Streptococcus pyogenes Cas9 displays biased one-dimensional diffusion on dsDNA to search for a target

The RNA-guided Streptococcus pyogenes Cas9 (spCas9) is a sequence-specific DNA endonuclease that works as one of the most powerful genetic editing tools. However, how the Cas9 locates its target among huge amounts of dsDNAs remains controversial. Here, combining biochemical and single-molecule assays, we revealed that Cas9 uses both three-dimensional and one-dimensional diffusion to find its target. We further observed a surprising biased one-dimensional diffusion of Cas9 from 39 to 59 end of the non-target strand under physiological salt condition, whereas low ionic concentration or mutations on PAM recognition residues induce unbiased one-dimensional diffusion of Cas9 along dsDNA. We quantified the diffusion length of 27 bp, which accelerates the target search efficiency of Cas9 by ~ 10 folds. Our results reveal a unique searching mechanism of Cas9 at physiological salt conditions, and provide important guidance for both in-vitro and in-vivo applications of Cas9.