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Paper test detects multiple diseases all at once!

CRISPR-based diagnostic tool on miniature paper test strips ...

Researchers who developed the CRISPR-based diagnostic tool, now developed a miniature paper test with 100-times greater sensitivity than a previous attempt. The new test strips can detect and identify multiple viruses in blood all at one time.

The team first unveiled a rapid, inexpensive, highly sensitive CRISPR-based diagnostic tool called SHERLOCK in 2017. Now, they greatly enhanced that into a miniature paper test with results that can be seen with the naked eye without expensive equipment.

SHERLOCK (shorthand for Specific High Sensitivity Reporter unLOCKing) now has a simple paper strip display of test results for a single genetic profile, borrowing from the presentation commonly seen in pregnancy tests. After dipping the paper strip into a processed sample, a line appears, indicating whether the target molecule was detected or not. This new feature helps pave the way for field use during an outbreak of disease. The team has also increased the sensitivity of SHERLOCK and added the capacity to accurately quantify the amount of target in a blood sample with the ability to test for multiple targets at once. All together, these advancements accelerate SHERLOCK's ability to quickly detect genetic profiles, including pathogens and tumor DNA.

Described in Science, the innovations build on the team's earlier version of SHERLOCK and add to a growing field of research that harnesses CRISPR systems for use beyond gene editing. The work, led by researchers from the Broad Institute of Massachusetts Institute of Technology (MIT), and Harvard has the potential for a transformative effect on research and global public health.
"SHERLOCK provides an inexpensive, easy-to-use, and sensitive diagnostic method to detect nucleic acid material - which can mean it detects a virus, tumor DNA, or many other targets. These improvements give us even more diagnostic information and put us closer to a tool that can be deployed in real-world applications."

Feng Zhang PhD, senior author and core institute member at the Broad Institute, investigator at the McGovern Institute for Brain Research at MIT, and the James and Patricia Poitras Professor in Neuroscience at Massachusetts Institute of Technology, MIT.

The researchers previously showcased SHERLOCK's utility by detecting cell-free tumor DNA in blood samples from lung cancer patients, also in detecting synthetic Zika and Dengue virus simultaneously.

Clear results on a paper strip

"The new paper readout for SHERLOCK lets you see whether your target was present in the sample, without instrumentation," explains co-first author Jonathan Gootenberg, a Harvard graduate student in Zhang's lab as well as the lab of Broad core institute member Aviv Regev. "This moves us much closer to a field-ready diagnostic."

The team envisions a wide range of uses for SHERLOCK, thanks to its versatility in nucleic acid target detection. "The technology demonstrates potential for many healthcare applications, including diagnosing infections in patients and detecting mutations that confer drug resistance or cause cancer, but it can also be used for industrial and agricultural applications where monitoring steps along the supply chain can reduce waste and improve safety," adds Zhang.

The basics

At the core of SHERLOCK's success is a CRISPR-associated protein called Cas13, which can be programmed to bind to a specific piece of RNA. Cas13's target can be any genetic sequence, including viral genomes, genes that confer antibiotic resistance in bacteria, or mutations that cause cancer. In certain circumstances, once Cas13 locates and cuts its specified target, the enzyme goes into overdrive, indiscriminately cutting other RNA nearby. To create SHERLOCK, the team harnessed this "off-target" activity and turned it to their advantage, engineering the system to be compatible with both DNA and RNA.

SHERLOCK's diagnostic potential relies on additional strands of synthetic RNA that are used to create a signal after being cleaved. Cas13 will chop up this RNA after it hits its original target, releasing the signaling molecule, which results in a readout that indicates the presence or absence of the target.

Detecting multiple targets

The SHERLOCK platform can now be adapted to test for multiple targets. SHERLOCK initially could only detect one nucleic acid sequence at a time, but now one analysis can give fluorescent signals for up to four different targets at once - meaning less sample is required to run through diagnostic panels. For example, the new version of SHERLOCK can determine in a single reaction whether a sample contains Zika or dengue virus particles, which both cause similar symptoms in patients. The platform uses Cas13 and Cas12a (previously known as Cpf1) enzymes from different species of bacteria to generate the additional signals.

Increased sensitivity

SHERLOCK's second iteration also uses an additional CRISPR-associated enzyme to amplify its detection signal, making the tool more sensitive than its predecessor. "With the original SHERLOCK, we were detecting a single molecule in a microliter, but now we can achieve 100-fold greater sensitivity," explains co-first author Omar Abudayyeh, an MIT graduate student in Zhang's lab at Broad. "That's especially important for applications like detecting cell-free tumor DNA in blood samples, where the concentration of your target might be extremely low. This next generation of features help make SHERLOCK a more precise system."

Rapid detection of nucleic acids is integral for clinical diagnostics and biotechnological applications. We recently developed a platform termed SHERLOCK (Specific High Sensitivity Enzymatic Reporter UnLOCKing) that combines isothermal pre-amplification with Cas13 to detect single molecules of RNA or DNA. Through characterization of CRISPR enzymology and application development, we report here four advances integrated into SHERLOCKv2: 1) 4-channel single reaction multiplexing using orthogonal CRISPR enzymes; 2) quantitative measurement of input down to 2 aM; 3) 3.5-fold increase in signal sensitivity by combining Cas13 with Csm6, an auxilary CRISPR-associated enzyme; and 4) lateral flow read-out. SHERLOCKv2 can detect Dengue or Zika virus ssRNA as well as mutations in patient liquid biopsy samples via lateral flow, highlighting its potential as a multiplexable, portable, rapid, and quantitative detection platform of nucleic acids.

Authors: Jonathan S. Gootenberg, Omar O. Abudayyeh, Max J. Kellner, Julia Joung, James J. Collins, Feng Zhang.

The authors have made their reagents available to the academic community through Addgene and their software tools can be accessed via the Zhang lab website and GitHub.

Paper cited: Gootenberg JS and Abudayyeh OO et al. Multiplexed and portable nucleic acid detection platform with Cas13, Cas12a, and Csm6. Science. Online February 15, 2018. DOI: 10.1126/science.aaq0179

This study was supported in part by the NIH (grants 1R01-HG009761, 1R01-MH110049, 1DP1-HL141201, F30 NRSA 1F30-CA210382); the Defense Threat Reduction Agency (grant HDTRA1-14-1-0006).

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Mar 6, 2018   Fetal Timeline   Maternal Timeline   News   News Archive

These SHERLOCK paper test strips developed by Feng Zhang's lab at the Broad Institute could be used to rapidly identify viruses in blood. Image credit: Zhang Laboratory, Broad Institute, MIT

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