NEB Podcast #26 -
COVID-19 Researcher Spotlight Series: Interview with the developers of STOPCovid

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Interviewers: Lydia Morrison, Marketing Communications Writer & Podcast Host, New England Biolabs, Inc.
Interviewee: Feng Zhang, Ph.D., The Broad Institute of MIT and Harvard, Omar Abudayyeh, Ph.D., The McGovern Institute of MIT, Jonathan Gootenberg, Ph.D., The McGovern Institute of MIT

Lydia Morrison: Welcome to the Lessons from Lab and Life Podcast. I'm your host Lydia Morrison, and I hope that this podcast offers you some new perspective. This is the second episode of our COVID-19 Researcher Spotlight Series, aimed at highlighting preprints, publications, protocols and other research related to COVID-19, all in 19 minutes or less.

Lydia Morrison: Today, I'm joined by molecular biologist Feng Zhang of the Broad Institute and his former students who now have a lab of their own, Jonathan Gootenberg and Omar Abudayyeh of the McGovern Institute for Brain Research at MIT. Feng, Omar and Jonathan thank you so much for joining me today.

Feng Zhang: Thank you for having us.

Lydia Morrison: I was wondering if we could start by your explaining a bit about your STOPCovid Research.

Feng Zhang: Sure. We started to work on STOPCovid when we first learned about the coronavirus pandemic. This was back in January and I remember reading an article in The New York Times saying that there's a new virus that's causing havoc for the public health system in China.

Feng Zhang: And we have been working on using CRISPR proteins for detecting nucleic acids and pathogens before we developed a technique called SHERLOCK and we thought maybe given this public health concern, we can start to repurpose what we developed before so that we can use it to detect coronavirus. And since then we've been working on trying to make the approach as sensitive and also as easy to use as possible. And that's what led to STOPCovid.

Lydia Morrison: How does this STOPCovid method work?

Feng Zhang: Omar or Jonathan, do you want to start with that?

Jonathan Gootenberg: Yeah. This is Jonathan. So STOPCovid relies on the basic technology of CRISPR diagnostics, which is essentially the use of these CRISPR enzymes and their interesting collateral effect to detect nucleic acids and then trigger an actual cleavage while the nucleic acid's in solution that allows us to produce a detectable signal.

Jonathan Gootenberg: So what happens is that when these CRISPR enzymes, and the CRISPR enzyme we're using in STOPCovid is Cas12b, but when it detects a sequence that it's programmed to target and this is a sequence that corresponds to a place in the SARS-CoV-2 genome, it becomes activated and actually will then cut a reporter that we've doped into the solution, which can produce a fluorescence or a lateral flow signal.

Jonathan Gootenberg: One of the interesting things about STOPCovid is that we've coupled in the same solution this CRISPR detection with an isothermal amplification. So that is an amplification similar in principle to PCR where we're getting exponential production of a DNA species, but it occurs at a single temperature. So we don't have to go through any cycling steps.

Jonathan Gootenberg: So that amplification, which is called LAMP, occurs at 60 degrees Celsius or 60 to 65 degrees Celsius. And we can do that in the same reaction as this CRISPR detection. So what essentially happens is that when we do the LAMP amplification on the RNA genome of SARS-CoV-2, then we can produce a lot of DNA that can then basically be very rapidly detected by the Cas12 to produce a fluorescence or a signal, lateral flow signal we can detect in the strip very easily. So it's a very simplified test for these molecular diagnostics that needs much less equipment.

Lydia Morrison: Less equipment sounds good, especially for places that don't have access to large instrumentation necessary to run like RT-PCR or sequencing. What are the other advantages of the STOPCovid test?

Omar Abudayyeh: Some of the main advantages of the STOPCovid tests are really the price point, as well as the portability of the test. And so a really big thing that we thought about when we were trying to simplify our original technology was how to limit the number of steps there are for the user in terms of fluid handling, as well as points where you could open up the tubes and cause spread of your reaction, which can cause false positives and future reactions that contamination actually really impacted the CDC test.

Omar Abudayyeh: And so the ability to make this into a single reaction with very limited steps for the user really makes us as portable as possible for being able to go into maybe people's homes or point of care settings in the future. And then of course the price is something we've been really thinking about. And so the components and the chemistries that go into it are readily scalable and will help to hopefully make this possible for daily testing or every other day testing in the future which is a real goal of ours.

Lydia Morrison: So what makes this test more scalable than those currently available?

Feng Zhang: The goal of developing STOPCovid is to make the reaction as easy to perform as possible. And also to have a high level of sensitivity. I think one of the advantages of STOPCovid is that the system can all work within a single step and the user will simply put a sample into the STOPCovid workflow and then be able to read out the result through this streamlined workflow process.

Feng Zhang: The reagents that go into creating a STOPCovid reaction is not that different than other amplification techniques like LAMP or even PCR, is a few proteins that will have to be prepared. And we've been getting quite a bit of help from NEB with purifying some of these proteins, which has been really helpful for us. And so once you have all that, then the ability to be able to run isothermal reaction, but with high levels of sensitivity, I think that's what makes STOPCovid particularly interesting.

Lydia Morrison: So I think Omar mentioned that this is a test that someone might be able to do at home, is that one of the future goals for the STOPCovid test? That this would be a home diagnostic like a pregnancy test that someone could take?

Feng Zhang: That is the ultimate goal of making the diagnostics something that people can use in the comfort of their own homes so that they don't have to travel to the hospital and if they are infected with the virus, during that trip there's the chance of them exposing other people to the virus. So that's the goal. There are few things that we'll have to work out in order to make this something that is truly a turnkey so that people can run in their homes. So we're working on figuring out how we might be able to develop a device that can make the process very streamlined and just one step and get it resolved in a very fail proof type of way.

Lydia Morrison: So how would those results be read out? How are the results delivered in the STOPCovid assay?

Jonathan Gootenberg: In our current a white paper that we released, we actually had the readout with a visual readout where the collateral activity cleaves a DNA species that can then be detected by a lateral flow strip, which essentially has the same readout properties as a pregnancy test. It's a line, but one of the very nice things about this technology is that it's very modular. So we can change the readout from a lateral flow readout to a fluorescent readout and vice versa. So for future iterations, each of these different readouts has different values. Obviously the lateral flow readout has the idea that you don't need any sort of readout instrumentation, you can do it by eye. But fluorescent readouts can be faster because you can read them in real time and that can also lead sometimes to more quantitative readout. So in future iterations of the chemistry and how the technology goes, we're envisioning either readout as potential options and looking at the positive negatives of both.

Lydia Morrison: And so I'm thinking about the utility of this as a home test and obviously a pregnancy test is easy to take, but performing a nasopharyngeal swab at home would obviously be a little bit more difficult and require a little bit more training. So what samples would this test work on?

Omar Abudayyeh: I think nasopharyngeal swabs have been used heavily in the past and yeah, they're not ideal for someone to take on their own because I think they're pretty invasive and uncomfortable. So, a big goal of STOPCovid was how do we port this over to other tests? And so there's been a number of papers on saliva being useful and having similar viral loads and maybe some people have made cases that might be even higher viral loads than other sample types.

Omar Abudayyeh: And of course, there's also anterior nare swabs which is basically a nasal swab. And so we've actually shown in the white paper that we can work from the saliva matrix and we're exploring saliva samples and the ultimate goal is hopefully to move towards nasal swabs because with saliva and nasal swabs someone can easily provide that sample type on their own.

Lydia Morrison: Absolutely. You've mentioned that isothermal amplification step, which is great because it occurs at one temperature and one temperature is probably something that someone could achieve at home with maybe not a cup of hot water, but maybe like a sous vide type instrument. What is the sensitivity of this test like? And how does it compare to some of the RT-qPCR tests that are currently available?

Jonathan Gootenberg: So isothermal amplifications are really great because they can be read out without having to change the temperature unlike PCR. And they're also very rapid and very sensitive. So in our current publication or I should say white paper that we released, we have a limited detection of a hundred viral copies and we're working to push that farther using additional methods. One thing is that if you compare these isothermal methods or I should say point of care methods to traditional readouts, like qPCR – in qPCR the current clinical protocol involves concentration and purification, which while very difficult to do in a point of care setting because it requires instrumentation and often expert handling of the sample, does have the value of taking potentially a lot of volume and putting a lot more concentrated viral sample into that. So one thing that we're looking to do in future iterations is potentially figure out ways that we can increase concentration and break the limit of what you can do without a concentrated sample. So that's one thing that we're thinking about.

Lydia Morrison: That's excellent. So could you guys tell me how you all got involved in this project together?

Feng Zhang: So Jonathan, Omar and I have been working together for many years now. They were graduate students in my lab and now they are independent PIs at MIT as junior fellows. When the coronavirus issue first came up in the news, we saw the news and realized that this is going to be a critical problem. And because we're having a working on SHERLOCK diagnostics before, we thought it would be great to develop SHERLOCK so that we can use it to detect coronavirus.

Feng Zhang: And as we started to develop the technique, we sent reagents out to a number of collaborators around the world. They have given us feedback that the reaction works well, it's sensitive and in fact one of our collaborators in Thailand has received approval so that they are using it in their hospitals to be able to screen new patients who were coming in for surgery so that they can triage them differently based on their infection status.

Feng Zhang: But one of the feedback that we received is also that the reaction is a little challenging to do because you have to run the amplification first and then combine it with the CRISPR detection step. And that transfer step can pose risk for a sample contamination. And so given all those feedback we then went back to the drawing board to think about how can we overcome these challenges and then we thought maybe we can develop a robust one pot reaction. And then that's when we thought maybe integrating the PreAmp and also with the new CRISPR protein that can be stable, and during the amplification process will help us get there.

Feng Zhang: We've sent that reagent out to a number of collaborators as well. And now one of the things that we are focused on is to make it more sensitive because as we try to develop the test to be broadly used, we want to make sure that it has as low of false negatives as possible. And that is something that we're focused on now. Hopefully it will have more improvements to report soon. And then simultaneously we're collaborating with others to see how we can best get us into a point of care or in a simple format so that people can use it in a much more widely distributed way.

Lydia Morrison: That's excellent. And I certainly look forward to seeing it be available for home use certainly many more diagnostics are going to be necessary in order for the globe really to start opening up, opening borders more and people to get back to life in a more natural, more normal way, the way that we were used to. And I really appreciate all the efforts from your group, all of your groups, as well as the collaborations. I think that this has been a great time to really emphasize the scientific collaborations that happen around the globe. And it's really refreshing I think to see these surrounding medical problems and solving these global health problems. And so hopefully this will continue into the future as well.

Feng Zhang: Definitely. And I think this has really been one of the most collaborative periods in science that we have all experienced. Scientists from around the world are really coming together recognizing that we are all universally faced with one common problem. And then by joining our efforts together we'll be able to get out of this sooner. But also I want to just thank you guys NEB has been a great partner working with us providing proteins and expertise, which has been very helpful as we are working in this challenging time to get access to reagents as quickly as possible. So thank you all for all of your help too.

Lydia Morrison: Well speaking on behalf of NEB, I can say that it is our honor and privilege to be able to help support your research. And we're very happy to do so. Just as a closing, could you tell us something good, do any of you have any good stories about maybe things that have happened or reflections that you've had during this time in quarantine?

Jonathan Gootenberg: It's been an exciting time to see, as Feng mentioned, people come together and be more collaborative and in the context of all of this, it's also still endurable to see the science of this discovery process. So the Cas12b enzyme that we're using here is one that's from a very thermophilic bacterium, that's actually spoils fruit juices, which is an interesting little anecdote about where these different enzymes come from.

Jonathan Gootenberg: So it's neat to still keep going back and mining these different enzymes for these properties. Similarly, during the process of this, we've done some optimizations that are just fun to do. We did a screening panel of different additives to increase the activity of the reaction. And the one that actually came out is the best was a taurine, which is in certain energy drinks and it's a neat stabilizing molecule. So it's neat to still be able to go back to the lab and tinker around and see these cool discoveries, even if it's in the context of everything that's going on. So you have to find these moments of joy in the backdrop of maybe a broader context.

Lydia Morrison: I'm glad that you've been able to find some moments of joy. I know that you've all been working very, very hard and diligently on this research on this STOPCovid kit and method. And it's wonderful to see all the results shared so quickly. It's nice to see the white papers and the preprints come out so regularly. It's nice to see this equal and open sharing of scientific information and knowledge. And it's certainly, I think that something that the public is really desirous of at this particular time. So thank you all so much for your work.

Feng Zhang: Thank you.

Jonathan Gootenberg: Thank you for having us.

Lydia Morrison: Thanks so much for joining me today as well. And hopefully we can speak again sometime.

Feng Zhang: Yeah. Take care, stay healthy in the meantime.

Lydia Morrison: Thank you. You as well. Thanks for tuning into this episode, be sure to check out the transcript of this podcast for helpful links to further resources and tune in for our COVID-19 Researcher Spotlight Series episode next week, which will actually be in an NEB TV episode in which I'm interviewing Nathan Tanner, a senior scientist here at NEB and our resident expert on Loop-mediated Isothermal Amplification, or LAMP technology. Nathan has a great perspective on lots of different applications of LAMP for SARS-CoV-2 diagnostics. So join us and find out how this single temperature amplification method is being used to create simple and robust COVID-19 diagnostics.

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