Reconstitution of B cell somatic hypermutation in yeast for directed evolution
Abstract/Contents
- Abstract
- Targeted mutagenesis is a powerful technique used by nature and scientists to generate biological diversity and evolve diverse biological properties of proteins such as enzymes and antibodies. In vivo targeted mutagenesis enables rapid and continuous evolution of desired phenotypes directly in a host organism. However, existing in vivo mutagenesis tools are limited by low mutation rates and arduous gene specific gRNA expression. We describe a new method for TaRgeted In vivo Diversification ENabled by T7 RNAP (TRIDENT). TRIDENT is a platform for targeted, continual, and inducible diversification at genes of interest in yeast at mutation rates up to one-million fold higher than natural genomic error rates. TRIDENT targets natural and engineered mutagenic deaminase enzymes to precise genetic loci by fusion to T7 RNA polymerase, resulting in mutation tracks of controllable size from tens to thousands of base pairs, dependent on the positions of a T7 promoter sequence. Mutation diversity is further increased by targeting of DNA repair factors to the sites of deaminase-driven mutation, enabling sustained mutation of all four DNA nucleotides at rates greater than one substitution mutations per thousand bp, a more than 10-fold improvement over recent state-of-the art in vivo mutagenesis systems. We demonstrate application of TRIDENT to entirely replace ex vivo mutagenesis by applying it to correct point mutations in enzymes, fluorescent proteins, and evolve a red-shifted fluorescent protein. TRIDENT is a powerful in vivo mutagenesis platform and will accelerate protein engineering.
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 | 2019; ©2019 |
| Publication date | 2019; 2019 |
| Issuance | monographic |
| Language | English |
Creators/Contributors
| Author | Cravens, Aaron James |
|---|---|
| Degree supervisor | Smolke, Christina D |
| Thesis advisor | Smolke, Christina D |
| Thesis advisor | Cochran, Jennifer R |
| Thesis advisor | Jarosz, Daniel |
| Degree committee member | Cochran, Jennifer R |
| Degree committee member | Jarosz, Daniel |
| Associated with | Stanford University, Department of Bioengineering. |
Subjects
| Genre | Theses |
|---|---|
| Genre | Text |
Bibliographic information
| Statement of responsibility | Aaron J. Cravens. |
|---|---|
| Note | Submitted to the Department of Bioengineering. |
| Thesis | Thesis Ph.D. Stanford University 2019. |
| Location | electronic resource |
Access conditions
- Copyright
- © 2019 by Aaron James Cravens
- License
- This work is licensed under a Creative Commons Attribution Non Commercial 3.0 Unported license (CC BY-NC).
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