Towards 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 103nucleotide 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 freerunning 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.

Single cell characterization of a synthetic bacterial clock with a hybrid feedback loop containing dCas9-sgRNA

“…Until now, CRISPRi has only rarely been used in the context of dynamical gene circuits, however. Notable exceptions are two recent examples of oscillating networks, in which three single guide RNAs (sgRNAs) cyclically repressed each other's transcription in the presence of dCas9 [20,21] or dCas12a [21]. In the present work, we took a hybrid approach -which we termed 'Rock Paper CRISPR' (RPC) oscillator-, in which only one of the transcriptional repressor links of a repressilator network was replaced by CRISPRi.…”