Catching up on CRISPR COVID-19 Diagnostics
SHERLOCK - The World’s First FDA-Approved CRISPR Application
In February 2020, US-based molecular diagnostics company Sherlock Biosciences dropped everything it had been doing to develop a new CRISPR-based test for the SARS-CoV-2 virus. Within just 3 months, the company was granted emergency use authorisation from the American Food and Drug Administration (FDA) for its novel Cas13-based test that can detect SARS-CoV-2 RNA in positive samples in just one hour. Later that month, we heard the inside story from Sherlock’s CEO Rahul Dhanda.
Rahul Dhanda told us that the company had been monitoring SARS-CoV-2 since it emerged in January 2020. Even though a pandemic wasn’t certain at that stage, the staff was concerned and began looking for SARS-CoV-2 sequences that could be used as detection targets for its Cas13-based detection platform, SHERLOCK.
SHERLOCK, for Specific High Sensitivity Reporter unLOCKing, is a highly sensitive and 100 % specific test i.e. no false positives, and it turns out results in about 1 hour. SHERLOCK relies on the ability of the RNA-targeting Cas13 enzyme to non-specifically cleave a fluorescently-labelled RNA reporter molecule upon recognition of SARS-CoV-2 sequences in positive samples. When the reporter molecule is cleaved, a fluorescence signal is released and this can easily be read on a plate reader.
SHERLOCK had already been used within research for the detection and genotyping of bacterial and viral pathogens and identification of antibiotic resistance genes. It was also successfully deployed to detect Zika and Dengue virus directly from patient urine and serum samples, demonstrating its field compatibility. The decision to go all-in and redesign the SHERLOCK platform for SARS-CoV-2 happened in February when the global situation was intensifying, about a month before the World Health Organisation (WHO) declared COVID-19 a pandemic.
Dhanda told us: »I was working with my board, and within 24 hours, I had a conversation with every board member independently. And they all felt very strongly that we should try to solve this, but solve it first and figure out what it means for the business later because this was such a critical problem. What we ended up doing is taking that decision to the company and pivoting the company to be focused 100% on COVID19.«
The company has since joined forces with others to manufacture and roll out the SARS-CoV-2 test on large scale, and is also working on a point-of-care test for rapid SARS-CoV-2 detection outside of medical institutions. The SHERLOCK story is a great reminder of the importance of having a vision and clear goal and what can be achieved when people work together.
CRISPR-Cas13 Surveillance Chip to Fight COVID-19 and Future Pandemics
The scientists at Sherlock Biosciences weren’t the only ones to redirect their Cas13 efforts to join the COVID-19 fight. In May, researchers from MIT and Harvard published their work on a rapid and cheap CRISPR-based system for massive scale viral detection. Paul Blainey, Associate Professor in Biological Engineering at MIT who led the team, told us that the group had been focused on developing a rapid detection platform for multiple viral pathogens but as the COVID-19 outbreak took hold, they shifted track to develop a dedicated assay for SARS-CoV-2.
The system, called CARMEN – Combinatorial Arrayed Reactions for Multiplexed Evaluation of Nucleic Acids - combines microfluidics, microarrays and the Cas13-based detection platform SHERLOCK. In essence, CARMEN runs thousands of nanolitre-scale SHERLOCK assays in parallel on a rubber chip similar to a smartphone in size.
A single CARMEN chip can run approx. 4,000 assays and gives results in a few hours. The assay matches the sensitivity of approved SARS-CoV-2 tests and can also discriminate between closely related viruses as well as drug-resistant forms of HIV. The researchers see CARMEN as an important surveillance tool for COVID-19 and believe that it will also play an important role in tackling future pandemics.
»We hope to live in a world where we can identify any kind of infectious illness, so we can begin to figure out how to respond more quickly. So we can prevent pandemics and not just react to them,« said Pardis Sabeti, senior author on the work who also participated in our interview.
CRISPR-Chip – a Quality Control Tool for CRISPR That Is Set to Revolutionise Biology
In July, we interviewed California-based Iranian Kiana Aran, co-founder of techbio company Cardea Bio and Assistant Professor at Keck Graduate Institute. Kiana Aran invented the CRISPR-Chip, an electronic sensor that uses CRISPR-Cas as a ‘DNA search engine’ that can scan genomes and nucleic acid samples for disease-associated mutations and infectious diseases such as SARS-CoV-2.
The potential for CRISPR-Chip extends far beyond diagnostics though. It can also be used to validate CRISPR-Cas complexes that are used in other applications, for example, to monitor the binding efficiencies of Cas variants and guide RNAs during optimisation, allowing researchers to find the best Cas variant for their application. This makes CRISPR-Chip a powerful upstream quality control tool to monitor safety and efficiency in clinical as well as research applications.
Aran and her colleagues have already demonstrated that the chip can detect the mutations associated with Duchenne muscular dystrophy and sickle cell anaemia, and the company is now collaborating with other scientists to develop a specific chip for SARS-CoV-2. Kiana Aran explained to us how the chip might be superior to the existing CRISPR-based approaches to COVID-19 surveillance:
»Whatever testing system we develop as we continue to fight this pandemic and prepare for the future ones, it has to be electronic, and the data has to be transferred without the user or doctors interfering, otherwise tracking will not be possible. It is not acceptable that the United States - the most modern society - still reports the COVID patient results on paper or pdf files to the agencies.«
CRISPR and PCR – The Best of Both Worlds for COVID-19 Mass Surveillance
For the last 6 months, a very diverse team of researchers from University of California, Santa Barbara has joined forces to develop a new low-cost SARS-CoV-2 surveillance protocol with potential for point-of-care and field use.
Explaining how the team came together in spite of their distinct research interests, Carolina Arias explained:
»When the pandemic was declared on March 11, I clearly remember having a meeting with Max Wilson and Diego Acosta-Alvear in one of our offices. We saw that the problem was growing completely out of proportion and that there was a huge void. That void was testing. We had the tools, the technical resources, and the molecular biology knowledge. We needed to try to implement some type of testing.«
First, the group developed CREST – Cas13-based, Rugged Equitable, Scalable Testing, which is a Cas13-based assay that begins with RNA isolated from human swab samples. The RNA is then reverse-transcribed to complementary DNA (cDNA) using standard reverse transcriptase enzyme and SARS-CoV-2-specific primers. The lab workhorse Taq polymerase is then used to amplify the SARS-CoV-2 cDNA to increase sensitivity and a final in vitro transcription step yields SARS-CoV-2 target RNA sequences that are then recognised by Cas13. Detection occurs via the off-target cleavage of a fluorescently-labelled RNA reporter molecule in a similar fashion to the SHERLOCK assay.
Unlike standard PCR- and emerging CRISPR-based tests for COVID-19, CREST is executed using cheap, portable instruments that are Bluetooth-enabled and battery operable for field use.
Having first-hand experience of the difficulties in procuring RNA isolation kits during the height of the pandemic, the group then developed a cheap RNA isolation protocol called PEARL – Precipitation Enhanced Analyte Retrieval – that is based on standard lab reagents and together with CREST matches the detection sensitivity of commercial kits.
The combined CREST and PEARL workflow overcomes the major hurdles associated with all other COVID-19 testing platforms; the protocols are cheaper to run and they don’t require difficult-to-source reagents such as the isothermal polymerase that is necessary for the SHERLOCK, CARMEN and other similar detection platforms based on CRISPR-Cas.
The group has already tested its workflow on real-life samples through a COVID-19 pilot surveillance programme for healthy volunteers from the local UC Santa Barbara campus community. The programme included two cohorts and the results highlight the importance of surveillance, as Carolina Arias explained:
»We tested the second cohort about three weeks after parts of Santa Barbara reopened and people could move around freely again. The results of our surveillance testing reflect perfectly what we are seeing in the community in terms of numbers of cases. So we picked up the leading edge of the outbreak. This shows the importance of expanding the capacity further and that is what we are trying to do.«
At the time of interview, the team was working on securing approval to roll out their assay in hard-to-reach areas in California.
The CRISPR Diagnostics Wave Continues
We continue to follow developments in CRISPR diagnostics for COVID-19 and we have far from covered all here. San Francisco-based Mammoth Biosciences is another major player within the field. Its CRISPR detection platform DETECTR - DNA Endonuclease Targeted CRISPR Trans Reporter - relies on the Cas12a nuclease, which is programmed to search for specific SARS-CoV-2 sequences in test samples. Similarly to Cas13, Cas12a also unleashes non-specific ssDNase activity upon target cleavage. This is exploted in the DETECTR assay, whereby fluorescence emitted from a cleaved ssDNA reporter molecule serves as the detection signal. In May 2020, Mammoth struck a deal with GlaxoSmithKline to develop a 20-minute CRISPR-based testfor SARS-CoV-2 and in July, the FDA granted UCSF an Emergency Use Authorization (EUA) to use DETECTR to test for the presence of SARS-CoV-2.
Companies and research groups all over the world have joined the fight against COVID-19 and as the pandemic continues, so too does the wave of innovative CRISPR-Cas diagnostics.
EdiGene (GuangZhou) Inc.
University of Pennsylvania