PT  - JOURNAL ARTICLE
AU  - Denes, Christopher E.
AU  - Cole, Alexander J.
AU  - Tran, Minh Thuan Nguyen
AU  - Mohd Khalid, Mohd Khairul Nizam
AU  - Hewitt, Alex W.
AU  - Hesselson, Daniel
AU  - Neely, G. Gregory
TI  - “Cheater” particles render the VEGAS platform unsuitable for mammalian directed evolution
AID  - 10.1101/2022.06.07.495067
DP  - 2022 Jan 01
TA  - bioRxiv
PG  - 2022.06.07.495067
4099  - http://biorxiv.org/content/early/2022/06/21/2022.06.07.495067.short
4100  - http://biorxiv.org/content/early/2022/06/21/2022.06.07.495067.full
AB  - 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 precluding its use for directed evolution. The primary technical issue with the VEGAS system is an immediate contamination with “cheater” particles that bypass directed evolution circuits. By sequencing we find these cheater particles contain Sindbis structural genes instead of the intended directed evolution target transgene. These cheaters outcompete the VEGAS transgenes within 2 rounds of transduction but cannot themselves activate synthetic circuits that drive expression of Sindbis structural genes, preventing directed evolution campaigns. Similar results have been obtained in independent labs. Taken together, the VEGAS system does not work as described and, without significant redesign to suppress cheaters, cannot be used for mammalian directed evolution campaigns.Competing Interest StatementThe authors have declared no competing interest.