Assaying the order of transcriptional events in cells through CRISPR/Cas9

Cells and organisms are complex systems whose life and development result from the action of entangled cellular pathways and machineries. Phenotypes are triggered by the order in which cellular events occur. Yet, while interrogating individual events is relatively straightforward, it is difficult to assess the specific order and relevance of events when several cooperate to induce different phenotypes. Recently, new techniques greatly improved our ability to obtain information from cells by enabling the longitudinal tracking of molecular events into genomic DNA. Yet, these tools are still unable to relay the order in which different events occur. During my PhD I set up a system to assess the order of two transcriptional events leading to a specific phenotype in mammalian cells. To this aim I am exploited the CRISPR/Cas9 system to trigger the recombination of an artificial DNA cassette in which barcode sequences are interspersed with two sets of guideRNA targets. The transcription of Cas9 and of two sgRNAs induces double-strand breaks and the recombination of the cassette, depending upon the order in which the sgRNAs have been transcribed. Sequencing of the recombined cassette thus allows to ascertain the sequence of recombinational events leading to the recombination (i.e., the order in which the sgRNAs were used to target the cassette), which in turn acts as a proxy of the order of the cellular events leading to the expression of the individual sgRNAs. I have obtained proof of principle of my assay. Having obtained a stable cell line carrying a single copy of the reporter cassette, I sequentially expressed in HEK293T cells the individual Cas9/sgRNA pairs, using different orders of expression. I then recovered the cells and amplified the reporter cassette: Sanger sequencing of the barcodes present in the recombined cassette confirmed that the system worked as intended. In order to simplify the system and make it more robust, I have redesigned the cassette by replacing the DNA barcodes with cassettes driving the expression of four fluorescent reporters. This allows me to visualize the recombination outcome by flow cytometry using live cells: only two fluorescent reporters are expressed in the cells in their initial status, and the sequential recombination of the artificial cassette will drive the expression of the other fluorescent proteins. Each combination of fluorescence reflects a well-defined order of transcriptional activation of the guideRNAs. Beyond making the procedure more straightforward, the use of fluorescent reporters and flow cytometry allow me to assess each cell individually. A defined order of activation for these factors could explain the differences observed in re-programmability among cells, and could be exploited to improve our reprogramming techniques.