Project Description

COVID-19 Testing Today

Since the beginning of the Sars-CoV-2 (COVID-19) pandemic in December 2019 through the beginning months of 2020, society has found it to be very difficult to contain the spread of the virus. The COVID-19 virus has infected nearly 30 million people worldwide and approximately 6.5 million people in the United States¹. Perhaps the biggest problem associated with this pandemic however is society's lack of an ability to test large portions of people rapidly in order to keep track of where the virus has been. Many colleges around the United States have tried enacting large-scale testing initiatives, but have a major weak point: a large window of opportunity between consecutive tests for virus contraction². Consequently, there is a lag between when patients contract the virus and when officials realize that action is required, leading towards a slower than the adequate rate of contact tracing.

Today, the gold standard for testing uses Reverse-Transcriptase Polymerase Chain Reaction (RT-PCR)³. In a RT-PCR test, samples of viral RNA are collected, usually by using a nasopharyngeal swab, and are then converted to DNA using the enzyme reverse transcriptase. Once that is done, that DNA is amplified using a PCR protocol to make many copies of itself in order to be processed correctly. While this method is good and provides accurate results, a major downside to it is that it requires expensive laboratory equipment, such as a thermocycler that many testing sites around the globe do not have access to. In addition, unless the user has a background in these molecular diagnostic techniques, it is very challenging to interpret the results using this method. Thus, a major bottleneck in the testing initiative has been produced: many testing sites have to delay the time they get their results because they have to send the samples they collect to a central laboratory instead of just running their analyses as soon as they collect their samples.

Taking all this into account, society needs a test that requires cheap equipment, that is user friendly, that is rapid, and that provides accurate results.

Current Alternatives to the RT-PCR test

There are alternatives to the gold standard test, such as a serological test that will search for antibodies in a patient’s bloodstream. While this test is good in the sense that it is accurate, a major disadvantage is that it is likely to produce false negatives during the early stages of virus incubation. This is because the patient hasn’t yet developed antibodies for the virus, so when the test screens for them, there will be no target to be found³.

Many companies and research groups have turned to developing a point-of-care (POC) test that doesn't require thermocycling, namely the SHERLOCK test. Instead of using RT-PCR as a means of amplification in the SHERLOCK test, the test instead uses RT-RPA (Reverse Transcriptase-Recombinase Polymerase Amplification). RT-RPA is an isothermal amplification method that essentially provides the same end result as RT-PCR, after which SHERLOCK processes the amplification by coupling small fluorescent molecules with CRISPR-Cas9 proteins to produce fluorescent signals wherever there is CRISPR-Cas9 cleavage activity⁴. The test concludes in a lateral flow strip readout that the user can use to determine a positive or

Our Approach to the Problem

As stated previously, many tests currently being developed are taking advantage of CRISPR technology to detect SARS-CoV2. However, all of these tests have been using Cas proteins that collaterally cleave once their targets have been found. This did not appear very advantageous to our team because it restricts the technology to be conducted in vitro since collateral cleavage in human cells could lead to many different complications and problems. Instead, our team wanted to look into a new type of Cas enzyme, the dCas enzyme.

In other fields of biological research dCas9 has been widely used in order to control gene expression. Why would that be, you may ask, since Cas proteins in the past have been used primarily for gene editing? The answer lies in the fact that dCas9 contains a de-activated cleavage domain such that when it binds to its target, it cannot cleave it⁵. As such, dCas9 can instead be used as a “shuttle” for other proteins that are attached to it such as enhancers or repressors that bind to nearby DNA sequences to control their expression. dCas9 has also been used by Peking 2019 iGEM to downregulate the origin of replication on bacteria cells in order to control their growth rate⁶.

Figure 1, taken from source 5. This schematic shows the overall schematic of using a dCas9 enzyme tied to an effector in order to regulate gene expression. As shown in this figure, dCas9 will bind to a target DNA sequence as specified by its respective guide RNA in order to perform its work. The effector can come in the form of either a transcriptional activator such as VP64 and EDLL, or as a repressor such as SRDX. However, effectors are not limited to just these proteins, nor are they limited to strictly transcriptional activators or repressors.

Our project takes advantage of this “deactivated Cas” property and applies it to Cas13b to produce dCas13b, a Cas enzyme that will bind to RNA without cleaving it. By doing so, we can specify a specific sequence in RNA for Cas enzymes to target. This concept lays the groundwork for our project: to detect viral RNA from SARS-CoV2 using a dCas13b protein.

The second part of our project involves using a fusion protein, 𝛃-lactamase, that is fused with dCas13b to produce a fluorescent signal when enzyme activity is executed. As demonstrated from the 2013 Calgary iGEM team, 𝛃-lactamase can be used to produce a colorimetric biosensing assay by hydrolyzing Nitrocefin and monitoring the change in color inside the reaction mixture⁷. The key feature of this enzyme, however, is that it is split into N-terminal and C-terminal ends and is rendered nonfunctional unless the two fragments fuse back together. This, as such, can be used to turn the project design into a biosensing device because there will be an “on” state and an “off” state depending on whether the two fragments have fused.

Figure 2, overall schematic for the A New CDC iGEM project. As shown in the figure above, two dCas13b enzymes will bind to a specific RNA sequence in close proximity to each other via their respective guide RNA’s, and will each have one-half of a split fusion enzyme, 𝛃-lactamase, attached to it. Once these two halves are reconstituted due to their proximity, they will generate enzyme activity which will produce a color change to signify a positive test result.

As shown in the figure above, both termini are fused to two separate dCas13b enzymes that bind to the target viral RNA sequence. One may ask, why did we choose to use two dCas13b enzymes instead of just one? The reason is because using two would reduce the probability of a false positive arising. If only one dCas13b enzyme binds somewhere on the viral RNA, then no signal would be produced. Just like two students checking over a math problem to verify that they have the correct answer, two dCas13b enzymes have to bind in close proximity to ensure that they have the right target. If they do, then enzyme activity will activate and one will see a color change contained within the test tube solution.

Human Practices and Outreach

In addition to developing this test, we also focused our human practices and outreach efforts not only in order to have a better understanding of COVID-19 testing, but also to serve the community, especially students on college campuses. To achieve this end, we developed a website, called SARS-CoV-2 Clearinghouse, to serve as COVID-19 information hub for college students. By providing useful information, resources, and student-written articles about issues surrounding the pandemic, the website is meant to help students get a better understanding of the pandemic and to clarify misconceptions. All of our posts have well-researched scientific backing, but are presented in a digestible way so that students who may not have a scientific background can understand them. Furthermore, these posts are focused to hit a lot of issues surrounding the pandemic that may have non-scientific components layered on top of them. For example, we wrote an article on the importance of mask wearing, and provide scientific justification as to why mask wearing is important with the intention to move past political ideas and towards improved public health. More information about our website, in addition to our collaboration efforts, and be found on the Human Practices page of the wiki.

Conclusion

As you will see on the other pages of this wiki, our goal is to design this test such that it can be carried out with cheap equipment while producing reliable results that can be easily interpreted by the user. By doing so, this test will provide not only an effective means to screen people, but will also allow society to act much sooner once the test results are learnt.

1: University, J. s. H. (2020). COVID-19 Map - Johns Hopkins Coronavirus Resource Center. John's Hopkins University. Retrieved October 7 2020 from https://coronavirus.jhu.edu/map.html

2: Barbaro, M. (2020). The Daily In Quarantine On a College Campus.

3: Cheng, M. P., Papenburg, J., Desjardins, M., Kanjilal, S., Quach, C., Libman, M., Dittrich, S., & Yansouni, C. P. (2020). Diagnostic Testing for Severe Acute Respiratory Syndrome-Related Coronavirus 2: A Narrative Review. Annals of internal medicine, 172(11), 726–734. >https://doi.org/10.7326/M20-1301

4: Zhang, F., Abudayyeh, O., & Gootenberg, J. (2020). A protocol for detection of COVID-19 using CRISPR diagnostics. Broad Institute.

5: Moradpour, M., & Abdulah, S. N. A. (2020). CRISPR/dCas9 platforms in plants: strategies and applications beyond genome editing. Plant Biotechnology Journal, 18(1). https://doi.org/https://doi.org/10.1111/pbi.13232

6: iGEM, T. P. (2019). A dCas9-Based DNA replication based control system. https://2019.igem.org/Team:Peking