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CRISPR's Future For Point-Of-Care Diagnostics

February 18, 2020 | CRISPR applications are exploding, and Can Dincer, with the Freiburg Center for Interactive Materials and Bioinspired Technologies (FIT) and Department of Microsystems Engineering (IMTEK) at the University of Freiburg in Germany, sees uniquely powerful applications for point-of-care diagnostics.  

Dincer’s group has developed what he says is the first CRISPR/Cas13a powered electrochemical microfluidic biosensor for on-site miRNA detection. Without any target amplification, it offers a low-cost, easily scalable, and multiplexed approach for nucleic acid diagnostics. 

On behalf of Diagnostics World News, Christina Lingham spoke with Dincer about the challenges in using nucleic acid testing at the point-of-care and the opportunities and challenges of CRISPR. 

Editor's Note: Lingham, Executive Director of Conferences and Fellow at Cambridge Healthtech Institute, is producing a track dedicated to Point-of-Care Diagnostics at the Diagnostics Innovation Summit in Lisbon, May 19-21, 2020. Dincer will be speaking on the program. Their conversation has been edited for length and clarity. 

Diagnostics World News: Can you tell us what some of the challenges with nucleic acid testing at the point-of-care are? 

Can Dincer: Nucleic acid testing is an established method in the laboratory, but if you go to the point-of-care, there are actually mainly two big obstacles. The first one is the isolation of the nucleic acids that you would like to analyze, and the second one is the target amplification. In a normal laboratory, you usually have a PCR device, which you can directly work with, but this can get difficult on-site because not every facility has the same capabilities as a laboratory, particular in some countries versus others.  

How have you come to engage CRISPR? Can you give us your perspective on how it's useful in the detection of nucleic acids at the point-of-care? 

We came up with the idea of using CRISPR in 2016 through brainstorming with a project partner. We were wondering how we could easily measure microRNA in a highly sensitive and specific manner, because in a project, we were targeting the on-site detection of different microRNAs, and we were searching for different amplification methods, as well as easily scalable systems.  

We saw the first CRISPR paper, which used Cas9 to detect some DNA and also RNA in a difficult way, and later on, the work with the Cas13a came up where we then directly saw that it's very easy to detect RNAs with this system, and it was also possible to measure short RNAs like micoRNAs, and to question why we use it.  

The main problem is not only at the point-of-care. It's also in the laboratory. If you want to do some RNA diagnostics, you have to transcribe this to DNA. This works for long RNA, but short RNA is known to be problematic. MicroRNAs have only 18-25 nucleic acids, but with Cas13a, you can analyze your sample without any transcription by directly measuring the RNA. It’s an enzyme, and if it binds to its analyte, it gets activated and starts to cleave surrounding RNAs. It's a time-dependent reaction, so the longer you wait, the more efficient the enzyme is. This means less signal because in that system, you are using CRISPR to cleave labeled reporter RNAs and if they are cleaved, your signals aren’t getting over.  

The CRISPR system allows you to only change by a single-stranded guide CRISPR RNA (crRNA), to use your system for these different analytes. For example, if you are targeting microRNA 19b and want to change it for another microRNA, the only thing in your system that actually changes is the crRNA, so your detection system, the chip, and its contents stay constant, which enables you to have a one-for-all chip. You only have to change what you are adding to your sample if you are giving into the chip, which makes this system actually very powerful, so it's really quite easy to extend the system for multiple RNAs.  

Now this isn’t proven, but to me, it is logical. We are talking about one enzyme and it always cuts the same reporter. The only thing that we are changing is the crRNA, which means that in an ideal way, the calibration curve, which you should get for different targets, should always be the same, as you are not changing the enzyme and the reporter. But there are some changes which we showed in our work. We believe that this comes from this single-stranded crRNA. Our future work is also dealing with optimizing this, which enables us to use, for example, one calibration for thousands of microRNA so long as we solve this problem with the crRNA. 

Can you talk about the advantage of the electrochemical CRISPR biosensor? 

All CRISPR-based systems have almost the same advantages, depending on whether it's optical or electrochemical. If you use CRISPR technology, you get these advantages, and you will get a signal amplification. It's easily scalable and theoretically it can give you a single-calibration curve, and you don't need this transcription of the RNA to the DNA. I would like to point out that the electrochemical system does show, using our system, that our limit-of-detection (LOD) is around five-to-fifty times better than the optical systems, which are using the same CRISPR/Cas13a system.  

What do you see as some of the earliest applications of this technology? 

I would like to use it to enable a multiplexed on-site testing for microRNAs. MicroRNA clusters, consisting of five-to-six microRNAs tend to be a good biomarker in diseases like Alzheimer’s or different types of cancer. The goal is to create a system which is capable without any target amplification, measuring different microRNAs on-site and simultaneously to enable an early diagnosis system.  

If I can comment on the current situation with the coronavirus outbreak in China, or actually now it's worldwide, this CRISPR-Cas13a can also be used for detecting the coronavirus because it's a single-stranded RNA virus. Each coronavirus has a specific part which defines it, so you can target this part of the virus using CRISPR, and you can get accelerated, and possibly on-site testing systems for such an infectious disease outbreak. 

Let’s talk about how CRISPR can help in the future with other infectious disease outbreaks.  

There are different enzymes of the CRISPR system which you can target, either single-stranded RNA or single-stranded DNA, which also relates to different infectious diseases. If you use Cas9, you can also target double-stranded DNA. This is your toolbox. Depending on the nucleic acid tests that you would like to create for a possible infectious disease, you can take the specific system you need from your toolbox. 

For example, with coronavirus, you can target the long, common corona-specific RNA part, and tell whether or not it is coronavirus, or you can differ between various coronavirus types such as SARS or Covid19. CRISPR will most probably help you with its specificity because this system is known to be not only sensitive, but also highly specific. You can even differentiate single mismatches in the nucleic acid. 

What are some of the limits to developing this technology, and how might we overcome them? 

The limiting part is that the achieved LOD is still not enough if you do not combine it with target amplification. For example, there are some groups in the United States who coupled this with either thermal or PCR amplification, and of course, you would like to get rid of the PCR, therefore you need to be able to work without any target amplification. Furthermore, for the optical one, the multiplexing is limited. If you would like to detect different microRNAs or RNAs from a single sample in the optical system, you will need either different fluorescent markers or Cas enzymes, which are also not indefinite, so you cannot measure, for example, eight RNAs in the same sample using the optical approaches. The electrochemical system, which we use for example, allows us more flexibility in that way. We are working now on a multiplexed version of our CRISPR-Biosensor, that can detect up to 8 RNAs in the single specimen. The multiplexing is a bottleneck that we are working to overcome. 

But we would also like to take an easier approach regarding signal amplification without any target amplification and go for electrochemical signal amplification. It is called redox cycling, where you have two finger structures which are very near each other. At one finger electrode, you are oxidizing a redox-active substance, and at the other one, you are reducing it back to its initial form again. Using this special technique and very small gaps—nanogaps—so you can get signal amplifications of more than 150. We are aiming to take the LOD to go from the picomolar range to the femtomolar range where you can also make more, not only semi-quantitative, also quantitative analysis.