Designing cell function: assembly of synthetic gene circuits for cell biology applications

Abstract

Synthetic biology is the discipline of engineering application-driven biological functionalities that were not evolved by nature. Early breakthroughs of cell engineering, which were based on ectopic (over)expression of single sets of transgenes, have already had a revolutionary impact on the biotechnology industry, regenerative medicine and blood transfusion therapies. Now, we require larger-scale, rationally assembled genetic circuits engineered to programme and control various human cell functions with high spatiotemporal precision in order to solve more complex problems in applied life sciences, biomedicine and environmental sciences. This will open new possibilities for employing synthetic biology to advance personalized medicine by converting cells into living therapeutics to combat hitherto intractable diseases.

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Fig. 1: Control of gene expression in human cells.
Fig. 2: Engineering of stimulus-responsive human cell behaviour.
Fig. 3: Spatiotemporal control.
Fig. 4: Prototype synthetic gene circuits.
Fig. 5: Present applications of synthetic gene circuits.
Fig. 6: Cell and gene therapy as next-generation precision therapeutics.

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Acknowledgements

The authors apologize to colleagues for work that they were unable to cite owing to space constraints. They thank H. Wang for critical comments on the manuscript. Synthetic biology research in the laboratory of M.F. is supported by the European Research Council (ERC ProNet advanced grant no. 321381) and by the Swiss National Centre of Competence in Research (NCCR) Molecular Systems Engineering.

Reviewer information

Nature Reviews Molecular Cell Biology thanks J. Collins, P. Silver and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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Both authors contributed equally to all aspects of article preparation, including researching data for the article, discussing content and writing and editing the manuscript.

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Correspondence to Martin Fussenegger.

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Glossary

Ectopic (over)expression

The forced expression of a particular gene in a cell type in which the gene is usually not expressed at a desired level.

Constitutive expression

The persistent production of a target protein by any cell that contains the encoding gene.

Gene switches

Any natural or synthetic system (for example, a promoter, an RNA molecule or a signalling pathway) that allows initiation, interruption or termination of target gene expression.

Trans-regulators

Chimeric regulatory proteins consisting of a trafficking domain (controls translocation to target DNA, RNA or protein destination) and a regulatory domain (specifies target-specific activity).

Episomal vectors

Carriers of coding genes that are not part of the endogenous chromosome, such as plasmid DNA, mini-circles or replicon RNA.

Guide RNAs

(gRNAs). Synthetic RNA molecules that bind and guide a specific Cas protein (CRISPR-associated protein) towards a gRNA-specific DNA or RNA target sequence through complementary base pairing; also known as single-guide RNAs (sgRNAs).

Aptamer

A single-stranded RNA or DNA sequence forming a secondary structure that undergoes a considerable conformational change upon binding to a specific chemical ligand (small molecules, ions or proteins) with high affinity.

Mutually orthogonal trans-regulators

A set of trans-regulators operating at parallel genetic targets that do not show cross-interaction in terms of direct binding or potential influence on each other’s downstream targets.

Riboswitches

Regulatory segments within an mRNA that bind to specific metabolites and modify the expression of the protein product of the riboswitch-containing mRNAs.

Self-cleaving aptazymes

Products of a chimeric fusion between an aptamer and a (self-cleaving) ribozyme in which the ligand-dependent conformational change of the aptamer is also propagated to affect the activity of the ribozyme.

Allosteric proteins

Proteins in which an active or inactive conformation is reversibly triggered by ligand binding or other stimuli (such as light).

Cryptochrome 2

(CRY2). A protein derived from Arabidopsis thaliana that undergoes reversible oligomerization or heterodimerization with CIBN (amino-terminal domain of cryptochrome-interacting basic helix–loop–helix) upon exposure to blue light.

Cre

A type I topoisomerase from bacteriophage P1 that catalyses site-specific recombination (inversion or deletion) of DNA between loxP sites

Memory buffers

Transient memory devices with a finite capacity for storing cellular information that ensure unperturbed functionality of a regulated subsystem when sufficiently charged.

Non-homologous end joining

(NHEJ). An error-prone endogenous DNA repair mechanism for double-stranded breaks that is usually initiated when a correct DNA template is not provided.

Boolean logic gates

Synthetic (bio)computing devices that convert multiple input signals into a smaller number of outputs according to a defined logic algorithm.

Quorum sensing

A cell–cell communication mechanism evolved in many bacterial species that allows specific (sub)populations to measure their local density (by production, release, accumulation and detection of a signalling molecule) and subsequently coordinate gene expression.

Intein

A protein motif that is irreversibly excised from a larger protein structure when specific peptide domains are brought into close proximity.

Epigenetic memory

A type of resettable memory of living cells that is based on inheritable post-translational protein modifications and DNA conformations rather than a change in nucleotide sequences.

Transient transfection

Delivery of foreign gene elements into host cells through episomal vectors that do not permanently integrate into the genome and therefore reside in the cells for only a few rounds of cell division.

Drug target mimetics

Simplified replicates of biological targets created outside of their natural environment that retain all basic molecular functionalities important for (high-throughput) drug screening and development.

Anti-targets

Biological targets that should not be activated by a potential drug candidate.

Biofilms

Sessile communities of virulent bacteria encased in an extracellular matrix adhering to a solid surface and showing increased survival compared with free-floating bacteria.

Gene drive systems

Systems that enable biased inheritance of a genetic element so that offspring within a population have a >50% chance of inheritance of a given trait.

Graft-versus-host reactions

Medical complications related to the immunological adverse effects in a patient (host) caused by implanted or infused therapeutics (graft).

Adiponectin

An adipocyte-derived protein hormone that increases insulin sensitivity in target tissues, such as fat, muscle or liver.

Graves disease

An autoimmune disorder characterized by hyperthyroidism in which autoantibodies constitutively trigger thyroid hormone production from the thyroid gland.

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Xie, M., Fussenegger, M. Designing cell function: assembly of synthetic gene circuits for cell biology applications. Nat Rev Mol Cell Biol 19, 507–525 (2018). https://doi.org/10.1038/s41580-018-0024-z

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