Eukaryotic transcription factors (TFs) perform complex and combinatorial functions within transcriptional networks. Here, we present a synthetic framework for systematically constructing eukaryotic ...transcription functions using artificial zinc fingers, modular DNA-binding domains found within many eukaryotic TFs. Utilizing this platform, we construct a library of orthogonal synthetic transcription factors (sTFs) and use these to wire synthetic transcriptional circuits in yeast. We engineer complex functions, such as tunable output strength and transcriptional cooperativity, by rationally adjusting a decomposed set of key component properties, e.g., DNA specificity, affinity, promoter design, protein-protein interactions. We show that subtle perturbations to these properties can transform an individual sTF between distinct roles (activator, cooperative factor, inhibitory factor) within a transcriptional complex, thus drastically altering the signal processing behavior of multi-input systems. This platform provides new genetic components for synthetic biology and enables bottom-up approaches to understanding the design principles of eukaryotic transcriptional complexes and networks.
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► Zinc fingers can be used to wire orthogonal connections in yeast synthetic circuits ► Zinc finger TF design permits adjustable component properties for modulating outputs ► Protein-protein interactions can be used to engineer cooperativity in zinc finger TFs ► TF component properties can be combinatorially adjusted to reshape signal integration
A library of synthetic eukaryotic transcription factors constructed from modifiable component properties of artificial zinc fingers is capable of modulating outputs and reshaping signal integration.
CRISPR loci are a cluster of repeats separated by short “spacer” sequences derived from prokaryotic viruses and plasmids that determine the targets of the host’s CRISPR-Cas immune response against ...its invaders. For type I and II CRISPR-Cas systems, single-nucleotide mutations in the seed or protospacer adjacent motif (PAM) of the target sequence cause immune failure and allow viral escape. This is overcome by the acquisition of multiple spacers that target the same invader. Here we show that targeting by the Staphylococcus epidermidis type III-A CRISPR-Cas system does not require PAM or seed sequences, and thus prevents viral escape via single-nucleotide substitutions. Instead, viral escapers can only arise through complete target deletion. Our work shows that, as opposed to type I and II systems, the relaxed specificity of type III CRISPR-Cas targeting provides robust immune responses that can lead to viral extinction with a single spacer targeting an essential phage sequence.
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•Type III CRISPR-Cas immunity does not require PAM or seed sequence motifs•Escape from type III immunity requires complete deletion of the target sequence•Type III targeting of an essential phage gene leads to phage extinction•The targeting flexibility of type III systems provides a robust immune response
Exploring the target specificity of type III-A CRISPR-Cas systems, Pyenson et al. find that most point mutations in the target region still allow robust immunity. As a consequence, viral escape from the type III-A CRISPR-Cas immune response requires the full deletion of the target, which is a very rare event.
Type II CRISPR-Cas systems provide immunity against phages and plasmids that infect bacteria through the insertion of a short sequence from the invader's genome, known as the 'spacer', into the ...CRISPR locus. Spacers are transcribed into guide RNAs that direct the Cas9 nuclease to its target on the invader. In liquid cultures, most bacteria acquire a single spacer. Multiple spacer integration is a rare event which significance for immunity is poorly understood. Here, we found that when phage infections occur on solid media, a high proportion of the surviving colonies display complex morphologies that contain cells with multiple spacers. This is the result of the viral-host co-evolution, in which the immunity provided by the initial acquired spacer is easily overcome by escaper phages. Our results reveal the versatility of CRISPR-Cas immunity, which can respond with both single or multiple spacer acquisition schemes to solve challenges presented by different environments.
Immune systems must recognize and destroy different pathogens that threaten the host. CRISPR-Cas immune systems protect prokaryotes from viral and plasmid infection utilizing small CRISPR RNAs that ...are complementary to the invader’s genome and specify the targets of RNA-guided Cas nucleases. Type III CRISPR-Cas immunity requires target transcription, and whereas genetic studies demonstrated DNA targeting, in vitro data have shown crRNA-guided RNA cleavage. The molecular mechanism behind these disparate activities is not known. Here, we show that transcription across the targets of the Staphylococcus epidermidis type III-A CRISPR-Cas system results in the cleavage of the target DNA and its transcripts, mediated by independent active sites within the Cas10-Csm ribonucleoprotein effector complex. Immunity against plasmids and DNA viruses requires DNA, but not RNA, cleavage activity. Our studies reveal a highly versatile mechanism of CRISPR immunity that can defend microorganisms against diverse DNA and RNA invaders.
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•Type III CRISPR-Cas systems provide immunity when the target DNA is transcribed•The type III Cas10-Csm complex performs co-transcriptional RNA-guided DNA cleavage•The complex is also capable of RNA-guided RNA cleavage•Both the target DNA and its transcript are subject to RNA-guided cleavage in vivo
The type III-A CRISPR-Cas system is capable of RNA-guided DNA and RNA cleavage in vivo, revealing a highly versatile mechanism of CRISPR immunity that protects microorganisms against diverse DNA and RNA invaders.
•Type III systems were the first to provide key aspects of the molecular mechanisms underlying CRISPR-Cas immunity.•Immunity only occurs if the target is transcribed.•The underlying molecular ...mechanism requires co-transcriptional cleavage of the target DNA and its transcripts.•Mobile genetic elements that silence their genomes are tolerated by Type III CRISPR-Cas immunity.
Type III CRISPR-Cas systems have a unique targeting mechanism that requires the transcription of the DNA target and results in the degradation of not only the genome of the invader but also its transcripts. Here we discuss the most recent studies describing dual DNA and RNA targeting by these systems, as well as the implications of this complex molecular mechanism for immunity in vivo.
Synthetic biology is increasingly used to develop sophisticated living devices for basic and applied research. Many of these genetic devices are engineered using multi-copy plasmids, but as the field ...progresses from proof-of-principle demonstrations to practical applications, it is important to develop single-copy synthetic modules that minimize consumption of cellular resources and can be stably maintained as genomic integrants. Here we use empirical design, mathematical modeling, and iterative construction and testing to build single-copy, bistable toggle switches with improved performance and reduced metabolic load that can be stably integrated into the host genome. Deterministic and stochastic models led us to focus on basal transcription to optimize circuit performance and helped to explain the resulting circuit robustness across a large range of component expression levels. The design parameters developed here provide important guidance for future efforts to convert functional multi-copy gene circuits into optimized single-copy circuits for practical, real-world use.
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•Reduced transcription leakage improves toggle robustness to parameter variation/noise•Mathematic models identify important design parameters that affect circuit stability•Empirical strategies for single-copy circuit construction are reported•Genome-integrated circuit places minimal metabolic burden on the host cell
Lee et al. (2016) use theoretical and empirical design and analysis to build a bistable, genome-integrated genetic toggle switch with reduced growth burden on the host cell. They demonstrate that reduced transcriptional leakage improves the robustness of the circuit to parameter variation and noise.
Organisms from every branch of life have evolved defenses against the viral parasites that infect them. For many bacteria and archaea, resistance to viruses is provided by Clustered Regularly ...Interspaced Short Palindromic Repeats (CRISPR). This system of proteins and RNAs provides sequence-specific targeting against foreign genetic elements like viruses. CRISPR loci are a cluster of repeats separated by short ‘‘spacer’’ sequences derived from prokaryotic viruses and plasmids that determine the targets of the host’s CRISPR-Cas immune response against its invaders. For type I and II CRISPR-Cas systems, single-nucleotide mutations in the seed or proto- spacer adjacent motif (PAM) of the target sequence cause immune failure and allow viral escape. This is overcome by the acquisition of multiple spacers that target the same invader. Here we show that targeting by the Staphylococcus epidermidis type III-A CRISPR-Cas system does not require PAM or seed sequences, and thus prevents viral escape via single-nucleotide substitutions. Instead, viral escapers can only arise through complete target deletion. Our work shows that, as opposed to type I and II systems, the relaxed specificity of type III CRISPR-Cas targeting provides robust immune responses that can lead to viral extinction with a single spacer targeting an essential phage sequence.Studies on the Type II system show that an infected population of bacteria will pick up a diverse spread of spacers from across the phage genome. In liquid culture, where cells constantly mix, a phage mutant can overcome the immunity of one spacer to kill the host, but upon infection of a neighboring cell they will encounter a different spacer and stop propagating. Whether and how this fail-safe mechanism works when the population is immobilized in solid media is not known. Here we show that when grown in top agar, a subset of cells within a single colony will acquire additional spacers that help combat resistant phage. This arms race between the host and virus changes the physical structure of the colony, as seen through segmentation of different sub-populations. Our work reveals how the dynamics of the CRISPR immune response critically depend on the ecology and interactions of the bacterial host.
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