A novel screening method using CRISPRa and FM 1-43 to identify cation channels
Pak, R.; Villarino, N.; Hung, K.; Wang, Y.; Patapoutian, A.
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The discovery of sensory ion channels, such as thermosensitive transient receptor potential (TRP) channels and mechanosensitive PIEZOs, have transformed our understanding of mammalian sensory biology. However, the sensory receptor landscape remains incomplete, as many physiologically relevant sensory stimuli still lack identified molecular targets. Here, we describe a novel screening strategy utilizing FM 1-43, a fluorescent marker for activity of various cation channels, with a CRISPRa library (MPCL) targeting multi-transmembrane domain proteins. We validate this method by focusing on allyl isothiocyanate (AITC) and its putative receptor TRPA1. Specifically, we show that CRISPRa-mediated overexpression of TRPA1 is sufficient for FM 1-43 labeling when co-treated with AITC. Furthermore, we show that using FM 1-43 and AITC, we can efficiently FACS enrich TRPA1-expressing cells from a pool of MPCL-expressing cells. Collectively, this presents a novel method for rapidly screening select cation-dependent sensory stimuli.
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