A dCas9 Proximity Reporter for Investigating DNA Linear and Rotational Dynamics

The dCas9 system has rapidly been developed into many tools to explore different aspects of the human genome; the high binding specificity, coupled with the inactive nuclease enzyme, allows for precise recruitment of molecules to specific sequences of DNA. We sought to exploit these capabilities to create a tool for assessing the real-time proximity of two DNA sequences within a biological system. By incorporating aptamers into gRNAs, dCas9 molecules can be used to recruit the {beta}9 or {beta}10 strands of split-NanoLuc(R) to specific DNA sequences and quantify the proximity of those sequences based on their ability to complex with the luciferase fragment ({Delta}11S) and produce luminescence. While many tools exist to detect a single DNA sequence, this system is uniquely capable of assessing how two DNA sequences interact with each other. As expected, we found that the interaction of two dCas9 molecules was affected by their linear distance from each other on dsDNA. Surprisingly, we found that their interaction was also strongly influenced by rotational orientation, even for sequences that are close together in linear space. This finding indicates that dCas9 rotational alignment is an important consideration for designing dCas9 systems that target multiple DNA sequences simultaneously. Beyond the findings presented herein, we believe this DNA proximity detection tool has the potential to be adapted for applications involving the proximity and orientation of two DNA sequences.