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 synthetic circuit construction. Here we use CRISPRi to build a synthetic oscillator (“CRISPRlator”), bistable network (toggle switch) and stripe pattern-forming incoherent feed-forward loop (IFFL). Our circuit designs, conceived to feature high predictability and orthogonality, as well as low metabolic burden and context-dependency, allow us to achieve robust circuit behaviors in Escherichia coli populations. Mathematical modeling suggests that unspecific binding in CRISPRi is essential to establish multistability. 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.
Highlights
Gene expression control based on CRISPR interference (CRISPRi) 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 synthetic circuit construction
Despite the enormous potential of CRISPRi for synthetic circuit design, the use of CRISPRi circuits in prokaryotes has been largely focused on logic gates and to the best of our knowledge none of the flagship circuits in synthetic biology have been re-constructed using CRISPRi
We fill this unaddressed gap by demonstrating that CRISPRi can be used for building some of the most notorious circuit topologies: the repressilator, a bistable toggle switch, and an incoherent feed-forward loop (IFFL, a.k.a. bandpass filter) that drives stripe pattern formation
Summary
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 synthetic circuit construction. Our circuit designs, conceived to feature high predictability and orthogonality, as well as low metabolic burden and contextdependency, allow us to achieve robust circuit behaviors in Escherichia coli populations. Due to its RNA-guided nature, CRISPRi is highly programmable[4], allows for easy design of sgRNAs that can be highly orthogonal[5] and whose behavior in different environments can be predicted in silico[6,7]. It imposes low burden on host cells[2] and is encoded in shorter sequences than protein-based repressors, thereby facilitating circuit handling and delivery and reducing cost. Mathematical modeling suggests that unspecific binding in CRISPRi is essential to establish multistability
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.