Neutralization of Competition Effects in CRISPRi-Based Genetic Circuits via a dCas9 Feedback Regulator

CRISPR interference (CRISPRi) has emerged as a powerful tool for modulating complex endogenous transcriptional programs and for building larger scale synthetic genetic circuits [1,2]. 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 reduces significantly when another sgRNA becomes expressed [3]. To solve this problem, decoupling sgRNA-mediated regulatory paths, we created a dCas9 concentration regulator that implements a negative feedback on dCas9 level [4]. 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. This recovery was confirmed even varying DNA copy numbers, strains and signaling molecules activating the systems; Our dCas9 generator is implemented in a dedicated plasmid, to be easily transported across compatible bacterial strains, thus enabling true scalability of CRISPRi-based transcriptional programs. We establish an important new tool of broad interest for CRISPRi users to either control endogenous transcriptional programs or to create new genetic circuits for any application. [1] Gao Y, Xiong X, Wong S, et al. Complex transcriptional modulation with orthogonal and inducible dCas9 regulators. Nat Methods 13, 12 (2016). [2] Kiani S, Beal J, Ebrahimkhani MR, et al. CRISPR transcriptional repression devices and layered circuits in mammalian cells. Nat Methods 11, 7 (2014). [3] Zhang S, Voigt CA. Engineered dCas9 with reduced toxicity in bacteria: implications for genetic circuit design. NAR 46, 20 (2018). [4] Huang H-H, Bellato M, Qian Y, et al. dCas9 regulator to neutralize competition in CRISPRi circuits. Nat Commun 12, 1692 (2021).