The Dr. John and Anne Chong Lab for Functional Genomics, Charles Perkins Centre and School of Life & Environmental Sciences, The University of Sydney, Sydney, NSW 2006, Australia.
Centenary Institute and Faculty of Medicine and Health, The University of Sydney, Sydney, NSW 2006, Australia.
ACS Synth Biol. 2022 Oct 21;11(10):3544-3549. doi: 10.1021/acssynbio.2c00460. Epub 2022 Oct 11.
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.
定向进化利用基因多样化和选择的循环来产生具有新特性的蛋白质。虽然传统的定向进化是在原核系统中进行的，但最近已经描述了一种哺乳动物定向进化系统（遗传激活序列的病毒进化，或“VEGAS”）。在这里，我们报告说 VEGAS 系统存在重大限制，使其无法用于定向进化。构成 VEGAS 系统的已解构的辛德比斯病毒（SINV）基因组不再能够促进 Sindbis 结构基因（SSG）依赖性病毒复制。此外，使用 VEGAS 系统产生的病毒颗粒迅速失去了目标定向进化转基因，并且取而代之的是，主要包含编码 SINV 结构成分的 RNA 的“作弊”颗粒出现了。通过测序，我们发现这种污染来自于最初 SINV 包装过程中提供的 RNA，而不是来自 VEGAS 系统的 RNA。值得注意的是，VEGAS 系统中使用的结构 RNA 和目标转基因都包含病毒包装序列。在稳健的 VEGAS 回路的背景下，SINV“作弊”颗粒的影响可能会被克服，但由于 VEGAS 系统中的 SSG 互补也存在缺陷，因此目前无法对真实的进化产物进行选择。在独立的实验室中也获得了类似的结果。总之，这些结果表明 VEGAS 系统不能按预期工作，并且在没有重大重新设计的情况下，不能用于哺乳动物定向进化活动。
