Engineering Cell Sensing and Responses Using a GPCR-coupled CRISPR-Cas System

Overview

Journal Nat Commun

Specialty Biology

Date 2017 Dec 22

PMID 29263378

Citations 46

Authors

Nathan H Kipniss

P C Dave P Dingal

Timothy R Abbott

Yuchen Gao

Haifeng Wang

Antonia A Dominguez

Louai Labanieh

Lei S Qi

Affiliations

Soon will be listed here.

Abstract

G-protein-coupled receptors (GPCRs) are the largest and most diverse group of membrane receptors in eukaryotes and detect a wide array of cues in the human body. Here we describe a molecular device that couples CRISPR-dCas9 genome regulation to diverse natural and synthetic extracellular signals via GPCRs. We generate alternative architectures for fusing CRISPR to GPCRs utilizing the previously reported design, Tango, and our design, ChaCha. Mathematical modeling suggests that for the CRISPR ChaCha design, multiple dCas9 molecules can be released across the lifetime of a GPCR. The CRISPR ChaCha is dose-dependent, reversible, and can activate multiple endogenous genes simultaneously in response to extracellular ligands. We adopt the design to diverse GPCRs that sense a broad spectrum of ligands, including synthetic compounds, chemokines, mitogens, fatty acids, and hormones. This toolkit of CRISPR-coupled GPCRs provides a modular platform for rewiring diverse ligand sensing to targeted genome regulation for engineering cellular functions.

Citing Articles

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References

Morsut L, Roybal K, Xiong X, Gordley R, Coyle S, Thomson M . Engineering Customized Cell Sensing and Response Behaviors Using Synthetic Notch Receptors. Cell. 2016; 164(4):780-91. PMC: 4752866. DOI: 10.1016/j.cell.2016.01.012. View

Tang W, Hu J, Liu D . Aptazyme-embedded guide RNAs enable ligand-responsive genome editing and transcriptional activation. Nat Commun. 2017; 8:15939. PMC: 5493748. DOI: 10.1038/ncomms15939. View

Rosenbaum D, Rasmussen S, Kobilka B . The structure and function of G-protein-coupled receptors. Nature. 2009; 459(7245):356-63. PMC: 3967846. DOI: 10.1038/nature08144. View

Mali P, Aach J, Stranges P, Esvelt K, Moosburner M, Kosuri S . CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nat Biotechnol. 2013; 31(9):833-8. PMC: 3818127. DOI: 10.1038/nbt.2675. View

Vardy E, Robinson J, Li C, Olsen R, DiBerto J, Giguere P . A New DREADD Facilitates the Multiplexed Chemogenetic Interrogation of Behavior. Neuron. 2015; 86(4):936-946. PMC: 4441592. DOI: 10.1016/j.neuron.2015.03.065. View

Jinek M, East A, Cheng A, Lin S, Ma E, Doudna J . RNA-programmed genome editing in human cells. Elife. 2013; 2:e00471. PMC: 3557905. DOI: 10.7554/eLife.00471. View

Schwarz K, Daringer N, Dolberg T, Leonard J . Rewiring human cellular input-output using modular extracellular sensors. Nat Chem Biol. 2016; 13(2):202-209. PMC: 11536266. DOI: 10.1038/nchembio.2253. View

Davila M, Riviere I, Wang X, Bartido S, Park J, Curran K . Efficacy and toxicity management of 19-28z CAR T cell therapy in B cell acute lymphoblastic leukemia. Sci Transl Med. 2014; 6(224):224ra25. PMC: 4684949. DOI: 10.1126/scitranslmed.3008226. View

Ran F, Cong L, Yan W, Scott D, Gootenberg J, Kriz A . In vivo genome editing using Staphylococcus aureus Cas9. Nature. 2015; 520(7546):186-91. PMC: 4393360. DOI: 10.1038/nature14299. View

10.

Baeumler T, Ahmed A, Fulga T . Engineering Synthetic Signaling Pathways with Programmable dCas9-Based Chimeric Receptors. Cell Rep. 2017; 20(11):2639-2653. PMC: 5608971. DOI: 10.1016/j.celrep.2017.08.044. View

11.

Armbruster B, Li X, Pausch M, Herlitze S, Roth B . Evolving the lock to fit the key to create a family of G protein-coupled receptors potently activated by an inert ligand. Proc Natl Acad Sci U S A. 2007; 104(12):5163-8. PMC: 1829280. DOI: 10.1073/pnas.0700293104. View

12.

Amabile A, Migliara A, Capasso P, Biffi M, Cittaro D, Naldini L . Inheritable Silencing of Endogenous Genes by Hit-and-Run Targeted Epigenetic Editing. Cell. 2016; 167(1):219-232.e14. PMC: 5039111. DOI: 10.1016/j.cell.2016.09.006. View

13.

Wacker D, Stevens R, Roth B . How Ligands Illuminate GPCR Molecular Pharmacology. Cell. 2017; 170(3):414-427. PMC: 5560499. DOI: 10.1016/j.cell.2017.07.009. View

14.

Congreve M, Marshall F . The impact of GPCR structures on pharmacology and structure-based drug design. Br J Pharmacol. 2009; 159(5):986-96. PMC: 2839258. DOI: 10.1111/j.1476-5381.2009.00476.x. View

15.

Ferry Q, Lyutova R, Fulga T . Rational design of inducible CRISPR guide RNAs for de novo assembly of transcriptional programs. Nat Commun. 2017; 8:14633. PMC: 5339017. DOI: 10.1038/ncomms14633. View

16.

Kapust R, Tozser J, Copeland T, Waugh D . The P1' specificity of tobacco etch virus protease. Biochem Biophys Res Commun. 2002; 294(5):949-55. DOI: 10.1016/S0006-291X(02)00574-0. View

17.

Boc A, Diallo A, Makarenkov V . T-REX: a web server for inferring, validating and visualizing phylogenetic trees and networks. Nucleic Acids Res. 2012; 40(Web Server issue):W573-9. PMC: 3394261. DOI: 10.1093/nar/gks485. View

18.

Zetsche B, Heidenreich M, Mohanraju P, Fedorova I, Kneppers J, DeGennaro E . Multiplex gene editing by CRISPR-Cpf1 using a single crRNA array. Nat Biotechnol. 2016; 35(1):31-34. PMC: 5225075. DOI: 10.1038/nbt.3737. View

19.

Isberg V, Mordalski S, Munk C, Rataj K, Harpsoe K, Hauser A . GPCRdb: an information system for G protein-coupled receptors. Nucleic Acids Res. 2016; 45(5):2936. PMC: 5389621. DOI: 10.1093/nar/gkw1218. View

20.

Langenhan T, Barr M, Bruchas M, Ewer J, Griffith L, Maiellaro I . Model Organisms in G Protein-Coupled Receptor Research. Mol Pharmacol. 2015; 88(3):596-603. PMC: 4551050. DOI: 10.1124/mol.115.098764. View