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. 2020 Jul 1;48(14):8165–8177. doi: 10.1093/nar/gkaa557

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© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

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Figure 4. — dCas12a RNA oscillator shows regularity. (A) An analogous RNA ring oscillator design replaces dCas9 with deactivated Cas12a (dCas12a). (B) Cas12a CRISPR RNA (crRNA) architecture. Designs use a 19-nt direct repeat dCas12a handle plus 20-nt targeting region. The PAM is ‘TTV’ for Francisella novicida Cas12a (FnCas12a). (C) Plasmid design of the dCas12a RNA oscillator circuit has a PA4-mVenus reporter, Tet-inducible dCas12a and crRNAs in a transcriptionally convergent direction. (D and E) Escherichia coli MG1655 cells with the circuit were grown in mother machine chips with EZ-RDM. (D) A kymograph of a single trench over time shows YFP reporter fluctuation. Each frame is 8 min, with one generation ∼25 min. Arrow marks induction for dCas12a production at frame 23 (184 min). (E) Example reporter traces show fluctuations resembling oscillations and a lower signal repressed state. Average generation ∼25 min. Pullout for trace viii zooms in on the peak shapes.