Sentinel cells enable genetic detection of SARS-CoV-2 Spike protein
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Abstract
The COVID-19 pandemic has demonstrated the need for exploring different diagnostic and therapeutic modalities to tackle future viral threats. In this vein, we propose the idea of sentinel cells, cellular biosensors capable of detecting viral antigens and responding to them with customizable responses. Using SARS-CoV-2 as a test case, we developed a live cell sensor (SARSNotch) using a de novo-designed protein binder against the SARS-CoV-2 Spike protein. SARSNotch is capable of driving custom genetically-encoded payloads in immortalized cell lines or in primary T lymphocytes in response to purified SARS-CoV-2 Spike or in the presence of Spike-expressing cells. Furthermore, SARSNotch is functional in a cellular system used in directed evolution platforms for development of better binders or therapeutics. In keeping with the rapid dissemination of scientific knowledge that has characterized the incredible scientific response to the ongoing pandemic, we extend an open invitation for others to make use of and improve SARSNotch sentinel cells in the hopes of unlocking the potential of the next generation of smart antiviral therapeutics.
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SciScore for 10.1101/2021.04.20.440678: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics IRB: All cells were maintained at 37°C with 5% CO2 Source of Primary Human T cells: Blood was obtained from Blood Centers of the Pacific (San Francisco, CA) as approved by the University Institutional Review Board. Sex as a biological variable not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources Antibodies: Surface expressed proteins were assayed for using Alexa Fluor 647 Anti-Myc tag antibody (Cell Signaling Technologies #2233S) Anti-Myc tagsuggested: NoneExperimental Models: Cell Lines Sentences Resources HEK293T cells expressing ACE2 and TMPRSS2, a generous gift of Hannah S … SciScore for 10.1101/2021.04.20.440678: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics IRB: All cells were maintained at 37°C with 5% CO2 Source of Primary Human T cells: Blood was obtained from Blood Centers of the Pacific (San Francisco, CA) as approved by the University Institutional Review Board. Sex as a biological variable not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources Antibodies: Surface expressed proteins were assayed for using Alexa Fluor 647 Anti-Myc tag antibody (Cell Signaling Technologies #2233S) Anti-Myc tagsuggested: NoneExperimental Models: Cell Lines Sentences Resources HEK293T cells expressing ACE2 and TMPRSS2, a generous gift of Hannah S Sperber and Dr. Satish Pillai, were cultured in DMEM High Glucose (Gibco #10569-010) supplemented with 10% FBS, 1% of Antibiotic-Antimycotic, HEK293Tsuggested: NoneGeneration of Stable Cell Lines: Lenti-X 293T cells (Takara Bio #632180) were seeded at approximately 7e5 cells/well in a 6-well plate to yield ~80% confluency the following day. 293Tsuggested: NoneMedia was changed after 24 hours, and at 48 hours the viral supernatant was filtered through a 0.45μm PVDF filter and added to Jurkat or K562 cells seeded at approximately 1e5 cells/well in a 12-well plate. Jurkatsuggested: NoneK562suggested: NonePrior to the flow cytometry, cells were seeded at densities described below in a 96 well plates, using flat-bottom plates (Falcon #353072) for experiments involving BHK-21 cells and U bottom plates for all other experiments (Falcon #877217) and incubated for 24-72 hours as specified by the experiment. BHK-21suggested: NoneRecombinant DNA Sentences Resources The following day cells were transfected with 1.5μg of transfer vector containing the desired expression cassette, and the lentiviral packaging plasmids pMD2 pMD2suggested: NoneG (170ng) and pCMV-dR8.91 (1.33μg) using 10μl of Lipofectamine 2000 (Invitrogen #11668-027) according to manufacturer protocols. pCMV-dR8.91suggested: NoneBriefly, 293T cells were transfected with plasmid DNA (340 ng of Spike vector, 1μg CMV-Gag-Pol (pCMV-dΔR8.91), 125 ng pAdvantage (Promega), 1 μg Luciferase reporter (per 6-well plate)) for 48 h. pCMV-dΔR8.91suggested: NoneFor positive control, Spike vector was replaced with pMD2.G and for negative control this vector was omitted. pMD2.Gsuggested: RRID:Addgene_12259)Software and Algorithms Sentences Resources For LCB1 and LCB3, protein sequences were translated to human optimized coding sequences using Benchling. Benchlingsuggested: (Benchling, RRID:SCR_013955)All data were collected using FACSDiva (BD Biosciences) Flow Cytometry: Flow cytometry was performed using a LSR-Fortessa (BD Biosciences). FACSDivasuggested: (BD FACSDiva Software, RRID:SCR_001456)The protein concentration was estimated based on the protein absorbance at 280nm with a spectrophotometer (Nanodrop One, Thermo), flash frozen, and stored in −80 °C. Thermosuggested: (Thermo Xcalibur, RRID:SCR_014593)All data analysis was conducted using custom Python scripts, available on github (https://github.com/weinberz/sarsnotch). Pythonsuggested: (IPython, RRID:SCR_001658)Analysis was conducted in Jupyter59 and relied on numpy60, matplotlib, seaborn, pandas, SciPy61, scikit-learn62 and fcsparser. matplotlibsuggested: (MatPlotLib, RRID:SCR_008624)Results from OddPub: Thank you for sharing your code and data.
Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We did not find any issues relating to the usage of bar graphs.
Results from JetFighter: We did not find any issues relating to colormaps.
Results from rtransparent:- Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
- Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
- No protocol registration statement was detected.
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