Date: 4th May 2020
The current pandemic highlights the present lack of broad, comprehensive diagnostic applications able to simultaneously detect many pathogens, or that are scalable to test many samples. Now researchers have developed a new diagnostic platform CARMEN, using CRISPR-based SHERLOCK detection technology along with microfluidic chips that can run thousands of tests simultaneously, and could potentially be used for public health efforts.
The diagnostic-field is ablaze with new CRISPR-based platforms and methods, emerging at a blistering rate. These platforms have the potential to play vital roles in healthcare not only in this current pandemic, but in future ones, and indeed for many other diseases and infections. However, currently CRISPR-based tests only detect one or at best a few pathogens in a given reaction.
Now a team of scientists, led by Pardis Sabeti and Paul Blainey, from the Broad Institute of Massachusetts Institute of Technology (MIT) and Harvard, US, have developed an innovative diagnostics platform that could be applied broadly in communities. The aim; to provide routine comprehensive diagnostic testing able to inform patients, healthcare workers, and policy makers in their efforts to supress and mitigate future outbreaks.
The obvious place to start for the team was to base their platform on CRISPR-Cas as they are highly programmable, sensitive, and easy to use. The team developed Combinatorial Arrayed Reactions for Multiplexed Evaluation of Nucleic acids (CARMEN), which flexibly scaled up CRISPR-based molecular diagnostics and combined it with an adapted microwell array system, which had been previously developed by Blainey and his the team.
The microarray system was created using rubber chips, rubber was poured over a mould, and contained tens of thousands of ‘microwells’, each able to hold a pair of nanolitre-sized droplets. One droplet would contain the amplified viral genetic material, and the other contained the CRISPR-detection reagents. The detection reagents were adapted from the CRISPR-based diagnostic SHERLOCK, which we have previously described. Both sample and detection droplets each contained distinct, solution-based fluorescent colour code that served as optical identifiers. Once loaded on to the chips, 2 droplets randomly self-organised into each well, and could be identified later by their colour-code (by fluorescent microscopy). Positive viral detection by Cas13 was determined by cleavage of a reporter molecule.
To assess the performance of the system the team initially designed a CARMEN- assay to test for 169 human-associated viruses against 58 plasma, serum, and throat/nasal swab samples from patients with a range of confirmed infections. As a gold-standard readout, next generation sequencing (NGS) was also performed, this was used to compare the accuracy of CARMEN and, impressively, a very high concordance of 99.7% was observed. Interestingly, both methods also detected the presence of undiagnosed viruses present in some sample, highlighting the power of this screening approach.
As the outbreak of COVID-19 emerged during the publication process, the team rapidly designed a new pan-coronavirus panel to include SARS-Cov-2. Using a single micro-chip over 400 samples could be tested in parallel against the coronavirus panel.
To further showcase CARMENs clinically relevance the team also designed an assay to detect viral mutations which confer drug resistance. 22 samples from patients were used to validate the assay, which showed 90% concordance with Sanger sequencing results.
Conclusion and future applications:
The team here demonstrate the flexibility, and scalability of CARMEN. This approach to diagnostics is resource-efficient and easy to implement and it is likely to be useful in the future for a broad set of uses. It can detect whether patients are harbouring multiple infections, to rule out a whole panel of diseases very quickly, or to test a large population of patients for a single (or limited range) of serious infection.
A single chip’s capacity ranged from detecting a single type of virus in more than 1,000 samples at a time to searching a small number of samples (~5) for more than 160 different viruses. With the entire protocol taking around 8 hours, and the ability to scale up testing – by adding more chips, the team could impressively run 4-5 chips per day. Their hope is that it offers a comprehensive disease surveillance platform which will improve patient care and public health.
CRISPR-based diagnostic applications have dramatically increased over the last months, many in response to COVID-19. One popular simple, low-cost readout is the lateral flow assay which has been applied to both COVID-19 and other viral infections.
Whilst, these applications hold huge potential for detecting one or two infections, with minimal requirements for specialised equipment, CARMEN is currently uniquely poised for high throughput both in terms of patient samples but also in the number of viral infection it can screen for.
In the future, the team imagine that region- and outbreak-specific detection panels could be deployed to test thousands of samples from selected populations, including animal vectors, animal reservoirs, or patients presenting with symptoms. Offering us exciting new capabilities to investigate both epidemiological and clinical questions.
For more information please see the press release from the Broad Institute.
Ackerman, C. M., C. Myhrvold, S. G. Thakku, C. A. Freije, H. C. Metsky, D. K. Yang, S. H. Ye, C. K. Boehm, T.-S. F. Kosoko-Thoroddsen, J. Kehe, T. G. Nguyen, A. Carter, A. Kulesa, J. R. Barnes, V. G. Dugan, D. T. Hung, P. C. Blainey and P. C. Sabeti (2020). “Massively multiplexed nucleic acid detection using Cas13.” Nature.
https://doi.org/10.1038/s41586-020-2279-8
