Real-space and real-time dynamics of CRISPR-Cas9 visualized by ...（二）
Results
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 MgCl2. 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 MgCl2. 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 MgCl2. The HNH domain fluctuations are indicated by magenta arrows
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 MgCl2. 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 MgCl2. 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 MgCl2, and from GFP-dCas9–RNA (n = 37). ND, not detected. d Binding position of Cas9–RNA after the product release (n = 181)
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.
