Kiattisewee Cholpisit, Karanjia Ava V, Cardiff Ryan A L, Olander Kira E, Leejareon Pansa, Alvi Sarah S, Carothers James M, Zalatan Jesse G
Molecular Engineering & Sciences Institute, University of Washington, Seattle, Washington 98195, United States.
Center for Synthetic Biology, University of Washington, Seattle, Washington 98195, United States.
ACS Synth Biol. 2025 Aug 15;14(8):3232-3244. doi: 10.1021/acssynbio.5c00358. Epub 2025 Jul 22.
CRISPR gene activation (CRISPRa) tools have shown great promise for bacterial strain engineering but often require customization for each intended application. Our goal is to create generalizable CRISPRa tools that can overcome previous limitations of gene activation in bacteria. In eukaryotic cells, multiple activators can be combined for synergistic gene activation. To identify potential effectors for synergistic activation in bacteria, we systematically characterized bacterial activator proteins with a set of engineered synthetic promoters. We found that optimal target sites for different activators could vary by up to 200 bases in the region upstream of the transcription start site (TSS). These optimal target sites qualitatively matched previous reports for each activator, but the precise targeting rules varied between different promoters. By characterizing targeting rules in the same promoter context, we were able to test activator combinations with each effector positioned at its optimal target site. We did not find any activator combinations that produced synergistic activation, and we found that many combinations were antagonistic. This systematic investigation highlights fundamental mechanistic differences between bacterial and eukaryotic transcriptional activation systems and suggests that alternative strategies will be necessary for strong bacterial gene activation at arbitrary endogenous targets.
CRISPR基因激活（CRISPRa）工具在细菌菌株工程中显示出巨大潜力，但通常需要针对每个预期应用进行定制。我们的目标是创建可通用的CRISPRa工具，以克服细菌中基因激活的先前限制。在真核细胞中，多种激活剂可组合用于协同基因激活。为了鉴定细菌中协同激活的潜在效应物，我们用一组工程化的合成启动子系统地表征了细菌激活蛋白。我们发现，不同激活剂的最佳靶位点在转录起始位点（TSS）上游区域最多可相差200个碱基。这些最佳靶位点在质量上与先前关于每种激活剂的报道相符，但不同启动子之间的精确靶向规则有所不同。通过在相同启动子背景下表征靶向规则，我们能够测试每种效应物位于其最佳靶位点时的激活剂组合。我们没有发现任何产生协同激活的激活剂组合，并且发现许多组合具有拮抗作用。这项系统研究突出了细菌和真核转录激活系统之间的基本机制差异，并表明对于在任意内源性靶标上进行强大的细菌基因激活，将需要替代策略。
