Biophysical Constraints Arising from Compositional Context in Synthetic Gene Networks

Yeung, Enoch; Dy, Aaron J.; Martin, Kyle B.; Ng, Andrew H.; Vecchio, Domitilla Del; Beck, James L.; Collins, James J.; Murray, Richard M.

doi:10.1016/j.cels.2017.06.001

Cited by 125 publications

(158 citation statements)

References 46 publications

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“…Circuit components were all expressed from a single vector (so-called "variable" vector) to avoid fluctuations in their stoichiometry. Circuit transcriptional units (TUs) were isolated from each other by strong transcriptional terminators and 200 bp spacer sequences, to avoid transcriptional readthrough and minimize compositional context 25 . To prevent mRNA context-dependency and provide transcriptional insulation within TUs, a 20 bp csy4 cleavage sequence 26 was used flanking sgRNAs and upstream of the ribosome binding sites (RBS) of the reporter genes.…”

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confidence: 99%

Multistable and dynamic CRISPRi-based synthetic circuits

Santos‐Moreno

Tasiudi

Stelling

et al. 2019

Preprint

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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

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mentioning

confidence: 99%

Multistable and dynamic CRISPRi-based synthetic circuits

Santos‐Moreno

Tasiudi

Stelling

et al. 2019

Preprint

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“…a TU assembled with an LS part 1 will always be the most upstream TU). Therefore, it is prudent to follow general design principles in order to maximize their repurposing 33 lacking. Responding to and containing such outbreaks is an important public health challenge that demands rapid production and iteration of naturally occurring viral circuits in mammalian host cells to enable discovery of inhibitors and investigation of the basic biology of the emerging pathogens.…”

Section: Discussionmentioning

confidence: 99%

A Toolkit for Rapid Modular Construction of Biological Circuits in Mammalian Cells

Fonseca

Bonny

Kumar

et al. 2018

Preprint

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The ability to rapidly assemble and prototype cellular circuits is vital for biological research and its applications in biotechnology and medicine. Current methods that permit the assembly of DNA circuits in mammalian cells are laborious, slow, expensive and mostly not permissive of rapid prototyping of constructs. Here we present the Mammalian ToolKit (MTK), a Golden Gate-based cloning toolkit for fast, reproducible and versatile assembly of large DNA vectors and their implementation in mammalian models. The MTK consists of a curated library of characterized, modular parts that can be easily mixed and matched to combinatorially assemble one transcriptional unit with different characteristics, or a hierarchy of transcriptional units weaved into complex circuits. MTK renders many cell engineering operations facile, as showcased by our ability to use the toolkit to generate single-integration landing pads, to create and deliver libraries of protein variants and sgRNAs, and to iterate through Cas9-based prototype circuits. As a biological proof of concept, we used the MTK to successfully design and rapidly construct in mammalian cells a challenging multicistronic circuit encoding the Ebola virus (EBOV) replication complex. This construct provides a non-infectious biosafety level 2 (BSL2) cellular assay for exploring the transcription and replication steps of the EBOV viral life cycle in its host. Its construction also demonstrates how the MTK can enable important and time sensitive applications such as the rapid testing of pharmacological inhibitors of emerging BSL4 viruses that pose a major threat to human health. and Extensible Mammalian Modular Assembly kit (EMMA) 3 based on Golden Gate assembly reactions were proposed. Both methods use sets of modular parts with given functionality, which can be assembled directionally into transcription units (TUs) by type-IIS restriction enzymes. While useful, all three methods have important shortcomings. GMAP requires sequencing in between all cloning steps, which makes the method slow and expensive. EMMA and mMoClo provide a method to hierarchically assemble DNA vectors that encode large circuits, but fall short on shareability, integration of Cas9 sgRNA cloning, a library of tested parts, and ease of use. While in yeast and plant systems, methods for the rapid, efficient and hierarchical assembly of genetic circuits from a large library of well described modular parts [8][9][10][11] exist, we are unaware that such a cloning toolkit for mammalian systems is readily available.In this work, we adapted the modular cloning strategy (MoClo) 12 strategy to build mammalian expression vectors. The Mammalian ToolKit (MTK) is a library of over 300 parts including vetted promoters, 3'UTRs, fluorescent proteins, insulator and P2A elements that can be combined to build large genetic circuits in a rapid and efficient fashion. These vectors can be delivered to a wide range of cell types, through viral, recombinase and CRISPR/Cas9 methods.As a proof of concept, we built a hAAVS1 landing p...

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“…where p(n) and κ x (n) are the steady state expression of p and the dissociation constant between ribosomes and protein p's mRNA transcript in the presence of n uORFs, respectively. Since we have derived from equation (6) that (i) Y max and Z 50 are both proportional to and hence proportional to κ x and that (ii) κ x (n) = (relative κ x )(n) × κ x (0) according to (7), our model predicts that Y max = Y max (n) and Z 50 = Z 50 (n) are both proportional to relative κ x .…”

Section: Primersmentioning

confidence: 96%

An endoribonuclease-based feedforward controller for decoupling resource-limited genetic modules in mammalian cells

Qian

Siciliano

DiAndreth

et al. 2019

Preprint

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Synthetic biology has the potential to bring forth advanced genetic devices for applications in healthcare and biotechnology. However, accurately predicting the behavior of engineered genetic devices remains difficult due to lack of modularity, wherein a device's output does not depend only on its intended inputs but also on its context. One contributor to lack of modularity is competition among genes for shared cellular resources, such as those required for transcription and translation, which can induce 'coupling' among otherwise independently-regulated genes. Here, we quantify the effects of resource sharing on engineered genetic systems in mammalian cells and develop an endoribonuclease-based incoherent feedforward loop (iFFL) to make gene expression levels robust to changes in resource availability. Our iFFL accurately controls gene expression levels in various cell lines and in the presence of significant resource sequestration by transcriptional activators. In addition to mitigating resource sharing, our iFFL also adapts gene expression to multiple log decades of DNA copy number variation, substantially improving upon previously-described miRNA-based iFFLs. Ultimately, our iFFL device will enable predictable, robust, and context-independent control of gene expression in mammalian cells.A promising strategy for engineering complex genetic devices is to compose together simpler systems that have been characterized in isolation 1-3 . A critical assumption of this modular design approach is that subsystems maintain their input/output (i/o) behavior when assembled into larger systems. However, this assumption often fails due to context dependence, i.e., the behavior of a module depends on the surrounding systems 2,4 . There are many sources of context-dependence, including unexpected off-target interactions between regulators and promoters 5 , transcription factor (TF) loading by DNA targets 6 , gene orientation 7 , and resource loading by expressed genes 8,9 . To date, much effort has gone into identifying and engineering gene regulators with unique binding specificity, e.g. between TFs and their DNA binding sites, with the goal of finding gene regulators that work orthogonally 5 . Nevertheless, even if subsystems are entirely composed of putatively orthogonal regulators, their gene expression levels can still become coupled to each other via competition for shared cellular resources 2,8-11 . For example, it has been demonstrated in prokaryotes that genes compete for the usage of ribosomes, such that increased expression from one gene decreases expression from others by sequestering (i.e. loading) ribosomes 8,9 . Little work has been done to understand how sharing of gene expression resources among genes affects engineered genetic devices in eukaryotic cells.Furthermore, while solutions to the ribosome sharing problem in bacterial cells have appeared recently 12-14 , solutions to resource sharing in mammalian cells are still lacking. Because the prokaryotic devices are either prokaryote-specific or only partia...

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Biophysical Constraints Arising from Compositional Context in Synthetic Gene Networks

Cited by 125 publications

References 46 publications

Multistable and dynamic CRISPRi-based synthetic circuits

Multistable and dynamic CRISPRi-based synthetic circuits

A Toolkit for Rapid Modular Construction of Biological Circuits in Mammalian Cells

An endoribonuclease-based feedforward controller for decoupling resource-limited genetic modules in mammalian cells

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