The post-PAM interaction of RNA-guided spCas9 with DNA dictates its target binding and dissociation

Abstract

Cas9 is an RNA-guided endonuclease that targets complementary DNA for cleavage and has been repurposed for many biological usages. Cas9 activities are governed by its direct interactions with DNA. However, information about this interplay and the mechanism involved in its direction of Cas9 activity remain obscure. Using a single-molecule approach, we probed Cas9/sgRNA/DNA interactions along the DNA sequence and found two stable interactions flanking the protospacer adjacent motif (PAM). Unexpectedly, one of them is located approximately 14 base pairs downstream of the PAM (post-PAM interaction), which is beyond the apparent footprint of Cas9 on DNA. Loss or occupation of this interaction site on DNA impairs Cas9 binding and cleavage. Consistently, a downstream helicase could readily displace DNA-bound Cas9 by disrupting this relatively weak post-PAM interaction. Our work identifies a critical interaction of Cas9 with DNA that dictates its binding and dissociation, which may suggest distinct strategies to modulate Cas9 activity.

Dynamics of Staphylococcus aureus Cas9 in DNA target Association and Dissociation

“…Instead, as indicated by the decreases in the DNA length upon the collisions, the exonuclease activity of Phi29 DNAP was more prone to being activated. It is noteworthy that these results are in stark contrast to our previous findings with dSpCas9, in which it could be disrupted by BLM and Phi29 DNAP from both directions (Zhang et al, 2019).…”

“…We recently reported similar interaction sites of SpCas9 with its DNA target (Zhang et al, 2019). However, dSaCas9 differs from dSpCas9 in that its pre-PAM interaction is significantly strong and is not affected by disruption from the post-PAM interaction in some cases ( Fig 1C).…”

“…According to the structural data (Nishimasu et al, 2015), this interaction is possibly achieved via extensive interactions among the RNA-DNA heteroduplex close to the PAM, the bridge helix and the REC lobe of the SaCas9 protein. Compared with SpCas9, wherein the disruption of its post-PAM interaction results in an immediate loss of its pre-PAM interaction and consequently its dissociation from the DNA (Zhang et al, 2019), the pre-PAM interaction of dSaCas9 is significantly strong and can be less affected by the disruption of its post-PAM interaction (Fig 1C). Moreover, we demonstrated that even though the post-PAM interaction of wildtype SaCas9 is stronger than that of dSaCas9 ( Figs 1D and EV4A), a downstream helicase can still displace it (Fig 5F).…”

“…Single-molecule DNA unzipping data were taken at 5 kHz and later filtered to 50 Hz. The acquired data signals were converted into force and DNA extension as described previously (Zhang et al, 2019). The elasticity parameters of both the dsDNA and ssDNA for data conversion were obtained from DNA force-extension measurements in previous studies (Zhang et al, 2019).…”

Spatiotemporal Control of CRISPR/Cas9 Function in Cells and Zebrafish using Light‐Activated Guide RNA

“…The disrupted interaction between dsDNAa nd the RNP complex is the result of blocked Watson-Crick base-pairing by the caging groups, [25] thus preventing further DNAbinding after recognition of the PA Ms equence and subsequent cleavage of the DNAt arget. [29] Notably,b oth non-caged and caged gRNAs bind to the Cas9 protein with similar affinity,d emonstrating that the caging of the protospacer region of the gRNAdoes not interfere with formation of the Cas9:…”