Competitive dCas9 binding as a mechanism for transcriptional control

Abstract

Catalytically dead Cas9 (dCas9) is a programmable transcription factor that can be targeted to promoters through the design of small guide RNAs (sgRNAs), where it can function as an activator or repressor. Natural promoters use overlapping binding sites as a mechanism for signal integration, where the binding of one can block, displace, or augment the activity of the other. Here, we implemented this strategy in Escherichia coli using pairs of sgRNAs designed to repress and then derepress transcription through competitive binding. When designed to target a promoter, this led to 27-fold repression and complete derepression. This system was also capable of ratiometric input comparison over two orders of magnitude. Additionally, we used this mechanism for promoter sequence-independent control by adopting it for elongation control, achieving 8-fold repression and 4-fold derepression. This work demonstrates a new genetic control mechanism that could be used to build analog circuit or implement cis-regulatory logic on CRISPRi-targeted native genes.

The Topological Characteristics of Biological Ratio-Sensing Networks

“…In another circuit, the output signal BFP was activated and inhibited by anhydrotetracycline and IPTG signals, respectively, as mediated by zinc finger transcriptional regulators [ 15 ]. Recently, the dCas9 transcriptional repressor was used to constructed a ratio-sensing circuit [ 16 ]. Instead of Topo_8383, the circuit adopted Topo_7627.…”

“… Examples of ratio sensing in natural and synthetic systems. ( A ) Example of a natural ratio sensing in the yeast galactose pathway; ( B ) Examples of synthetic ratio sensing as summarized from references [ 14 , 15 , 16 ]. Arab, arabinose; AHL, acyl-homoserine lactone; AraC, the AraC protein; LacI, the repressor LacI protein; LuxR, the LuxR protein; GFP, the green fluorescent protein; mCherry, the red fluorescent protein; aTc, anhydrotetracycline; IPTG, isopropyl β-D-1-thiogalactopyranoside; ZF, the Zinc finger protein; VP16, a kind of transcriptional activating domain; BFP, YFP, and RFP are the blue, yellow, and red fluorescent protein, respectively; sgRP2 and sgDP5, sgRNAs guiding the dCas9 repressor protein.…”

“…On the other hand, synthetic regulatory circuits were constructed in Escherichia coli and yeast cells to perform ratio sensing as a demonstration of cellular analog computing. These circuits were implemented with competitive transcription factors [ 14 , 15 ] or RNA-guided dCas9 transcriptional regulators [ 16 ].…”

Incoherent merger network for robust ratiometric gene expression response

“…A well known molecular mechanisms for ratiometric sensing identified in natural systems is competitive binding of two input molecules to an integrator molecule, such as ligands binding to a receptor or regulators binding to a promoter [1][3]. This mechanism was leveraged to design a synthetic reporter that responds to the ratio ATP/ADP with a Michaelis-Menten function [5] and was later implemented with single guide RNAs (sgRNAs) for integrating the signals from two competing sgRNAs into one reporter output [41]. In these designs, the molecule that integrates the signals (a promoter) is different from the molecule that responds to the signal ratio (the output protein), therefore the output response will vary as the level of global cellular resources implicated in gene expression change.…”

From DNA-protein interactions to the genetic circuit design using CRISPR-dCas systems

“…However, such a design had significant levels of gene activation even without guanine ( Tang et al, 2017 ). Finally, competition of two dCas-sgRNA complexes that target neighboring DNA sites has been used to expand the flexibility of dCas-based circuits ( Anderson and Voigt, 2021 ) ( Figure 1F ). Such a design is good for ratiometric signal processing when the relative strength of two signals is used as an input to the circuit.…”