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

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-nt guide RNAs showed irregular fluctuations with a wide distribution of peak-to-peak period lengths averaging approximately nine generations, a dCas12a oscillator design with 40-nt 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+ h 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.

journal_name

Nucleic Acids Res

journal_title

Nucleic acids research

authors

Kuo J,Yuan R,Sánchez C,Paulsson J,Silver PA

doi

10.1093/nar/gkaa557

subject

Has Abstract

pub_date

2020-08-20 00:00:00

pages

8165-8177

issue

14

eissn

0305-1048

issn

1362-4962

pii

5866101

journal_volume

48

pub_type

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