Biology subjects

Fusco, V.

Publications and source records attributed to Fusco, V..

4 recordsLinked to original sources

bioRxiv2024

The CRISPRaTOR: a biomolecular circuit for Automatic Gene Regulation in Mammalian Cells with CRISPR technology.

We introduce the CRISPRaTOR, a biomolecular circuit for precise control of gene expression in mammalian cells. The CRISPRaTOR leverages the stochiometric interaction between the artificial transcription factor VPR-dCas9, and the anti-CRISPR protein AcrIIA4, enhanced with synthetic coiled-coil domains to boost their interaction, to maintain the expression of a reporter protein constant across diverse experimental conditions, including fluctuations in protein degradation rates and plasmid concentrations, by automatically adjusting its mRNA level. This capability, known as Robust Perfect Adaptation (RPA), is crucial for the stable functioning of biological systems and has wide-ranging implications for biotechnological applications. The CRISPRaTOR belongs to a class of biomolecular circuits named antithetic integral controllers, and it can be easily adapted to regulate any endogenous transcription factor thanks to the versatility of CRISPR-Cas system. Finally, we show that RPA holds also in cells genomically integrated with the CRISPRaTOR, thus paving the way for practical applications in biotechnology that require stable cell lines.

synthetic biology↗

bioRxiv2024

A general strategy to engineer high-performance mammalian Whole-Cell Biosensors

Transcription-based whole-cell biosensors (WCBs) are cells engineered with an analyte-responsive promoter driving the transcription of a reporter gene. WCBs can sense and report on bioactive molecules (analytes) relevant to human health. Designing an analyte-sensitive promoter requires a cumbersome trial-and-error approach and usually results in biosensors with poor performance. Here, we integrated Synthetic Biology with Control Engineering to design, computationally model, and experimentally implement high-performance biosensors in mammalian cells. Our approach, unlike traditional methods, does not rely on optimizing individual components such as promoters and transcription factors. Instead, it uses biomolecular circuits to enhance the biosensors performance despite inherent component flaws. We experimentally implemented eight different biosensors by employing CRISPR-Cas systems, then quantitatively compared their performance and identified one configuration, which we named CASense, that overcomes the limitations of current biosensors. Our approach is generalisable and can be adapted to sense any analyte of interest for which there is an analyte-sensitive promoter, making it a versatile tool for multiple applications. As a proof of concept, we engineered a high-performance biosensor of intracellular copper due to the critical role that copper plays in human health and disease, and to the lack of techniques able to measure intracellular copper levels in living cells. The significance of our work lies in its potential to impact the monitoring of bioactive molecules and chemicals both in vitro and in vivo, which is crucial in areas such as toxicology, drug discovery, disease diagnosis and therapy.

synthetic biology↗

bioRxiv2023

The CASwitch: a synthetic biology solution for high-performance inducible gene expression systems in biotechnology

Inducible gene expression systems can be used to control the expression of a gene of interest by means of a small-molecule. One of the most common designs involves engineering a small-molecule responsive transcription factor (TF) and its cognate promoter, which often results in a compromise between minimal uninduced background expression (leakiness) and maximal induced expression. Here, we focussed on an alternative strategy using quantitative synthetic biology to mitigate leakiness while maintaining high expression, without modifying neither the TF nor the promoter. Through mathematical modelling and experimental validations, we designed the CASwitch, a mammalian synthetic gene circuit based on combining two well-known network motifs: the Coherent Feed-Forward Loop (CFFL) and the Mutual Inhibition (MI). The CASwitch combines the CRISPR-Cas endoribonuclease CasRx with the state-of-the-art Tet-On3G inducible gene system to achieve high performances. To demonstrate the potentialities of the CASwitch, we applied it to three different scenarios: enhancing a whole-cell biosensor, controlling expression of a toxic gene and inducible production of Adeno-Associated Virus (AAV) vectors.

synthetic biology↗

bioRxiv2021

Embedded control of cell growth using tunable genetic systems

We present an embedded feedback control strategy to control the density of a bacterial population, allowing cells to self-regulate their growth rate so as to reach a desired density at steady state. We consider a static culture condition, where cells are provided with a limited amount of space and nutrients. The control strategy is built using a tunable expression system (TES), which controls the production of a growth inhibitor protein, complemented with a quorum sensing mechanism for the sensing of the population density. We show on a simplified population-level model that the TES endows the control system with additional flexibility by allowing the set-point to be changed online. Finally, we validate the effectiveness of the proposed control strategy by means of realistic in silico experiments conducted in BSim, an agent-based simulator explicitly designed to simulate bacterial populations, and we test the robustness of our design to disturbances and parameters variations due, for instance, to cell-to-cell variability.

synthetic biology↗