Tailoring the CRISPR system to transactivate coagulation gene promoters in normal and mutated contexts

Publication date: Available online 18 April 2019Source: Biochimica et Biophysica Acta (BBA) - Gene Regulatory MechanismsAuthor(s): Silvia Pignani, Federico Zappaterra, Elena Barbon, Antonia Follenzi, Matteo Bovolenta, Francesco Bernardi, Alessio Branchini, Mirko PinottiAbstractEngineered transcription factors (TF) have expanded our ability to modulate gene expression and hold great promise as bio-therapeutics. The first-generation TF, based on Zinc Fingers or Transcription-Activator-like Effectors (TALE), required complex and time-consuming assembly protocols, and were indeed replaced in recent years by the CRISPR activation (CRISPRa) technology. Here, with coagulation F7/F8 gene promoters as models, we exploited a CRISPRa system based on deactivated (d)Cas9, fused with a transcriptional activator (VPR), which is driven to its target by a single guide (sg)RNA.Reporter gene assays in hepatoma cells identified a sgRNA (sgRNAF7.5) triggering a ~35-fold increase in the activity of F7 promoter, either wild-type, or defective due to the c.-61T>G mutation. The effect was higher (~15-fold) than that of an engineered TALE-TF (TF4) targeting the same promoter region. Noticeably, when challenged on the endogenous F7 gene, the dCas9-VPR/sgRNAF7.5 combination was more efficient (~6.5-fold) in promoting factor VII (FVII) protein secretion/activity than TF4 (~3.8-fold). The approach was translated to the promoter of F8, whose reduced expression causes hemophilia A. Reporter gene assays in h...
Source: Biochimica et Biophysica Acta (BBA) Gene Regulatory Mechanisms - Category: Genetics & Stem Cells Source Type: research