This post was reviewed by one of our subject matter experts, Dr. Scott Covey of UBC.
This study was conducted by researchers from UC San Francisco and Mammoth Biosciences
The paper we’ll be demystifying today can be found here, if you’d like to follow along.
The research team behind this research hails from San Francisco, California. It includes researchers from industry (Mammoth Biosciences) and academia (UCSF), and is a great example of how collaboration is the root of all great science. In this paper the authors describe how they worked to develop a new way of testing for SARS-CoV-2 infection that can give a clear yes or no answer within 40 minutes.
When it comes to combating COVID-19, testing is key. When doctors suspect that their patient might be suffering from COVID-19, they take a swab sample and send it to a lab for testing. The test approved by the CDC uses RT-qPCR (read more about it here) to detect viral RNA in patient swab samples. This assay is sensitive, but has long turnaround times (>24 hours) and requires specialized equipment. Serology assays (assays that look for antibodies in the blood) are a cool alternative but might not be able to catch early-stage SARS-CoV-2 infection. This is because it takes a few days for the body to mount an antibody response.
The authors of this paper set out to find a new way of testing for SARS-CoV-2. Their new assay is based on two relatively new technologies: RT-LAMP and CRISPR-Cas12.
CRISPR-Cas12 is a cousin of CRISPR-Cas9, a tool for powerful gene editing. You can think of CRISPR-Cas9 as a very accurate, very easily targeted pair of scissors that can make cuts in DNA at very specific points.
This technology was developed from a bacterial defence system and was first commercialized back in 2012. “CRISPR” is an acronym that stands for ‘clustered regularly interspaced short palindromic repeats.’ Bacteria generate CRISPR arrays by cutting up the DNA of any invading pathogens. If that pathogen infects again, the bacteria uses those CRISPR arrays to create guide RNAs for Cas9 enzymes. The guide RNAs point the Cas9 enzymes at any DNA sequences that look like the pathogen those guide RNAs originated from. Thanks to this, the Cas9 enzymes can now seek and destroy any genetic material belonging to the invading pathogen.
Cas9 is the name for an enzyme that acts as scissors to cut apart DNA. The Cas9 enzyme is moved to the right spot in the DNA by the short guide RNA sequence. A team of very smart researchers saw this bacterial defence system as a chance to improve our existing gene editing technology. They realized that by creating guide RNAs that target the Cas9 enzyme to a specific portion of DNA within a particular organism, they could make targeted changes to any genome. While the first reported CRISPR systems used Cas9, other Cas enzymes were discovered shortly after. These included Cas12 and Cas13. Each Cas enzyme has its own sets of advantages and disadvantages when it comes to gene editing. For this particular experiment, the authors chose to use Cas12. This lead to the name – CRISPR-Cas12.
The next piece of new technology is RT-LAMP. When a swab sample is taken from a patient who is positive for SARS-CoV-2, that swab sample will likely have some virus RNA. The amount of virus RNA in a single sample won’t be enough for any assay to detect- the scientists testing for the virus have to somehow make this tiny amount of RNA into enough to be picked up by their assay. Thankfully, technologies such as RT-PCR and RT-LAMP exist. The RT part stands for ‘reverse transcription,’ and refers to the process of converting the RNA within a sample into DNA. Once the scientists have this DNA, they can go about making many more copies of it using PCR (polymerase chain reaction) or LAMP. We’ve talked a lot about PCR before- in this post, for example.
Briefly, PCR makes many more copies of a target DNA sequence. It does this by using two ‘primers,’ which sit on either side of the target DNA sequence. When these primers bind they act as a landing dock for a polymerase. A polymerase is an enzyme that builds new DNA piece by piece, using the original DNA strand as a template. In order for PCR to work, the DNA has to be heated to a very high temperature- 98ºC- to separate the double stranded DNA into single strands (denatured). Once the DNA has been denatured, the temperature is lowered until the primers can bind to the correct sequences (anneal). Now it’s time for extension- the process of making more DNA. The extension step is carried out by DNA polymerase and generally happens at a temperature slightly higher than the temperature where the primers bind. These temperature changes repeat several times, cycling over and over again. Traditional PCR has to be carried out in a machine called a thermocycler. A thermocycler has to constantly change its internal temperature to allow for the various steps of PCR to take place.
Now what about RT-LAMP?
LAMP stands for ‘loop-mediated isothermal amplification.’ That’s a mouthful, but the main thing to know is that RT-LAMP is similar to RT-PCR, with some key differences:
- It all happens at one temperature, unlike PCR which oscillates between very high temperatures and lower temperatures.
- It uses 4 primers to recognize 6 different elements of a target DNA sequence, whereas RT-PCR uses 3 primers to recognize two markers flanking the target DNA sequence
The isothermal (iso= same, thermal = temperature, so isothermal = all one temperature) part of LAMP makes it fast and also eliminates the need for thermocyclers (which are bulky and very expensive). RT-LAMP reactions can be carried out in a single tube placed on a heat block, and the LAMP reaction used in this article only takes 20-30 minutes to complete.
In additional to being fast and more accessible, RT-LAMP can be more specific than traditional PCR. This is because it needs the six elements of the target sequence to match up with four primers rather than just two. These advantages allowed the authors of this paper to develop an assay that quickly and selectively makes many more copies of (amplifies) SARS-CoV-2 genetic material.
Now that we’ve talked about the technology, let’s look at the assay. The researchers named their new assay SARS-CoV-2 DNA Endonuclease-Targeted CRISPR Trans Reporter, or DETECTR.
Scientists love their clever names, as you can tell.
Towards the LAMP part of the assay, the researchers had to design new primers. These primers target the envelope (E) and nucleoprotein (N) genes of SARS-CoV-2. For the CRISPR-Cas12 step the researchers designed guide RNAs that bind to the genomes of three different SARS-like coronaviruses: SARS-CoV-2, bat SARS-like coronavirus, and SARS-CoV. The guide RNAs they made detect the E gene of all three viruses but only detect the N gene of SARS-CoV-2. This mimics the designs used by the WHO and US CDC assays. Both the WHO and CDC assays use multiple probes that are either specific to only SARS-CoV-2 or can detect multiple SARS-like coronaviruses.
In brief, the DETECTR assay begins with a reverse-transcriptase step to convert the RNA to cDNA. Directly after this the resulting cDNA is put through LAMP to make more copies of the DNA. The LAMP step is allowed to take place for 20-30 minutes. In the meantime the researchers combine Cas12 enzyme with various guide RNAs (targeting either the E or N gene) and incubate the mixture until enzyme-RNA complexes are formed. To these enzyme-RNA complexes is added something called a lateral flow cleavage reporter.
A lot of words, I know, but the payoff is coming. For a flowchart of how the assay works, check out panel d of Figure 1 of the paper.
After the LAMP step is completed, some of that amplified DNA is added to the Cas12/guide RNA/cleavage reporter mixture. This entire mix is then put onto a lateral flow strip. A lateral flow strip is like a pregnancy test- it shows one line for a negative result and two lines for a positive one. This allows for a quick visual answer to ‘do they have the virus?’ From swab to result, the entire assay takes about 1 hour to run. The patient is considered positive for the virus if the assay detects both the E and N genes of SARS-CoV-2. If only one of the two genes is detected, the patient would be labelled as a possible positive. This is the same as with the other CDC approved assays out there.
The researchers used SARS-CoV-2 RNA that was made in cells to confirm that the assay works, which is called validating. Once the DETECTR assay was successfully developed and validated, the scientists wanted to compare it to the RT-qPCR assays already in use. They found that the lowest amount of virus RNA that the assay can accurately detect (limit of detection) of RT-qPCR is 1 virus copy per uL (1/1000th of a mL). The limit of detection of DETECTR is slightly higher at 10 virus copies per uL. This means DETECTR is slightly less sensitive.
They then moved to actual patient swab samples- comparing RT-qPCR with their DETECTR assay to see how well the two assays agreed on whether a patient had the virus or not. They found that the percentage of times both assays agreed that the patient had the virus (positive agreement rate) was 95%. The percentage of times both assays agreed that the patient did not have the virus (negative agreement rate) was 100%.
This assay has its own drawbacks. Since the DETECTR assay uses the same methods to collect samples and isolate RNA as the CDC assays, it has some of the same limitations. These limitations include the low availability of PPE, extraction kits and other key materials. It’s also important to note that this assay is not ready to be deployed in the field until it receives the proper approval. While these are valid drawbacks we shouldn’t discount the enormous amount of scientific effort that was put into this study, or the importance of the results.
The researchers conclude by highlighting the importance of their achievement: they have developed an assay that has similar accuracy to RT-qPCR. Their assay uses common methods and easily available materials. Major advantages of the DETECTR assay over RT-qPCR are that it is faster, is highly specific and doesn’t require complicated lab equipment to run. The authors suggest that similar technology could be used to develop portable tests for use in airports, emergency departments and medical clinics in the near future. Testing is our main defence against the COVID-19 pandemic. The DETECTR assay may prove to be our next leap forward.