A new era of biological engineering is emerging in which living cells are used as building blocks to address therapeutic challenges. These efforts are distinct from traditional molecular ...engineering—their focus is not on optimizing individual genes and proteins as therapeutics, but rather on using molecular components as modules to reprogram how cells make decisions and communicate to achieve higher-order physiological functions in vivo. This cell-centric approach is enabled by a growing tool kit of components that can synthetically control core cell-level functional outputs, such as where in the body a cell should go, what other cells it should interact with, and what messages it should transmit or receive. The power of cell engineering has been clinically validated by the development of immune cells designed to kill cancer. This same tool kit for rewiring cell connectivity is beginning to be used to engineer cell therapies for a host of other diseases and to program the self-organization of tissues and organs. By forcing the conceptual distillation of complex biological functions into a finite set of instructions that operate at the cell level, these efforts also shed light on the fundamental hierarchical logic that links molecular components to higher-order physiological function.
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.
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•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.
Chimeric antigen receptor (CAR) T cells have proven that engineered immune cells can serve as a powerful new class of cancer therapeutics. Clinical experience has helped to define the major ...challenges that must be met to make engineered T cells a reliable, safe, and effective platform that can be deployed against a broad range of tumors. The emergence of synthetic biology approaches for cellular engineering is providing us with a broadly expanded set of tools for programming immune cells. We discuss how these tools could be used to design the next generation of smart T cell precision therapeutics.
The emergence of synthetic biology approaches for cellular engineering is providing us with a broadly expanded set of tools that can be used to design the next generation of smart T cell precision therapeutics for cancer.
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.
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.
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► 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.
Living cells have evolved a broad array of complex signalling responses, which enables them to survive diverse environmental challenges and execute specific physiological functions. Our increasingly ...sophisticated understanding of the molecular mechanisms of cell signalling networks in eukaryotes has revealed a remarkably modular organization and synthetic biologists are exploring how this can be exploited to engineer cells with novel signalling behaviours. This approach is beginning to reveal the logic of how cells might evolve innovative new functions and moves us towards the exciting possibility of engineering custom cells with precise sensing-response functions that could be useful in medicine and biotechnology.
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.
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•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.
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► 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.
The bacterial CRISPR-Cas9 system has emerged as a multifunctional platform for sequence-specific regulation of gene expression. This Review describes the development of technologies based on ...nuclease-deactivated Cas9, termed dCas9, for RNA-guided genomic transcription regulation, both by repression through CRISPR interference (CRISPRi) and by activation through CRISPR activation (CRISPRa). We highlight different uses in diverse organisms, including bacterial and eukaryotic cells, and summarize current applications of harnessing CRISPR-dCas9 for multiplexed, inducible gene regulation, genome-wide screens and cell fate engineering. We also provide a perspective on future developments of the technology and its applications in biomedical research and clinical studies.
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