Investigation of the Cas9 Mediated DNA Cleavage using Time-Lapse AFM Imaging

Abstract

Bacteria and archaea have a defense mechanism for the viral DNA invasion that is facilitated by the clustered regularly interspaced short palindromic repeats (CRISPR), and the CRISPR associated (Cas) proteins. The viral infections trigger their adaptive immune system by insertion the foreign DNA segments into the CRISPR loci followed by transcribing the small RNAs with Cas proteins. In particular, type II system cas protein (cas9) associates with the guide RNA to target and silence the viral infections by cleaving the both strands of DNA. The ability of substituting the viral specific RNA with another desired guide RNA makes cas9:RNA system an interest of not only bacterial immunity, but also genetic engineering. Recently, several reports demonstrated the successful repression of genes by means of cas9 enzyme when the target sequence is closer to the promoter site. Despite rapidly increasing number of studies related to the cas9 application, kinetics of cas9 based cleavage mechanism is weakly understood. Using Atomic Force Microscopy (AFM), we monitored the cas9 cleavage activity of the target DNA upon introducing the cas9:RNA complexes to the aqueous environment. We will present our kinetic analysis of cas9 nuclease activity. Our approach may shed light on the possible single-turnover kinetic feature of the cas9 enzyme and help improve cas9-mediated gene editing and therapeutic developments.