Decipher the complexity of cis-regulatory regions by a modified Cas9

Background Understanding complex mechanisms of human transcriptional regulation remains a major challenge. Classical reporter studies already enabled the discovery ofcis-regulatory elements within the non-coding DNA; however, the influence of genomic context and potential interactions are still largely unknown. Using a modified Cas9 activation complex we explore the complexity ofrenintranscription in its native genomic context. Methods With the help of genomic editing, we stably tagged the nativereninon chromosome 1 with thefirefly luciferaseand stably integrated a programmable modified Cas9 basedtrans-activation complex (SAM-complex) by lentiviral transduction into human cells. By delivering five specific guide-RNA homologous to specific promoter regions ofreninwe were able to guide this SAM-complex to these regions of interest. We measured gene expression and generated and compared computational models. Results SAM complexes induced activation ofreninin our cells afterreninspecific guide-RNA had been provided. All possible combinations of the five guides were subjected to model analysis in linear models. Quantifying the prediction error and the calculation of an estimator of the relative quality of the statistical models for our given set of data revealed that a model incorporating interactions in the proximal promoter is the superior model for explanation of the data. Conclusion By applying our combined experimental and modelling approach we can show that interactions occur within the selected sequences of the proximalreninpromoter region. This combined approach might potentially be useful to investigate other genomic regions. Our findings may help to better understand the transcriptional regulation of humanrenin.