A CRISPR-based flow cytometric approach to to assess the order of transcriptional events

Recently many devices have been developed to record and store information in living cells, however most of them are limited by their use only in prokaryotes or only allow us to obtain snapshots of cellular events at a given time. Many efforts are ongoing to develop systems to test the order in which different events occur in a mammalian cell system to obtain a specific phenotype. Based on this, I set up an assay to verify a posteriori the order in which two different transcriptional events occur in a mammalian cell through a genome editing tool based on the CRISPR/Cas9 system. Artificial DNA targets for a pair of sgRNAs and Cas9 have been interspersed in a cassette containing the coding sequence of four fluorescent proteins. Transcription of Cas9 and of a specific sgRNAs will trigger double strand breaks in the cassette and its recombination: from an initial state in which two fluorescent proteins are expressed, the recombination of the cassette will result in the loss/acquisition of the other fluorescent proteins in combinations dependent from the order in which the sgRNAs are transcribed.