Sentinel cells enable genetic detection of SARS-CoV-2 Spike protein

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.

De novo-designed minibinders expand the synthetic biology sensing repertoire

“…We previously demonstrated that the de-novo designed LCB1 and LCB3 minibinders for SARS-CoV-2 Spike protein could be used as functional antigen sensors when integrated with synNotch 18 . These anti-SARS Notch constructs successfully detected both purified Spike protein and Spike expressed on the surface of mammalian cells in a model of virally-infected cells to drive a synthetic transcriptional output.…”

“…See Supplemental Figure 1A for schema of output circuit and SNIPR expression constructs. We incubated a polyclonal population of SNIPRexpressing cells with either untransduced K562 cells or K562s stably expressing a prefusion stabilized version of the SARS-CoV-2 Spike ectodomain 41 displayed on a PDGFR transmembrane domain as previously described 18 (Figure 1B). After 72 hours, we assessed our output circuit-only expressing Jurkat cells (No Notch) and LCB1and LCB3-SNIPR expressing cells for BFP expression (Figure 1C).…”

Computational pipeline provides mechanistic understanding of Omicron variant of concern neutralizing engineered ACE2 receptor traps

“…WT-fl-Spike protein was purified based on the previously described protocol (Weinberg et al, 2021) 30ml of ExpiCHO-S cells at 6M cells/mL were transfected with 1ug/mL of the spike WT using the ExpiCHO TM Expression System Kit (Gibco, catalog # A29133) following the manufacturer’s Standard protocol. Briefly, the transfected cells were shaken at 37°C and 8% CO 2 and 18 hours post-transfection, the cultures were supplemented with ExpiCHO TM Feed and ExpiFectamine TM CHO Enhancer and continued to be shaken at 37°C and 8% CO 2 for up to 10 days.…”

Small molecule and cell contact-inducible systems for controlling expression and differentiation in stem cells

“…Cells were co-cultured for 72 hours and the BFP expression and surface expression of receptors and ALFA-tag were conducted using flow cytometry. synNotch and SNIPR activation was assessed as described previously by fitting a two-component Gaussian mixture model to BFP expression data and estimating the fraction of the population in the 'off' and 'on' components 41 . SNIPR Activation-Ngn2 Differentiation Assay mESCs expressing SNIPR constructs were seeded at a density of 1e4 cells/well in a lowattachment round bottom 96-well plate.…”