Problem and Background

Most diagnostics platforms for detecting nucleic acids of SARS-CoV-2 require an amplification step. To ensure that amplicons are generated from the viral genome rather than an amplification carryover contamination, several common methods have been proposed, including the use of molecular beacons with fluorophores and expensive quenchers.

The aim of this project is to design and test a set of toehold switches and engineered ribozymes that will interact sequence-specifically with Sars-CoV-2 in cell-free systems generating a colorimetric output. The final design requires that the target regions be compatible with existing isothermal amplification methods, such as NASBA and RPA, however for this current stage of the project we will only focus on the design of functional toehold and ribozyme sensors.

Toehold and ribozyme sensors have been implemented in vitro using commercial cell-free systems that rely on purified individual components for transcription, translation and energy regeneration (PURExpress from NEB). Here we propose to validate designs for cell-free toehold and ribozyme sensors in low-cost DIY cell lysates produced using a widely implemented open protocol published on the protocols.io platform.

Proposed solution

We propose to develop a set of RNA structures that can interact with SARS-CoV-2 sequences to generate a colorimetric or fluorescent change for diagnostic use. Two kinds of RNA structures will be evaluated: Toehold and Ribozyme sensors. Both aim to repress the expression of a reporter gene unless an interaction with a trigger RNA sequence takes place. These methods will be implemented in vitro in ideal conditions using purified components required for transcription, translation and energy regeneration; and also in low-cost DIY implementations of these systems. This approach is invaluable because it will allow decentralized accessibility by eliminating the necessity of costly kits. 

 In-silico designs will rely on open source software, available on github. Viral target regions that are good candidates for isothermal amplification techniques will be chosen. Although the amplification step is beyond the scope of this proposal, validated designs could be tested after amplification steps in further projects.  

We hope to help in the development of an easily executable SARS-CoV-2 detection test for use in laboratories that are not equipped for currently accepted detection methods such as RT-qPCR. Our solution consists of developing a cell-free sensor that is sequence-specific and capable of generating a visible colorimetric output. In the future, these sensors can be coupled to isothermal amplification to detect clinically relevant samples.

If you are interested in designing and/or testing designs using cell-free systems please join us in this project or in our slack channel (https://open-covid19.slack.com/archives/C010MRZM95Z) :)

Neither diagnosis of cases of SARS-CoV-2 nor detection of environmental contamination by the virus are yet simple. Complicated techniques of molecular amplification, requiring serious infrastructure and biosafety procedures, limit people everywhere from knowing whether the virus is really in their environment or not.

Our focus for this proposal is the design of a set of toehold and ribozyme sensors which detect SARS-CoV-2 RNA sequences in cell-free expression systems. Toehold sensors are riboregulators that inhibit translation of a reporter gene via RBS sequestration in the absence of an interaction with a cognate sequence called the trigger RNA(1–3). Ribozyme sensors are transcription riboregulators which self-cleave in cis or in trans upon recognition of an RNA target sequence(4). For this project, toehold and ribozyme-sensors will be designed using Nupack and Ribosoft softwares, respectively, to recognize sequences specific to Sars-CoV-2 and not other respiratory viruses to allow the expression of a reporter gene only when interaction with the RNA target has occurred.

The genetic circuit designs consist of a T7 transcriptional unit regulating the transcription of a SARS-CoV-2-targeting toehold switch or a ribozyme regulating a reporter gene (lacZ or sfGFP).These designs will be rapidly produced by PCR and prototyped in commercial cell-free systems engineered for linear DNA expression (AccuRapid Cell-Free Protein Expression Kit or MyTxTl) and in a low-cost DIY alternative done by producing engineered cell lysates in-house.

Objective 1: In-silico selection of best candidate sequences for isothermal amplification of SARS-CoV-2.

Objective 2: In-silico design of toehold switches and ribozymes that interact with sequences selected in Objective 1.

Objective 3: Generate all materials needed: Toehold sensors, ribozyme sensors and reporters by PCR; DIY cell-free systems

Objective 4: Test the sensor designs in ideal sensing conditions (RNA transcribed in vitro) in commercial and DIY cell-free systems.

Methodology

Objective 1: In-silico selection of optimal candidates for isothermal amplifications will be done using python scripts. Conserved regions of the Sars-CoV-2 genome will be analyzed and sub-sequences will be ranked according to previously published parameters for optimal NASBA amplification(2).  

Objective 2: The sequences found in Objective 1 will be parsed in two different softwares: Toehold designer and RiboSoft for designing toehold sensors and ribozymes respectively. 

Objective 3:

Primers will be designed according to the best hits in Objective 1 and 2 and synthesized by IDT or local oligo providers. Positive controls, and other reporters will be distributed amongst team members via FedEX shipping. 

Linear DNA genetic circuit sequences will be made by PCR amplification, gel electrophoresis and purification. Our workplaces are equipped for these experiments.

Cell-free DIY systems will be done in Chile in a BSL2 laboratory.

Objective 4:

In vitro transcription and RNA purification will be done using Qiagen commercial kits. 

Due to the capabilities and resources of the team members, we will divide the testing accordingly. In Spain, Francisco will test toeholds and Ribozymes using the GFP reporter in the AccuRapid Cell-Free Protein Expression Kit. In Canada, Vesta will test toehold and Ribozymes using GFP output in MyTxTl. Anibal in Chile, will test best performing toeholds switches and ribozymes in a DIY cell-free system using GFP and LacZ.

In Chile we have access to a plate reader so fluorescence and absorbance data will be gathered from there. In Spain and Canada we will be able to test outputs by eye and by taking quality images with cell-phones.

Expected results

Objective 1: We expect to have a table with the list of sequences that are optimal for NASBA amplification techniques with the value of their parameters. We will also indicate in that table whether that sequence is being used already in a test, such as in the NEB colorimetric LAMP, q-RT-PCR and others.

Objective 2: We expect to have two tables, one for each design tool. For the toehold designer, we will have a ranking of the toehold sensors along with their value in each evaluated parameter, and the sequence they should target, a similar table will be the output for the ribozyme designs.

Objective 3: Purified linear DNA (about 20 uL at 100ng/uL) of every design will be tested, as well as positive controls (about 100 uL at 100 ng/uL).

Objective 4: First result will be a data set with all the raw results from different sources such as cell-phone images and plate reader files, and a well-documented report with every experiment and observations. Specifically, a characterization of several toehold switches for SARS-CoV-2, and the selection of the best performing candidates amongst all tested conditions. Also, a characterization of several ribozymes for SARS-CoV-2, and the selection of the best performing candidate. Comparison of the ribozyme and toehold efficiencies, and a characterization across the three different cell-free systems including a DIY cell lysate, patent-free and reproducible by anyone that wants to experiment with cell-free sensors.

References

1. Green, A. A., Silver, P. A., Collins, J. J. & Yin, P. Toehold Switches: De-Novo-Designed Regulators of Gene Expression. Cell 159, 925–939 (2014).

2. Pardee, K. et al. Rapid, Low-Cost Detection of Zika Virus Using Programmable Biomolecular Components. Cell 165, 1255–1266 (2016).

3. Ma, D., Shen, L., Wu, K., Diehnelt, C. W. & Green, A. A. Low-cost detection of norovirus using paper-based cell-free systems and synbody-based viral enrichment. Synth. Biol. 3, (2018).

4. Kharma, N. et al. Automated design of hammerhead ribozymes and validation by targeting the PABPN1 gene transcript. Nucleic Acids Res. 44, e39–e39 (2016).

5. Broughton, J. P., Deng, W., Fasching, C. L., Singh, J. & Chen, J. S. A protocol for rapid detection of the 2019 novel coronavirus SARS-CoV-2 using CRISPR diagnostics: SARS-CoV-2 DETECTR. 9.

6. Jung, Y. J. et al. Comparative analysis of primer-probe sets for the laboratory confirmation of SARS-CoV-2. http://biorxiv.org/lookup/doi/10.1101/2020.02.25.964775 (2020) doi:10.1101/2020.02.25.964775.

Timeline

[Day 1] In-silico design toeholds and ribozymes for Sars-Cov2.

[Day 1] Ordering of reagents (primers, enzymes, buffers, positive control DNA/RNA, kits).

[Week 1] Preparing in-House cell-free extracts suitable for the project.

[Week 1 and 2] Testing Toeholds and ribozymes in cell-free systems in ideal conditions ( Purified DNA, RNA from in vitro transcription).

[Week 2 and 3] Testing Toeholds and ribozymes in in-house DIY cell-free systems in ideal conditions ( Purified DNA, RNA from in vitro transcription).

[Week 4] Conclusions and broad dissemination of the open info.

Our progress

A great deal of information about the virus was already available openly, and close examination of available sequences and preprints confirmed several sets of promising primers to detect conserved COVID-19 viral targets. We have identified several that look like they would be suitable for this method, and one set has already been subjected to initial tests in the partner project #proj-neb-lamp-test. Optimization and determination of the best primer sets for inclusion in the #CoronaDetective ‘test kit’ are the key project aims, once funding enables us to get necessary reagents to project participants.

The impact is potentially very large, as diagnostic tools are only available in the specialised laboratory setting now. To our knowledge this is the first open source covid19 cell free sensor designed, all the documentation produced will be extremely helpful for comparing this strategy with other projects and sensors currently developed, seeing the main advantages and drawbacks. 

We expect that this system would be one of the most affordable strategies, without compromising the quality of the results in terms of Limit of Detection and false positives rate. Once we validate the system with commercial reagents, we will optimize the system to make anyone with scarce resources able to easily replicate the system.

Therefore the impact we want to adress is huge, as we are describing a system capable of doing environmental / DIY testing of SARS-cov-2 in a low resources setting. 

Biosafety

WARNING! This sensor is intended for working with in vitro-prepared short nucleic sequences.

No human or clinical samples should be handled outside of appropriate biosafety facilities. OpenCovid-19 Initiative's Biosafety and Biosecurity Guidelines will be applied to this project.

For our experiments no live or attenuated virus will be used. Only parts of genes will be used as the positive controls and these parts will never contain more than 2/3 of the viral genome.

While we will be using synthetic genetic constructs, the use of cell-free systems ensures that no GMOs will be released into the environment. Furthermore, no toxin-related genes will be used in the constructs.

All the activities will be performed according to local Biosafety guidelines for BSL1 (Canada and Spain) or BLS2 (Chile) labs.

Team

Anibal Arce Medina: Biochemist by training. PhD(c) in molecular biology in PUC, Chile. In his thesis he is developing ultra-low-cost cell-free technologies, implementing Toehold RNA sensing reactions for local interest such as the PVY virus. He is also interested in measuring sources of variability in the cell-free systems using ratiometric fluorescent reports. He has done several internships in Microsoft Research Institute, Cambridge UK; Keith Pardee’s lab in Toronto among others. Anibal will design toehold sensors and implement both toeholds and ribozymes sensors in low-cost DIY cell-free systems.

Francisco Javier Quero: Biologist by training. Tsinghua University Master Student and part of the managing team of openFIESTA's community lab. Two years working with synthetic biology as the coordinator of Madrid iGEM teams 2018 and 2019. Experience developing isothermal amplifications for infectious diseases. Francisco will validate toehold and ribozyme designs regulating GFP using the AccuRapid Cell-Free Protein Expression Kit cell-free system.

Vesta Korniakova is a Msc. Student in Applied Microbiology at INRS-Institut Armand Frappier, in Montreal, Canada currently working with riboswitches. She is also a member of Brico.bio and Foulab, two community labs in Montreal. For this project, Vesta will design and validate toehold and ribozyme sensors regulating sfGFP from a home lab using a commercial cell-free system provided by Arbor Biosciences.

Fernan Federici is a researcher at iBio institute and PUC Chile. Fernan is a biologist by training. He has worked on developmental biology, open source technology, DNA assembly and educational resources. Fernán is a member of TECNOx, GOSH and CYTED-reGOSH open technology movements. His group also collaborates with OpenPlant and OpenBioEconomy Lab. More info at: https://federicilab.org/

We have donations of myTxTl cell-free Mastermix from Arbor Biosciences (thank you Khalid for your help with this!), and some local resources in Chile from iBio for implementation of low-cost cell-free systems in Chile.

We are looking for further funding for obtaining the remainder of necessary materials for continuing this project from the individual labs located in Chile, Spain and Canada.

A complete list of costs is available here.