Systematic Mapping of Bacterial CRISPRa Systems for Synergistic Gene Activation Reveals Antagonistic Effects
1
Issued Date
2025-08-15
Resource Type
eISSN
21615063
Scopus ID
2-s2.0-105013560315
Pubmed ID
40693287
Journal Title
ACS Synthetic Biology
Volume
14
Issue
8
Start Page
3232
End Page
3244
Rights Holder(s)
SCOPUS
Bibliographic Citation
ACS Synthetic Biology Vol.14 No.8 (2025) , 3232-3244
Suggested Citation
Kiattisewee C., Karanjia A.V., Cardiff R.A.L., Olander K.E., Leejareon P., Alvi S.S., Carothers J.M., Zalatan J.G. Systematic Mapping of Bacterial CRISPRa Systems for Synergistic Gene Activation Reveals Antagonistic Effects. ACS Synthetic Biology Vol.14 No.8 (2025) , 3232-3244. 3244. doi:10.1021/acssynbio.5c00358 Retrieved from: https://repository.li.mahidol.ac.th/handle/123456789/111822
Title
Systematic Mapping of Bacterial CRISPRa Systems for Synergistic Gene Activation Reveals Antagonistic Effects
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Corresponding Author(s)
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Abstract
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.