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 the time course of several days. Through statistical analysis of the single cell data, the potential for the circuit to act as a synthetic pacemaker for cellular processes is evaluated. The use of CRISPRi in the context of an oscillator circuit is found to have profound effects on its dynamics. Specifically, irreversible binding of dCas9-sgRNA appears to prolong the period of the oscillator. Further, we demonstrate that the oscillator affects 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).…”

CRISPR-based gene expression control for synthetic gene circuits

“…In this setup, the dCas12a-based circuit displayed more regular oscillations than the one relying on dCas9 [128]. Additionally, a new preprint explored a hybrid oscillator in which one of the three initial TFs of the repressilator is replaced by a dCas9-gRNA complex [129]. In this study, decoy binding sites for the dCas9-gRNA complex have been suggested to play a role in generating the required non-linearity in the absence of cooperativity.…”

“…Indeed, a lack of non-linearity poses major challenges for building important synthetic networks such as oscillating or multistable circuits, which require non-linear gene expression control to operate [124][125][126][127]. However, recent results demonstrate that it is actually possible to build CRISPR-based multistable and dynamic circuits such as a toggle switch and an oscillator (see 'CRISPR-based synthetic circuits' section for details) [69,128,129].…”

“…Instead, mathematical modeling suggests that the repeated weak binding of dCas9 to the numerous PAM sites during its target search could provide the non-linearity required for these circuits to work [69]. Nonetheless, the conditions that determine the linear [41] versus non-linear [69,128,129] behavior of CRISPR gene regulation remain yet to be uncovered.…”

Overcoming leak sensitivity in CRISPRi circuits using antisense RNA sequestration and regulatory feedback

“…However, despite successes in creating dCas-based endpoint 3,13,14 and dynamic [15][16][17] circuits, challenges remain when circuits are scaled up from just a few CRISPRi ele-ments. 18 In fact, CRISPR's reprogrammability is also the source of its greatest weaknesses: because a shared pool of dCas proteins is drawn on simultaneously from all active elements, or nodes, in a circuit, it is possible for downstream nodes to interfere with the regulatory activity of those further upstream, an effect called retroactivity.…”