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05/27/2017

    CRISPR-Based Nucleic Acid Detection




    A CRISPR-based diagnostic tool can detect pathogens, identify cancerous mutations, and genotype human DNA, researchers reported on April 13 in Science.


    “This project is the culmination of a lot of work we’ve been doing in the lab,” study coauthor Omar Abudayyeh, a MD/PhD student at Harvard Medical School and a member of Feng Zhang’s lab at the Broad Institute, told The Scientist. “We’ve been interested in mining bacterial diversity in nature, to try to uncover new tools and proteins that can be used to better public health and society.”


    A few years ago, while searching through bacterial genomes for new CRISPR enzymes, Omar Abudayyeh, coauthor of this study and a member of Feng Zhang’s lab at the Broad Institute and colleagues discovered C2c2 (now known as Cas13a). The enzyme has unique properties—not only does it target RNA instead of DNA, is also cleaves nearby RNA sequences, the researchers found. “When it recognizes its target, it becomes crazy and will start chewing up anything else in the tube,” Abudayyeh said. “We call [this] collateral activity.”


    Collateral activity is a key characteristic of the newly developed nucleic acid detection platform, SHERLOCK (Specific High Sensitivity Enzymatic Reporter UnLOCKing), which also includes a reporter RNA strand that fluoresces when cleaved. When Cas13a detects the targeted RNA sequence, its unbiased RNAse activity will slice the reporter sequence, releasing a detectable fluorescent signal.


    “We knew that Cas13a had sensitive collateral activity, but when we characterized it initially, we found that it wasn’t sensitive enough,” said coauthor Jonathan Gootenberg, a graduate student in Zhang’s lab. To circumvent this issue, the group collaborated with James Collins, a biological engineering professor at MIT, to incorporate isothermal RNA amplification, which Collins’s team had previously used to create a paper-based Zika test.


    Combined, this system was capable of detecting single RNA and DNA molecules at attomolar concentrations. Adding the amplification step helped “increase the sensitivity of the test a million-fold, and also [allowed] freeze drying and other applications that really made it into a robust tool that we have now,” Gootenberg said. “We can do detections for samples in as little as an hour, it's portable—we can freeze dry it and reconstitute it—and it’s very low-cost.”


    The team tested SHERLOCK across a number of applications. The tool was able to identify Zika virus RNA in human serum, urine, and saliva, and could also discriminate between Zika and dengue, a related flavivirus. It was also able to differentiate between various bacterial pathogens and identify single nucleotide polymorphisms (SNPs) in human DNA samples.


    Jennifer Doudna, a biochemist at the University of California, Berkeley, and colleagues have also previously tested C2c2’s use in detecting RNA. “The new method improves the limit of detection for C2c2 by pre-amplifying the nucleic acid target,” Doudna wrote in an email. “The limitation is that it still requires the nucleic acid amplification steps . . . . This could be challenging to scale into a point-of-care application.” (UC Berkeley and the Broad are currently involved in a patent dispute regarding CRISPR gene editing.)


    The authors estimated that a paper-based SHERLOCK assay would cost around $0.61 per test. “Working with the Broad Institute, we’re exploring spinning out technology, either in a startup or with various parties,” Collins said. “[We] have already generated considerable interest from different groups on the outside.”


    J.S. Gootenberg et al., “Nucleic acid detection with CRISPR-Cas13a/C2c2,” Science, doi:10.1126/science.aam9321, 2017.