Engineering cell sensing and responses using a GPCR-coupled CRISPR-Cas system

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Synthetic biology is changing the way we engineer living systems by providing frameworks to manage information flow and designed system complexity. Applying these concepts to engineer useful input-output devices in human cells has paved the way for engineered receptor technologies. A particular subset of these, Chimeric Antigen Receptors (CARs), are FDA approved for treat cancer. CAR therapy functions by enabling engineered T-cells to sense a surface marker (or cue) on cancer and convert that into an anti-tumoral program. Yet, this technology cannot interpret soluble markers that can be hallmarks of the disease, especially those from G-protein coupled receptor (GPCR) signaling systems. The CAR technology also cannot execute arbitrary gene programs to modulate either tumor cell behavior, or host T cell behavior. In this thesis, to bridge these technological gaps, I develop a new molecular input-output device, CRISPR-ChaCha. The technology leverages the ligand sensing diversity of GPCRs with the genome targeting flexibility of CRISPR-Cas systems. I first determine a functional architecture to fuse GPCR signaling proteins and CRISPR-Cas activators. I then show that the CRISPR ChaCha is dose-dependent, reversible, and can activate multiple endogenous genes simultaneously in response to extracellular ligands. The system displays a high degree of modularity, which is the ability to swap components and maintain function. I adopt this modular design to diverse GPCRs that sense a broad spectrum of ligands and to function with an additional CRISPR-Cas protein. I also make perturbations to device function to derive CRISPR-ChaCha design rules, to later build next generation devices with little optimization required. We hope that this flexible input-output device can be used for "smart" therapeutics, and rational engineering of cellular behavior.


Type of resource text
Form electronic resource; remote; computer; online resource
Extent 1 online resource.
Place California
Place [Stanford, California]
Publisher [Stanford University]
Copyright date 2019; ©2019
Publication date 2019; 2019
Issuance monographic
Language English


Author Kipniss, Nathan Hilton
Degree supervisor Qi, Lei, (Professor of Bioengineering)
Thesis advisor Qi, Lei, (Professor of Bioengineering)
Thesis advisor Cochran, Jennifer R
Thesis advisor Kobilka, Brian K
Degree committee member Cochran, Jennifer R
Degree committee member Kobilka, Brian K
Associated with Stanford University, Department of Bioengineering.


Genre Theses
Genre Text

Bibliographic information

Statement of responsibility Nathan Hilton Kipniss.
Note Submitted to the Department of Bioengineering.
Thesis Thesis Ph.D. Stanford University 2019.
Location electronic resource

Access conditions

© 2019 by Nathan Hilton Kipniss
This work is licensed under a Creative Commons Attribution Non Commercial 3.0 Unported license (CC BY-NC).

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