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Broad Researchers Develop Protocol to Use CRISPR-Based Diagnostic Tool for Zika Testing

NEW YORK (GenomeWeb) – A team led by researchers at the Broad Institute have demonstrated that the CRISPR-based SHERLOCK (Specific High Sensitivity Enzymatic Reporter UnLOCKing) platform developed in the lab of Feng Zhang can be deployed as a diagnostic tool to detect Zika virus in the field.

In a study published today in Science, the researchers demonstrated that SHERLOCK can detect Zika virus (ZIKV) and dengue virus (DENV) in patient samples at concentrations down to 1 copy per μl. They also developed a protocol they called HUDSON (Heating Unextracted Diagnostic Samples to Obliterate Nucleases), which pairs with SHERLOCK to detect viruses directly in bodily fluids, enabling instrument-free detection of DENV directly from patient samples in less than two hours. The team further showed that SHERLOCK can distinguish between the four DENV serotypes as well as region-specific strains of ZIKV from the 2015 to 2016 pandemic.

SHERLOCK was first described in a paper in Science in April 2017 by members of the Zhang lab. This March, the same team published a second paper describing new improvements on the technology. It now has four-channel single reaction multiplexing using orthogonal CRISPR enzymes, quantitative measurement of input down to 2 attomolar concentration, a 3.5-fold increase in signal sensitivity through a combination of Cas13 with auxiliary CRISPR-associated enzyme Csm6, and a 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," the authors wrote in that paper.

For this new study, the Broad's Pardis Sabeti and her colleagues designed a ZIKV assay for SHERLOCK with a single-copy sensitivity when tested on seedstock cDNA. They evaluated its performance on 40 cDNAs derived from samples collected during the 2015 to 2016 ZIKV pandemic, 37 from patient samples with suspected ZIKV infections, and three from mosquito pools. For 16 samples from these patients, they benchmarked SHERLOCK by comparing its sensitivity and specificity to other nucleic acid amplification tests including the commercially available Altona Realstar Zika Virus RT-PCR assay.

"Of the 10 samples tested positive by the Altona assay, all 10 were detected by SHERLOCK (100 percent sensitivity); the other six samples were negative by both assays (100 percent specificity, 100 percent concordance)," the team wrote. "Our ZIKV assay had no false positives when tested on healthy urine and water. We then validated the ability of SHERLOCK to detect DENV, a related but more diverse flavivirus with similar symptoms to ZIKV infection. All 24 RT-PCR-positive DENV RNA samples were confirmed DENV positive after 1 hour of detection."

In order to be able to use SHERLOCK in the field, however, the researchers determined that it should not require an extraction step to detect viral nucleic acid in bodily fluids. So, they developed HUDSON, a method to lyse viral particles and inactivate the high levels of RNases found in bodily fluids using heat and chemical reduction. They found that HUDSON-treated urine or saliva could be directly added to RPA reactions without dilution or purification, without inhibiting subsequent amplification or detection.

"To mimic clinical infection, where viral nucleic acid is encapsulated in infections particles, we spiked infectious ZIKV particles into bodily fluids," the authors wrote. HUDSON combined with SHERLOCK permitted sensitive detection of ZIKV RNA from infectious particles at 45 cp/μl in whole blood or serum, ~1 cp/μl in saliva, and 10 cp/μl in urine. Total turnaround time was less than two hours with fluorescent and colorimetric readout."

The researchers then went on to design diagnostics for three region-specific SNPs from the 2015 to 2016 ZIKV pandemic and identified these SNPs in synthetic targets, a viral seedstock, and cDNA samples from Honduras, the Dominican Republic, and the US. They also developed multiple SHERLOCK assays for a mutation in S139N, which was recently associated with fetal microcephaly, and found they could identify the mutation in patient samples from the 2015 to 2016 ZIKV pandemic with a visual readout.

They further designed and tested assays in one week for the six most commonly observed drug-resistance mutations in HIV reverse transcriptase.

Indeed, as Zhang lab researchers Jonathan Gootenberg and Omar Abudayyeh noted to GenomeWeb in March, rapid design of new diagnostic assays that can be plugged into the SHERLOCK platform is one of the goals for the technology so that it can be used in the field. Gootenberg, Abudayyeh, and Zhang were all coauthors on the new study.

"The really great thing about the SHERLOCK assay is how easy it is to redesign and deploy," Abudayyeh said in March. "We've shown that we can detect dozens of different targets, and we can design these assays in as little as a week. That speaks to how robust it is, and how well it works for each given set of CRISPR RNAs. It's as easy as designing a new CRISPR RNA and then showing that it works."

The approach of combining HUDSON and SHERLOCK can be easily adapted to "detect virtually any virus present in bodily fluids, scaled to enable multiplexed detection, and the reagents can be lyophilized for cold-chain independence," Sabeti and her colleagues concluded in their study. "Cas13-based detection is a promising next-generation diagnostic strategy with the potential to be implemented almost anywhere in the world to enable effective, rapid diagnosis of viral infections."

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