Coming to you LIVE from the 5th annual RNA Symposium: Processing RNA. Follow us on Twitter or the tag #umichrna!
Live blogger: Alyse Krausz
Editor: Logan Walker
CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats) is well-known as “molecular scissors” that enables scientists to edit DNA. But there’s more to CRISPR technology than just cutting and pasting DNA! In bacteria, the many CRISPR-Cas systems provide a defense system against viral infections, and viruses use DNA or RNA as their genetic material. Nature has evolved some CRISPR-Cas systems that target DNA, such as CRISPR-Cas9, and others that target RNA, such as CRISPR-Cas13. Dr. Feng Zhang, a Professor of Brain and Cognitive Sciences and of Biological Engineering at MIT, and his lab have discovered and developed the CRISPR-Cas13a system for use as a diagnostic tool. Their lab has harnessed CRISPR-Cas13a as a biotechnology to create a molecular detection platform called SHERLOCK (Specific High-sensitivity Enzymatic Reporter unLOCKing) capable of detecting RNA or DNA with high sensitivity and specificity.
The SHERLOCK System
SHERLOCK is based on CRISPR-Cas13a, which unlike CRISPR-Cas9, can cut RNA. To detect a virus, such as Zika, Cas13a is programmed with CRISPR RNA (crRNA) that recognizes an RNA sequence that is specific to the virus of interest. Notably, this allows it to be rapidly “reprogrammed” or developed into a new tool by simply replacing the crRNA. Viral RNA from a biological sample, such as serum or urine, is amplified using RT-RPA (reverse transcription recombinase polymerase amplification) and transcribed back into RNA. CRISPR-Cas13a then binds to the Zika virus target sequence, which activates the complex’s RNA cutting abilities. The activated form of CRISPR-Cas13a is interesting because it degrades all RNA it can find, including so-called “collateral RNA”, which was not explicitly targeted. This activated CRISPR-Cas13a complex cuts reporter molecules that consist of a fluorophore (green dot in Figure 1) and a quencher (grey dot in Figure 1). When the fluorophore and quencher are connected, no fluorescent signal is emitted, but once the connection is cleaved, the fluorophore fluoresces. The fluorescence intensity is related to the concentration of viral RNA in the sample. The SHERLOCK system can be readily adapted to a lateral flow readout on a paper strip (similar to a pregnancy test), which allows virus tests to be used wherever they are needed. In the lateral flow format, Zhang could detect Zika virus within 1 hour at attomolar concentrations.
The RNA cleavage preferences of Cas13 can be harnessed to achieve multiplexing of SHERLOCK-based viral tests. Rather than cleaving the RNA at a specific site, Cas13 recognizes specific functional motifs, such as a uracil nucleotide in a single-stranded region of RNA. A multiplex assay for different viral pathogens can be created by using different Cas13 proteins that cleave different reporter sequences. Exploiting this fact, Zhang and his lab successfully created a multiplex assay for Zika, Dengue, and synthetic RNA, simply by changing the Cas13s and reporter sequences used for each assay. A multiplexed SHERLOCK assay would be particularly useful for distinguishing between different viral infections that present with the same clinical symptoms (e.g. SARS-CoV-2, influenza, and RSV).
From SHERLOCK to STOPCovid
When the SARS-CoV-2 pandemic emerged, Zhang and his lab quickly adapted the SHERLOCK system to detect COVID-19 infections and sent test kits out to collaborators around the world to help them detect SARS-CoV-2 in their patient samples. A collaborator based in Thailand used the test in a clinical trial and showed that SHERLOCK has 93% sensitivity and 100% specificity. Based on the clinical trial data, this collaborator received permission from the Thai government to use the SHERLOCK test to screen COVID-19 patients.
SHERLOCK was able to detect SARS-CoV-2, but it was a two-step process of isothermal amplification followed by CRISPR detection. The two-step process necessitated the opening of the amplification reaction vial to introduce the CRISPR reagents, which had the potential to introduce contaminants and invalidate the results of the test. To address this shortcoming, Zhang and his lab created the STOPCovid system, which is a one-step reaction that combines the amplification reaction with the CRISPR detection. To make this one-pot reaction a reality, they had to avoid the need for sample extraction. They ended up using QuickExtract to lyse the cells in nasal swab samples and access the viral RNA. They also had to change the amplification type they were using to RT-LAMP (reverse transcription loop-mediated isothermal amplification) instead of RT-RPA. LAMP runs at a higher temperature than RT-RPA (60°C vs 37-42°C), so they switched to Cas12b, which is stable at 60°C, unlike Cas13a. They also included reaction additives to increase the signal of the reaction. The combination of all these factors (LAMP, Cas12b, and reaction additives) allowed STOPCovid to eliminate LAMP background signal, preventing false positives. They additionally showed that the LAMP reaction could be run using a sous-vide to control the temperature, which is a much more economical solution than the thermocycler machines typically used!
In the one-step STOPCovid reaction, Zhang discovered that the sensitivity was not as good as the original two-step SHERLOCK reaction. To get around this, Zhang and his lab introduced magnetic beads to capture the RNA and concentrate it. This simple addition allowed them to rival the sensitivity of qPCR (which has been used in many other clinical tests) for detecting SARS-CoV-2. The improved STOPCovid.v2 system achieved 93% sensitivity and 98.5% specificity on patient samples spanning the full viral load of SARS-CoV-2 (qPCR cycle threshold of 15 to 40). The STOPCovid.v2 assay works with both nasopharyngeal and anterior nasal swabs. The Zhang lab is currently developing a small device to run the STOPCovid test so that it can be easily used not only for SARS-CoV-2 but for future viral pathogens.
Zhang ended his talk by advocating for continued exploration of the natural diversity of bacteria as the existing CRISPR systems are the tip of the iceberg!
Feng Zhang attended Harvard University as an undergraduate, where he earned an A.B. in Chemistry and Physics in 2004. He transitioned to Stanford for his Ph.D. in Chemistry and Biophysics, which he obtained in 2009 while working in Karl Deisseroth’s research group on his pioneering optogenetics work in conjunction with Ed Boyden. After a postdoc in George Church’s lab, he moved to MIT in January of 2011, where he is now both an Associate Professor of Biological Engineering and a Core Institute Member of the Broad Institute of MIT and Harvard.
Feng Zhang has received a number of honors, including the NIH Pioneer Award and the Perl-UNC Neuroscience Prize, along with Ed Boyden and thesis advisor Deisseroth. He is additionally a Searle Scholar.