclease substrate preferences
• Cas13a subfamilies also use orthogonal crRNAs
• crRNA processing is not essential for ssRNA targeting by Cas13a

Summary
CRISPR adaptive immunity pathways protect prokaryotic cells against foreign nucleic acids using CRISPR RNA (crRNA)-guided nucleases. In type VI-A CRISPR-Cas systems, the signature protein Cas13a (formerly C2c2) contains two separate ribonuclease activities that catalyze crRNA maturation and ssRNA degradation. The Cas13a protein family occurs across different bacterial phyla and varies widely in both protein sequence and corresponding crRNA sequence conservation. Although grouped phylogenetically together, we show that the Cas13a enzyme family comprises two distinct functional groups that recognize orthogonal sets of crRNAs and possess different ssRNA cleavage specificities. These functional distinctions could not be bioinformatically predicted, suggesting more subtle co-evolution of Cas13a enzymes. Additionally, we find that Cas13a pre-crRNA processing is not essential for ssRNA cleavage, although it enhances ssRNA targeting for crRNAs encoded internally within the CRISPR array. We define two Cas13a protein subfamilies that can operate in parallel for RNA detection both in bacteria and for diagnostic applications.

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).
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May 11, 2017   Fetal Timeline   Maternal Timeline   News   News Archive   



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

 


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