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Detecting Cas9-sgRNA Complex Interactions with DNA via Fluorescent Microscopy: Computer Simulations of Experimental Designs
At the center of the modern CRISPR/Cas genome editing revolution is the engineered ribonucleoprotein complex of the Cas9 protein with sgRNA that is able to bind and cleave DNA at specific loci. The targeted activity is conferred by the ability of the complex to bind to the DNA regions which are complementary to the parts of the sequence encoded by the sgRNA. Apart from DNA cleavage activity the selective binding activity of CRISPR/Cas complexes alone have been exploited in many technological applications to target certain compounds to specific DNA loci (e.g. for detecting certain DNA sequences, marking up certain portions of the genome with fluorescent labels, altering epigenetic state of the genetic loci, etc.). The most common version of the CRISPR/Cas editing system is currently based on the spCas9 protein obtained from S. pyogenes. However, search for other systems which are more effective and have less off-target activity is under way. Particularly, Cas proteins from other species including alternative CRISPR systems (e.g. recently characterized CasX system) combined by artificially engineered mutations can be attempted[1,2]. Hence, an effective in vitro assay to characterize the binding affinity of the Cas- based ribonucleoprotein complex to its target DNA sequence is of high methodological importance. An effective way of measuring the biomolecular complex affinity can be based on using the FRET microscopy by monitoring the increase of the FRET signal as the two molecules labeled with a corresponding pair of fluorescent dyes bind together [3,4]. In this work by employing atomistic molecular simulations we show that such measurements are possible to study the binding of Cas9-sgRNA complex to its target DNA by attaching Cy3 and Cy5 labels to specific sites at DNA and RNA molecules. We simulate various attachment possibilities for optimal experimental designs.