DNA Unwinding by CRISPR-Cas9 Studied using Site-Directed Spin Labeling

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 sequence-specifically. In recent years, the CRISPR-Cas9 system has been successfully adapted for genome engineering in a wide range of organisms; however, its mechanism of function at the molecular level remains to be fully understood. A key step in Cas9 target selection is the unwinding of the target DNA duplex to allow formation of a three-stranded R-loop, in which the guide segment of the RNA is base-paired to the protospacer segment residing at one of the DNA strands. Here, we investigate DNA deformation and unwinding in Streptococcus pyogenes Cas9 (SpyCas9) complexes using the method of site-directed spin labeling (SDSL) with electron paramagnetic resonance (EPR) spectroscopy. Nucleotide-independent nitroxide labels were attached at selected sites of a target DNA duplex, and labeled DNAs were assembled with SpyCas9 and the corresponding RNA, resulting in minimal perturbation of native activity. EPR measurements revealed distinct increases in inter-strand distances along the target DNA duplex in the Cas9 complex, consistent with DNA deformation and unwinding. Importantly, the inter-strand distances increased non-uniformly along the protospacer segment of the DNA, indicating a varying degree of DNA unwinding within the region complementary to the guide RNA. Additional work is ongoing to more clearly understand the mechanism of DNA unwinding and its connection with target cleavage by Cas9.