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Towards a translationally-independent RNA-based synthetic oscillator using deactivated CRISPR-Cas

James Kuo, Ruoshi Yuan, Carlos Sánchez, Johan Paulsson, Pamela A. Silver
doi: https://doi.org/10.1101/2020.05.13.094730
James Kuo
aDepartment of Systems Biology, Blavatnik Institute at Harvard Medical School, Boston, MA 02115, USA
bWyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA 02115, USA
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Ruoshi Yuan
aDepartment of Systems Biology, Blavatnik Institute at Harvard Medical School, Boston, MA 02115, USA
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Carlos Sánchez
aDepartment of Systems Biology, Blavatnik Institute at Harvard Medical School, Boston, MA 02115, USA
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Johan Paulsson
aDepartment of Systems Biology, Blavatnik Institute at Harvard Medical School, Boston, MA 02115, USA
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Pamela A. Silver
aDepartment of Systems Biology, Blavatnik Institute at Harvard Medical School, Boston, MA 02115, USA
bWyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA 02115, USA
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  • For correspondence: pamela_silver@hms.harvard.edu
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Abstract

In synthetic circuits, CRISPR-Cas systems have been used effectively for endpoint changes from an initial state to a final state, such as in logic gates. Here, we use deactivated Cas9 (dCas9) and deactivated Cas12a (dCas12a) to construct dynamic RNA ring oscillators that cycle continuously between states over time in bacterial cells. While our dCas9 circuits using 103-nucleotide guide RNAs showed irregular fluctuations with a wide distribution of peak-to-peak period lengths averaging ∼9 generations, a dCas12a oscillator design with 40-nucleotide CRISPR RNAs performed much better, having a strongly repressed off-state, distinct autocorrelation function peaks, and an average peak-to-peak period length of ∼7.5 generations. Along with free-running oscillator circuits, we measure repression response times in open-loop systems with inducible RNA steps to compare with oscillator period times. We track thousands of cells for 24+ hours at the single-cell level using a microfluidic device. In creating a circuit with nearly translationally-independent behavior, as the RNAs control each others’ transcription, we present the possibility for a synthetic oscillator generalizable across many organisms and readily linkable for transcriptional control.

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 May 14, 2020.
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Towards a translationally-independent RNA-based synthetic oscillator using deactivated CRISPR-Cas
James Kuo, Ruoshi Yuan, Carlos Sánchez, Johan Paulsson, Pamela A. Silver
bioRxiv 2020.05.13.094730; doi: https://doi.org/10.1101/2020.05.13.094730
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Towards a translationally-independent RNA-based synthetic oscillator using deactivated CRISPR-Cas
James Kuo, Ruoshi Yuan, Carlos Sánchez, Johan Paulsson, Pamela A. Silver
bioRxiv 2020.05.13.094730; doi: https://doi.org/10.1101/2020.05.13.094730

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