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.
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.
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.
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.
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.