Transcriptional and position effect contributions to recombinant adeno-associated virus-mediated homologous recombination
Abstract/Contents
- Abstract
- Recombinant AAV vectors have the unique ability to promote targeted integration of transgenes via homologous integration at specified genomic sites reaching frequencies of 0.1-1%. We studied genomic parameters that influence targeting efficiencies on a large scale. To do this, we generated more than 1000 engineered, doxycycline-inducible target sites in the human HAP1 cell line and infected this polyclonal population with a library of AAV-DJ targeting vectors each carrying a unique barcode. The heterogeneity of barcode integration at each target site provided an assessment of targeting efficiency at that locus. We compared targeting efficiency with and without target site transcription for identical chromosomal positions, finding that targeting efficiency was enhanced by target site transcription. Chromatin states associated with transcription and epigenetic measures reflecting accessible chromatin and a depletion of heterochromatin were also predictive of higher targeting efficiency. Furthermore, there was a positive effect on the amenability of a site to targeting due to other factors such as the level of endogenous transcription from intersecting genes and, in the absence of target site transcription, intersecting LINE-1 elements and accessible chromatin. These results define important parameters that may not only assist in designing optimal targeting vectors for genome editing, but also provide new insights into the mechanism of AAV-mediated homologous recombination
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 | Spector, Laura Pauline |
|---|---|
| Degree supervisor | Kay, Mark Allan |
| Thesis advisor | Kay, Mark Allan |
| Thesis advisor | Fire, Andrew Zachary |
| Thesis advisor | Winslow, Monte |
| Thesis advisor | Wysocka, Joanna, Ph. D |
| Degree committee member | Fire, Andrew Zachary |
| Degree committee member | Winslow, Monte |
| Degree committee member | Wysocka, Joanna, Ph. D |
| Associated with | Stanford University, Department of Genetics. |
Subjects
| Genre | Theses |
|---|---|
| Genre | Text |
Bibliographic information
| Statement of responsibility | Laura P. Spector |
|---|---|
| Note | Submitted to the Department of Genetics |
| Thesis | Thesis Ph.D. Stanford University 2020 |
| Location | electronic resource |
Access conditions
- Copyright
- © 2020 by Laura Pauline Spector
- License
- This work is licensed under a Creative Commons Attribution Non Commercial 3.0 Unported license (CC BY-NC).
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