I was recently involved in a collaboration between the Zhang and Collins labs at MIT to use the RNA-targeting CRISPR protein Cas13a/C2c2 to detect either DNA or RNA from pathogens. By combining the use of Cas13a/C2c2 as a detector with isothermal amplification of the DNA or RNA targets, we were able to get down to attomolar detection. You can read the full paper over at Science but here I can give some of my own experience with and views on the platform we’re calling SHERLOCK (Specific High Sensitivity Enzymatic Reporter UnLOCKing).
To be clear, I’m the 5th author on this paper and definitely agree that the four people ahead of me did more of the work. I am not the expert on Cas13a/C2c2. However, I did help some to develop SHERLOCK as a diagnostic system and use it enough to get a handle for how it works with different targets. I also tried it with another diagnostic project and have found it easy to work with.
How it works
Cas13a/C2c2 has two RNA cutting abilities. The first is that it cuts the RNA that you target with theCRISPR guide RNA (crRNA) much like Cas9 cuts DNA that you directly target. The other RNA cutting is more broad and is activated after finding Cas13a/C2c2 finds its target RNA. This broad RNA cutting activity acts like an RNase and will cut many RNAs present in the reaction (or in the cell). In a 2016 Nature paper, the Doudna lab showed that the RNase activity of Cas13a/C2c2 could be used to detect picomolar levels of RNA. They found that Cas13a/C2c2 was able to do at least 104 turnovers per target RNA recognized. That catalytic activity of Cas13a/C2c2 gives a strong output signal for even s small input of target RNA.
So Cas13a/C2c2 can be turned into a diagnostic using just a crRNA and a fluorescent RNA probe. After the Cas13a/C2c2 finds its target it starts cutting other RNAs, including the probe, and separates a fluorophore from its quencher. It’s that separation of fluorophore and quencher that gives the fluorescent signal.
To boost the natural sensitivity of Cas13a/C2c2 as a diagnostic, we paired it with isothermal amplification of DNA or RNA. Isothermal amplification methods amplify nucleic acids similar to polymerase chain reaction (PCR) but instead use enzyme mixes that can do the job at a single temperature. SHERLOCK makes use of recombinase polymerase amplification (RPA) that can work between 37-42˚C. This allows both amplification reactions and Cas13a/C2c2 reactions at 37˚C and means that there is no need for expensive machinery to precisely cycle temperatures like PCR.
The level of sensitivity we got certainly jumps off of the page. As mentioned in the last section, Cas13a/C2c2 is itself quite sensitive to RNA molecules and its collateral RNase activity can cut many probe RNAs. It binds to its target RNA and then quickly generates signal through its general RNA cutting activity. As a detector it’s ~1000 times more sensitive than another detector, the RNA toehold switch, that we’ve used in the Collins lab to things like detect Zika.
Similar to the paper-based Zika detection, SHERLOCK was able to be freeze-dried for room-temperature storage, used with minimal hardware, and rapidly reprogrammed rapidly to target almost any sequence. But in addition to the sensitivity advantage, SHERLOCK was able to detect single base mutations. As many important mutations in humans or the pathogens that infect humans are only single bases, the ability to distinguish those small changes would be a major achievement for a cheap diagnostic. Some screening has to be done to find crRNAs that work best for a given mutation, but in general a few variants should be enough.
This is still early days for Cas13a/C2c2 based CRISPR diagnostics, so there will be more challenges to be addressed in academic labs and in a company setting. While we showed freeze-drying on glass fiber paper and adding RNase inhibitor worked without producing much background signal, samples that contain many RNases could create false positives. A negative control that lacks Cas13a/C2c2or crRNA could inform you of the problem but the RNase containing sample likely couldn’t give you an accurate read of how much nucleic acid is actually present. At the lab bench, we didn’t have problems with background signal but working somewhere like a remote community health center would probably bring less controlled conditions. Rigorous tests will need to be done to make sure that the freeze-dried tests can last out in different conditions.
Other improvements could include a good way to change from a fluorescent output to a color change as the output. This would allow easy readout by eye like a pregnancy test and reduce equipment costs. A color readout can be done by anyone without risk of equipment malfunction. Reducing the equipment and technical skill needed for a diagnostic is key to how easily it can actually be deployed in areas that need it.
Future for CRISPR diagnostics
The variety of CRISPR proteins that target DNA or RNA and can be easily programmed to cut or bind to nearly any sequence. Cas13a/C2c2 is nice because it comes with a secondary activity (general RNA cutting) that can readily be turned into a fluorescent readout. However, CRISPR-Cas9 can also be used for diagnostics when cleverly combined with a way of detecting its targeted DNA cutting. Overall, CRISPR proteins are poised to get integrated into nucleic acid diagnostics as they provide programmable detection and more specificity than traditional nucleic acid amplification-based techniques.
Conflicts of interest:
Since I am an author on the paper being discussed, I am mostly certainly biased in favor of its significance. While co-authors may have patent and potential financial stakes in work that follows, I do not personally have a stake in any of these ventures. However, I do work for MIT and have Broad Institute affiliations so I do have institutional conflicts of interest.