RNA-induced structural stabilization in Cas9

We first observed apo-Cas9 and pre-assembled Cas9–RNA on a mica surface treated with 3-aminopropyl-triethoxysilane (AP-mica). Unexpectedly, the HS-AFM movies revealed that apo-Cas9 adopts flexible modular conformations, unlike the stable closed conformation observed in the crystal structure12 (Fig. 1b, c, Supplementary Movie 1). In contrast, the HS-AFM movies of Cas9–RNA showed a stable bilobed architecture, consistent with the crystal structure13 (Fig. 1b, d, Supplementary Movie 2). The correlation coefficients for the sequential HS-AFM images highlighted the substantial differences in the conformational flexibilities between apo-Cas9 and Cas9–RNA (Fig. 1e, Supplementary Fig. 2a, b). A structural comparison between apo-Cas912 and Cas9–RNA13 indicated that the three domains (REC1–3) in the REC lobe adopt distinct arrangements, whereas the RuvC domain interacts similarly with the HNH and PAM-interacting (PI) domains to form the NUC lobe structure (Fig. 1b). This supports the notion that the three REC domains of apo-Cas9 adopt flexible conformations in solution, although apo-Cas9 adopted a closed conformation in the crystal structure, probably due to crystal packing interactions. Together, our HS-AFM data reveal the unexpected conformational flexibility of apo-Cas9, and highlight the guide-RNA-mediated stabilization of the REC lobe conformation and induction of structural rearrangements in the Cas9 protein.

PAM-dependent DNA targeting by Cas9–RNA

We next sought to visualize the binding of Cas9–RNA to the target DNA. To avoid Mg2+-dependent DNA cleavage by Cas97, we incubated the pre-assembled Cas9–RNA and a 600-bp dsDNA containing a 20-nt target site with the TGG PAM 400-bp downstream from its 5′ end, in the absence of Mg2+ (Fig. 2a). We then adsorbed the Cas9–RNA–DNA complex on the AP-mica surface, and performed HS-AFM observations. The HS-AFM movies revealed that Cas9–RNA specifically binds to the expected target site in the DNA (Fig. 2b, c, Supplementary Movie 3). An analysis of the HS-AFM images confirmed the specific binding of Cas9–RNA to the target site in all of the observed DNA molecules (Fig. 2d, Supplementary Fig. 3a). In contrast, Cas9–RNA did not bind to the target DNA containing TTT, rather than TGG, as the PAM (Supplementary Fig. 3b), consistent with the observation that Cas9 requires the NGG sequence as the PAM for DNA recognition7,11. These results demonstrate that our HS-AFM movies faithfully recapitulate the PAM-dependent target recognition by Cas9–RNA.

Fig. 2 HS-AFM observations of Cas9–RNA–DNA. a Schematic of the dsDNA substrate. The target site and the PAM are colored blue and purple, respectively. The sites cleaved by the RuvC and HNH domains are indicated by the cyan and magenta triangles, respectively. TS, target strand; NTS, non-target strand. b HS-AFM image of Cas9–RNA–DNA in the absence of MgCl 2 . The scale bar is 20 nm. c Cross-sectional profile along the DNA in a representative HS-AFM image of Cas9–RNA–DNA. d Distribution of the height peaks in the HS-AFM images of Cas9–RNA–DNA (n = 65). The peak distribution fits a Gaussian curve, with the peak corresponding to the target site. e Sequential HS-AFM images of Cas9–RNA–DNA in the absence of MgCl 2 . The HNH domain is indicated by white arrows, whereas its disappearance (fluctuation) is indicated by magenta arrows. The scale bar is 10 nm. f Close-up view of a representative HS-AFM image of Cas9–RNA–DNA. The scale bar is 10 nm. g Time courses of correlation coefficients for the individual domains between the sequential HS-AFM images of Cas9–RNA–DNA in the absence of MgCl 2 . The HNH domain fluctuations are indicated by magenta arrows Full size image

In the HS-AFM movies of Cas9–RNA–DNA, we observed a prominent protrusion between the two lobes, which is not discernible in the Cas9–RNA movies (Fig. 2e, Supplementary Movie 3). A comparison with the crystal structures13,14,15,16 indicated that this protrusion corresponds to the HNH nuclease domain (Figs. 1b and 2f). The domain assignment is further supported by the HS-AFM images of N-terminal GFP-fused dCas9(D10A/H840A)–RNA bound to the DNA (Supplementary Fig. 3c, d, Supplementary Movie 4). We observed that Cas9–RNA binding induces ~ 30° local bending in the target DNA, consistent with the crystal structures of Cas9–RNA–DNA14,16. Notably, the protrusion frequently disappeared for a short time during the HS-AFM imaging (Fig. 2e, Supplementary Movie 3). A time course of the correlation coefficients calculated for a limited area on the three regions (REC, HNH and RuvC-PI) showed that the HNH domain fluctuates in the Cas9–RNA–DNA complex, unlike the other domains (Fig. 2g, Supplementary Fig. 3e). Thus, these HS-AFM data provide direct visualizations of the conformational dynamics of the HNH domain upon DNA binding, as suggested by previous structural studies15,16 and FRET experiments17,18.

Target DNA cleavage by Cas9–RNA

We next sought to observe the target DNA cleavage by Cas9–RNA. To this end, we mixed pre-assembled Cas9–RNA with the target DNA in the absence of Mg2+, adsorbed the complex on the AP-mica surface, and then initiated the cleavage reaction by the addition of Mg2+. The HS-AFM movies revealed that the HNH domain also fluctuates in the presence of Mg2+ (Fig. 3a, b, Supplementary Fig. 4, Supplementary Movie 5). Notably, in the presence of Mg2+, the HNH domain remained in a low-height state after several fluctuations, followed by the release of the DNA from the Cas9–RNA complex (Fig. 3a, b, Supplementary Fig. 4, Supplementary Movie 5). The DNA release was not observed in the absence of Mg2+ (Fig. 3c). We observed the binding of nuclease-inactive dCas9–RNA to the target DNA, but the DNA was not released from the complex (Fig. 3c). These results confirmed that the released DNA represents a cleavage product, and indicated that, in the low-height state, the HNH active site is located near the scissile phosphate of the target strand to accomplish the DNA cleavage.

Fig. 3 HS-AFM observations of DNA cleavage by Cas9–RNA. a Sequential HS-AFM images of Cas9–RNA–DNA in the presence of MgCl 2 . The HNH domains in the inactive (high-height) and active (low-height) states are indicated by white and magenta arrows, respectively. The scale bar is 10 nm. b Time courses of correlation coefficients for the individual domains between the sequential HS-AFM images of Cas9–RNA–DNA in the presence of MgCl 2 . The HNH domain fluctuations are indicated by magenta arrows. The release of the cleavage product is indicated by a blue line. c Rates of the cleavage product release from Cas9–RNA in the presence (n = 361) and absence (n = 36) of MgCl 2 , and from GFP-dCas9–RNA (n = 37). ND, not detected. d Binding position of Cas9–RNA after the product release (n = 181) Full size image

Conformational dynamics of the HNH domain

In the available Cas9–RNA–DNA structures, the HNH domain adopts catalytically inactive conformations and is not located near the scissile phosphate in the target DNA strand14,15,16 (Supplementary Fig. 5a), suggesting that the HNH domain must undergo structural rearrangements to approach the cleavage site. Consistently, bulk and single-molecule FRET studies indicated that the HNH domain adopts three major conformations: R, I, and D states17,18. The R and I conformations are consistent with the crystal structures of the Cas9–RNA13 and Cas9–RNA–DNA14,15 complexes, respectively (Supplementary Fig. 5a). A structure in the D conformation has not been determined, but was predicted by modeling17 (Supplementary Fig. 5b). In addition, structural and functional studies revealed that the L1 and L2 linker regions between the HNH and RuvC domains play a pivotal role in the conformational rearrangements of the HNH domain15,16,17 (Supplementary Fig. 5c). Notably, the high- and low-height states observed in our HS-AFM images are in agreement with the I and D conformations, respectively (Fig. 4a, b). The height differences of the HNH domain in the two states (0.8 ± 0.2 nm, n = 14) are likely to reflect the HNH displacement toward the target DNA for the cleavage reaction (Supplementary Fig. 5d). Thus, our HS-AFM movies directly visualized the catalytically active D state of Cas9, and revealed the conformational dynamics of the HNH domain during DNA cleavage.

Fig. 4 Structural rearrangement of the HNH domain. a, b HS-AFM images of the HNH domain in the high-height (a) and low-height (b) states. The mean center positions of the HNH and REC1 domains are indicated by dots. Red and blue lines indicate the cross-sectional position used for the height distribution analysis in Supplementary Fig. 5d. For comparison, the Cas9–RNA–DNA models in the I and D states are shown below the respective images. The structural models consist of Cas9–RNA (PDB: 4OO8) and DNA (PDB: 5F9R), and the D state model was built as described previously17. The scale bars are 5 nm Full size image

DNA release after cleavage

Our HS-AFM movies revealed that most of the Cas9–RNA molecules remain bound to the PAM-distal region (the non-PAM side) of the cleaved DNA after the release of the PAM-containing region (the PAM side) (104.50 s; Fig. 3a, d, Supplementary Fig. 6a, b, Supplementary Movie 5). The dwell time of the low-height state before the DNA release ranged widely from 0.4 to 29.2 s, whereas previous biochemical experiments showed that Cas9–RNA remains tightly bound to the DNA even after cleavage11,26. This discrepancy suggests that the physical contacts with the AFM probe facilitate the dissociation of Cas9–RNA from the DNA after cleavage. We observed some Cas9–RNA molecules that remained bound to the PAM side of the cleaved DNA after the release of the non-PAM side (Fig. 3d). Although this would require the unwinding of the RNA–DNA heteroduplex, it is unclear how the non-PAM side is released from the complex, due to the limited resolution of the HS-AFM imaging. The release of the non-PAM side was not observed in a previous DNA-curtain assay11, and this discrepancy may also be derived from the effects of the contacts with the AFM probe. The HS-AFM movies showed that the released DNAs on the PAM side are apparently longer by ~ 2.7 nm (n = 14), as compared with those before the release (Supplementary Fig. 6b, c). Given that the 8-bp PAM DNA duplex (0.34 nm/bp × 8 bp = 2.7 nm) is accommodated between the REC1 and PI domains in the crystal structure14 (Supplementary Fig. 6d), this apparent extension of the PAM-side DNA is likely due to the release of the PAM-containing region, which is bound inside the Cas9–RNA molecule before the release.

Target DNA search by Cas9–RNA

Previous DNA-curtain assays11 and single-particle tracking analyses27 suggested that Cas9–RNA interrogates the target sites via three-dimensional diffusion in vitro and in mammalian cells, respectively. Using HS-AFM, we sought to visualize the target interrogation by the Cas9–RNA complex. However, we failed to observe the movement of Cas9–RNA along the DNA, since the strong interactions between Cas9–RNA and the AP-mica surface suppress the free diffusion of the complexes. In contrast, Cas9–RNA can diffuse more freely on a mica-supported lipid bilayer, thus allowing the HS-AFM observations of the Cas9–RNA movement along the DNA. We adsorbed the 600-bp dsDNA containing a 20-nt target site with the TGG PAM on the mica-supported lipid bilayer, and then added apo-Cas9 or the pre-assembled Cas9–RNA complex (Fig. 5a, b). The HS-AFM movies revealed that multiple apo-Cas9 molecules bind and slide along the DNA (Fig. 5a, Supplementary Movie 6). An analysis of the HS-AFM movies confirmed that apo-Cas9 binds to the DNA in a non-specific manner (Fig. 5c, Supplementary Fig. 7a), consistent with the DNA-curtain study11. A time course analysis of the DNA-bound Cas9 positions confirmed that apo-Cas9 slides along the DNA (Fig. 5d). In contrast, the HS-AFM movies revealed that the Cas9–RNA complexes do not slide along the DNA, and rapidly bind to the target site in a specific manner (Fig. 5b, Supplementary Movie 7). An analysis of the HS-AFM images confirmed the specific binding of Cas9–RNA to the target site (Fig. 5c, d, Supplementary Fig. 7b).

Fig. 5 HS-AFM observations of target interrogation by Cas9–RNA. a, b Sequential HS-AFM images of the DNA after addition of apo-Cas9 (a) and Cas9–RNA (b) on the lipid bilayer. Apo-Cas9 and Cas9–RNA are indicated by white arrows. The scale bars are 50 nm. c Binding distributions of apo-Cas9 (n = 69) and Cas9–RNA (n = 61). The binding distribution of Cas9–RNA fits a Gaussian curve, with the peak corresponding to the target site. d Time courses of the binding positions of apo-Cas9 and Cas9–RNA. The distances from one end of the DNA were measured for five representative apo-Cas9 (left) and Cas9–RNA (right) molecules. Blue lines indicate the positions 200 and 400 bp from one end of the DNA (the potential target sites). Blue arrows indicate the binding of Cas9–RNA to the target site Full size image

Intriguingly, we observed short-lived bright spots (less than 3 ms) on the DNA (Fig. 6a, Supplementary Movie 8). These spots on the DNA were only observed in the presence of the Cas9–RNA complex, but not in its absence (Fig. 6b–d), suggesting that the observed short-lived spots represent the transient binding of Cas9–RNA to non-target sites. The lifetime of the non-target binding was estimated to be ~1 ms (Fig. 6e). Given that this lifetime is much shorter than the reported value (~3.3 s) from the DNA-curtain study11, it is possible that the dissociation of the Cas9–RNA complex was facilitated by the contacts with the AFM probe. On the basis of these HS-AFM data, we conclude that Cas9–RNA searches for the target sites by three-dimensional diffusion, rather than one-dimensional sliding, consistent with the DNA-curtain study11.