Site-Directed Spin Labeling Studies of Nucleic Acid Dependent Conformational Changes in CRISPR-Cas9

Abstract

In a type II clustered, regularly interspersed, short palindromic repeats (CRISPR) system, RNAs derived from the CRISPR locus complex with the CRISPR-associated (Cas) protein Cas9 to form an RNA-guided nuclease that cleaves double-stranded DNAs based on specificity dictated by base-pairing between RNA and DNA. In recent years, the CRISPR-Cas9 system has been successfully adapted for genome engineering in a wide range of organisms. Studies have indicated that a series of conformational changes in Cas9, dictated by the RNA and the target DNA, directs Cas9 into its active conformation, yet, details on these conformational changes, as well as their roles in the mechanism of function of Cas9, remains to be elucidated. Here, we investigate nucleic acid dependent conformational changes in Streptococcus pyogenes Cas9 (SpyCas9) using the method of site-directed spin labeling (SDSL). SDSL monitors site-specifically attached stable radicals (e.g., nitroxide spin labels) using electron paramagnetic resonance (EPR) spectroscopy, and provides structural (e.g., distance constraints) and dynamic (e.g., motions at the labeling site) information on the parent molecule. Using nitroxide spin labels attached to selective sites of Cas9, we were able to monitor conformational changes of Cas9 upon binding to its RNA partner. Work is ongoing to dissect the details of these conformational changes, as well as the critical elements dictating such changes. The results will provide key information for understanding the mechanism of Cas9 function.