Engineering alternative splicing devices for mammalian synthetic biology
Abstract/Contents
- Abstract
- From the approximately 20,000 genes embedded in DNA genomes to the functional proteins they encode for, much of the functional diversity of mammals can be attributed to alternative splicing, a pre-messenger RNA processing mechanism that produces multiple proteins from a single gene through careful arrangement of exons into a final transcript. While synthetic biology harnesses natural sequences refined by genetic evolution to build new functions from modular DNA components in engineered biological systems, few systems have leveraged alternative splicing as a precision tool for genetic control in mammalian systems. Combining the evolved mechanisms of splicing regulation with the ability to produce multiple, distinct proteins from a single gene, alternative splicing in synthetic genetic devices expands our capacity to control mammalian systems for medical, industrial, and biological discovery platforms. We have developed a class of mutually exclusive alternative splicing devices that can express two distinct isoforms based on which one of two internal exons is included in the final RNA molecule. In our efforts to engineer these devices for novel functions to control biological systems, we expanded the functional outputs of our alternative splicing devices using modular protein domains and fluorescent proteins, in addition to updating previous design considerations that limited our initial efforts, such as size and sequence constraints. Our ability to regulate outcomes from alternative splicing devices is important for their use as genetic transcriptional tools for mammalian synthetic biology. We modify our intron framework to support the production of both isoforms at various ratios based on mutations and truncations to consensus elements responsible for alterna- tive splicing. We explored synthetic approaches to controlling isoforms, such as the CRISPR-Cas13 RNA-targeting system and the MS2 protein aptamer, that allowed us to shift splicing patterns in a dynamic fashion. Our strategy to controlling isoform outcomes and protein functionality replicates how evolution evolved RNA splicing for a synthetic alternative splicing device. We present the design, construction, regulation, and versatility of alternative splicing devices. Their unique ability to compress genetic information combined with transcriptional regulation tools, both naturally encoded and artificially introduced, advances the novel mechanism we can harness to engineer biology
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 | 2020; ©2020 |
| Publication date | 2020; 2020 |
| Issuance | monographic |
| Language | English |
Creators/Contributors
| Author | Kim, Cameron Michael |
|---|---|
| Degree supervisor | Smolke, Christina D |
| Thesis advisor | Smolke, Christina D |
| Thesis advisor | Qi, Lei, (Professor of Bioengineering) |
| Thesis advisor | Swartz, James R |
| Degree committee member | Qi, Lei, (Professor of Bioengineering) |
| Degree committee member | Swartz, James R |
| Associated with | Stanford University, Department of Bioengineering. |
Subjects
| Genre | Theses |
|---|---|
| Genre | Text |
Bibliographic information
| Statement of responsibility | Cameron Michael Kim |
|---|---|
| Note | Submitted to the Department of Bioengineering |
| Thesis | Thesis Ph.D. Stanford University 2020 |
| Location | electronic resource |
Access conditions
- Copyright
- © 2020 by Cameron Michael Kim
- License
- This work is licensed under a Creative Commons Attribution Non Commercial 3.0 Unported license (CC BY-NC).
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