Site-directed spin labeling studies of target DNA recognition by a CRISPR nuclease

2018-07-15Clustered, regularly-interspaced, palindromic repeats (CRISPR) systems derived from bacterial immunity are well-known for their nuclease activity. In a widely-studied type II CRISPR system, two short RNAs complex with the CRISPR-associated protein Cas9 and serve as guides for a sequence-specific duplex DNA target. A CRISPR-Cas9 system using a single guide RNA as a substitute for the native short RNAs has been used for genome editing in various cell types across multiple species. The tunable nature, fidelity, and versatility of CRISPR-Cas9 have contributed to its central role in the CRISPR revolution in genome engineering. Many studies have focused on the structure and mechanism of function of Cas9, and although a great deal has been reported in recent years, questions about the specific process of DNA targeting still remain unanswered. The work presented in this thesis uses the method of site-directed spin labeling (SDSL) to attach stable nitroxide radical probes along a DNA backbone in a sequence-independent scheme, which are monitored by electron paramagnetic resonance (EPR) spectroscopy. Continuous-wave (cw) EPR reports dynamic information at the nitroxide labeling site, and double electron-electron resonance (DEER) spectroscopy measures distances between pairs of nitroxide labels on the molecule of interest. In these studies, nitroxide spin probes were attached at several sites along a CRISPR-Cas9 target DNA duplex, and both cwEPR and DEER were performed to directly monitor DNA unwinding and inform on DNA conformation within a CRISPR-Cas9 complex. The results validate that SDSL is a valuable method to provide structural information on CRISPR-Cas9 target DNA unwinding and reveal significant guide RNA-dependent DNA conformational features, towards elucidating the intricate mechanism of CRISPR-Cas9 DNA targeting