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Synthetic Circuits Based on Split CAS9 to Detect Cellular Events

View ORCID ProfileAlicja Przybyszewska-Podstawka, View ORCID ProfileJakub Czapiński, View ORCID ProfileJoanna Kałafut, View ORCID ProfileAdolfo Rivero-Müller
doi: https://doi.org/10.1101/2023.03.16.533022
Alicja Przybyszewska-Podstawka
1Department of Biochemistry and Molecular Biology, Medical University of Lublin, 20-093, Lublin, Poland
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  • ORCID record for Alicja Przybyszewska-Podstawka
Jakub Czapiński
1Department of Biochemistry and Molecular Biology, Medical University of Lublin, 20-093, Lublin, Poland
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Joanna Kałafut
1Department of Biochemistry and Molecular Biology, Medical University of Lublin, 20-093, Lublin, Poland
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Adolfo Rivero-Müller
1Department of Biochemistry and Molecular Biology, Medical University of Lublin, 20-093, Lublin, Poland
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  • For correspondence: a.rivero@umlub.pl
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Abstract

Synthetic biology involves the generation of logic circuits to create or control biological functions and behaviors by engineering interconnected genetic elements such as promoters, repressors, and transcriptional activators. CRISPR discovery, and its adaptations to mammalian cells, has made it the tool of choice in molecular biology and revolutionized genome engineering in biomedical sciences. Here, we describe an adaptation of a split Cas9 to generate synthetic logic gates to sense biological events. As proof-of-concept, the complementing halves of split Cas9 were placed under different promoters, one unique to cancer cells of epithelial origin (phCEA) and one universal promoter (pCMV). We used self-assembling inteins to reunite the halves when co-expressed. Only cancer cells with epithelial origin expressed both halves and activated a reporter becoming green fluorescent. We then investigated whether we could apply this system to the detection of biological processes such as epithelial to mesenchymal transition (EMT). We designed another logic gate where one halve is expressed only by cancer cells of epithelial origin, while the other is activated during EMT – under the control of TWIST1. Indeed, cells undergoing EMT were detected by the activation of the reporter. Finally, the split-Cas9 logic gate was applied as a sensor to detect cell-cell fusion events in multiple cell lines. Each cell type expressed only one halve of split Cas9, and only induction of fusion resulted in the appearance of multinucleated syncytia and the expression of the reporter system. The simplicity and flexibility of the split Cas9 system reported here can be integrated to many other cellular processes, not only as a sensor but as an actuator.

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Competing Interest Statement

The authors have declared no competing interest.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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Posted March 17, 2023.
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Synthetic Circuits Based on Split CAS9 to Detect Cellular Events
Alicja Przybyszewska-Podstawka, Jakub Czapiński, Joanna Kałafut, Adolfo Rivero-Müller
bioRxiv 2023.03.16.533022; doi: https://doi.org/10.1101/2023.03.16.533022
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Synthetic Circuits Based on Split CAS9 to Detect Cellular Events
Alicja Przybyszewska-Podstawka, Jakub Czapiński, Joanna Kałafut, Adolfo Rivero-Müller
bioRxiv 2023.03.16.533022; doi: https://doi.org/10.1101/2023.03.16.533022

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