Optogenetic single-cell control of transcription achieves mRNA tunability and reduced variability

The study of gene expression at the single-cell level has exposed the importance of stochasticity for the behavior of cellular systems. Research on cellular variability has mostly relied on observing expression either in response to natural stimuli or to constant gene regulators. However, the ability to probe cells individually can lead to a deeper understanding of the underlying process. Here, we propose an experimental platform for optogenetic feedback control of individual cells. It consists of a digital micromirror device that, coupled to a microscope, can target light-responsive cells with individualized illumination profiles, thereby exploiting the good spatial resolution of optogenetic induction. Together with an automated software pipeline for segmentation, quantification and tracking of single cells, the platform enables independent and real-time control of numerous cells. We demonstrate our platform by regulating transcription in over a hundred yeast cells simultaneously, while achieving tunability of mRNA abundance. Using a novel technique to measure extrinsic variation, we further show that single cell feedback regulation of this highly stochastic process achieves a 10-fold reduction of extrinsic variation in nascent mRNA over population control, with superior control loop properties. Our platform establishes a new, flexible method for studying transcriptional dynamics in single cells.