Sophisticated mammalian cell engineering with CRISPR/Cas12a
Abstract/Contents
- Abstract
- The application of CRISPR/Cas systems for programmable DNA manipulation has revolutionized the field of genome engineering. In addition to their use for cutting DNA, nuclease-dead versions of Cas proteins fused to different effectors are a powerful tool for cell engineering and synthetic biology. In this thesis, I will discuss two projects from my Ph.D., both demonstrating how Cas12a can be used for constructing next-generation genetic circuits in human cells. For my first project I developed a split Cas12a protein, which spontaneously re-assembles into a functional gene-activating effector when all components are present. Therapeutic cell engineering, in which cells are programmed to sense and respond to complex disease states, requires integration of information about the cellular environment and execution of a functional response only in response to the specified combination of inputs. By linking the expression of individual components of the split Cas12a system to different input signals, it forms the functional core of multi-input, multi-output logic circuits in mammalian cells. The system is highly programmable and can generate expandable AND gates, as well as incorporate NOT logic by using anti-CRISPR proteins as an OFF switch. For my second project I established a platform for parallel signal recording using a hyper-efficient Cas12a base editor. Biological signal recording enables the study of molecular inputs experienced throughout cellular history, by encoding a transient signal as a permanent, targeted DNA mutation that can be read out at a later time by sequencing. However, current methods are often limited in their ability to scale up beyond a single biologically relevant signal in mammalian contexts. This novel implementation of Cas12a for signal recording provides a powerful platform for scalable recording, demonstrated by the recording of up to four signals in parallel in human cells. This method is plug-and-play with diverse inputs in different cell types, including tumor antigen recording in CAR-T cells. It can also be easily extended for more sophisticated applications, such as time-delimited recording and dual recorder-effector cells. Overall, these two projects demonstrate the utility of Cas-based effectors in complex genetic circuits, for both understand-ing fundamental biological processes as well as constructing smarter cellular therapeutics.
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 | 2022; ©2022 |
| Publication date | 2022; 2022 |
| Issuance | monographic |
| Language | English |
Creators/Contributors
| Author | Kempton, Hannah Rose |
|---|---|
| Degree supervisor | Qi, Lei, (Professor of Bioengineering) |
| Thesis advisor | Qi, Lei, (Professor of Bioengineering) |
| Thesis advisor | Cochran, Jennifer R |
| Thesis advisor | Covert, Markus |
| Degree committee member | Cochran, Jennifer R |
| Degree committee member | Covert, Markus |
| Associated with | Stanford University, Department of Bioengineering |
Subjects
| Genre | Theses |
|---|---|
| Genre | Text |
Bibliographic information
| Statement of responsibility | Hannah Kempton. |
|---|---|
| Note | Submitted to the Department of Bioengineering. |
| Thesis | Thesis Ph.D. Stanford University 2022. |
| Location | https://purl.stanford.edu/tv892pk5937 |
Access conditions
- Copyright
- © 2022 by Hannah Rose Kempton
- License
- This work is licensed under a Creative Commons Attribution Non Commercial 3.0 Unported license (CC BY-NC).
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