Synthetic circuits for cell ratio control | Nature

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Subjects

- Genetic circuit engineering

- Multicellular systems

- Synthetic biology

Abstract

Recent advances in genetic engineering have provided diverse tools for artificially diversifying both prokaryotic and eukaryotic cell populations1,2,3,4,5,6. However, achieving precise control over the ratios of multiple cell types within a population derived from a single founder remains a major challenge. Here we introduce a suite of recombinase-mediated genetic devices designed to accurately control population ratios, enabling the distribution of distinct functionalities across multiple cell types. We systematically evaluated key parameters that influence recombination efficiency and developed data-driven models to reliably predict binary differentiation outcomes. Using these devices, we constructed parallel and series circuit topologies to implement user-defined, multistep cell-fate branching programs. The branching devices facilitated the autonomous differentiation of precision fermentation consortia from a single founder yeast strain, optimizing cell-type ratios for applications such as pigmentation and cellulose degradation. Similar effects were obtained with mammalian cells. We also engineered multicellular aggregates with genetically encoded morphologies by coordinating self-organization through cell adhesion molecules. Our work provides a comprehensive characterization of recombinase-based cell-fate branching mechanisms and introduces an approach for constructing synthetic consortia and multicellular assemblies.

Main

In multicellular organisms, stem cells undergo asymmetric cell divisions, giving rise to progeny with distinct phenotypes7,8. By orchestrating complex gene-regulatory networks, eukaryotic stem cells differentiate into specialized cell lineages that form functional tissues or organs. This differentiation process reflects the genetic intricacy of multicellular eukaryotes, contributing to their evolutionary success8. Parallel phenomena in microorganisms include asymmetric division in Caulobacter crescentus, which produces motile cells that lack reproductive capabilities and sessile cells that are capable of division but not motility9. Similarly, under nitrogen-limiting conditions, the central cells in Anabaena filaments divide asymmetrically, generating progeny specialized in nitrogen fixation or photosynthesis, and ensuring colony survival10. These cellular communities accomplish sophisticated tasks that are unattainable by a single cell type11.

By mimicking the differentiation and cooperative division of labour that characterize natural selection, synthetic biology is unlocking the reprogramming of cellular functions, facilitating the emergence of diverse artificial cell states2,3,4,5,6,12,13. A foundational milestone was the genetic toggle switch, which allowed reversible switching between two distinct states in Escherichia coli14. Other genetic tools for engineering bacterial ‘stem cells’ include partitioning the bacterial chromosome (Par), inducing asymmetric cell division and generating progeny with distinct genetic content3. Epigenetic tools, including phase-separated and scaffolding proteins such as PopZ, have advanced the control of microbial cell differentiation2,6. Multicellular eukaryotic systems have also been engineered; for example, the MultiFate circuit produces multiple stable phenotypes in Chinese hamster ovary (CHO) cells1. Although genetic circuits can potentially program and rewrite cell fates, challenges remain—in particular, scaling genetic toggle switches and DNA partitioning systems with increasing numbers of cell states and quantitatively controlling the ratio of specific cell types in offspring populations. Most artificial systems do not autonomously differentiate and so do not generate complex multicellular systems from a single founder cell type. These limitations constrain the potential applications of synthetic communities to biomanufacturing, regenerative medicine and therapeutic development.

Recombinases—enzymes that catalyse recombination between specific DNA sequences—offer a promising solution. These enzymes target attB (bacterial) and attP (phage) sites, and the recombination outcome (excision or inversion) is determined by the number and orientation of these sites15. Serine recombinases are widely used in constructing Boolean logic gates16, amplification circuits17 and information storage systems18, and multiple att sites can be strategically positioned to expand the toolbox for cell-state programming18,19.

Here we present a suite of recombinase-based cell-fate branching devices that generate diverse progeny cell types with user-defined ratios and enable quantitative control over multicellular morphology. By tuning DNA sequences between att sites and exploiting mutated att variants, we achieve precise control of binary population ratios spanning 0.1% to 99.9% from a single founder. These branching devices can be implemented in parallel