dCas9 regulator to neutralize competition in CRISPRi circuits
Name
s41467-021-21772-6.pdf
Description
Published version
Size
1.19 MB
Format
Adobe PDF
Checksum (MD5)
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Author(s)
Huang, Hsin-Ho
Bellato, Massimo
Qian, Yili
Cárdenas, Pablo
Pasotti, Lorenzo
Magni, Paolo
Del Vecchio, Domitilla
Date Issued
2021
Journal
Nature Communications
Publisher
Springer Science and Business Media LLC
Citation
Huang, Hsin-Ho, Bellato, Massimo, Qian, Yili, Cárdenas, Pablo, Pasotti, Lorenzo et al. 2021. "dCas9 regulator to neutralize competition in CRISPRi circuits." Nature Communications, 12 (1).
Version
Final published version
Abstract
CRISPRi-mediated gene regulation allows simultaneous control of many genes. However, highly specific sgRNA-promoter binding is, alone, insufficient to achieve independent transcriptional regulation of multiple targets. Indeed, due to competition for dCas9, the repression ability of one sgRNA changes significantly when another sgRNA becomes expressed. To solve this problem and decouple sgRNA-mediated regulatory paths, we create a dCas9 concentration regulator that implements negative feedback on dCas9 level. This allows any sgRNA to maintain an approximately constant dose-response curve, independent of other sgRNAs. We demonstrate the regulator performance on both single-stage and layered CRISPRi-based genetic circuits, zeroing competition effects of up to 15-fold changes in circuit I/O response encountered without the dCas9 regulator. The dCas9 regulator decouples sgRNA-mediated regulatory paths, enabling concurrent and independent regulation of multiple genes. This allows predictable composition of CRISPRi-based genetic modules, which is essential in the design of larger scale synthetic genetic circuits.
MIT Department
Massachusetts Institute of Technology. Department of Mechanical Engineering
Massachusetts Institute of Technology. Department of Biological Engineering
Massachusetts Institute of Technology. Synthetic Biology Center
Terms of Use
Creative Commons Attribution 4.0 International license
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DOI of Published Version
https://doi.org/10.1038/S41467-021-21772-6
