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Broad Institute Team Engineers Sherlock, Powered by CRISPR, Isothermal Amplification

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NEW YORK (GenomeWeb) – A star-studded team of Broad Institute researchers has developed a new nucleic acid detection technology that combines isothermal amplification and a new breed of CRISPR enzyme that targets RNA.

"It's a different way of using CRISPR, not for editing the genome, but to detect and diagnose biological material," Broad researcher Feng Zhang said in a statement.

In a paper published today in Science, the team — led by co-first authors Jonathan Gootenberg and Omar Abudayyeh, of the Zhang lab, and senior authors Zhang and Jim Collins, also of the Broad Institute — described the technology, dubbed "Sherlock," and its application in detecting Zika and dengue viruses, bacteria and their antibiotic resistance genes, human SNPs, and mutations in cell-free tumor DNA.

Specifically, the researchers leveraged a special property of the CRISPR protein formerly known as C2c2, now called Cas13a, an enzyme discovered in 2015 by the Zhang lab. Like many CRISPR proteins, Cas13a can target, bind, and cleave a nucleic acid target with great specificity; however, once activated, this protein will frenetically chop up just about any RNA oligonucleotide, even those that don't match the original target sequence.

This "collateral cleavage" is a capability that no other CRISPR proteins seem to have, Gootenberg said. "It's a unique quality that enables us to use it as a detection tool. It means we can use many advantages of CRISPR systems, like re-programmability and specificity, and leverage them into diagnostic applications."

Sherlock boasts a ton of features for a diagnostic platform, including sensitivity, specificity, speed, portability, and low cost. It's a promising bit of chemistry, but turning it into a widely available commercial platform, as the researchers hope to do, will a challenge.

Cas13a is a one of several CRISPR proteins recently discovered through a collaboration between Zhang and Eugene Koonin of the National Center for Biotechnology Information, looking through metagenomic databases for genomic loci related to CRISPR systems that have been developed into genome editing tools. Cas13a, however, was different from Cas9 and the other CRISPR proteins, with hints that it targeted and cleaved RNA, rather than DNA.

In 2016, Gootenberg and Abudayyeh led an effort to characterize the Cas13a protein and its RNA-cleaving abilities, including collateral cleavage. "When we first realized it was going crazy in the tube, we were really surprised and almost didn't believe it at first," Abudayyeh said.

At the time, Zhang suggested that it might be applied in an RNA interference-like way. To this end, his lab started collaborating with Broad infectious disease researcher Pardis Sabeti on developing the enzyme for use as an RNA virus therapeutic.

As Gootenberg and Abudayyeh were tinkering with Cas13a, they said they realized that it could be used to measure lentivirus infection in cells. They weren't the only ones who figured out how to use Cas13a for RNA detection. In October, 2016, researchers led by University of California, Berkeley Professor Jennifer Doudna described the two different RNA-cleaving mechanisms present in Cas13a. Among their observations was that an RNA reporter molecule with a quenched fluorescent signal, which would be unquenched in the case of collateral cleavage, could be used as a readout for target detection.

But Cas13a by itself doesn't have the sensitivity necessary for detecting viruses like Zika. "For many diagnostic applications you only have 10 to 100 copies per milliliter," Abudayyeh said," That requires attomolar sensitivity."

To boost sensitivity, the Zhang team looked for ways to pre-amplify nucleic acids. They hit upon Collins' work using isothermal amplification in order to generate target RNAs for Cas13a to detect.

Recombinase polymerase amplification, an alternative to PCR, offered several other advantages besides upping the sensitivity by a million-fold.

"You go from just RNA detection to having both RNA and DNA detection," Abudayyeh said. The method includes another amplification step using T7 polymerase to turn DNA in to RNA. "You have Cas13a recognition of RNA that's already undergone two amplification steps. It's the combo of these three amp steps that comprise what Sherlock is, and gives you the power to do all the applications we described."

Yet another advantage of isothermal amplification — it can be run off of human body heat, reducing the need for a thermocycler — makes Sherlock promising for Zika or dengue detection. "That makes it much easier to adapt for low-cost or point-of-care applications, like in resource-poor areas where Zika or Ebola might be spreading," Gootenberg said. "It makes the tech more versatile and we're excited about applying the tech to these emerging problems of global health."

Sabeti, who's actively researching Zika virus, said it's hard to detect because it has a low copy number in blood or urine. "Having something with very high sensitivity is very important and technology that you can use without preprocessing the sample ahead of time is very valuable," she said.

Abudayyeh and Gootenberg also performed proof-of-concept experiments detecting bacterial DNA and antibiotic resistance genes. "As we kept working on the technology, we kept seeing more and more applications," Gootenberg said. In addition to human SNP genotyping and cancer mutations in mock-cell-free DNA, which the team did explore, they've got ideas on how to apply Sherlock to metagenomics, viral mutation tracking, and point-of-care testing.

While there's a lot of potential, there's no guarantee it will be realized. All those amplification steps add up, increasing complexity, Doudna said in an email. "This many-step process could be challenging to scale into a point-of-care application," she said.

There's also a hitch: RPA isn't available yet for clinical testing and its commercial use is controlled by TwistDx, an Alere subsidiary. For TwistDx, it's a validation that RPA can be "elegantly" run alongside other chemistries, CEO Niall Armes said. If Sherlock is to be advanced, a collaboration would need to occur or a new amplification method used.

And bringing a neat chemistry into a commercially viable platform, especially one intended for use in resource-poor settings, is a challenge, Armes said.

"Economic challenges are not well understood by people in academia. To reach those markets and be viable, you need low manufacturing costs," he said. "For point-of-care testing, we're only just now seeing uptake in developed markets. It's going to be another set of years again before those products can be supported and sustained in these markets that have an even more aggressive requirement for low pricing."

Finally, there's the issue of inventorship. In their respective 2016 and 2017 papers, both Doudna and the Broad said they'd applied for patents based on the RNA-detection application of Cas13a. Doudna published first, but the Broad seems to have engineered the system to make the most lucrative applications feasible.

Zhang has long talked about finding new CRISPR enzymes to add to the genome editing toolbox. Over the last year, leading researchers have found a whole new toolbox to add CRISPR to.

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