Mechanism of Duplex DNA Destabilization by RNA-Guided Cas9 Nuclease during DNA Interrogation

Abstract

The Cas9 endonuclease cleaves double-stranded DNA bearing sequences complementary to a 20-nucleotide segment in the guide RNA (gRNA), which are flanked by a conserved PAM sequence. The Cas9-gRNA system has been successfully used for genome editing in various organisms. The Cas9-gRNA effector complex locates the target site by scanning and interrogating the genomic DNA. Unpairing of a short PAM-proximal segment of DNA is a critical event in DNA interrogation that may determine the overall rate of target location. We sought to elucidate mechanism of initial DNA interrogation steps that precede the pairing of target DNA with gRNA. Using fluorometric and biochemical assays, we studied Cas9-gRNA complexes with model DNA substrates likely mimicking early intermediates on the pathway to the final Cas9-gRNA-DNA R-loop complex. The results show that Cas9-gRNA binding to PAM favors separation of few nearest to PAM base pairs but causes no significant effect on DNA duplex stability in the protospacer region distant from PAM. The destabilization of DNA duplex is achieved primarily through interactions of Cas9-gRNA with non-target DNA strand nucleotides and hindering of duplex DNA entry into the Cas9-gRNA interior. The data also suggest that interaction of the target DNA strand with the “phosphate lock” loop of Cas9 is subsequent to the initial destabilization step and is coupled with the DNA-gRNA pairing.