The COMET toolkit for composing customizable genetic programs in mammalian cells

Donahue, Patrick S.; Draut, Joseph W.; Muldoon, Joseph J.; Edelstein, Hailey I.; Bagheri, Neda; Leonard, Joshua N.

doi:10.1038/s41467-019-14147-5

Cited by 61 publications

(81 citation statements)

References 54 publications

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“…MESA receptors were cloned into pcDNA backbones to confer high expression in HEK293FT cells. These plasmid backbones are versions of the pcDNA3.1/Hygro(+) Mammalian Expression Vector (Thermo Fisher #V87020), modified by our laboratory in previous work (Addgene #138749) ( 32 ). In general, restriction sites were chosen to facilitate modular swapping of parts via restriction enzyme cloning.…”

Section: Methodsmentioning

confidence: 99%

Elucidation and refinement of synthetic receptor mechanisms

et al. 2020

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Synthetic receptors are powerful tools for engineering mammalian cell-based devices. These biosensors enable cell-based therapies to perform complex tasks such as regulating therapeutic gene expression in response to sensing physiological cues. Although multiple synthetic receptor systems now exist, many aspects of receptor performance are poorly understood. In general, it would be useful to understand how receptor design choices influence performance characteristics. In this study, we examined the modular extracellular sensor architecture (MESA) and systematically evaluated previously unexamined design choices, yielding substantially improved receptors. A key finding that might extend to other receptor systems is that the choice of transmembrane domain (TMD) is important for generating high-performing receptors. To provide mechanistic insights, we adopted and employed a Förster resonance energy transfer (FRET)-based assay to elucidate how TMDs affect receptor complex formation and connected these observations to functional performance. To build further insight into these phenomena, we developed a library of new MESA receptors that sense an expanded set of ligands. Based upon these explorations, we conclude that TMDs affect signaling primarily by modulating intracellular domain geometry. Finally, to guide the design of future receptors, we propose general principles for linking design choices to biophysical mechanisms and performance characteristics.

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Section: Methodsmentioning

confidence: 99%

Elucidation and refinement of synthetic receptor mechanisms

et al. 2020

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“…Plasmid DNA used for transfection was prepared using the PEG precipitation method, which was previously described in detail. 27…”

Section: Methodsmentioning

confidence: 99%

Computation-guided optimization of split protein systems

Dolberg¹,

Meger²,

Boucher³

et al. 2019

Preprint

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25Splitting bioactive proteins, such as enzymes or fluorescent reporters, into conditionally reconstituting 26 fragments is a powerful strategy for building tools to study and control biochemical systems. However, split 27 proteins often exhibit a high propensity to reconstitute even in the absence of the conditional trigger, which 28 limits their utility. Current approaches for tuning reconstitution propensity are laborious, context-specific, or 29 often ineffective. Here, we report a computational design-driven strategy that is grounded in fundamental 30 protein biophysics and which guides the experimental evaluation of a focused, sparse set of mutants-31 which vary in the degree of interfacial destabilization while preserving features such as stability and catalytic 32 activity-to identify an optimal functional window. We validate our method by solving two distinct split 33 protein design challenges, generating both broad insights and new technology platforms. This method will 34 streamline the generation and use of split protein systems for diverse applications. 35 36 KEYWORDS: synthetic biology, split proteins, computational protein design, protein engineering 37 38

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“…S4B). Following early structural work and recent advances in zinc finger engineering (52,(57)(58)(59), we incorporated arginine-to-alanine mutations at key positions in the ZF known to impact DNA binding, which decreased background monomeric activity without reducing foreground homodimer activity (bars within red square in Fig. 2A, Fig.…”

Section: Engineered Zinc Fingers Enable Homodimer-dependent Activatiomentioning

confidence: 99%

Synthetic multistability in mammalian cells

Zhu

García-Ojalvo

et al. 2021

Preprint

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In multicellular organisms, gene regulatory circuits generate thousands of molecularly distinct, mitotically heritable states, through the property of multistability. Designing synthetic multistable circuits would provide insight into natural cell fate control circuit architectures and allow engineering of multicellular programs that require interactions among cells in distinct states. Here we introduce MultiFate, a naturally-inspired, synthetic circuit that supports long-term, controllable, and expandable multistability in mammalian cells. MultiFate uses engineered zinc finger transcription factors that transcriptionally self-activate as homodimers and mutually inhibit one another through heterodimerization. Using model-based design, we engineered MultiFate circuits that generate up to seven states, each stable for at least 18 days. MultiFate permits controlled state-switching and modulation of state stability through external inputs, and can be easily expanded with additional transcription factors. Together, these results provide a foundation for engineering multicellular behaviors in mammalian cells.

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The COMET toolkit for composing customizable genetic programs in mammalian cells

Cited by 61 publications

References 54 publications

Elucidation and refinement of synthetic receptor mechanisms

Elucidation and refinement of synthetic receptor mechanisms

Computation-guided optimization of split protein systems

Synthetic multistability in mammalian cells

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