Rueff, Anne-Stéphanievan Raaphorst, RenskeAggarwal, Surya D.Santos Moreno, JavierLaloux, GéraldineSchaerli, YolandaWeiser, Jeffrey N.Veening, Jan-Willem2025-04-162025-04-162023Rueff AS, van Raaphorst R, Aggarwal SD, Santos-Moreno J, Laloux G, Schaerli Y, et al. Synthetic genetic oscillators demonstrate the functional importance of phenotypic variation in pneumococcal-host interactions. Nat Commun. 2023 Nov 17;14(1):7454. DOI: 10.1038/s41467-023-43241-y2041-1723http://hdl.handle.net/10230/70145Phenotypic variation is the phenomenon in which clonal cells display different traits even under identical environmental conditions. This plasticity is thought to be important for processes including bacterial virulence, but direct evidence for its relevance is often lacking. For instance, variation in capsule production in the human pathogen Streptococcus pneumoniae has been linked to different clinical outcomes, but the exact relationship between variation and pathogenesis is not well understood due to complex natural regulation. In this study, we use synthetic oscillatory gene regulatory networks (GRNs) based on CRISPR interference (CRISPRi) together with live cell imaging and cell tracking within microfluidics devices to mimic and test the biological function of bacterial phenotypic variation. We provide a universally applicable approach for engineering intricate GRNs using only two components: dCas9 and extended sgRNAs (ext-sgRNAs). Our findings demonstrate that variation in capsule production is beneficial for pneumococcal fitness in traits associated with pathogenesis providing conclusive evidence for this longstanding question.application/pdfeng© The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.info:eu-repo/semantics/articlehttp://dx.doi.org/10.1038/s41467-023-43241-yInfectionMicrobial geneticsPathogensSynthetic biologyTranscriptioninfo:eu-repo/semantics/openAccess