Heat-Triggered Remote Control of CRISPR-dCas9 for Tunable Transcriptional Modulation

• Type of Material: Article
• Authors: Lena Gamboa, Georgia Institute of Technology; Erick Phung, Georgia Institute of Technology; Haoxin Li, Georgia Institute of Technology; Jared P. Meyers, Georgia Institute of Technology; Anna C. Hart, Georgia Institute of Technology; Ian C. Miller, Georgia Institute of Technology; Gabe Kwong, Emory University
• Language: English
• Date: 2020-02-01
• Publisher: AMER CHEMICAL SOC
• Publication Version: Author Accepted Manuscript (After Peer Review)
• Copyright Statement: © 2020 American Chemical Society.
• Final Published Version (URL): http://dx.doi.org/10.1021/acschembio.9b01005
• Title of Journal or Parent Work: ACS CHEMICAL BIOLOGY
• Volume: 15
• Issue: 2
• Start Page: 533
• End Page: 542
• Grant/Funding Information: L.G. is supported by the Alfred P. Sloan Foundation, the NIH GT BioMAT Training Grant (5T32EB006343) and the NSF GRFP (DGE-1451512).; This work was funded by the NIH Director’s New Innovator Award (DP2HD091793), the National Center for Advancing Translational Sciences (UL1TR000454), the Shurl and Kay Curci Foundation, and the NSF (ECCS-1542174).; G.A.K. holds a Career Award at the Scientific Interface from the Burroughs Wellcome Fund.
• Keywords: ACTIVATION; INDUCIBLE CRISPR-CAS9 SYSTEM; Science & Technology; HYPERTHERMIA; GENE-EXPRESSION; REGULATOR; THERAPY; REPRESSION; Biochemistry & Molecular Biology; MECHANISMS; DNA-BINDING; KNOCKOUT; Life Sciences & Biomedicine
• Research Categories: Biology, Genetics; Chemistry, Biochemistry

Abstract

CRISPR-associated proteins (Cas) are enabling powerful new approaches to control mammalian cell functions, yet the lack of spatially defined, noninvasive modalities limits their use as biological tools. Here, we integrate thermal gene switches with dCas9 complexes to confer remote control of gene activation and suppression with short pulses of heat. Using a thermal switch constructed from the heat shock protein A6 (HSPA6) locus, we show that a single heat pulse 3-5 °C above basal temperature is sufficient to trigger expression of dCas9 complexes. We demonstrate that dCas9 fused to the transcriptional activator VP64 is functional after heat activation, and, depending on the number of heat pulses, drives transcription of endogenous genes GzmB and CCL21 to levels equivalent to that achieved by a constitutive viral promoter. Across a range of input temperatures, we find that downstream protein expression of GzmB closely correlates with transcript levels (R2 = 0.99). Using dCas9 fused with the transcriptional suppressor KRAB, we show that longitudinal suppression of the reporter d2GFP depends on key thermal input parameters including pulse magnitude, number of pulses, and dose fractionation. In living mice, we extend our study using photothermal heating to spatially target implanted cells to suppress d2GFP in vivo. Our study establishes a noninvasive and targeted approach to harness Cas-based proteins for modulation of gene expression to complement current methods for remote control of cell function.