RNA‐guided transcriptional activation of TIMP2 utilizing CRISPR:Cas9 (1148.13)

The clustered, regularly interspaced palindromic repeat (CRISPR) system is capable of genome‐specific targeting utilizing the CRISPR associated 9 (Cas9) endoribonuclease and effector targeting by a single guide RNA (sgRNA). We show that fusion of the transcriptional activating viral protein, VP64, to an enzymatically dead form of Cas9 (dCas9) is capable of enhancing gene‐targeted transcriptional activity. Using a luciferase reporter assay we show that a N‐terminal fusion of VP64 to dCas9 (dCas9.nVP64) results in higher transcriptional activity compared to the C‐terminal or flanked VP64 fusion to dCas9. Additionally, we find that there are optimal targets within the murine Tissue Inhibitor of Metalloproteinase 2 (TIMP2) promoter that results in maximal transcriptional activation and that multiple sgRNAs expressed simultaneously results in a synergistic enhancement of promoter activity. Finally, using a genome‐wide bioinformatics approach we identified a number of potential off target sites for a given sgRNA‐target within the promoter of other genes. Selective activation of TIMP2 with the CRISPR‐Cas9 system opens up a strategy for treating diseases associated with over activity of the Matrix Metalloproteinases. Grant Funding Source : Supported by the Training Program in Translational Cardiovascular Science, 5 T32 HL 7936‐12