Cas9-Mediated Genome Editing in Mouse Primary Cells
Abstract/Contents
- Abstract
- Gene targeting through Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9-mediated homologous recombination is a novel and powerful technique with promising applications in human disease modeling and therapeutic development. However, one major factor limiting genome editing efficiency is the time and difficulty of obtaining a transgenic animal, which may require several generations of breeding and expensive maintenance. While the establishment of an animal line with endogenous Cas9 nuclease expression could potentially accelerate the study of both gene knock-out and overexpression models, particularly in primary cells, previous work has yet to test the feasibility of such a system. This project provided the first in vitro proof of concept for a Cas9 transgenic mouse line as a research tool through the generation and characterization of Cas9-expressing mouse cells. In NIH3T3 cells, constitutive expression of either the Cas9 nuclease or nickase resulted in toxicity, with high levels of wild-type Cas9 expression appearing to be lethal over time. Following delivery of a CRISPR guide RNA sequence into cultured Cas9-expressing mouse cells via lentivirus, efficient genome editing was also observed. These findings demonstrate the prospect of high-speed multiplex genome engineering in mouse primary cells with future clinical implications such as cancer modeling and gene therapy. Moving forward, the generation of an inducible Cas9 transgenic animal should be pursued.
Description
| Type of resource | text |
|---|---|
| Date created | June 15, 2014 |
Creators/Contributors
| Author | Wang, Sean K | |
|---|---|---|
| Degree granting institution | Stanford University, Department of Biology, 2014 | |
| Primary advisor | Weissman, Irving L. | |
| Advisor | Gozani, Or |
Subjects
| Subject | CRISPR |
|---|---|
| Subject | Cas9 |
| Subject | guide RNA |
| Subject | genome editing |
| Subject | embryonic stem cells |
| Subject | toxicity |
| Subject | Biology |
| Subject | Stanford |
| Genre | Thesis |
Bibliographic information
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- User agrees that, where applicable, content will not be used to identify or to otherwise infringe the privacy or confidentiality rights of individuals. Content distributed via the Stanford Digital Repository may be subject to additional license and use restrictions applied by the depositor.
- License
- This work is licensed under a Creative Commons Attribution Share Alike 3.0 Unported license (CC BY-SA).
Preferred citation
- Preferred Citation
- Wang, Sean K. (2014). Cas9-Mediated Genome Editing in Mouse Primary Cells. Stanford Digital Repository. Available at: http://purl.stanford.edu/fx203jc8736
Collection
Undergraduate Theses, Department of Biology, 2013-2014
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- seankw@stanford.edu
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