Abstract
AbstractStreptococcus pyogenesCRISPR-Cas9 (SpCas9) stabilizes each strand of a DNA bubble via distinct interactions, forming a stable ~20 bp R-loop on the complementary strand but relying on narrower protein-DNA interactions to bind the non-complementary strand with 5’ NGG protospacer adjacent motif (PAM). The enzyme’s HNH endonuclease domain cleaves DNA within the R-loop, and its RuvC endonuclease cleaves 5’ to the NGG and opposite the HNH cleavage site to generate blunt DNA ends. We show that this nucleoprotein complex can be mechanically strained by shifting the RNA:DNA hybrid and that the HNH cleavage site tracks this shift but the RuvC cleavage site does not, resulting in overhanging DNA ends. This is observed using cleavage in living cells and sequencing assays to characterize overhangs generated by stressed complexes, as well as single-molecule cleavage assays to characterize differential cleavage kinetics of HNH and RuvC endonucleases in stressed complexes as well as complex disassembly.Using Sanger sequencing and high-throughput genome sequencing of DNA cleavage reactions, we find that the SpCas9 complex responds to internal mechanical strain by robustly generating overhanging, rather than blunt, DNA ends. Internal mechanical strain is generated by increasing or decreasing the spacing between the RNA:DNA hybrid and the downstream canonical PAM. Up to two-base 3’ overhangs can be robustly generated via a two-base increase in the distance between hybrid and PAM. We also use single-molecule experiments to reconstruct the full course of the CRISPR-SpCas9 reaction in real-time, monitoring and quantifying the R-loop formation, the first and second DNA incision events, and the post-catalytic complex dissociation. Complex dissociation and release of broken DNA ends appears to be a rate-limiting step of the reaction, and strained SpCas9 are sufficiently destabilized so as to rapidly dissociate after formation of broken DNA ends.
Publisher
Cold Spring Harbor Laboratory