Engineering cell sensing and responses using a GPCR-coupled CRISPR-Cas system
Abstract/Contents
- Abstract
- 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.
Description
| 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 |
Creators/Contributors
| 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. |
Subjects
| 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
- Copyright
- © 2019 by Nathan Hilton Kipniss
- License
- This work is licensed under a Creative Commons Attribution Non Commercial 3.0 Unported license (CC BY-NC).
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