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The VEGAS Platform is unsuitable for Mammalian Directed Evolution
Citation
Denes, CE and Cole, AJ and Nguyen Tran, MT and Mohd Khalid, MKN and Hewitt, AH and Hesselson, D and Hewitt, A, The VEGAS Platform is unsuitable for Mammalian Directed Evolution, ACS Synthetic Biology, 11, (10) pp. 3544-3549. ISSN 2161-5063 (2022) [Refereed Article]
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DOI: doi:10.1021/acssynbio.2c00460
Abstract
Directed evolution uses cycles of gene diversification and selection to generate proteins with novel properties. While traditionally directed evolution is performed in prokaryotic systems, recently a mammalian directed evolution system (viral evolution of genetically actuating sequences, or "VEGAS") has been described. Here we report that the VEGAS system has major limitations that preclude its use for directed evolution. The deconstructed Sindbis virus (SINV) genome that comprises the VEGAS system could no longer promote Sindbis structural gene (SSG)-dependent viral replication. Moreover, viral particles generated using the VEGAS system rapidly lost the target directed evolution transgene, and instead, "cheater" particles, primarily containing RNA encoding SINV structural components, arose. By sequencing, we found that this contamination came from RNA provided during initial SINV packaging, not RNA derived from the VEGAS system. Of note, both the structural RNA and target transgenes used in the VEGAS system contain viral packaging sequences. The impact of SINV "cheater" particles could be potentially overcome in the context of a robust VEGAS circuit, but since SSG complementation is also defective in the VEGAS system, selection for authentic evolution products is not currently possible. Similar results have been obtained in independent laboratories. Taken together, these results show that the VEGAS system does not work as described and, without significant redesign, cannot be used for mammalian directed evolution campaigns.
Item Details
| Item Type: | Refereed Article |
|---|---|
| Keywords: | Sindbis virus; VEGAS; directed evolution; replicability; reproducibility. |
| Research Division: | Biomedical and Clinical Sciences |
| Research Group: | Reproductive medicine |
| Research Field: | Reproduction |
| Objective Division: | Health |
| Objective Group: | Evaluation of health and support services |
| Objective Field: | Evaluation of health outcomes |
| UTAS Author: | Nguyen Tran, MT (Mr Minh Thuan Nguyen Tran) |
| UTAS Author: | Mohd Khalid, MKN (Mr Mohd Mohd Khalid) |
| UTAS Author: | Hewitt, AH (Professor Alex Hewitt) |
| ID Code: | 155587 |
| Year Published: | 2022 |
| Deposited By: | Menzies Institute for Medical Research |
| Deposited On: | 2023-03-01 |
| Last Modified: | 2023-03-01 |
| Downloads: | 0 |
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