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The VEGAS Platform is unsuitable for Mammalian Directed Evolution

journal contribution
posted on 2023-05-21, 16:46 authored by Denes, CE, Cole, AJ, Minh Thuan Nguyen Tran, Mohd Khalid, MKN, Alexander HewittAlexander Hewitt, Hesselson, D, Hewitt, A
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.

History

Publication title

ACS Synthetic Biology

Volume

11

Issue

10

Pagination

3544-3549

ISSN

2161-5063

Department/School

Menzies Institute for Medical Research

Publisher

American Chemical Society

Place of publication

Washington, DC

Rights statement

© 2022 American Chemical Society.

Repository Status

  • Restricted

Socio-economic Objectives

Evaluation of health outcomes

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