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| NOR gate-based logic circuits. (a–f) Six different two-input logic circuits constructed by interconnecting NOR gates. For each of the four input possibilities ( ÀÀ , À þ , þ À , and þ þ ), a distinct strain was constructed with the corresponding inputs expressed off of constitutive promoters (for logical þ ), or not integrated at all (for logical À ). Fluorescence values were collected using flow cytometry of cells growing in log phase. The histograms represent population fraction from three different biological replicates measured during a single experiment and were normalized so that area sums to unity. Fluorescence population ratios of the circuits are included in the Supplementary Table 3.
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Natural genetic circuits enable cells to make sophisticated digital decisions. Building equally complex synthetic circuits in eukaryotes remains difficult, however, because commonly used components leak transcriptionally, do not arbitrarily interconnect or do not have digital responses. Here, we designed dCas9-Mxi1-based NOR gates in Saccharomyces...
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... that most circuits in Figs 3 and 4 required that only a handful of gate combinations be screened to identify a functional design, and others required only one. Table 1 compares our technology with selected published circuits. We measured circuit complexity with a combination of two metrics: the number of gates and the number of connections among gates, allowing us to locate circuits in a two-dimensional plot ( Supplementary Fig. 14). We can calculate a complexity score using the two metrics, complexity ¼ (gates 2 þ connections 2 ) 1/2 . For example, the XOR gate had five gates and four connections, producing a complexity of (5 2 þ 4 2 ) 1/2 ¼ 6.4, while the cascade has a complexity of (7 2 þ 1 2 ) 1/2 ¼ 9.2. These complexities compare well with gene circuits developed in Escherichia coli, for example. Our NOR gates enabled extremely simple design and construction of large gene circuits. Before genetic circuits can be made much larger, however, many factors that influence the size and complexity of synthetic genetic circuits must be ...
Citations
... An alternative to the use of recombinases and TFs for creating synthetic circuits is CRISPR (clustered regularly interspaced short palindromic repeats) interference (CRISPRi), which uses a nuclease-dead Cas9 (dCas9) protein to inhibit target promoter activity 29 . Although circuits based on CRISPRi are highly programmable [30][31][32][33][34][35] , a variety of factors can affect their activity. Circuit kinetics is influenced by the slower target search speed of dCas9, which can be ameliorated by its intracellular concentration 36,37 . ...
... One potential strategy to modulate this involves the fusion of repression domains to dCas9, which can lead to increased repression efficiency. The amount of repression is a crucial factor in constructing multilayered circuits, as insufficient repression can cause transcriptional leakage, which may impair circuit performance 30 . While CRISPRi has been demonstrated to work for transcriptional regulation in plants [39][40][41][42][43] , to date, it has not been leveraged to construct synthetic gene circuits. ...
... As TFs and the RNA polymerase II holoenzyme complex generally bind to the promoter region surrounding the TATA box and transcriptional start site (TSS), this region should provide an effective target for achieving maximum repression with CRISPRi, as previously demonstrated in other species [30][31][32] . Thus, we wanted to confirm whether all four copies of both sgRNA-A-binding and sgRNA-B-binding sites in the version B integrator were required for effective CRISPRi. ...
The construction of synthetic gene circuits in plants has been limited by a lack of orthogonal and modular parts. Here, we implement a CRISPR (clustered regularly interspaced short palindromic repeats) interference (CRISPRi)-based reversible gene circuit platform in plants. We create a toolkit of engineered repressible promoters of different strengths and construct NOT and NOR gates in Arabidopsis thaliana protoplasts. We determine the optimal processing system to express single guide RNAs from RNA Pol II promoters to introduce NOR gate programmability for interfacing with host regulatory sequences. The performance of a NOR gate in stably transformed Arabidopsis plants demonstrates the system’s programmability and reversibility in a complex multicellular organism. Furthermore, cross-species activity of CRISPRi-based logic gates is shown in Physcomitrium patens, Triticum aestivum and Brassica napus protoplasts. Layering multiple NOR gates together creates OR, NIMPLY and AND logic functions, highlighting the modularity of our system. Our CRISPRi circuits are orthogonal, compact, reversible, programmable and modular and provide a platform for sophisticated spatiotemporal control of gene expression in plants.
... However, examples of engineered genetic circuits capable of multiplexing stimuli and regulating multiple genes are limited. This paucity can be attributed to the limited number of suitable components for implementing scalable circuitry (1,(233)(234)(235) and to the difficulty of sequentially combining components into larger, high-level circuits (236)(237)(238)(239). Due to the ease of designing and tuning new components that can be interconnected, CRISPR-based tools have rapidly become a suitable framework for building complex genetic circuits. ...
... That is, the output transcription levels of upstream nodes must be matched to the relevant transcriptional input range of downstream nodes (Figure 4c). Compared to other circuitry systems, the CRISPRa/i framework facilitates the circuit design and level-matching process because many orthogonal and high-performing gRNAs and promoters can be readily constructed (208), and it has already been shown to enable deep and wide circuits with up to ten nodes of regulation (48,118,237) (Figure 4f ). Several factors must be considered when assembling multi-gRNA circuits for CFES or bacteria. ...
In the past decades, the broad selection of CRISPR-Cas systems has revolutionized biotechnology by enabling multimodal genetic manipulation in diverse organisms. Rooted in a molecular engineering perspective, we recapitulate the different CRISPR components and how they can be designed for specific genetic engineering applications. We first introduce the repertoire of Cas proteins and tethered effectors used to program new biological functions through gene editing and gene regulation. We review current guide RNA (gRNA) design strategies and computational tools and how CRISPR-based genetic circuits can be constructed through regulated gRNA expression. Then, we present recent advances in CRISPR-based biosensing, bioproduction, and biotherapeutics across in vitro and in vivo prokaryotic systems. Finally, we discuss forthcoming applications in prokaryotic CRISPR technology that will transform synthetic biology principles in the near future.
... Different host organisms are used for this purpose, often chosen based on the specific application. For example, genetic circuits are now routinely engineered in bacteria [8], yeast [9], plants [10], or mammalian cells [11]. ...
Genetic circuits confer computing abilities to living cells, performing novel transformations of input stimuli into output responses. These genetic circuits are routinely engineered for insertion into bacterial plasmids and chromosomes, using a design paradigm whose only spatial consideration is a linear ordering of the individual components. However, chromosomal DNA has a complex three dimensional conformation which alters the mechanics of gene expression, leading to dynamics that are specific to chromosomal location. Here we demonstrate that because of this, position in the bacterial chromosome is crucial to the function of synthetic genetic circuits, and that three dimensional space should not be overlooked in their design. Our results show that genetically identical circuits can be reprogrammed to produce different outputs by changing their spatial positioning and configuration. We engineer 221 spatially unique genetic circuits of four different types, three regulatory cascades and a toggle switch, by either inserting the entire circuit in a specific chromosomal position or separating and distributing circuit modules. Their analysis reveals that spatial positioning can be used not only to optimize circuits but also to switch circuits between modes of operation, giving rise to new functions. Alongside a comprehensive characterization of chromosomal space using single-cell RNA-seq profiles and Hi-C interaction maps, we offer baseline information for leveraging intracellular space as a design parameter in bioengineering.
... Synthetic biologists have developed separate technologies to emulate decision-making [1][2][3][4][5][6][7][8][9] , intercellular communication [10][11][12][13][14][15][16] , and the equivalent of memory [17][18][19][20][21][22][23][24][25] in myriad chassis cells. We posited that all three properties can be unified in a single chassis cell to form an intelligent synthetic biological system (see Fig. 1, Supplementary Note 1). ...
Synthetic biologists seek to engineer intelligent living systems capable of decision-making, communication, and memory. Separate technologies exist for each tenet of intelligence; however, the unification of all three properties in a living system has not been achieved. Here, we engineer completely intelligent Escherichia coli strains that harbor six orthogonal and inducible genome-integrated recombinases, forming Molecularly Encoded Memory via an Orthogonal Recombinase arraY (MEMORY). MEMORY chassis cells facilitate intelligence via the discrete multi-input regulation of recombinase functions enabling inheritable DNA inversions, deletions, and genomic insertions. MEMORY cells can achieve programmable and permanent gain (or loss) of functions extrachromosomally or from a specific genomic locus, without the loss or modification of the MEMORY platform – enabling the sequential programming and reprogramming of DNA circuits within the cell. We demonstrate all three tenets of intelligence via a probiotic (Nissle 1917) MEMORY strain capable of information exchange with the gastrointestinal commensal Bacteroides thetaiotaomicron .
... Another benefit of using dCas9 is that it can direct its regulatory effect to a promoter instead of requiring the insertion of an operator sequence to control a native gene (16)(17)(18). It has been reported that targeting with multiple gRNAs in overlapping or different positions of a single promoter results in mutually exclusive binding that either recruits or blocks RNAP (13, [19][20][21][22]. Although there are numerous in cellulo and in vitro examples of transcription regulation enacted by targeting one or more sites with dCas9-gRNA, to our knowledge there are no examples of mechanistic details how dCas9 behaves in the context of DNA secondary structures, such as the G-quadruplex (GQ). ...
Putative G-quadruplex forming sequences (PQS) have been identified in promoter sequences of prominent genes that are implicated among others in cancer and neurological disorders. We explored mechanistic aspects of CRISPR-dCas9-mediated gene expression regulation, which is transient and sequence specific unlike alternative approaches that lack such specificity or create permanent mutations, using the PQS in tyrosine hydroxylase (TH) and c-Myc promoters as model systems. We performed in vitro ensemble and single molecule investigations to study whether G-quadruplex (GQ) structures or dCas9 impede T7 RNA polymerase (RNAP) elongation process and whether orientation of these factors is significant. Our results demonstrate that dCas9 is more likely to block RNAP progression when the non-template strand is targeted. While the GQ in TH promoter was effectively destabilized when the dCas9 target site partially overlapped with the PQS, the c-Myc GQ remained folded and stalled RNAP elongation. We also determined that a minimum separation between the transcription start site and the dCas9 target site is required for effective stalling of RNAP by dCas9. Our study provides significant insights about the factors that impact dCas9-mediated transcription regulation when dCas9 targets the vicinity of sequences that form secondary structures and provides practical guidelines for designing guide RNA sequences.
... Another example is CRISPR-based logic circuits. The CRISPRibased NOT and NOR gates have been interconnected into two-input logic gates, among which the AND and NAND gates were built from six sgRNAs 109 . However, these gates can be constructed using fewer sgRNAs by incorporating crRNA-tracrRNA interaction with CRISPRa 110 or protein-splicing with CRISPRi systems 69 . ...
As synthetic biology permeates society, the signal processing circuits in engineered living systems must be customized to meet practical demands. Towards this mission, novel regulatory mechanisms and genetic circuits with unprecedented complexity have been implemented over the past decade. These regulatory mechanisms, such as transcription and translation control, could be integrated into hybrid circuits termed “multi-level circuits”. The multi-level circuit design will tremendously benefit the current genetic circuit design paradigm, from modifying basic circuit dynamics to facilitating real-world applications, unleashing our capabilities to customize cellular signal processing and address global challenges through synthetic biology.
... Synthetic biology's field of biosensors, or genetically encoded sensors, has the potential to revolutionize agriculture (Williams et al., 2017). To produce elicit results, a biosensor can be coupled with a variety of genetically encoded components, such as new receptors, deactivated Cas9 and derivatives (capable of binding but not cleaving DNA), and transcription factors (Gander et al., 2017;Kim et al., 2018). For instance, plants can be designed to stimuli to various environmental pollutants, nutrients, and abiotic stress factors (Pouvreau et al., 2018). ...
Synthetic biology (SynBio), an emerging interdisciplinary scientific field, is known for designing and building new artificial biological pathways, creatures, or devices, as well as reworking existing natural biological systems. It is therefore unsurpassed as “an umbrella phrase… that encompasses activity spanning from the basic biology to creative technologies, instead of being a new conceptual framework” (Li et al., 2021). SynBio became popular in the late 2000s when DNA sequencing and synthesis became affordable and fast enough which revolutionized molecular sciences. The U.S. Department of Defense recognized synthetic biology as one of the top six disruptive technologies for development in the 21st century (U.S. Department of Defense, 2014). The development of advanced technologies for reading and writing DNA has resulted in groundbreaking progress in the design, assembly, manipulation of genes, materials, circuits, and metabolic pathways. These progressions have enabled scientists to manipulate biological systems and organisms to a greater extent than ever before. Examples of this include the production of compounds like leghemoglobin, sitagliptin, and diamines by engineered cells, which were awarded the 2018 Nobel Prize. Meanwhile, the 2020 Nobel Prize was awarded for the engineering of modified cells themselves, such as bacteria, CAR-Ts, and genome-edited soy (Cumbers, 2020; Voigt, 2020). Recent developments in information technology (bioinformatics and design tools) and biotechnology (genome sequencing, genome editing, gene synthesis, and biofoundries) concomitantly artificial intelligence (machine learning) accelerated the discovery and optimization of metabolic pathways through the Design-Build-Test-Learn cycle.
... Previous literature has stated the advantages of employing programmability of CRISPR-based devices. For instance, programmable devices with a unified mechanism can simplify the engineering process and improve the predictability of their functions; the programmability also supports highly orthogonal biological functions to enhance the robustness of devices and support multiplexed and scalable engineering [39,[49][50][51][52][53][54]. ...
As the most valuable feature of the CRISPR system, the programmability based on Watson–Crick base pairing has been widely exploited in engineering RNA sensors. The base pairing in these systems offers a connection between the RNA of interest and the CRISPR effector, providing a highly specific mechanism for RNA detection both in vivo and in vitro. In the last decade, despite the many successful RNA sensing approaches developed during the era of CRISPR explosion, a deeper understanding of the characteristics of CRISPR systems and the continuous expansion of the CRISPR family members indicates that the CRISPR-based RNA sensor remains a promising area from which a variety of new functions and applications can be engineered. Here, we present a systematic overview of the various strategies of engineering CRISPR gRNA for programmable RNA detection with an aim to clarify the role of gRNA's programmability among the present limitations and future development of CRISPR-enabled RNA sensors.
... The field first gained attention through early biological implementations of two simple genetic circuits; the genetic toggle switch, which is capable of flipping between two stable states (Gardner et al, 2000); and the repressilator, a negative feedback loop composed of three repressor genes capable of oscillations (Elowitz and Leibler, 2000). Since then, synthetic biology has been used to design more complex systems such as biosensors and cell computers (Shaw et al, 2019;Gander et al, 2017). To scale up from simple to complex systems, synthetic biology employs a modular approach from engineering. ...
... Thus, standard modules (genetic constructs that have been designed according to specifications) can be re-used as part of a more complex system. With this approach, even relatively simple genetic circuits can be assembled into biological logic gates, and ultimately biological devices capable of making complex decisions (Gander et al, 2017). ...
Synthetic biology aims to engineer novel functionalities into biological systems. While the approach has been widely applied to single cells, the scale of synthetic circuits designed in this way is limited by factors such as resource competition and retroactivity. Synthetic biology of cell populations has the potential to overcome some of these limitations by physically isolating synthetic genes from each other. To rationally design cell populations, we require mathematical models that link between intracellular biochemistry and intercellular interactions. The interfacing of agent-based models with systems biology models is particularly important in understanding the effects of cell heterogeneity and cell-to-cell interactions.
In this study, we develop a model of gene expression that is suitable for incorporation into agent-based models of cell populations. To be scalable to large cell populations, models of gene expression should be both computationally efficient and compliant with the laws of physics. We satisfy the first requirement by applying a model reduction scheme to translation, and the second requirement by formulating models using bond graphs. We show that the reduced model of translation faithfully reproduces the behaviour of the full model at steady state. The reduced model is benchmarked against the full model, and we find a substantial speedup at realistic protein lengths. Using the modularity of the bond graph approach, we couple separate models of gene expression to build models of the toggle switch and repressilator. With these models, we explore the effects of resource availability and cell-to-cell heterogeneity on circuit behaviour. The modelling approaches developed in this study are a bridge towards rationally designing collective cell behaviours such as synchronisation and division of labour.
... Synthetic biology approaches to create Boolean logic gates of genes have shown high flexibility of genetic networks in prokaryotes and the practical use of such systems as biosensors [4,5]. Similar approaches have also emerged for eukaryotic organisms by utilizing CRISPR/Cas9 or inducible gene regulation [6,7]. ...
Inducible gene regulation methods are indispensable in diverse biological applications, yet many of them have severe limitations in their applicability. These include inducer toxicity, a limited variety of organisms the given system can be used in, and side effects of the induction method. In this study, a novel inducible system, the RuX system, was created using a mutant ligand-binding domain of the glucocorticoid receptor (CS1/CD), used together with various genetic elements such as the Gal4 DNA-binding domain or Cre recombinase. The RuX system is shown to be capable of over 1000-fold inducibility, has flexible applications, and is offered for use in cell cultures.
