The complex, interconnected architecture of cell-signaling networks makes it challenging to disentangle how cells process extracellular information to make decisions. We have developed an optogenetic ...approach to selectively activate isolated intracellular signaling nodes with light and use this method to follow the flow of information from the signaling protein Ras. By measuring dose and frequency responses in single cells, we characterize the precision, timing, and efficiency with which signals are transmitted from Ras to Erk. Moreover, we elucidate how a single pathway can specify distinct physiological outcomes: by combining distinct temporal patterns of stimulation with proteomic profiling, we identify signaling programs that differentially respond to Ras dynamics, including a paracrine circuit that activates STAT3 only after persistent (>1 hr) Ras activation. Optogenetic stimulation provides a powerful tool for analyzing the intrinsic transmission properties of pathway modules and identifying how they dynamically encode distinct outcomes.
Display omitted
•Optogenetic inputs can directly control the activity of the Ras/Erk module•Individual cells exhibit precise and reversible Ras/Erk dose-response behavior•Ras efficiently transmits signals to Erk with dynamics ranging from minutes to hours•An optogenetic/proteomic screen identifies dynamically gated Ras response modules
An engineered optogenetic control shows that the Ras/Erk pathway can efficiently transmit signals with timescales ranging from four minutes to several hours and identifies dynamically regulated downstream targets that differentially respond to transient versus sustained signaling.
Cells that secrete and sense the same signaling molecule are ubiquitous. To uncover the functional capabilities of the core "secrete-and-sense" circuit motif shared by these cells, we engineered ...yeast to secrete and sense the mating pheromone. Perturbing each circuit element revealed parameters that control the degree to which the cell communicated with itself versus with its neighbors. This tunable interplay of self-communication and neighbor communication enables cells to span a diverse repertoire of cellular behaviors. These include a cell being asocial by responding only to itself and social through quorum sensing, and an isogenic population of cells splitting into social and asocial subpopulations. A mathematical model explained these behaviors. The versatility of the secrete-and-sense circuit motif may explain its recurrence across species.
The genetic interrogation and reprogramming of cells requires methods for robust and precise targeting of genes for expression or repression. The CRISPR-associated catalytically inactive dCas9 ...protein offers a general platform for RNA-guided DNA targeting. Here, we show that fusion of dCas9 to effector domains with distinct regulatory functions enables stable and efficient transcriptional repression or activation in human and yeast cells, with the site of delivery determined solely by a coexpressed short guide (sg)RNA. Coupling of dCas9 to a transcriptional repressor domain can robustly silence expression of multiple endogenous genes. RNA-seq analysis indicates that CRISPR interference (CRISPRi)-mediated transcriptional repression is highly specific. Our results establish that the CRISPR system can be used as a modular and flexible DNA-binding platform for the recruitment of proteins to a target DNA sequence, revealing the potential of CRISPRi as a general tool for the precise regulation of gene expression in eukaryotic cells.
Display omitted
•CRISPRi enables robust gene repression and activation in human cells•CRISPRi knockdown is specific with minimal off-target effects in human cells•CRISPRi can effectively repress endogenous genes in human and yeast•dCas9 enables modular and programmable RNA-guided genome regulation in eukaryotes
Catalytically inactive CRISPR can be targeted to specific loci in human and yeast cells to specifically repress and activate transcription. The study demonstrates the potential for adapting CRISPRi for multiple modes of transcriptional control, chromatin modification, and regulatory element mapping in a broad range of eukaryotes.
Targeted gene regulation on a genome-wide scale is a powerful strategy for interrogating, perturbing, and engineering cellular systems. Here, we develop a method for controlling gene expression based ...on Cas9, an RNA-guided DNA endonuclease from a type II CRISPR system. We show that a catalytically dead Cas9 lacking endonuclease activity, when coexpressed with a guide RNA, generates a DNA recognition complex that can specifically interfere with transcriptional elongation, RNA polymerase binding, or transcription factor binding. This system, which we call CRISPR interference (CRISPRi), can efficiently repress expression of targeted genes in Escherichia coli, with no detectable off-target effects. CRISPRi can be used to repress multiple target genes simultaneously, and its effects are reversible. We also show evidence that the system can be adapted for gene repression in mammalian cells. This RNA-guided DNA recognition platform provides a simple approach for selectively perturbing gene expression on a genome-wide scale.
Display omitted
► Inactive CRISPR associated 9 protein (dCas9) is repurposed for genome engineering ► dCas9 and a complementary short guide RNA can target specific genomic sites ► CRISPR interference (CRISPRi) can regulate multiple genes without off-target effects ► CRISPRi is compact and can be ported to bacterial and mammalian cells
The authors have developed a CRISPR interference system in which a catalytically dead Cas9 protein can be targeted to a specific genomic site through a complementary small guide RNA, allowing systematic perturbation of gene transcription in bacteria and mammalian cells.
The spatial and temporal organization of molecules within a cell is critical for coordinating the many distinct activities carried out by the cell. In an increasing number of biological signaling ...processes, scaffold proteins have been found to play a central role in physically assembling the relevant molecular components. Although most scaffolds use a simple tethering mechanism to increase the efficiency of interaction between individual partner molecules, these proteins can also exert complex allosteric control over their partners and are themselves the target of regulation. Scaffold proteins offer a simple, flexible strategy for regulating selectivity in pathways, shaping output behaviors, and achieving new responses from preexisting signaling components. As a result, scaffold proteins have been exploited by evolution, pathogens, and cellular engineers to reshape cellular behavior.
Protein function is often regulated by posttranslational modifications (PTMs), and recent advances in mass spectrometry have resulted in an exponential increase in PTM identification. However, the ...functional significance of the vast majority of these modifications remains unknown. To address this problem, we compiled nearly 200,000 phosphorylation, acetylation, and ubiquitination sites from 11 eukaryotic species, including 2,500 newly identified ubiquitylation sites for Saccharomyces cerevisiae. We developed methods to prioritize the functional relevance of these PTMs by predicting those that likely participate in cross-regulatory events, regulate domain activity, or mediate protein-protein interactions. PTM conservation within domain families identifies regulatory “hot spots” that overlap with functionally important regions, a concept that we experimentally validated on the HSP70 domain family. Finally, our analysis of the evolution of PTM regulation highlights potential routes for neutral drift in regulatory interactions and suggests that only a fraction of modification sites are likely to have a significant biological role.
Display omitted
► We compiled a resource of nearly 200,000 PTMs across 11 eukaryotic species ► Computational methods were developed to assign function to PTMs ► Conservation studies identify regulatory regions within domain families ► Evolutionary analysis suggests that only a fraction of PTMs are functionally important
Analysis of 200,000 PTMs across 11 species in the light of structural and interaction data reveals ground rules for establishing whether a PTM is likely to impact protein function and suggests that only a fraction of modification sites are likely to be functionally important.
T cells can be re-directed to kill cancer cells using chimeric antigen receptors (CARs) or T cell receptors (TCRs). This approach, however, is constrained by the rarity of tumor-specific single ...antigens. Targeting antigens also found on bystander tissues can cause life-threatening adverse effects. A powerful way to enhance ON-target activity of therapeutic T cells is to engineer them to require combinatorial antigens. Here, we engineer a combinatorially activated T cell circuit in which a synthetic Notch receptor for one antigen induces the expression of a CAR for a second antigen. These dual-receptor AND-gate T cells are only armed and activated in the presence of dual antigen tumor cells. These T cells show precise therapeutic discrimination in vivo—sparing single antigen “bystander” tumors while efficiently clearing combinatorial antigen “disease” tumors. This type of precision dual-receptor circuit opens the door to immune recognition of a wider range of tumors.
Display omitted
Display omitted
•Engineering of AND-gate T cells activated only by dual antigen recognition•The synNotch receptor (senses antigen 1) induces CAR expression (senses antigen 2)•AND-gate T cells spare single antigen cells but kill dual antigen tumors in vivo•SynNotch/CAR circuits expand set of antigens that can be targeted by immunotherapy
T cells engineered with dual-receptor circuits that recognize combinations of antigens can efficiently kill target tumor cells in vivo, while sparing bystander cells.
The Notch protein is one of the most mechanistically direct transmembrane receptors—the intracellular domain contains a transcriptional regulator that is released from the membrane when engagement of ...the cognate extracellular ligand induces intramembrane proteolysis. We find that chimeric forms of Notch, in which both the extracellular sensor module and the intracellular transcriptional module are replaced with heterologous protein domains, can serve as a general platform for generating novel cell-cell contact signaling pathways. Synthetic Notch (synNotch) pathways can drive user-defined functional responses in diverse mammalian cell types. Because individual synNotch pathways do not share common signaling intermediates, the pathways are functionally orthogonal. Thus, multiple synNotch receptors can be used in the same cell to achieve combinatorial integration of environmental cues, including Boolean response programs, multi-cellular signaling cascades, and self-organized cellular patterns. SynNotch receptors provide extraordinary flexibility in engineering cells with customized sensing/response behaviors to user-specified extracellular cues.
Display omitted
Display omitted
•SynNotch receptors with altered extra- and intracellular domains yield new responses•SynNotch pathways drive diverse user-defined responses in many mammalian cell types•Multiple synNotch pathways are functionally orthogonal to one another•SynNotch pathways control differentiation, spatial patterning, and Boolean decisions
Modular synthetic Notch receptors (synNotch) provide extraordinary flexibility in engineering mammalian cells with customized sensing/response behaviors to achieve combinatorial integration of user-specified environmental cues.
Two decades ago, the pharmaceutical industry-long dominated by small-molecule drugs-was revolutionized by the the advent of biologics. Today, biomedicine sits on the cusp of a new revolution: the use ...of microbial and human cells as versatile therapeutic engines. Here, we discuss the promise of this "third pillar" of therapeutics in the context of current scientific, regulatory, economic, and perceptual challenges. History suggests that the advent of cellular medicines will require the development of a foundational cellular engineering science that provides a systematic framework for safely and predictably altering and regulating cellular behaviors.
Immunotherapies with chimeric antigen receptor (CAR) T cells and checkpoint inhibitors (including antibodies that antagonize programmed cell death protein 1 PD-1) have both opened new avenues for ...cancer treatment, but the clinical potential of combined disruption of inhibitory checkpoints and CAR T cell therapy remains incompletely explored. Here we show that programmed death ligand 1 (PD-L1) expression on tumor cells can render human CAR T cells (anti-CD19 4-1BBζ) hypo-functional, resulting in impaired tumor clearance in a sub-cutaneous xenograft model. To overcome this suppressed anti-tumor response, we developed a protocol for combined Cas9 ribonucleoprotein (Cas9 RNP)-mediated gene editing and lentiviral transduction to generate PD-1 deficient anti-CD19 CAR T cells. Pdcd1 (PD-1) disruption augmented CAR T cell mediated killing of tumor cells in vitro and enhanced clearance of PD-L1+ tumor xenografts in vivo. This study demonstrates improved therapeutic efficacy of Cas9-edited CAR T cells and highlights the potential of precision genome engineering to enhance next-generation cell therapies.
PDF
