Engineering Synthetic Signaling Pathways with Programmable dCas9-Based Chimeric Receptors

• Published in: Cell reports (Cambridge) Vol. 20; no. 11; pp. 2639 - 2653
• Main Authors: Baeumler, Toni A., Ahmed, Ahmed Ashour, Fulga, Tudor A.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 12.09.2017; Cell Press; Elsevier
• Subjects: Cell Membrane - metabolism; chimeric receptors; CRISPR; CRISPR-Cas Systems - genetics; dCas9-synR; dCas9-VP64; Gene Expression; Genetic Engineering; genome engineering; GPCR; HEK293 Cells; Humans; Models, Biological; Peptide Hydrolases - metabolism; Receptors, G-Protein-Coupled - metabolism; Recombinant Proteins - metabolism; RTK; Signal Transduction; split Cas9; synthetic receptors; Transcription Factors - metabolism; Transcriptional Activation - genetics; transcriptional programs; CRISPR; chimeric receptors; dCas9-VP64; dCas9-synR; RTK; synthetic receptors; GPCR; split Cas9; transcriptional programs; genome engineering
• ISSN: 2211-1247; 2211-1247
• DOI: 10.1016/j.celrep.2017.08.044

Synthetic receptors provide a powerful experimental tool for generation of designer cells capable of monitoring the environment, sensing specific input signals, and executing diverse custom response programs. To advance the promise of cellular engineering, we have developed a class of chimeric receptors that integrate a highly programmable and portable nuclease-deficient CRISPR/Cas9 (dCas9) signal transduction module. We demonstrate that the core dCas9 synthetic receptor (dCas9-synR) architecture can be readily adapted to various classes of native ectodomain scaffolds, linking their natural inputs with orthogonal output functions. Importantly, these receptors achieved stringent OFF/ON state transition characteristics, showed agonist-mediated dose-dependent activation, and could be programmed to couple specific disease markers with diverse, therapeutically relevant multi-gene expression circuits. The modular dCas9-synR platform developed here provides a generalizable blueprint for designing next generations of synthetic receptors, which will enable the implementation of highly complex combinatorial functions in cellular engineering. [Display omitted] •dCas9-synRs based on a highly programmable split-dCas9 signal transduction module•Core architecture can be standardized across multiple classes of sensing domains•Integration of user-controlled AND gates in the basic dCas9-synR architecture•dCas9-synRs enable implementation of complex combinatorial therapeutic responses Using a highly programmable split-dCas9-based signal transduction module, Baeumler et al. have created a novel class of synthetic receptors (dCas9-synRs) capable of coupling biologically relevant input signals with the direct activation of custom user-defined output response programs. dCas9-synRs expand the promise of cellular engineering for research and therapeutic applications.