Shifted PAMs generate DNA overhangs and enhance SpCas9 post-catalytic complex dissociation

Using Sanger sequencing and high-throughput genome sequencing of DNA cleavage reactions, we find that the Streptococcus pyogenes SpCas9 complex responds to internal mechanical strain by robustly generating a distribution of overhanging, rather than blunt, DNA ends. Internal mechanical strain is generated by shifting (increasing or decreasing) the spacing between the RNA-DNA hybrid and the downstream canonical PAM. Up to 2-base 3 ' overhangs can be robustly generated via a 2-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, structurally and kinetically monitoring and quantifying R-loop formation, the first and second DNA-incision events, and dissociation of the post-catalytic complex. Complex dissociation and release of broken DNA ends is a rate-limiting step of the reaction, and shifted SpCas9 is sufficiently destabilized so as to rapidly dissociate after formation of broken DNA ends. Here, using single-molecule experiments, the authors show that SpCas9 responds to shifted PAMs by inducing overhanging DNA ends. Such shift-PAM targeting is enhanced by physiological levels of DNA supercoiling and in turn promotes dissociation of the complex after catalysis.

Automated summary

Using Sanger sequencing, high-throughput genome sequencing, and single-molecule experiments, the authors observe that the Streptococcus pyogenes SpCas9 complex generates overhanging DNA ends in response to internal mechanical strain. They also find that the dissociation of the complex and release of broken DNA ends are rate-limiting steps of the reaction.