Drug targets, off-targets, and nuclease-induced toxicity
Abstract/Contents
- Abstract
- High-throughput functional screens in mammalian cells allow for the quantitative interrogation of a wide variety of biological problems. By systematically perturbing genes and coupling this to a measurement of phenotype, these screens have revealed new functional information and allowed us to characterize many genes in the genome. The technology for performing these screens has advanced rapidly, particularly in the last five years with the advent of CRISPR and related technologies. In Chapter 1, we briefly overview the historical uses of RNAi in functional screens, focusing on the issues and lessons learned from this technology. In Chapter 2, we directly compare an RNAi to a CRISPR cutting screen, looking for genes in the human leukemia line K562. We develop a new computational approach allowing the combination of information from both screens, and identify major differences between these technologies. In Chapter 3, we focus on CRISPR cutting screens and two sources of those differences: DNA damage caused by CRISPR/Cas9 nuclease and off-target cutting. In Chapter 4, we apply CRISPR interference technology to knockdown pairs of genes, allowing the identification of the mechanism of action of a small molecule Retro-2. In a combination of cellular, biochemical, and genetic approaches, we confirm the proposed activity of Retro-2, opening the possibility of using Retro-2 or other compounds with similar mechanisms for broad-spectrum antivirals. Finally, in Chapter 5, we discuss recent studies and their relevance to our work in Chapters 2 and 3, enumerate other studies that have relied on the technology developed in Chapters 2 and 3, and finally discuss future possibilities for the approach we used in Chapter 4. Taken together, these chapters represent key insights, improvements, and applications of high-throughput functional screens in mammalian cells.
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 | 2018; ©2018 |
| Publication date | 2018; 2018 |
| Issuance | monographic |
| Language | English |
Creators/Contributors
| Author | Morgens, David Warren |
|---|---|
| Degree supervisor | Bassik, Michael |
| Thesis advisor | Bassik, Michael |
| Thesis advisor | Altman, Russ |
| Thesis advisor | Dixon, Scott |
| Thesis advisor | Sherlock, Gavin |
| Degree committee member | Altman, Russ |
| Degree committee member | Dixon, Scott |
| Degree committee member | Sherlock, Gavin |
| Associated with | Stanford University, Department of Genetics. |
Subjects
| Genre | Theses |
|---|---|
| Genre | Text |
Bibliographic information
| Statement of responsibility | David Warren Morgens. |
|---|---|
| Note | Submitted to the Department of Genetics. |
| Thesis | Thesis Ph.D. Stanford University 2018. |
| Location | electronic resource |
Access conditions
- Copyright
- © 2018 by David Warren Morgens
- License
- This work is licensed under a Creative Commons Attribution Non Commercial 3.0 Unported license (CC BY-NC).
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