De Carluccio, Giuliano (2023) The CASwitch : a C oherent Feed Forward Loop synthetic gene circuit for tight multi level regulation of gene expression. [Tesi di dottorato]

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Synthetic biology is now an established biological engineering discipline that combines molecular biology and engineering knowledge with the ultimate goal of programming new cell functions using sophisticated gene circuits. During the last two decades, synthetic biology has grown into a thriving field that deploys an extensive toolkit of knowledge, devices, design strategies, and that holds great promise for the future. Despite the progress made, most current developments are not immediately translatable to “outside-the-lab” contexts. This thesis focuses on the use of synthetic biology to improve current state-of-the-art inducible systems for controlled modulation of gene expression. Inducible gene expression systems allow to switch on, or off, transcription of a gene of interest by treating cells or even whole organism with a chemical compound. As such, they are indispensable research tools in the life sciences and in biotechnology. However, state-of-the-art inducible gene expression systems all exhibit unintended basal gene expression, commonly known as leakiness, which affects their modularity and increases the likelihood of accidental activation. This makes it difficult to predict their behavior and limits their potential applications in biotechnological and clinical settings. The goal of this thesis is to develop a novel inducible gene expression system that goes beyond the state-of-the-art Tet-On3G system, the most broadly used inducible gene system in mammals, by means of a synthetic biology approach. To date, the Tet-On3G encompasses more than 7000 publications and several implementations in inducible animal models in research contexts. However, its deployment for applications “out-side the lab” is severely hampered by its high level of leakiness. In this thesis, I built on the Tet-On3G, by wiring it into a multi-level regulated synthetic gene circuit implementing a coherent feed forward regulatory motif (CFFL-4), endowing the Tet-On3G with quasi-zero leakiness while retaining high maximum inducible expression, resulting in more than one order of magnitude increase in fold induction levels compared to the current state-of-the-art. This resulted in the generation of a new tight inducible gene system in mammalian cells that I called it the CASwitch, for its capacity to switch gene expression off or on at will by means of a CRISPR-Cas13d endoribonuclease. To prove the usefulness of the CASwitch in applications of practical interest, I first modified the CASwitch to build two whole-cell biosensors: a copper biosensor able to detect copper in growing medium, and lysosomal stress biosensor able to sense the activation of the stress-responsive transcription factor TFEB. Both biosensors resulted in considerable improvements in dynamic range, detection resolution, and response reliability, as compared to their state-of-the-art counterparts. Finally, I also applied the CASwitch to enable the development of factory cells able to produce Adeno Associated Virus vectors (AAVs) on demand. Specifically, I modified the CASwitch to achieve tight but inducible control of toxic viral gene expression, essential for AAV production and demonstrated inducible production of AAVs by means of doxycycline. These results holds great promise for further feasible engineering of a stable AAV producer HEK293 cell line.

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