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. 2023 Nov 17;14:7454. doi: 10.1038/s41467-023-43241-y

Fig. 1. Design and characterization of a three-node ring oscillator ‘CRISPRlator’ using extended sgRNAs.

Fig. 1

a Schematic overview of the use of ext-sgRNAs to construct a three-node ring oscillator. Ext-sgRNA1 has a spacer sequence (sgRNA6) that is complementary to binding site 6 (BS6) of the non-template strand of the DNA sequence encoding ext-sgRNA2. Ext-sgRNA2 has a spacer (sgRNA3) targeting BS3 present on the DNA sequence of ext-sgRNA3. Ext-sgRNA3 has a spacer (sgRNA2) targeting BS2 present on the DNA sequence of ext-sgRNA1. For clarity, only the variable 5’ region of ext-sgRNAs sequences is shown (the dCas9-binding handle and transcription terminator are not shown). b Graphical representation of the three-node ring oscillator called CRISPRlator. Expression of ext-sgRNA1 represses transcription of the mScarlet-I reporter (red); ext-sgRNA2 represses mTurquoise2 (blue) and ext-sgRNA3 represses mNeonGreen (green). As the ext-sgRNAs also repress each other’s transcription (a), this GRN is expected to periodically express the three reporters in the order red->green->blue. c Snapshots with 3h45min intervals of a typical microfluidics experiment of the CRISPRlator strain are shown. Scale bar 2 µm. Time in hours and minutes. d Autocorrelation function (ACF) of the CRISRlator cells grown in mother machine channels. ACF calculated in one mother machine lane for individual cells (thin lines) and averaged (fat line with black outlines) shows oscillations for all three fluorescent signals. e Progression of cell length (left) and progression of normalized fluorescent signals (right) of one individual mother cell in the mother machine over time. Source data for d and e are provided as a Source Data file.