Engineered CRISPR/dCas9 systems to dynamically control transcription and chromatin organization
Abstract/Contents
- Abstract
- The discovery and development of CRISPR/Cas9 technologies have revolutionized the ways in which fundamental cellular processes like gene function and chromatin organization can be studied. Transcriptional regulators based on nuclease-deficient Cas9 (dCas9) allow fine-tuned expression control of virtually any gene in the genome, providing new ways to control cell fate and to identify the genetic drivers of cellular phenotypes. An ever-expanding array of dCas9-based chromatin modifying proteins offers the ability to directly probe the causal role of individual features of chromatin toward regulating nearby and distant regions of the genome. In this dissertation, I present two sets of engineered dCas9-based systems for the dynamic regulation of gene expression and heterochromatin formation, respectively. First, I build and characterize a set of dCas9 transcriptional activators and repressors whose activities are controlled by chemical and light inducers. From a screen of chemical- and light-inducible dimerization systems, I identify two potent chemical inducers that mediate efficient and reversible gene activation and repression in mammalian cells. Different inducer systems can be paired with orthogonal dCas9 transcriptional regulators to independently control expression of different genes within the same cell. Furthermore, using this platform, I devise Boolean AND, OR, NAND, and NOR dCas9 logic processors and a diametric regulator that activates gene expression with one inducer and represses with another. This modular dCas9 platform provides a robust approach for enacting complex transcriptional programs in mammalian cells suitable for controlling directed differentiation and building synthetic gene circuits. Second, I adapt the chemically-inducible dCas9 platform for the conversion of chromatin states across tens of kilobases of genomic DNA, an approach I name CRISPR-Engineered Chromatin Organization (EChO). I show that CRISPR-EChO can be used to recruit heterochromatin components, in particular human Heterochromatin Protein 1α (HP1α), in high numbers to genomic targets on different chromosomes to form discrete structures visible by confocal microscopy. Synthetically tethered HP1α causes the bound genomic region to form novel contacts with other regions of heterochromatin and to reversibly compact into a condensed state. The condensed state induces transcriptional repression across a 600 kilobase genomic region and exhibits delayed disassembly kinetics. These findings collectively support an HP1α polymer model for the regulation of heterochromatin organization and function within the nucleus. Together, these two bodies of work represent divergent applications of dCas9 technology for mammalian synthetic biology that operate at different genomic scales and highlight the distinct advantages of dynamic and targeted control of nuclear processes for studying biological questions
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 | Gao, Yuchen (Tony) |
|---|---|
| Degree supervisor | Qi, Lei, (Professor of Bioengineering) |
| Thesis advisor | Qi, Lei, (Professor of Bioengineering) |
| Thesis advisor | Bhutani, Nidhi |
| Thesis advisor | Porteus, Matthew H |
| Thesis advisor | Wysocka, Joanna, Ph. D |
| Degree committee member | Bhutani, Nidhi |
| Degree committee member | Porteus, Matthew H |
| Degree committee member | Wysocka, Joanna, Ph. D |
| Associated with | Stanford University, Cancer Biology Program. |
Subjects
| Genre | Theses |
|---|---|
| Genre | Text |
Bibliographic information
| Statement of responsibility | Yuchen Gao |
|---|---|
| Note | Submitted to the Cancer Biology Program |
| Thesis | Thesis Ph.D. Stanford University 2020 |
| Location | electronic resource |
Access conditions
- Copyright
- © 2020 by Yuchen Gao
- License
- This work is licensed under a Creative Commons Attribution Non Commercial Share Alike 3.0 Unported license (CC BY-NC-SA).
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