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Diagnostic detection with SHERLOCK
04-25-2017
by Kelsey Kaustinen  |  Email the author
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CAMBRIDGE, Mass.—The potential of CRISPR/Cas9 has turned the system into one of the latest research sweethearts given the options it could open up in gene editing, and some approaches are already beginning clinical trials. But a combined team of researchers from the Massachusetts Institute of Technology (MIT) and Harvard is taking a different tack—using CRISPR for diagnosis rather than genetic editing, thanks to a protein that targets RNA instead of DNA.
 
The work comes from scientists at the Broad Institute of MIT and Harvard, the McGovern Institute for Brain Research at MIT, the Institute for Medical Engineering & Science at MIT and the Wyss Institute for Biologically Inspired Engineering at Harvard University. The details were published in Science in a study titled “Nucleic acid detection with CRISPR-Cas13a/C2c2.”
 
As noted in the abstract, “The RNA-guided, RNA-targeting CRISPR effector Cas13a (previously known as C2c2) exhibits a 'collateral effect' of promiscuous RNAse activity upon target recognition. We combine the collateral effect of Cas13a with isothermal amplification to establish a CRISPR-based diagnostic (CRISPR-Dx), providing rapid DNA or RNA detection with attomolar sensitivity and single-base mismatch specificity … Furthermore, SHERLOCK reaction reagents can be lyophilized for cold-chain independence and long-term storage, and readily reconstituted on paper for field applications.”
 
“It’s exciting that the Cas13a enzyme, which was originally identified in our collaboration with Eugene Koonin to study the basic biology of bacterial immunity, which can be harnessed to achieve such extraordinary sensitivity, which will be powerful for both science and clinical medicine,” said Feng Zhang, core institute member of the Broad Institute, an investigator at the McGovern Institute for Brain Research at MIT, and the James and Patricia Poitras '63 Professor in Neuroscience and Associate Professor in Brain and Cognitive Sciences and Biological Engineering at MIT.
 
Feng Zhang, a core institute member of the Broad Institute and an investigator at the McGovern Institute for Brain Research at MIT, and colleagues first characterized the Cas13a (previously known as C2c2) in June 2016. This enzyme differs from CRISPR enzymes such as Cas9 and Cpf1 in several ways; in addition to targeting RNA rather than DNA, Cas13a can remain active even after cutting its target and can demonstrate “promiscuous” behavior in which it cuts other non-targeted RNA, something the team noted as collateral cleavage activity.
 
While a team from UC Berkeley described the use of this trait for RNA in a September 2016 paper in Nature, that method demanded millions of molecules and was not sufficiently sensitive. The latest method, according to a press release, is “a million-fold more sensitive,” thanks to a collaboration between Zhang and his team and Jim Collins of the Broad. Collins and his colleagues at the Wyss Institute had created a rapid synthetic paper-based test for the Ebola virus back in 2014 that can be shipped and stored at room temperature, and also modified it to detect Zika virus. The team boosted the sensitivity by upping the RNA in the sample through apply low levels of heat. Zhang, Collins and their teams used a different process that used body heat to increase the levels of DNA or RNA, and once the levels were sufficient, applied another amplification step to convert DNA to RNA.
 
As noted in the Science study, this approach can now detect the presence of a single molecule of an RNA or DNA target. Omar Abudayyeh and Jonathan Gootenberg, co-authors and graduate students at MIT and Harvard, respectively, gave the tool the name SHERLOCK (Specific High-sensitivity Enzymatic Reporter unLOCKing).
 
“We can now effectively and readily make sensors for any nucleic acid, which is incredibly powerful when you think of diagnostics and research applications,” said Collins. “This tool offers the sensitivity that could detect an extremely small amount of cancer DNA in a patient’s blood sample, for example, which would help researchers understand how cancer mutates over time. For public health, it could help researchers monitor the frequency of antibiotic-resistant bacteria in a population. The scientific possibilities get very exciting very quickly.”
 
The researchers tested SHERLOCK on several disease types, and were able to detect Zika virus in blood or urine samples within hours, distinguish between different strains of Zika virus, determine specific types of bacteria, detect antibiotic resistance genes, identify cancerous mutations in simulated cell-free DNA fragments and quickly read a patient's genetic information (i.e. heart disease risk) from a saliva sample.
 
“One thing that’s especially powerful about SHERLOCK is its ability to start testing without a lot of complicated and time-consuming upstream experimental work,” said Pardis Sabeti, co-author in the paper. “This ability to take raw samples and immediately start processing could transform the diagnosis of Zika and a boundless number of other infectious diseases. This is just the beginning.”
 
 
SOURCE: Broad Institute press release
 
Code: E04261701

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