In the past few weeks, we’ve received over 2,600 COVID19 related plasmid requests. These plasmids have been heading to research labs in 43 countries.
COVID-19 research is happening fast and we’re glad to join these efforts and help scientists through plasmid sharing and more. These include plasmids that have been in the repository for many years, and new plasmids that scientists have deposited in the last several weeks. We've grown the collection of COVID-19 related plasmids to over 500 plasmids and our mission to accelerate research through sharing reagents has never been so important as scientists work around the clock to find solutions for COVID-19. Sharing speeds science.
|Figure 1: SARS-CoV-2 proteins and other components. Image from Maya Kostman for the IGI.|
Not only are we supporting academic scientists during this time, we're offering some of these plasmids to industry scientists as well… and that collection is always expanding! All COVID-19 plasmids available to industry are clearly marked in our COVID-19 plasmids and resources page. And, to make it easier for you to find new plasmids for COVID-19 research, the page is updated daily.
Here are some highlights from the collection. (We are also sharing a large set of plasmids from Ginkgo Bioworks, which will be highlighted in a future blog post.)
Plasmids deposited from recent COVID-19 research
Receptor recognition by SARS-CoV-2
Fang Li’s lab determined the crystal structure of the interaction between the receptor-binding domain of the spike protein from the virus and ACE2. Previously, the spike protein from SARS-CoV (the virus behind the 2003 SARS outbreak) was found to bind ACE2. The lab examined this interaction for SARS-CoV-2, SARS-CoV, and RaTG13, a bat coronavirus closely related to SARS-CoV-2. Their deposit includes plasmids expressing ACE2 and the spike protein from SARS-CoV and SARS-CoV-2.
Molecular decoys prevent S protein binding on human ACE2 receptors
The SARS-CoV-2 spike protein S binds ACE2 receptors on human cells to gain access to the cells’ interior. But if it can’t bind ACE2 receptors, it can’t enter human cells. Erik Procko’s lab set out to find a molecular decoy that would lure the virus away from binding human ACE2 receptors. They created variants of ACE2 and found out which variants bound the S protein more than the wild type. From successful single mutants, they combined mutations which led to a better binding. The lab also created a fusion of ACE2 to Fc of IgG or IgA classes which could provide a boost in avidity while recruiting immune effector functions and increasing serum stability.
Mapping the SARS-CoV-2-Human Protein-Protein Interaction
The Krogan lab generated lentiviral vectors expressing SARS-CoV-2 open reading frames and identified interactions of these proteins with human proteins. Their deposited plasmids expressed 26 of the 29 SARS-CoV-2 proteins which all but one are cloned into pLVX vectors (for lentiviral packaging) and contain an IRES-Puro marker for stable cell clone selection. For more information on these plasmids and a behind-the-scenes glimpse of their work, head over to their blog post.
Codon-optimized SARS-CoV-2 open reading frames
Fritz Roth’s lab created plasmids containing codon-optimized coding sequences for SARS-CoV-2 open reading frames in Gateway-compatible entry vectors. This enables the transfer into a variety of expression and tagging vectors.
Developing a one-enzyme RT-qPCR assay to detect SARS-CoV-2
Four years ago, Andrew Ellington’s lab evolved a reverse transcription xenopolymerase (RTX) that can be used for single-enzyme reverse transcription-polymerase chain reaction without complementary DNA isolation. This development allowed them to quickly adapt the enzyme for single-enzyme RT-qPCR assays to detect SARS-CoV-2. They’ve detailed their protocol on bioRxiv and deposited the plasmid for IPTG-inducible expression of RTX.
Previously deposited plasmids speed COVID-19 research
Many years prior to the COVID-19 pandemic, scientists have deposited plasmids from other research projects. These deposits have helped researchers get started quickly on SARS-CoV-2 research.
A plasmid from a zebrafish study becomes useful for COVID-19 research
In a study unrelated to COVID, or even infectious disease, Roger Reeves’s lab created a library of 164 cDNAs of conserved protein coding genes to study in zebrafish embryos. One of these plasmids is now being used in COVID-19 research and has been requested over 50 times.
At the time, the Reeves lab was examining how overexpression of genes from human chromosome 21 contribute to early embryogenesis. One of these coding sequences was the serine protease TMPRSS2. During SARS-CoV-2 and SARS-CoV infection, TMPRSS2 cleaves the S protein, which is required for viral and cellular membrane fusion. When TMPRSS2 is inhibited, the virus cannot enter. This plasmid is a great example of how two seemingly disparate fields share reagents.
The SARS-CoV spike protein binds ACE2
During the 2003 SARS outbreak, Hyeryun Choe’s lab identified that ACE2 is the functional receptor for SARS-CoV. This became a great starting point for SARS-CoV-2 researchers to investigate how the virus interacts with the receptor. They also found that when 293T cells expressed ACE2, SARS-CoV could replicate, but the virus could not replicate in mock-transfected cells.
The protease activity of cathepsins L is required to infect ACE-2 expressing cells
Shortly after the SARS outbreak, Hyeryun Choe’s lab found that cathepsins play a role in SARS-CoV infection. In 2008, another group of researchers later found that cathepsin L triggers proteolysis of the SARS-CoV spike protein, which activates its membrane fusion function. Now, the Choe lab found that SARS-CoV-2 infection also requires the cysteine protease activity of cathepsin L to infect ACE2-expressing cells. The lab deposited plasmids containing the human cathepsins S, L, and B.
Additional resources on the Addgene blog
- Find COVID-19 resources on our COVID-19 and Coronavirus Plasmids & Resources page
- Stay up-to-date on Addgene's operations during the pandemic
- View our latest newsletter to learn more about how we're accelerating COVID-19 research
Resources on Addgene.org
- Learn more about mapping the SARS-CoV-2 - human protein-protein interaction
- Find tips for remote collaborations
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