In this work, our goal is to construct a biological odor sensor in an easily cultured eukaryotic cells (HEK293) by use of a CRISPR/Cas9-activated olfactory receptor signaling transduction system with enhanced sensitivity and better controllability, which we call it “iSmeller” for such an intensified biological nose. It could be applied for the detection of pathological odors (e.g. cancer), food preservation and water quality etc.
Once a specific odor binds to the olfactory receptor on the cell membrane, it leads to a series of signaling rally and consequently generates a flux of cAMP (cyclic AMP) which could be detected by a cAMP-activated reporter gene system. Complete olfactory receptor signaling exists primarily in the olfactory epithelia cells which are not easily cultured and amplified to generate the ex vivo biosensor, and the mixed expression of different endogenous olfactory receptors in these cells may generate high background or confounding noises to detect a specific odor from the engineering point of view. Eukaryotic HEK293 cells could overcome these pitfalls and allows us to introduce highly specific olfactory receptor signaling within those easily engineered cells. Moreover, such eukaryotic cell-based biosensor can better mimic the powerful mammalian nose by the combinatorial olfactory receptor design, compared to the prokaryote or cell-free based biosensor systems.
As a proof-of-principle, we choose two odor compound/receptor pairs (β-citronellol/OR1A1 and bourgeonal/OR1D2) to construct the iSmeller odor sensors and evaluate their sensitivity and specificity. Since HEK293 cells do not express any olfactory receptors, we first exogenously express the targeted olfactory receptors on the cell membrane. To achieve an intensified downstream signaling to enhance the detection sensitivity, we employed the CRISPR activation (CRISPRa) system to simultaneously increase the expression of the core components of olfactory receptor signaling via lentiviral transduction, including GNAL, RTP1 and RIC8B. At the endpoint, a cAMP-activated reporter gene (luciferase) is transfected to read out the signaling strength in response to specific and different range of odors.
1. Clone human OR1A1 and OR1D2 with a rho tag and express them on the membrane of HEK293 cells. Test the sensitivity and specificity of this basic odor biosensors with their cognate odors β-citronellol or bourgeonal.
2. Construct signaling amplification circuit with CRISPRa systems. Design multiple sgRNAs targeting GNAL, RTP1 and RIC8B and evaluate their activation performance in HEK293 cells to pick out the most effective ones.
3. Introduce the pooled lentiviral CRISPRa-based signaling amplification components into the basic odor biosensors generated in step 1, and evaluate the performance of such iSmeller in contrast to the basic version. We expect that this iSmeller will significantly increase the sensitivity and/or specificity of the odor biosensor.
