Multistable and dynamic CRISPRi-based synthetic circuits

Abstract

Gene expression control based on CRISPRi (clustered regularly interspaced short palindromic repeats interference) has emerged as a powerful tool for creating synthetic gene circuits, both in prokaryotes and in eukaryotes; yet, its lack of cooperativity has been pointed out as a potential obstacle for dynamic or multistable circuit construction, raising the question of whether CRISPRi is widely applicable for synthetic circuit design.Here we use CRISPRi to build prominent synthetic gene circuits that accurately govern temporal and spatial gene expression in Escherichia coli. We report the first-ever CRSPRi oscillator ("CRISPRlator"), bistable network and stripe pattern-forming incoherent feedforward loop (IFFL). Our circuit designs, conceived to feature high predictability and orthogonality and low metabolic burden and context-dependency, allowed us to achieve robust circuit behaviors (e.g. synchronous oscillations) and to expand the IFFL into a twice as complex, two-stripe patterning system. Our work demonstrates the wide applicability of CRISPRi in synthetic circuits and paves the way for future efforts towards engineering more complex synthetic networks, boosted by the advantages of CRISPR technology.Synthetic biology aims to build artificial decision-making circuits that are programmable, predictable and perform a specific function 1 . Since the rise of synthetic biology in the 2000s, most synthetic circuits have been governed by protein-based regulators. Recently, however, there has been growing interest in circuits based on RNA regulators as a means to overcome some of the intrinsic limitations of protein regulators 2 .The prokaryotic adaptive immunity system CRISPR constitutes a powerful platform for the construction of RNA-driven synthetic circuits 3 . The catalytically-dead mutant dCas9 can be easily directed to virtually any sequence by a single-guide RNA molecule (sgRNA). When a prokaryotic promoter (or downstream) region is targeted, steric hindrance by the dCas9-sgRNA complex results in transcriptional repression -an approach known as CRISPR interference (CRISPRi). CRISPRi offers several advantages over protein regulators for synthetic circuit

Towards a translationally-independent RNA-based synthetic oscillator using deactivated CRISPR-Cas

“…In this manner, we present one of the first dynamic uses of CRISPR-Cas systems over time. The only other example we are aware of is an experimental effort to make a dCas9-based CRISPR interference oscillator (Santos-Moreno et al 2020), where their periods were much longer at 14-17 generations, as well as a modeling-only exploration (Clamons and Murray 2017). Most previous CRISPR component circuits, while impressive, used endpoint as the behavior, showing before-and-after changes from incubations of several hours or more.…”

Toward a translationally independent RNA-based synthetic oscillator using deactivated CRISPR-Cas

“…We present construction of new translationally independent synthetic oscillators based on transcription of CRISPR-associated RNAs, a dynamic use of CRISPR-Cas systems over time. The only other example we are aware of is an experimental effort to make a dCas9-based CRISPR interference oscillator ( 30 ), where their periods were much longer at 14–17 generations, as well as a modeling-only exploration ( 49 ). Most previous CRISPR component circuits, while impressive, used endpoint as the behavior, showing before-and-after changes from incubations of several hours or more.…”

“…The dCas12a RNA oscillator may also be an interesting model system for further study in itself, as the oscillatory behavior is seen even without direct binding cooperativity in CRISPR systems ( 16 , 30 ). However, the system likely has non-linearity from other mechanisms, such as bound-repressor degradation ( 52 ), with DNA-bound dCas12a-crRNA removed by dilution from cell division and possibly protein degradation, or Michaelis–Menten degradation of the RNAs ( 53 ).…”

“…Our free-running dCas12a RNA oscillator fluctuates far more regularly than our dCas9-based designs and shows oscillatory behavior, with significant autocorrelation function (ACF) peaks for the population average. Along with another recent effort building dynamic CRISPR-based circuits ( 30 ), our circuits represent foundational work toward development of an even more regular oscillator using CRISPR-Cas parts and has the potential to be generalizable in a variety of host organisms across kingdoms.…”

Controlling spatiotemporal pattern formation in a concentration gradient with a synthetic toggle switch

“…Overall, our results underscore the relevance of studying the dynamical context of a gene regulatory network in order to understand patterning processes, not only of synthetic circuits but also of developmental systems (Ferrell, 2002, Sagner and. Future work will reveal if the TS properties are conserved when incorporated in more complex (synthetic) gene regulatory networks, for example when combined with the repressilator (Elowitz and Leibler, 2000, Potvin-Trottier et al, 2016, Santos-Moreno and Schaerli, 2019a to yield the AC-DC network (Perez-Carrasco et al, 2018, Verd et al, 2019, Balaskas et al, 2012, Panovska-Griffiths et al, 2013. Moreover, the here established engineering guidelines on how to control patterning with a synthetic TS will be valuable for future synthetic pattern formation, for example in the context of engineered living materials based on bacterial biofilms (Gilbert and Ellis, 2018, Nguyen et al, 2018, Moser et al, 2019, Cao et al, 2017.…”