5 min readTwo New Diagnostic CRISPR-Based Tools Revealed Independently

Cambridge, MA and Chevy Chase, MD – Teams of researchers from Broad Institute and Howard Hughes Medical Institute have independently revealed two new diagnostic tools based on CRISPR technology – called SHERLOCK and DETECTR. Both teams reported their achievements in the latest issue of the journal Science.

SHERLOCK (Specific High Sensitivity Enzymatic Reporter UnLOCKing) – created in a Broad Institute of MIT and Harvard lab

The SHERLOCK team developed a simple paper strip to display test results for a single genetic signature, borrowing from the visual cues common in pregnancy tests. After dipping the paper strip into a processed sample, a line appears, indicating whether the target molecule was detected or not.

These are co-first authors Jonathan Gootenberg (left) and Omar Abudayyeh (right) holding a paper strip that displays a positive SHERLOCK readout. Image: Zhang lab, Broad Institute of MIT and Harvard

This new feature helps pave the way for field use, such as during an outbreak. The team has also increased the sensitivity of SHERLOCK and added the capacity to accurately quantify the amount of target in a sample and test for multiple targets at once. All together, these advancements accelerate SHERLOCK’s ability to quickly and precisely detect genetic signatures — including pathogens and tumour DNA — in samples.

“SHERLOCK provides an inexpensive, easy-to-use, and sensitive diagnostic method for detecting nucleic acid material — and that can mean a virus, tumour DNA, and many other targets,” said senior author Feng Zhang, 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 MIT. “The SHERLOCK improvements now give us even more diagnostic information and put us closer to a tool that can be deployed in real-world applications.”

The researchers previously showcased SHERLOCK’s utility for a range of applications. In the new study, the team uses SHERLOCK to detect cell-free tumour DNA in blood samples from lung cancer patients and to detect synthetic Zika and Dengue virus simultaneously, in addition to other demonstrations.

“The new paper readout for SHERLOCK lets you see whether your target was present in the sample, without instrumentation,” said 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,” added Zhang.

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 signalling molecule, which results in a readout that indicates the presence or absence of the target.

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.

 

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,” explained 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 tumour 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.”

Publication: Multiplexed and portable nucleic acid detection platform with Cas13, Cas12a, and Csm6. Gootenberg, JS et al. Science (15 February 2018). Click here to view.

DETECTR (DNA Endonuclease Targeted CRISPR Trans Reporter) – created in a Howard Hughes Medical Institute lab

The new method DETECTR, developed in a laboratory of HHMI Investigator Jennifer Doudna, combines the capabilities of CRISPR with a molecular flare gun and was able to spot two types of cancer-causing human papillomavirus, or HPV, in human samples. Doudna and colleagues first reported their results in a preprint posted on bioRxiv.org in November 2017; the peer-reviewed report appears February 15, 2018, in the journal Science.

HHMI Investigator Jennifer Doudna and colleagues have created DETECTR, a DNA-detection system based on CRISPR biology. The new technology could lead to fast, reliable medical tests. Image: Ryan Anson

DETECTR relies on Cas12a, an enzyme described in 2015. Like its molecular cousin Cas9, which Doudna and colleague Emmanuelle Charpentier turned into a genome editing tool in 2012, Cas12a snips DNA. But instead of snipping only the DNA strand it binds, Cas12a chops other DNA, too. “We started to see this surprising activity where it would just start cutting random stuff,” says study coauthor Lucas Harrington, a graduate student in Doudna’s lab.

Under certain circumstances, the enzyme turns into a DNA shredding machine, slicing up any single-stranded DNA nearby, the researchers saw. But this wasn’t indiscriminate destruction. For the machete action to begin, Cas12a first has to find a precise DNA target. Researchers can program that target by adding a guide – an RNA molecule that tells Cas12a what to look for. “It’s so easy to reprogram this to find any piece of DNA that you want to detect,” Harrington says.

Once Cas12a locks onto and snips the target, it then begins shredding all of the single-stranded DNA it can find. But for the system to be useful, Doudna and colleagues needed a way to see when Cas12a starts this molecular mayhem, signaling that it has found its target. So the researchers used a glowing molecule – an easy-to-spot flare – linked by a single strand of DNA to a suppressor molecule that prevents the glow. When Cas12a turns into a machete, it slices the DNA strand that links these two molecules together. This removes the suppressor, letting the glowing molecule shine ­­– a signal researchers can detect.

The team then put their DNA detective to the test. Working with Dr. Joel Palefsky and his team at the University of California, San Francisco, they hunted for DNA signals from two types of cancer-causing HPV: type 16 and type 18. Researchers obtained 25 DNA samples taken from people who had no HPV infection, one type of virus, or both types. For HPV16, DETECTR made the right call for all 25 of the samples. For HPV18, DETECTR got it right for 23 of 25 samples. The ones it missed gave weak signals that can likely be improved with different guide RNA design, Doudna says.

Compared with current methods to detect HPV, DETECTR is “simpler, quicker, and does not require specialized equipment,” Harrington says. That could make the system useful in resource-limited health clinics and for point-of-care diagnostics.

About 79 million Americans carry HPV, the Centers for Disease Control and Prevention estimates, and more than 4,000 women die from cervical cancer each year. Together, HPV16 and HPV18 cause 70 percent of cervical cancers and precancerous lesions, according to the World Health Organization.

Doudna’s team’s method could easily be applied to other types of viral or bacterial infections, and even cancer markers, chromosomal abnormalities, or other genetic signals, Harrington says. More generally, the results highlight the promise of basic biology. Basic research on an ancient bacterial defense system keeps turning up new surprises, and new potential uses. CRISPR “is a treasure chest that we keep digging into and finding new things,” Harrington says.

Publication: CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity. Chen, JS et al. Science (15 February 2018). Click here to view.

 

Biotechnology, CRISPR-Cas9, Diagnostics, DNA sequence, Gene Technology, Laboratory device, Molecular Genetics

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