CRISPR /Cas9 searches for a protospacer adjacent motif by lateral diffusion

Abstract

The Streptococcus pyogenes CRISPR/Cas9 (SpCas9) nuclease has been widely applied in genetic engineering. Despite its importance in genome editing, aspects of the precise molecular mechanism of Cas9 activity remain ambiguous. In particular, because of the lack of a method with high spatio‐temporal resolution, transient interactions between Cas9 and DNA could not be reliably investigated. It therefore remains controversial how Cas9 searches for protospacer adjacent motif (PAM) sequences. We have developed single‐molecule Förster resonance energy transfer (smFRET) assays to monitor transient interactions of Cas9 and DNA in real time. Our study shows that Cas9 interacts with the PAM sequence weakly, yet probing neighboring sequences via facilitated diffusion. This dynamic mode of interactions leads to translocation of Cas9 to another PAM nearby and consequently an on‐target sequence. We propose a model in which lateral diffusion competes with three‐dimensional diffusion and thus is involved in PAM finding and consequently on‐target binding. Our results imply that the neighboring sequences can be very important when choosing a target in genetic engineering applications.

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

“…When Cas9 encounters the PAM site of its cognate target, it initiates RNA strand invasion and R-loop expansion (Gorski et al, 2017;Jiang and Doudna, 2017). On the other hand, Cas9 continues its 1D diffusion on dsDNA after transient binding with PAM sites flanking nontarget sequence, which is consistent with previous reports (Globyte et al, 2019). Lastly, we quantified that 1D diffusion facilitates target search of Cas9 by ~ 10 folds ( Fig.…”

“…Strikingly, extension of dsDNA on either 5' or 3' end of NT strand increased rates of Cas9 target search, suggesting an unbiased 1-D diffusion mechanisms at low salt condition. Our findings agreed with previous smFRET measurements in the same low-salt buffer (Globyte et al, 2019).…”

“…We quantified that the bias 1D diffusion length of Cas9 was 27 ± 7 bp ( Fig. 3g), which is close to the previously reported ~ 20 bp of the diffusion length (Globyte et al, 2019).…”

“…DNA curtain experiments proposed a random collision (threedimensional diffusion, 3D diffusion) model of Cas9 to search for its targets . With improved spatial resolution, single-molecule fluorescence resonance energy transfer (smFRET) assays under low salt conditions capture one-dimensional (1D) diffusion of Cas9 among multiple PAM sites on the same dsDNA (Globyte et al, 2019). However, how Cas9 finds its target under physiological salt conditions remains largely unknown.…”

Argonaute bypasses cellular obstacles without hindrance during target search

“…Previous studies have shown that certain DNA/RNA-guided proteins interact with DNA through non-specific electrostatic interactions 22-24 , but none so far have studied the nature of these interactions and their contribution on lateral diffusion. Since these lived 12,22,27-29 , we require a method offering high spatiotemporal resolution.…”