dCas9 regulator to neutralize competition in CRISPRi circuits

Hsin-Ho Huang, Massimo Bellato, Yili Qian, Pablo Cárdenas, Lorenzo Pasotti, Paolo Magni and Domitilla Del Vecchio ()
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Hsin-Ho Huang: Massachusetts Institute of Technology
Massimo Bellato: University of Pavia
Yili Qian: Massachusetts Institute of Technology
Pablo Cárdenas: Massachusetts Institute of Technology
Lorenzo Pasotti: University of Pavia
Paolo Magni: University of Pavia
Domitilla Del Vecchio: Massachusetts Institute of Technology

Nature Communications, 2021, vol. 12, issue 1, 1-7

Abstract: 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.

Date: 2021
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Persistent link: https://EconPapers.repec.org/RePEc:nat:natcom:v:12:y:2021:i:1:d:10.1038_s41467-021-21772-6

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DOI: 10.1038/s41467-021-21772-6

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