CRISPR-Cas systems offer versatile technologies for genome engineering, yet their implementation has been outpaced by ongoing discoveries of new Cas nucleases and anti-CRISPR proteins. Here, we ...present the use of E. coli cell-free transcription-translation (TXTL) systems to vastly improve the speed and scalability of CRISPR characterization and validation. TXTL can express active CRISPR machinery from added plasmids and linear DNA, and TXTL can output quantitative dynamics of DNA cleavage and gene repression—all without protein purification or live cells. We used TXTL to measure the dynamics of DNA cleavage and gene repression for single- and multi-effector CRISPR nucleases, predict gene repression strength in E. coli, determine the specificities of 24 diverse anti-CRISPR proteins, and develop a fast and scalable screen for protospacer-adjacent motifs that was successfully applied to five uncharacterized Cpf1 nucleases. These examples underscore how TXTL can facilitate the characterization and application of CRISPR technologies across their many uses.
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•Active expression of multiple CRISPR nucleases and gRNAs in an E. coli TXTL system•Repression activity of dCas9 strongly correlated between TXTL and E. coli•A TXTL-based PAM assay allowed characterization of five Cpf1 nucleases•Determined specificities of 24 anti-CRISPR proteins against five Cas9 nucleases
Marshall et al. demonstrate that an E. coli cell-free transcription-translation (TXTL) system can be used to improve the speed and scalability of characterizing CRISPR nucleases and their accessory factors. The method will facilitate the discovery of uncharacterized CRISPR nucleases and anti-CRISPR proteins and aid the validation of designed gRNAs.
The role of phenotypic plasticity in the evolution of new traits is controversial due to a lack of direct evidence. Phage host range becomes plastic in the presence of restriction-modification (R-M) ...systems in their hosts. I modeled the evolution of phage host range in the presence of R-M systems. The model makes two main predictions. The first prediction is that the offspring of the first phage to gain a new methylation pattern by infecting a new host make up a disproportionate fraction of the subsequent specialist population, indicating that the plastically produced phenotype is highly predictive of evolutionary outcome. The second prediction is that the first phage to gain this pattern is not always genetically distinct from other phages in the population. Taken together, these results suggest that plasticity could play a causal role on par with mutation during the evolution of phage host range. This uniquely tractable system could enable the first direct test of "plasticity first" evolution.
•We detail a rapid method of elucidating PAMs using a cell-free TXTL system.•Cas:gRNA RNPs are produced, assembled, and assayed in a single reaction.•PAMs are recognized by their depletion from a ...randomized library.•The method is demonstrated using the Neisseria meningitidis Cas9.
The RNA-guided nucleases derived from the CRISPR-Cas systems in bacteria and archaea have found numerous applications in biotechnology, including genome editing, imaging, and gene regulation. However, the discovery of novel Cas nucleases has outpaced their characterization and subsequent exploitation. A key step in characterizing Cas nucleases is determining which protospacer-adjacent motif (PAM) sequences they recognize. Here, we report advances to an in vitro method based on an E. coli cell-free transcription-translation system (TXTL) to rapidly elucidate PAMs recognized by Cas nucleases. The method obviates the need for cloning Cas nucleases or gRNAs, does not require the purification of protein or RNA, and can be performed in less than a day. To advance our previously published method, we incorporated an internal GFP cleavage control to assess the extent of library cleavage as well as Sanger sequencing of the cleaved library to assess PAM depletion prior to next-generation sequencing. We also detail the methods needed to construct all relevant DNA constructs, and how to troubleshoot the assay. We finally demonstrate the technique by determining PAM sequences recognized by the Neisseria meningitidis Cas9, revealing subtle sequence requirements of this highly specific PAM. The overall method offers a rapid means to identify PAMs recognized by diverse CRISPR nucleases, with the potential to greatly accelerate our ability to characterize and harness novel CRISPR nucleases across their many uses.
To build complex genetic networks with predictable behaviors, synthetic biologists use libraries of modular parts that can be characterized in isolation and assembled together to create programmable ...higher‐order functions. Characterization experiments and computational models for gene regulatory parts operating in isolation are routinely used to predict the dynamics of interconnected parts and guide the construction of new synthetic devices. Here, we individually characterize two modes of RNA‐based transcriptional regulation, using small transcription activating RNAs (STARs) and clustered regularly interspaced short palindromic repeats interference (CRISPRi), and show how their distinct regulatory timescales can be used to engineer a composed feedforward loop that creates a pulse of gene expression. We use a cell‐free transcription‐translation system (TXTL) to rapidly characterize the system, and we apply Bayesian inference to extract kinetic parameters for an ordinary differential equation‐based mechanistic model. We then demonstrate in simulation and verify with TXTL experiments that the simultaneous regulation of a single gene target with STARs and CRISPRi leads to a pulse of gene expression. Our results suggest the modularity of the two regulators in an integrated genetic circuit, and we anticipate that construction and modeling frameworks that can leverage this modularity will become increasingly important as synthetic circuits increase in complexity.
Modular genetic regulators are vital for the construction of predictable synthetic networks. In this study, two RNA‐based genetic regulators were individually characterized and parameterized revealing a timescale mismatch. Simulations and experiments demonstrated these regulators could modularly combine to create a pulse generator network in a cell‐free transcription‐translation system.
Fluctuations in nutrient availability profoundly impact gene expression. Previous work revealed postrecruitment regulation of RNA polymerase II (Pol II) during starvation and recovery in ...Caenorhabditis elegans, suggesting that promoter-proximal pausing promotes rapid response to feeding. To test this hypothesis, we measured Pol II elongation genome wide by two complementary approaches and analyzed elongation in conjunction with Pol II binding and expression. We confirmed bona fide pausing during starvation and also discovered Pol II docking. Pausing occurs at active stress-response genes that become downregulated in response to feeding. In contrast, “docked” Pol II accumulates without initiating upstream of inactive growth genes that become rapidly upregulated upon feeding. Beyond differences in function and expression, these two sets of genes have different core promoter motifs, suggesting alternative transcriptional machinery. Our work suggests that growth and stress genes are both regulated postrecruitment during starvation but at initiation and elongation, respectively, coordinating gene expression with nutrient availability.
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•Pol II pausing and backtracking occur during starvation in C. elegans•Pol II also binds upstream of TSSs without initiating transcription•Docking and pausing occur at growth and stress genes, respectively•Docked genes are TATA-less and are upregulated in response to feeding
Baugh and colleagues show that RNA polymerase II pauses and backtracks during early elongation in C. elegans. A key factor that promotes pausing in other animals is lacking in the worm, implying variation in regulation of early elongation. They also made the surprising discovery that the polymerase is recruited to the DNA upstream of many growth genes during starvation without initiation. The authors propose that postrecruitment control of initiation coordinates growth with nutrient availability.
Nutrient availability has profound influence on development. In the nematode C. elegans, nutrient availability governs post-embryonic development. L1-stage larvae remain in a state of developmental ...arrest after hatching until they feed. This "L1 arrest" (or "L1 diapause") is associated with increased stress resistance, supporting starvation survival. Loss of the transcription factor daf-16/FOXO, an effector of insulin/IGF signaling, results in arrest-defective and starvation-sensitive phenotypes. We show that daf-16/FOXO regulates L1 arrest cell-nonautonomously, suggesting that insulin/IGF signaling regulates at least one additional signaling pathway. We used mRNA-seq to identify candidate signaling molecules affected by daf-16/FOXO during L1 arrest. dbl-1/TGF-β, a ligand for the Sma/Mab pathway, daf-12/NHR and daf-36/oxygenase, an upstream component of the daf-12 steroid hormone signaling pathway, were up-regulated during L1 arrest in a daf-16/FOXO mutant. Using genetic epistasis analysis, we show that dbl-1/TGF-β and daf-12/NHR steroid hormone signaling pathways are required for the daf-16/FOXO arrest-defective phenotype, suggesting that daf-16/FOXO represses dbl-1/TGF-β, daf-12/NHR and daf-36/oxygenase. The dbl-1/TGF-β and daf-12/NHR pathways have not previously been shown to affect L1 development, but we found that disruption of these pathways delayed L1 development in fed larvae, consistent with these pathways promoting development in starved daf-16/FOXO mutants. Though the dbl-1/TGF-β and daf-12/NHR pathways are epistatic to daf-16/FOXO for the arrest-defective phenotype, disruption of these pathways does not suppress starvation sensitivity of daf-16/FOXO mutants. This observation uncouples starvation survival from developmental arrest, indicating that DAF-16/FOXO targets distinct effectors for each phenotype and revealing that inappropriate development during starvation does not cause the early demise of daf-16/FOXO mutants. Overall, this study shows that daf-16/FOXO promotes developmental arrest cell-nonautonomously by repressing pathways that promote larval development.
When sensing is gambling Maxwell, Colin S.; Magwene, Paul M.
Evolution,
April 2017, Letnik:
71, Številka:
4
Journal Article
Recenzirano
Odprti dostop
Genotypes can persist in unpredictable environments by “hedging their bets” and producing diverse phenotypes. Theoretical studies have shown that the phenotypic variability needed for a bet-hedging ...strategy can be generated by factors either inside or outside an organism. However, sensing the environment and bet hedging are frequently treated as distinct evolutionary strategies. Furthermore, nearly all empirical studies of the molecular underpinnings of bet-hedging strategies to date have focused on internal sources of variability. We took a synthetic approach and constructed an experimental system where a phenotypic trade-off is mediated by actively sensing a cue present in the environment. We show that active sensing can generate a diversified bethedging strategy. Mutations affecting the norm of reaction to the cue alter the diversification strategy, indicating that bet hedging by active sensing is evolvable. Our results indicate that a broader class of biological systems should be considered as potential examples of bet-hedging strategies, and that research into the structure of environmental variability is needed to distinguish bet-hedging strategies from adaptive plasticity.
The
insulin-like signaling network supports homeostasis and developmental plasticity. The genome encodes 40 insulin-like peptides and one known receptor. Feedback regulation has been reported, but ...the extent of feedback and its effect on signaling dynamics in response to changes in nutrient availability has not been determined. We measured messenger RNA expression for each insulin-like peptide, the receptor
, components of the PI3K pathway, and its transcriptional effectors
/FoxO and
/Nrf at high temporal resolution during transition from a starved, quiescent state to a fed, growing state in wild type and mutants affecting
/InsR and
/FoxO. We also analyzed the effect of temperature on insulin-like gene expression. We found that most PI3K pathway components and insulin-like peptides are affected by signaling activity, revealing pervasive positive and negative feedback regulation at intra- and intercellular levels. Reporter gene analysis demonstrated that the
/InsR agonist
positively regulates its own transcription and that the putative agonist
cross-regulates DAF-28 protein expression through feedback. Our results show that positive and negative feedback regulation of insulin-like signaling is widespread, giving rise to an organismal FoxO-to-FoxO signaling network that supports homeostasis during fluctuations in nutrient availability.
The roundworm C. elegans reversibly arrests larval development during starvation 1, but extended early-life starvation reduces reproductive success 2, 3. Maternal dietary restriction (DR) buffers ...progeny from starvation as young larvae, preserving reproductive success 4. However, the developmental basis of reduced fertility following early-life starvation is unknown, and it is unclear how maternal diet modifies developmental physiology in progeny. We show here that extended starvation in first-stage (L1) larvae followed by unrestricted feeding results in a variety of developmental abnormalities in the reproductive system, including proliferative germ-cell tumors and uterine masses that express neuronal and epidermal cell fate markers. We found that maternal DR and reduced maternal insulin/insulin-like growth factor (IGF) signaling (IIS) increase oocyte provisioning of vitellogenin lipoprotein, reducing penetrance of starvation-induced abnormalities in progeny, including tumors. Furthermore, we show that maternal DR and reduced maternal IIS reduce IIS in progeny. daf-16/FoxO and skn-1/Nrf, transcriptional effectors of IIS, are required in progeny for maternal DR and increased vitellogenin provisioning to suppress starvation-induced abnormalities. daf-16/FoxO activity in somatic tissues is sufficient to suppress starvation-induced abnormalities, suggesting cell-nonautonomous regulation of reproductive system development. This work reveals that early-life starvation compromises reproductive development and that vitellogenin-mediated intergenerational insulin/IGF-to-insulin/IGF signaling mediates adaptation to nutrient availability.
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•Early-life starvation and unrestricted feeding result in reproductive abnormalities•Maternal dietary restriction protects progeny from starvation-induced abnormalities•Maternal diet and insulin/IGF signaling affect vitellogenin oocyte provisioning•Vitellogenin oocyte provisioning affects progeny insulin/IGF signaling
Jordan et al. show that early-life starvation causes reproductive abnormalities in C. elegans but that maternal dietary restriction increases progeny starvation resistance. Dietary restriction and reduced insulin/IGF signaling increase maternal vitellogenin provisioning, which activates daf-16/FoxO in progeny to confer protection from starvation.
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