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

• Published in: PloS one 2020-07, Vol.15 (7), p.e0235530-e0235530
• Main Authors: Kirchner, Steven, Reuter, Stefanie, Westphal, Anika, Mrowka, Ralf
• Format: Article
• Language: English
• Subjects: Activation; Binding sites; Biology and Life Sciences; Chromosome 1; Chromosomes; Complexity; Computer applications; Context; CRISPR-Cas systems; CRISPR-Cas Systems - genetics; Deoxyribonucleic acid; DNA; DNA binding proteins; Experiments; Fireflies; Gene Editing; Gene expression; Gene regulation; Genes, Reporter; Genetic aspects; Genetic research; Genomics; HEK293 Cells; Homology; Humans; Hypertension; Luciferases, Firefly - genetics; Luciferases, Firefly - metabolism; Mathematical models; Mutation; Nephrology; Promoter Regions, Genetic; Promoters (Genetics); Proteins; Regulatory sequences; Regulatory Sequences, Nucleic Acid - genetics; Renin; Renin - genetics; Research and Analysis Methods; Ribonucleic acid; RNA; RNA polymerase; RNA, Guide, CRISPR-Cas Systems; Statistical analysis; Statistical modelling; Statistical models; Studies; Transcription; Transcription (Genetics); Transcription factors; Transgenic animals
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0235530

Understanding complex mechanisms of human transcriptional regulation remains a major challenge. Classical reporter studies already enabled the discovery of cis-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 of renin transcription in its native genomic context. With the help of genomic editing, we stably tagged the native renin on chromosome 1 with the firefly luciferase and stably integrated a programmable modified Cas9 based trans-activation complex (SAM-complex) by lentiviral transduction into human cells. By delivering five specific guide-RNA homologous to specific promoter regions of renin we were able to guide this SAM-complex to these regions of interest. We measured gene expression and generated and compared computational models. SAM complexes induced activation of renin in our cells after renin specific 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. By applying our combined experimental and modelling approach we can show that interactions occur within the selected sequences of the proximal renin promoter region. This combined approach might potentially be useful to investigate other genomic regions. Our findings may help to better understand the transcriptional regulation of human renin.