Single Cell Characterization of a Synthetic Bacterial Clock with a Hybrid Feedback Loop Containing dCas9-sgRNA

Abstract

Genetic networks that generate oscillations in gene expression activity are found in a wide range of organisms throughout all kingdoms of life. Oscillatory dynamics facilitates the temporal orchestration of metabolic and growth processes inside cells and organisms, as well as the synchronization of such processes with periodically occurring changes in the environment. Synthetic oscillator gene circuits such as the “repressilator” can perform similar functions in bacteria. Until recently, such circuits were mainly based on a relatively small set of well-characterized transcriptional repressors and activators. A promising, sequence-programmable alternative for gene regulation is given by CRISPR interference (CRISPRi), which enables transcriptional repression of nearly arbitrary gene targets directed by short guide RNA molecules. In order to demonstrate the use of CRISPRi in the context of dynamic gene circuits, we here replaced one of the nodes of a repressilator circuit by the RNA-guided dCas9 protein. Using single cell experiments in microfluidic reactors we show that this system displays robust relaxation oscillations over multiple periods and over several days. With a period of ≈14 bacterial generations, our oscillator is similar in speed as previously reported oscillators. Using an information-theoretic approach for the analysis of the single cell data, the potential of the circuit to act as a synthetic pacemaker for cellular processes is evaluated. We also observe that the oscillator appears to affect cellular growth, leading to variations in growth rate with the oscillator’s frequency.

Competitive dCas9 binding as a mechanism for transcriptional control

“…CRISPRi has been rapidly adopted because it can exert regulatory control without having to mutate the target; however, it only imparts a single on/off signal. To this end, depression/derepression can be used to inactivate the effects of CRISPRi in the control of native genes (Nielsen & Voigt, 2014; Weinberg et al , 2017; Moser et al , 2018; Henningsen et al , 2020). For example, CRISPRi/a has been used to dynamically repress or activate enzymes to control carbon flux in metabolic engineering applications (Moser et al , 2018; Peng et al , 2018; Lu et al , 2019; Tian et al , 2019; Fontana et al , 2020b).…”

Bacterial degrons in synthetic circuits

“…These effector molecules replace the role of natural TFs in synthetic circuits [ 188 ]. A modified repressilator was designed that uses the dCas9 protein to replace the role of LacI in the original design [ 187 ]. A single-guide RNA (sgRNA) binds to the P lac promoter and imitates the repressive function of LacI (negative feedback loop).…”

CRISPR-Powered Microfluidics in Diagnostics: A Review of Main Applications

“…In this technique, a catalytically inactivated Cas9 enzyme (dCas9) is used. Henningsen et al introduced a so-called "Rock Paper CRISPR" (RPC) as a hybrid method, replacing one of the transcriptional repressors of repressilator by CRISPRi [54]. Using this approach, they managed to achieve stable temporal fluctuations to monitor 100 bacteria in a microfluidic machine.…”