ARTICLE
DOI: 10.1038/s41467-017-01466-8

OPEN

Real-space and real-time dynamics of CRISPR-Cas9
visualized by high-speed atomic force microscopy
Mikihiro Shibata1,2, Hiroshi Nishimasu3,4, Noriyuki Kodera2,4, Seiichi Hirano3, Toshio Ando2,5,Takayuki Uchihashi2,5,6,7 & Osamu Nureki 3

The CRISPR-associated endonuclease Cas9 binds to a guide RNA and cleaves doublestranded DNA with a sequence complementary to the RNA guide. The Cas9–RNA
system has been harnessed for numerous applications, such as genome editing. Here we use
high-speed atomic force microscopy (HS-AFM) to visualize the real-space and real-time
dynamics of CRISPR-Cas9 in action. HS-AFM movies indicate that, whereas apo-Cas9
adopts unexpected ﬂexible conformations, Cas9–RNA forms a stable bilobed structure andinterrogates target sites on the DNA by three-dimensional diffusion. These movies also
provide real-time visualization of the Cas9-mediated DNA cleavage process. Notably, theCas9 HNH nuclease domain ﬂuctuates upon DNA binding, and subsequently adopts an active
conformation, where the HNH active site is docked at the cleavage site in the target
DNA. Collectively, our HS-AFM data extend our understanding of the action mechanism of
CRISPR-Cas9.

1 High-speed AFM for Biological Application Unit, Institute for Frontier Science Initiative, Kanazawa University, Kakuma, Kanazawa 920-1192, Japan.
2 Bio-AFM Frontier Research Center, Kanazawa University, Kakuma, Kanazawa 920-1192, Japan. 3 Department of Biological Sciences, Graduate School of
Science, The University of Tokyo, 2-11-16 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan. 4 JST, PRESTO, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan.
5 CREST/JST, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan. 6 Department of Physics, Kanazawa University, Kakuma, Kanazawa 920-1192, Japan.
7
Present address: Department of Physics, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8602, Japan. Mikihiro Shibata and Hiroshi Nishimasu
contributed equally to this work. Correspondence and requests for materials should be addressed to M.S. (email: msshibata@staff.kanazawa-u.ac.jp)
or to H.N. (email: nisimasu@bs.s.u-tokyo.ac.jp) or to O.N. (email: nureki@bs.s.u-tokyo.ac.jp)

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NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-01466-8

In the microbial CRISPR-Cas (clustered regularly interspaced
short palindromic repeats-CRISPR-associated proteins) adaptive immune system1–3, the RNA-guided DNA endonuclease
Cas9 associates with a CRISPR RNA (crRNA) and a trans-activating crRNA (tracrRNA), and cleaves the double-stranded (ds)
DNA complementary to the crRNA guide4–7 (Supplementary
Fig. 1a). Cas9 can function with a single-guide RNA (sgRNA), in
which the crRNA and the tracrRNA are fused with an artiﬁcial
linker7. The two-component system, consisting of Cas9 from
Streptococcus pyogenes and the sgRNA, has been harnessed forgenome-engineering technologies in a variety of cell types andorganisms8–10. Cas9–RNA ﬁrst recognizes a short nucleotide
sequence (NGG for S. pyogenes Cas9; N represents any nucleotide) next to a target sequence in the dsDNA, called a protospacer
adjacent motif (PAM). Cas9–RNA then initiates DNA unwindingat the PAM-proximal region, which is followed by the directional
formation of an R-loop, consisting of the RNA–DNA hybrid andthe displaced non-target DNA strand11. After the R-loop formation, the HNH and RuvC nuclease domains of Cas9 cleave thetarget DNA strand (complementary to the RNA guide) and thenon-target DNA strand, respectively6,7.
Previous structural studies showed that apo-Cas9 adopts a
closed auto-inhibited conformation12, whereas the Cas9–RNA
binary complex adopts a bilobed architecture comprising an
α-helical recognition (REC) lobe and a nuclease (NUC) lobe13(Fig. 1a, b). A comparison between these structures suggests that
Cas9 undergoes a closed-to-open structural rearrangement uponbinding the guide RNA. Moreover, the crystal structures of
Cas9–RNA bound to the target DNA provided mechanistic

a

PI

RuvC
-III

L2

L1

RuvC
-II

HNH

1368
REC3

REC2

BH

REC1

REC1

REC lobe

1
RuvC
-I

insights into RNA-guided DNA targeting by Cas914,15 (Fig. 1b).
Furthermore, the Cas9 R-loop complex structure revealed
drastic conformational changes in the linker regions between
the RuvC and HNH nuclease domains, thereby translocating
the HNH domain closer to the target DNA strand16 (Fig. 1b,
Supplementary Fig. 1b). Consistently, bulk and single-molecule
Förster resonance energy transfer (FRET) studies indicated thatthe HNH domain undergoes a structural transition during DNA
cleavage and adopts three major conformations: RNA-bound (R),intermediate (I) and active docked (D) states17,18. The R and I
conformations predominantly correspond to the crystal structures of Cas9–RNA13 and Cas9–RNA–DNA14,15, respectively. A
structure of the D conformation was predicted by modeling17,18,
but it has not been determined, although the D conformation
approximates the crystal structure of the Cas9 R-loop complex16.
These structural and imaging studies provided mechanistic
insights into the Cas9-mediated DNA recognition and cleavage,
but its action mechanism has not been fully clariﬁed. It is
unknown how apo-Cas9 in the closed conformation assembles
with the guide RNA to form an effector complex. In addition,
Cas9 in the catalytically-active D conformation has not beenvisualized.
High-speed atomic force microscopy (HS-AFM) is a powerful
technique that enables real-space and real-time observations ofmacromolecules, which are not feasible by other techniques19.
HS-AFM imaging has elucidated the dynamics of various proteins
with unprecedented details; for example, the photo-induced
conformational change of bacteriorhodopsin20, myosin V walking
on an actin ﬁlament21, cellulose degradation by a cellulase

NUC lobe
apo-Cas9(PDB: 4CMP)

Cas9–RNA(PDB: 4ZT0)

Cas9–RNA–DNA(PDB: 4OO8)

Cas9–RNA–DNA(PDB: 4UN3)

Cas9–RNA–DNA(PDB: 5F9R)

NUC

REC

b

c apo-Cas9
0.25 s0.50 s0.75 s

4.3 s

6.3 s

17.5 s24.3 s

4.2
0
(nm)

10 nm

d Cas9–RNA
0s

0.20 s0.60 s

1.4 s

5.4 s

9.4 s

17.2 s25.6 s

5.8
0
(nm)

10 nm

apo-Cas9

Correlation coefficients0seCas9–RNA

0.2

0

10

20
30
Time (s)40

Fig. 1 HS-AFM observations of apo-Cas9 and Cas9–RNA. a Domain structure of S. pyogenes Cas9. BH, Bridge helix. b Crystal structures of apo-Cas9(PDB: 4CMP)12, Cas9–RNA (PDB: 4ZT0)13, Cas9–RNA bound to its single-stranded DNA target (PDB: 4OO8)15, Cas9–RNA bound to a partial DNA duplex(PDB: 4UN3)14 and Cas9–RNA bound to its dsDNA target (a Cas9 R-loop complex) (PDB: 5F9R)16. The guide RNA and the target DNA are colored red
and yellow, respectively. The PAM is colored purple. The 98-nt guide RNA (PDB: 4OO8) was used for HS-AFM observations. c, d Sequential HS-AFM
images of apo-Cas9 (c) and Cas9–RNA (d) on the AP-mica surface. The color (from black to white) corresponds to the height. The scale bars are 10 nm.
e Time courses of correlation coefﬁcients between the sequential HS-AFM images of apo-Cas9 and Cas9–RNA
2

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NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-01466-8a

400 bp200 bpNTS 5′
TS 3′

3′
5′
Target siteb

PAM (TGG)c
Height (nm)Cas9–RNA–DNA (− MgCl2)
Cas9–RNA

6
3
0
0

40

80
120
DNA length (nm)

0

20

40
60
DNA length (%)

6.4 s

3′

20 nm

24

Cas9–RNA count

d

5′

DNA

160

16
8
0
80

100

e Cas9–RNA–DNA (− MgCl2)
0.2 s

4.0 s

5.0 s

67.0 s70.6 s72.0 s

NUC

REC

0s

10 nm HNH
64.2 s

8.0

0
(nm)

f

g
10 nm

Cas9–RNA–DNA (− MgCl2)

DNA

0.2

PI

RuvC

REC
HNH

Correlation coefficients

REC

HNH

RuvC-PI

0

10

20

30

Time (s)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

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NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-01466-8aCas9–RNA–DNA (+ MgCl2)

NUC31.75 s39.00 s62.00 s63.75 s84.50 s96.00 s104.50 s

NUC

REC

HNH
10 nm REC

64.25 sHNH

73.75 sCleaved
DNA

8.2

0
(nm)

c
Cas9
DNA release (%)

Correlation coefficientsd
GFPdCas9

100

REC

HNH

RuvC-PI

75
50
25
ND

50
25

gC

l

l

2

2

N

M
+

gC
M

–

gC

l

2

0

90

M

60
Time (s)

+

30

75

ND

0
0

100

P
si AM
de

0.2

on
si -PAde M

Cas9–RNA–DNA (+ MgCl2)

Bound Cas9 (%)

b

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 thecleavage 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)

enzyme22, rotary catalysis of F1-ATPase23, lipid membrane
remodeling by ESCRT-III polymerization24 and lipid membranestabilization by annexin V25.
In this study, we employed HS-AFM to visualize the real-spaceand real-time dynamics of CRISPR-Cas9 in action, at nanometer
resolution. HS-AFM movies revealed that apo-Cas9 adopts ﬂexible conformations, whereas Cas9–RNA forms a stable bilobed
architecture. Furthermore, the HS-AFM movies directly visualized the Cas9-mediated DNA cleavage reaction accompanied by
the drastic structural transition of the HNH nuclease domain.
Overall, the HS-AFM movies provided distinct scenes of Cas9 inaction, comprising the complex assembly, target search, target
cleavage and product release, thus substantially improving ourmechanistic understanding of CRISPR-Cas9.
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 withthe 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) in4the 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, andhighlight the guide-RNA-mediated stabilization of the REC lobe
conformation and induction of structural rearrangements in theCas9 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 incubatedthe 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, andperformed 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

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NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-01466-8ab

Cas9–RNA–DNA(high-height I state)

Cas9–RNA–DNA(low-height D state)0.25 s

0s

B

B
A

A

5 nm

5 nm
REC2

REC2
REC1

REC1
REC3

REC3
RNA

RNA

HNH

HNH

DNA

RuvC

PI

DNA

RuvC

PI

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 meancenter 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 nmfaithfully recapitulate the PAM-dependent target recognition by
Cas9–RNA.
In the HS-AFM movies of Cas9–RNA–DNA, we observed
a prominent protrusion between the two lobes, which is notdiscernible in the Cas9–RNA movies (Fig. 2e, Supplementary
Movie 3). A comparison with the crystal structures13–16 indicatedthat 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 timecourse of the correlation coefﬁcients calculated for a limited areaon 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 absenceof Mg2+, adsorbed the complex on the AP-mica surface, andthen 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,
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Supplementary Movie 5). Notably, in the presence of Mg2+, theHNH 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 thereleased DNA represents a cleavage product, and indicated that, inthe low-height state, the HNH active site is located near the scissile
phosphate of the target strand to accomplish the DNA cleavage.
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 strand14–16 (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 predictedby modeling17 (Supplementary Fig. 5b). In addition, structuraland functional studies revealed that the L1 and L2 linker regions
between the HNH and RuvC domains play a pivotal role inthe conformational rearrangements of the HNH domain15–17(Supplementary Fig. 5c). Notably, the high- and low-height statesobserved in our HS-AFM images are in agreement with the I and| DOI: 10.1038/s41467-017-01466-8 | www.nature.com/naturecommunications

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a apo-Cas9 + DNA
0s

0.30 s0.45 s0.75 s2.25 s20 min

DNA
5.6

0
(nm)

50 nm

b Cas9–RNA + DNA
0s

0.15 s6.75 s8.10 s12.75 s20 min
7.3

DNA

0
(nm)

50 nm

c

d
apo-Cas9 + DNA

100 nmapo-Cas9 + DNA

8

0

16
Cas9–RNA countCas9–RNA + DNA

Distance from one end of the DNA (nm)apo-Cas9 count16

Cas9–RNA + DNA

8

Mol 1

Mol 1

Mol 2

Mol 2

Mol 3

Mol 3

Mol 4

Mol 4

Mol 5

Mol 5

0
0

20

40
60
DNA length (%)

80

100

0

5

10
Time (s)

15

0

5

10
Time (s)15

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) andCas9–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 Timecourses 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

D conformations, respectively (Fig. 4a, b). The height differencesof the HNH domain in the two states (0.8 ± 0.2 nm, n = 14) arelikely to reﬂect the HNH displacement toward the target DNA forthe cleavage reaction (Supplementary Fig. 5d). Thus, our HSAFM movies directly visualized the catalytically active D state ofCas9, and revealed the conformational dynamics of the HNH
domain during DNA cleavage.
DNA release after cleavage. Our HS-AFM movies revealed thatmost of the Cas9–RNA molecules remain bound to the PAMdistal 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 thephysical contacts with the AFM probe facilitate the dissociation of6

Cas9–RNA from the DNA after cleavage. We observed someCas9–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 aprevious DNA-curtain assay11, and this discrepancy may also bederived 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 withthose 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 extensionof the PAM-side DNA is likely due to the release of the PAMcontaining region, which is bound inside the Cas9–RNA molecule
before the release.

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NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-01466-8

a Cas9–RNA + DNA
0s

0.15 s0.30 s0.45 s

0
(nm)

50 nm

b

5

10 15 20
Time (s)Cas9–RNA + DNA
5.0 nmHeight difference (nm)5.0 nmHeight difference (nm)

0

25

5

10 15 20
Time (s)

25

e

10

Cas9–RNA count

600

8
6
4
2
ND

D

N

A

on

ly

0

400

200

0
C
as
+ 9–R
D
N NA
A

No. of frames (%)

c

DNA only

0

d

4.0

0

5
10
Time (ms)15

Fig. 6 HS-AFM observations of the non-speciﬁc transient binding of
Cas9–RNA. a Sequential HS-AFM images of Cas9–RNA molecules
transiently bound to non-target sites of the DNA. Transient binding of
Cas9–RNA appears as a bright spot in the images, as indicated by the whitearrows. Most of the binding events were completed within a single-line
scanning time (1.9 ms; 150 ms / 80 lines). The scale bar is 50 nm.
b, c Differences between the lowest and highest heights in each image
during the HS-AFM observations of DNA only (b) and Cas9–RNA–DNA(c). In c, the spikes indicate the transient binding of Cas9–RNA to the DNA.
d, e Frequency (d) and lifetime (e) of the transient binding of Cas9–RNA to
the DNA. The lifetime was estimated by counting the successive line
numbers on which the spike-like spots were continuously seen. Spike-likespots with heights over 3.5 nm were judged as Cas9–RNA molecules. The
lifetime was ﬁtted by the 1st order exponential decay, with a time constantof 0.98 ± 0.02 ms (n = 656)

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 theCas9–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 freediffusion 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 theDNA. We adsorbed the 600-bp dsDNA containing a 20-nt target
site with the TGG PAM on the mica-supported lipid bilayer, andthen 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,
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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 thetarget 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).
Intriguingly, we observed short-lived bright spots (less than 3ms) 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 theobserved 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 thislifetime is much shorter than the reported value (~3.3 s) from the
DNA-curtain study11, it is possible that the dissociation of theCas9–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.
Discussion
HS-AFM enables the direct visualization of the structures anddynamics of intact molecules, in contrast to other single-molecule
imaging methods, in which a molecule of interest must be labeledwith ﬂuorescent probes. Using HS-AFM, we visualized the realspace and real-time dynamics of CRISPR-Cas9 in action, thereby
improving our mechanistic understanding of the RNA-guided
DNA cleavage by Cas9. Although our HS-AFM data areessentially consistent with previous ﬂuorescence-based imaging
studies11,17,18, there are some discrepancies between these studies.
For example, a recent single-molecule FRET study reported thatthe majority (90%) of Cas9–RNA stably adopt the D state upon
DNA binding, while only 3% of the complexes undergo transitions between the I and D states18. In contrast, our HS-AFM data
showed that most of the Cas9–RNA complexes ﬂuctuate between
the I and D states after DNA binding. This discrepancy is likely
due to the differences in the imaging techniques and experimental
conditions. On the basis of our HS-AFM data, together withprevious structural, biochemical and biophysical data11–18,28, we
propose a model for the Cas9–RNA-mediated DNA cleavage(Fig. 7a–c). Apo-Cas9 adopts a ﬂexible modular architecture, andassembles with the guide RNA to form the stable Cas9–RNA
effector complex. The Cas9–RNA complex interrogates the target
sites on the DNA via three-dimensional diffusion, and recognizes
the complementary target site with the NGG PAM. Cas9–RNA
then unwinds the dsDNA target to form the R-loop. The HNH
domain undergoes conformational ﬂuctuations upon R-loop
formation, and then adopts the catalytically-active docked
conformation to cleave the target strand, while the RuvC domain
cleaves the non-target strand. Overall, this study provides
unprecedented details about the functional dynamics of CRISPRCas9, and highlights the potential of HS-AFM to elucidate
the action mechanisms of RNA-guided effector nucleases fromdistinct CRISPR-Cas systems29.
Methods
Sample preparation. Wild-type S. pyogenes Cas9 and GFP-dCas9(D10A/C81L/
C574E/H840A) were expressed in Escherichia coli Rosetta2 (DE3), and thenpuriﬁed to homogeneity by column chromatography, as described with minor
modiﬁcations15. Brieﬂy, the Cas9 protein was expressed in E. coli Rosetta2 (DE3)(Novagen), and was puriﬁed by chromatography on Ni-NTA Superﬂow(QIAGEN), HiTrap SP HP (GE Healthcare), and HiLoad Superdex 200 16/60(GE Healthcare) columns. The 98-nt guide RNA was transcribed in vitro,| DOI: 10.1038/s41467-017-01466-8 | www.nature.com/naturecommunications

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NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-01466-8

a
REC2
REC3

Guide

RNA

RNA scaffold

REC1
HNH
RuvC

PI
Cas9–RNA

apo-Cas9b

DNA
Target site

PAM

cREC3 REC2

TS
NTS

REC1

HNH
RuvC

PI

PAM recognition
DNA unwinding

R-loop formation
HNH fluctuation

HNH translocation
DNA cleavage

Fig. 7 Model of CRISPR-Cas9 action. a Assembly of the Cas9–RNA binary complex. b Target DNA interrogation by Cas9–RNA via three-dimensional
diffusion. c DNA recognition and cleavage by Cas9–RNA. DNA cleavages by the RuvC and HNH domains are indicated by the cyan and magenta triangles,
respectively
and then puriﬁed by denaturing urea polyacrylamide gel electrophoresis, as
described15. The 600-bp target DNA was PCR-ampliﬁed using the pUC119
plasmid containing the 20-nt target sequence and the TGG PAM30 as the template,
and then puriﬁed using a Wizard DNA Clean-Up System (Promega). The
sequences of Cas9, the guide RNA and the target DNA are provided in Supplementary Data 1.
HS-AFM observations on an AP-mica surface. The laboratory-built high-speed
AFM was used in the tapping mode31. The cantilever deﬂection was detected with
an optical beam deﬂection detector, on which a 0.7 mW, 780 nm infrared laser was
mounted. The infrared laser beam was focused onto the back side of the cantilever(Olympus: BL-AC7DS-KU4) through a ×60 objective lens (Nikon: CFI S Plan
Fluor ELWD 60×). The reﬂected laser from the cantilever was detected witha two-segmented PIN photodiode. The spring constant of the cantilever was~100 pN nm−1. The resonant frequency and the quality factor of the cantilever inliquid were ~800 kHz and ~2, respectively. An amorphous carbon tip was fabricated on the original AFM tip by electron beam deposition (EBD). The length of
the additional AFM tip was ~500 nm, and the radius of the apex of the tip was~4 nm. The free oscillation amplitude of the cantilever was ~1 nm and the set-point
amplitude was set to 90% of the free amplitude. For HS-AFM observations of Cas9,
a mica surface was treated for 3 min with 0.011% (3-aminopropyl)triethoxysilane(APTES) (Sigma-Aldrich). The complex of Cas9, RNA and DNA was preassembled (Cas9:RNA:DNA = 1:1:1 mole ratio) in AFM-imaging buffer. HS-AFM
observations of apo-Cas9 and Cas9–RNA were performed in buffer, consisting of
20 mM Tris-HCl, pH 8.0, 100 mM KCl and 0.01 mM EDTA. HS-AFM observationsof the Cas9–RNA–DNA and GFP-dCas9–RNA–DNA complexes were performed
in buffer, consisting of 20 mM Tris-HCl, pH 8.0, 30 mM KCl and 0.01 mM EDTA.
All HS-AFM experiments were performed at room temperature.
HS-AFM observations on a lipid bilayer. The DNA was loosely immobilized on amica-supported lipid bilayer31. In brief, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC; Avanti Polar Lipids) and 1,2-dipalmitoyl-3-trimethylammonium-propane (DPTAP; Avanti Polar Lipids) were suspended in chloroform, and1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(cap biotinyl) (biotin-capDPPE; Avanti Polar Lipids) was suspended in a mixture of chloroform:methanol:
water = 65:35:8 (volume ratio). The lipid solutions were mixed at a weight ratio of
DPPC:DPTAP:biotin-cap-DPPE = 90:5:5. Small unilamellar vesicles (SUVs) of the8lipid mixture, at 0.2 mg ml−1 in 10 mM MgCl2, were prepared by sonication for
1 min, using a bus-sonicator (AS ONE). A mica disk (1.0 mm in diameter) glued on
the sample stage of the HS-AFM was freshly cleaved, and the mica-supported lipid
bilayer was formed on it by depositing 2 μl of the SUVs solution for 3 min, followedby 1 μl of 20 mM Tris-HCl, pH 8.8, for over 10 min. The sample surface was rinsed
with drops of Milli-Q water (20 μl × 5) to remove the excess SUVs, and then thesolution was replaced with buffer A (20 mM Tris-HCl, pH 8.0, 30 mM KCl,
0.01 mM EDTA). DNA (2 μl, 1 ng μl−1) in buffer A was deposited on the surface
for 3 min. After rinsing the surface with buffer B (20 mM Tris-HCl, pH 8.0, 30 mM
KCl, 2 mM MgCl2), the sample stage was immersed in a liquid cell ﬁlled with buffer
B (~55 μl), and HS-AFM observations were performed. During the HS-AFM
observations, a drop (~5 μl) of either apo-Cas9 or Cas9–RNA (molar ratio of Cas9:
RNA = 1:2) was added to the liquid cell, at a ﬁnal concentration of ~80 nM.
Correlation analysis of HS-AFM images. 2D correlation coefﬁcients were
calculated between the HS-AFM images of the ﬁrst frame and each of the frames
within the Region of Interest (ROI) (i.e., the ﬁrst frame is the reference)23. The sizesof the ROIs for apo-Cas9 and Cas9–RNA that enclosed the whole Cas9 molecule
were about 27 × 24 nm2. For the Cas9–RNA–DNA complex, the sizes of the ROIs
for the REC, HNH and RuvC-PI domains that enclosed the whole region ofeach domain were about 13 × 10 nm2, 7 × 7 nm2 and 13 × 10 nm2, respectively.
The 2D correlation coefﬁcient was calculated frame by frame for each ROI.
The 2D correlation coefﬁcient is deﬁned as,


P P 
m
n Hmn  H Rmn  R
ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ
r ¼ r

2 P P 
2 
P P 
m
n Hmn  Hmn Rmn  R
in which Hmn and Rmn are the heights at the pixel point ðm; nÞ in the ROI to beanalyzed and the reference ROI of the reference frame, respectively. H and R are
the mean values of the height matrices H and R, respectively.
Calculation of the center positions of HS-AFM images. The center positions of
the HNH and REC domains were calculated, as described below. First, the ROIs
that enclosed the whole HNH and REC domains were about 7 × 7 nm2, andthen the center positions of the ROIs were calculated by the X, Y, and Z data of the
HS-AFM images. The X and Y data correspond to the lateral coordinates, while the

NATURE COMMUNICATIONS | 8: 1430

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ARTICLE

NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-01466-8

Z data correspond to the height. After the conformational change in the HNH
domain, the identical ROIs were used for the calculation of the center positions.
Data availability. The data sets generated during the current study are available
from the corresponding author upon request.

Received: 13 June 2017 Accepted: 18 September 2017

References
1. Hsu, P. D., Lander, E. S. & Zhang, F. Development and applications of CRISPRCas9 for genome engineering. Cell 157, 1262–1278 (2014).
2. Marrafﬁni, L. A. CRISPR-Cas immunity in prokaryotes. Nature 526, 55–61(2015).
3. Wright, A. V., Nunez, J. K. & Doudna, J. A. Biology and applications of CRISPR
systems: harnessing Nature’s toolbox for genome engineering. Cell 164, 29–44(2016).
4. Deltcheva, E. et al. CRISPR RNA maturation by trans-encoded small RNA andhost factor RNase III. Nature 471, 602–607 (2011).
5. Garneau, J. E. et al. The CRISPR/Cas bacterial immune system cleaves
bacteriophage and plasmid DNA. Nature 468, 67–71 (2010).
6. Gasiunas, G., Barrangou, R., Horvath, P. & Siksnys, V. Cas9-crRNAribonucleoprotein complex mediates speciﬁc DNA cleavage for adaptive
immunity in bacteria. Proc. Natl Acad. Sci. USA 109, E2579–2586 (2012).
7. Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease inadaptive bacterial immunity. Science 337, 816–821 (2012).
8. Cong, L. et al. Multiplex genome engineering using CRISPR/Cas systems.
Science 339, 819–823 (2013).
9. Jinek, M. et al. RNA-programmed genome editing in human cells. Elife 2,
e00471 (2013).
10. Mali, P. et al. RNA-guided human genome engineering via Cas9. Science 339,
823–826 (2013).
11. Sternberg, S. H., Redding, S., Jinek, M., Greene, E. C. & Doudna, J. A. DNA
interrogation by the CRISPR RNA-guided endonuclease Cas9. Nature 507,
62–67 (2014).
12. Jinek, M. et al. Structures of Cas9 endonucleases reveal RNA-mediatedconformational activation. Science 343, 1247997 (2014).
13. Jiang, F., Zhou, K., Ma, L., Gressel, S. & Doudna, J. A. A Cas9-guide RNAcomplex preorganized for target DNA recognition. Science 348, 1477–1481(2015).
14. Anders, C., Niewoehner, O., Duerst, A. & Jinek, M. Structural basis of PAMdependent target DNA recognition by the Cas9 endonuclease. Nature 513,
569–573 (2014).
15. Nishimasu, H. et al. Crystal structure of Cas9 in complex with guide RNA andtarget DNA. Cell 156, 935–949 (2014).
16. Jiang, F. et al. Structures of a CRISPR-Cas9 R-loop complex primed for DNA
cleavage. Science 351, 867–871 (2016).
17. Sternberg, S. H., LaFrance, B., Kaplan, M. & Doudna, J. A. Conformational
control of DNA target cleavage by CRISPR-Cas9. Nature 527, 110–113 (2015).
18. Dagdas, Y. S., Chen, J. S., Sternberg, S. H., Doudna, J. A. & Yildiz, A. A
conformational checkpoint between DNA binding and cleavage by CRISPRCas9. Sci. Adv. 3, eaao0027 (2017).
19. Ando, T., Uchihashi, T. & Scheuring, S. Filming biomolecular processes byhigh-speed atomic force microscopy. Chem. Rev. 114, 3120–3188 (2014).
20. Shibata, M., Yamashita, H., Uchihashi, T., Kandori, H. & Ando, T. High-speed
atomic force microscopy shows dynamic molecular processes in photoactivatedbacteriorhodopsin. Nat. Nanotech. 5, 208–212 (2010).
21. Kodera, N., Yamamoto, D., Ishikawa, R. & Ando, T. Video imaging ofwalking myosin V by high-speed atomic force microscopy. Nature 468, 72–76(2010).
22. Igarashi, K. et al. Trafﬁc jams reduce hydrolytic efﬁciency of cellulase on
cellulose surface. Science 333, 1279–1282 (2011).
23. Uchihashi, T., Iino, R., Ando, T. & Noji, H. High-speed atomic force
microscopy reveals rotary catalysis of rotorless F1-ATPase. Science 333,
755–758 (2011).

NATURE COMMUNICATIONS | 8: 1430

24. Chiaruttini, N. et al. Relaxation of loaded ESCRT-III spiral springs drives
membrane deformation. Cell 163, 866–879 (2015).
25. Miyagi, A., Chipot, C., Rangl, M. & Scheuring, S. High-speed atomic force
microscopy shows that annexin V stabilizes membranes on the second
timescale. Nat. Nanotech. 11, 783–790 (2016).
26. Richardson, C. D., Ray, G. J., DeWitt, M. A., Curie, G. L. & Corn, J. E.
Enhancing homology-directed genome editing by catalytically active andinactive CRISPR-Cas9 using asymmetric donor DNA. Nat. Biotechnol. 34,
339–344 (2016).
27. Knight, S. C. et al. Dynamics of CRISPR-Cas9 genome interrogation in living
cells. Science 350, 823–826 (2015).
28. Singh, D., Sternberg, S. H., Fei, J., Doudna, J. A. & Ha, T. Real-time observationof DNA recognition and rejection by the RNA-guided endonuclease Cas9. Nat.
Commun. 7, 12778 (2016).
29. Mohanraju, P. et al. Diverse evolutionary roots and mechanistic variations of
the CRISPR-Cas systems. Science 353, aad5147 (2016).
30. Nishimasu, H. et al. Crystal structure of Staphylococcus aureus Cas9. Cell 162,
1113–1126 (2015).
31. Uchihashi, T., Kodera, N. & Ando, T. Guide to video recording of structure
dynamics and dynamic processes of proteins by high-speed atomic force
microscopy. Nat. Protoc. 7, 1193–1206 (2012).

Acknowledgements
This work was supported by the Kao Foundation for Arts and Science (M.S.), the Brain
Science Foundation (M.S.), JST/PRESTO (H.N. (JPMJPR13L8) and N.K. (JPMJPR13L4)),
JST/CREST (T.A. (JPMJCR13M1)), the Basic Science and Platform Technology Programfor Innovative Biological Medicine from the Japan Agency for Medical Research andDevelopment, AMED (O.N.), and JSPS KAKENHI Grant Numbers JP16K18523 (M.S.),26291010 and 15H01463 (H.N.), 15H04360 (N.K.), 24227005 and 26119003 (T.A.), and
16H00830, 16H00758 and 15H03540 (T.U.).

Author contributions
M.S. and H.N. conceived and designed the experiments; M.S. performed HS-AFM
experiments on the AP-mica surface; N.K. performed HS-AFM experiments on the lipid
bilayer; M.S. analyzed the data; H.N. and S.H. prepared Cas9, RNA and DNA; T.A.
developed HS-AFM techniques; T.U. developed the software for the HS-AFM instrument
and data analysis; M.S. and H.N. wrote the paper; and T.A., O.N. and T.U. supervised the
project. All authors discussed the results and commented on the manuscript.

Additional information
Supplementary Information accompanies this paper at doi:10.1038/s41467-017-01466-8.
Competing interests: The authors declare no competing ﬁnancial interests.
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