Biology subjects

Ploessl, D. S.

Publications and source records attributed to Ploessl, D. S..

5 recordsLinked to original sources

bioRxiv2026

Programmable nanobody circuits for cell selection

Efficient and scalable isolation of specific cell populations remains a central bottleneck for genome engineering, pooled screening, and cell therapy manufacturing. Here, we present DASIT (Destabilized-nanobody Antigen Selection and Identification Tool), a protein-based circuit for antigen-specific cell selection. DASIT uses a destabilized nanobody fused to an antibiotic resistance protein. In cells expressing the target antigen, binding of the nanobody fusion to the cognate antigen stabilizes DASIT, thereby coupling the presence of an antigen to a selectable signal. We developed DASIT circuits that enable robust selection of antigen-expressing cells and show that they can be designed to target distinct antigen classes and perform across cell types. Because DASIT operates at the protein level, it supports both stable integration and transient delivery, enabling recyclable selection without permanent genomic integration of resistance markers. We demonstrate scalable, FACS-free enrichment in three challenging applications: multiplexed, logic-gated integration of landing pads in human iPSCs, high-throughput CRISPR screening, and phenotypic selection of in vitro-derived neurons at transplantation scale. By decoupling selection from vector integration, DASIT establishes an automation-compatible architecture for multistep genome engineering, high-throughput library screening, and large-scale cell manufacturing. HighlightsDASIT enriches for antigen-positive cells across multiple selection markers and antigens Intermediate levels of DASIT expression support selection across stable and transient delivery modalities Logic-gated, precision genome engineering of human iPSCs via DASIT selection DASIT enables scalable activity-based selection for high-throughput base editing screens DASIT-selected engineered motor neurons survive grafting into acute spinal cord injury

synthetic biology↗

bioRxiv2025

Self-amplifying RNA enables rapid, durable, integration-free programming of hiPSCs

Genetic modification of human induced pluripotent stem cells (hiPSCs) is a powerful approach to measure and manipulate the cellular processes underlying differentiation and disease. Conventional genetic engineering of hiPSC lines requires a laborious process involving transfection, selection and expansion that can result in karyotypic abnormalities or transgene silencing during differentiation, limiting their applications. Self-amplifying RNA (saRNA) delivery is a potential alternative integration-free method for durable expression of transgenes. Here, we used saRNA to deliver transcription factors and functional reporters in hiPSCs and demonstrate that expression can persist for weeks. Specifically, saRNA delivery enables highly efficient forward programming to Ngn2-induced neurons and enables measurement of functional reporters over time. We show that a single transfection of saRNA encoded jRCaMP1b reporter in hiPSCs generates sustained expression throughout differentiation to 3D cardiac spheroids. The persistence of the reporter allows measurement of calcium dynamics at a single-cell and population level over weeks, allowing tracking of cardiomyocyte maturation and drug responses. Together, our systematic analysis shows that saRNA provides sustained transgene expression in hiPSCs, supporting integration- free cell-fate programming and measurement of functional reporters in clinically relevant model systems. HighlightsO_LIA single saRNA transfection generates durable transgene expression C_LIO_LIsaRNA transfection of Ngn2 in hiPSCs results in robust neuronal differentiation C_LIO_LIsaRNA-delivery of functional reporters enables single-cell analysis of primary and hiPSC-derived cells C_LIO_LIsaRNA-based sensor allows monitoring of maturation and drug responses in 3D cardiac spheroids C_LI

bioengineering↗

bioRxiv2025

Gene syntax defines supercoiling-mediated transcriptional feedback

Gene syntax--the order and arrangement of genes and their regulatory elements--shapes the dynamic coordination of both natural and synthetic gene circuits. Transcription at one locus profoundly impacts the transcription of nearby adjacent genes, but the molecular basis of this effect remains poorly understood. Here, using integrated reporter circuits in human cells, we show that the reciprocal effects of transcription and DNA supercoiling, which we term supercoiling-mediated feedback, regulates expression of adjacent genes in a syntax-specific manner. Using a suite of chromatin state assays, we measure syntax-and induction-dependent formation of chromatin structures in human induced pluripotent stem cells. Applying syntax as a design parameter and without altering sequence or copy number, we built compact gene circuits, tuning the expression mean, noise, and stoichiometry across diverse delivery methods and cell types. Integrating supercoiling-mediated feedback into models of gene regulation will expand our understanding of native systems and enhance the design of synthetic gene circuits.

synthetic biology↗

bioRxiv2024

High-resolution profiling reveals coupled transcriptional and translational regulation of transgenes

Concentrations of RNAs and proteins provide important determinants of cell fate. Robust gene circuit design requires an understanding of how the combined actions of individual genetic components influence both mRNA and protein levels. Here, we simultaneously measure mRNA and protein levels in single cells using HCR Flow-FISH for a set of commonly used synthetic promoters. We find that promoters generate differences in both the mRNA abundance and the effective translation rate of these transcripts. Stronger promoters not only transcribe more RNA but also show higher effective translation rates. While the strength of the promoter is largely preserved upon genome integration with identical elements, the choice of polyadenylation signal and coding sequence can generate large differences in the profiles of the mRNAs and proteins. We used long-read direct RNA sequencing to characterize full-length mRNA isoforms and observe remarkable uniformity of mRNA isoforms from the transgenic units. Together, our high-resolution profiling of transgenic mRNAs and proteins offers insight into the impact of common synthetic genetic components on transcriptional and translational mechanisms. By developing a novel framework for quantifying expression profiles of transgenes, we have established a system for comparing native and synthetic gene regulation and for building more robust transgenic systems.

synthetic biology↗

bioRxiv2024

Programmable promoter editing for precise control of transgene expression

Subtle changes in gene expression direct cells to distinct cellular states. Identifying and controlling dose-dependent transgenes require tools for precisely titrating expression. To this end, we developed a highly modular, extensible framework called DIAL for building editable promoters that allow for fine-scale, heritable changes in transgene expression. Using DIAL, we increase expression by recombinase-mediated excision of spacers between the binding sites of a synthetic zinc finger transcription factor and the core promoter. By nesting varying numbers and lengths of spacers, DIAL generates a tunable range of unimodal setpoints from a single promoter. Through small-molecule control of transcription factors and recombinases, DIAL supports temporally defined, user-guided control of transgene expression that is extensible to additional transcription factors. Lentiviral delivery of DIAL generates multiple setpoints in primary cells and iPSCs. As promoter editing generates stable states, DIAL setpoints are heritable, facilitating mapping of transgene levels to phenotypes. The DIAL framework opens new opportunities for tailoring transgene expression and improving the predictability and performance of gene circuits across diverse applications. HighlightsPromoter editing generates unimodal setpoints from a single synthetic promoter DIAL setpoints are tuned by the length of an excisable spacer within the promoter DIAL setpoints are uniform and robust to varying levels of the transcription factor DIAL transmits transient inputs into heritable promoter states The TET-DIAL system enables small-molecule activation at defined setpoints DIAL regulates physiologically-relevant transgenes and performs in primary cells and iPSCs One Sentence SummaryDIAL offers an extensible framework for designing synthetic promoters that perform across a range of cell types to generate heritable setpoints of gene expression.

synthetic biology↗