SCIENCE ADVANCES | RESEARCH ARTICLE
BIOPHYSICS

Programming PAM antennae for efficient CRISPR-Cas9
DNA editing
Fei Wang1,2, Yaya Hao2, Qian Li2, Jiang Li3,4, Honglu Zhang5, Xueli Zhang1, Lihua Wang3,6,
Carlos Bustamante5*, Chunhai Fan2*

INTRODUCTIONCRISPR-Cas9 has become a versatile tool for genome editing, diagnosis,
and therapeutics (1–3). With a single-guide RNA (sgRNA), Cas9
can recognize a specific 20–base pair (bp) sequence flanked by a
3-base protospacer-adjacent motif (PAM) (4). Genome editing withCRISPR-Cas9 has been performed on bacteria (5), plants (6), animals(7), and even humans (8), holding great promise of future treatmentof human diseases. Despite the enormous progress achieved so far,a critical technical hurdle is the target site editing efficiency, especiallyon large genomes (9, 10). To this end, many efforts have been
focused on sequence optimization, chemical modifications of sgRNA,or reengineering of Cas nucleases (11–16). For example, introducing
chemical modifications on sgRNA increased gene-editing activity
via the reduction of susceptibility of this component to nuclease degradation (15). Algorithms have also been developed to consider target
sequence features such as its position within the gene or chromatin(11). A number of reengineered versions of Cas nucleases have also beenreported to have better targeting ability and cleavage efficiency (14, 17).
Recruitment of sgRNA/Cas9 to target sites by PAM and its activation of the nuclease domain of Cas9 are indispensable for CRISPR
editing (18–21). Variation of the PAM density has been shown toaffect target binding by Cas9, but modulation of the PAM microenvironment has been less explored for gene editing. Studies of the1

Joint Research Center for Precision Medicine, Shanghai Jiao Tong University AffiliatedSixth People's Hospital South Campus, Southern Medical University Affiliated Fengxian
Hospital, Shanghai 201499, China. 2School of Chemistry and Chemical Engineering,
Frontiers Science Center of Transformative Molecules, and Institute of Molecular
Medicine, Renji Hospital, School of Medicine, Shanghai Jiao Tong University,
Shanghai 200240, China. 3CAS Key Laboratory of Interfacial Physics and Technology,
Shanghai Institute of Applied Physics, Chinese Academy of Sciences, Shanghai
201800, China. 4Shanghai Synchrotron Radiation Facility, Zhangjiang Laboratory,
Shanghai Advanced Research Institute, Chinese Academy of Sciences, Shanghai
201210, China. 5Department of Chemistry, Institute for Quantitative Biosciences(QB3), Department of Physics, Department of Molecular and Cell Biology, Kavli Energy
Nanosciences Institute at Berkeley, Howard Hughes Medical Institute, Universityof California, Berkeley, Berkeley, CA 94720, USA. 6Shanghai Key Laboratory ofGreen Chemistry and Chemical Processes, School of Chemistry and Molecular Engineering, East China Normal University, 500 Dongchuan Road, Shanghai, 200241, China.
*Corresponding author. Email: carlosb@berkeley.edu (C.B.); fanchunhai@sjtu.
edu.cn (C.F.)
Wang et al., Sci. Adv. 2020; 6 : eaay9948

8 May 2020

effect of PAM’s spatial distribution and density may lead to alternative strategies to engineer CRISPR editing and contribute to better
understanding of gene editing in complex environments (e.g., inchromatin).
DNA origami technology provides a powerful means to organize
nanoscale objects with molecular and submolecular precision andaddressability (22, 23). Using this approach, individual molecules ofnucleic acids and proteins can be site-specifically deposited on a
“canvas” over distances ranging from tens to hundreds of nanometers,
and their reactions can be easily monitored with optical or atomic
force microscopy (AFM) (24–26). Origami-based platforms are thus
particularly useful for studying biomolecular interactions at the single-­molecule level (27, 28). In this work, we design DNA origami–­
confined PAM antennae in the vicinity of a target, providing a
microenvironment to investigate the impact of the spatial distributionof PAM on target cleavage efficiency. We study the dynamic processof sgRNA/Cas9 binding in the presence/absence of PAM antennae
and program the PAM antennae for more efficient DNA editing.
RESULTS

PAM antenna on DNA origami
To create a local PAM environment for CRISPR-Cas9–based binding
and target cleavage in vitro, we used a two-dimensional, rectangular-­shaped DNA origami as a template to site-specifically arrange thesgRNA/Cas9 components (Fig. 1A and figs. S1 and S2). The double-­stranded DNA (dsDNA) target was placed at the center of theorigami (Fig. 1B, black area). To arrange the PAM antenna, we
extended eight staple strands adjacent to the target site from the 3′
end, followed by hybridization with a short single-stranded DNA(ssDNA) containing six PAM sites (Fig. 1B), forming a PAM-richregion of ~12 nm by 11 nm (referred to as origami P). Four biotin
molecules were modified on the backside of the DNA origami toimmobilize it on the avidin-coated surface of glass slides. Controls
were prepared by assembling blank origami (referred to as origami B)
with the target alone. AFM topography images revealed the presenceof a PAM island at the center of origami P, with a full width at half
maximum of ~16 nm (Fig. 1, C and D, and fig. S3), consistent with
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Bacterial CRISPR-Cas9 nucleases have been repurposed as powerful genome editing tools. Whereas engineering
guide RNAs or Cas nucleases have proven to improve the efficiency of CRISPR editing, modulation of protospacer-­
adjacent motif (PAM), indispensable for CRISPR, has been less explored. Here, we develop a DNA origami–based
platform to program a PAM antenna microenvironment and address its performance at the single-molecule levelwith submolecular resolution. To mimic spatially controlled in vivo PAM distribution as may occur in chromatin, weinvestigate the effect of PAM antennae surrounding target DNA. We find that PAM antennae effectively sensitize the
DNA cleavage by recruiting Cas9 molecules. Super-resolution tracking of single single-guide RNA/Cas9s reveals
localized translocation of Cas9 among spatially proximal PAMs. We find that the introduction of the PAM antennae
effectively modulates the microenvironment for enhanced target cleavage (up to ~50%). These results provide
insight into factors that promote more efficient genome editing.

Copyright © 2020
The Authors, somerights reserved;
exclusive licensee
American Association
for the Advancementof Science. No claim to
original U.S. Government
Works. Distributed
under a Creative
Commons Attribution
NonCommercial
License 4.0 (CC BY-NC).

SCIENCE ADVANCES | RESEARCH ARTICLE
A

B

DNA origami

Target
PAM

12 nm

PAM

sgRNA/Cas9

10.88 nmD
Origami P

4
Height (nm)Origami B

3

1.37nm

16 nm

2
1

B

0

P
0

50
100
Distance (nm)Fig. 1. Structural design of PAM antenna on DNA origami. (A) Schematic illustration of a local PAM environment that could recruit sgRNA/Cas9 molecule on a rectangular DNA origami. (B) Schematics for design and construction of origami P with a PAM antenna (yellow dots) surrounding the target (red dots). Target dsDNA has a 20-bp
target sequence for hybridization with sgRNA (red) and a 3-bp PAM sequence (highlighted in yellow). Each antenna duplex containing six PAM sites (5′-NGG-3′) protrudes
from a staple strand, which can bind to sgRNA/Cas9 transiently. (C) AFM images of origami B (left) and origami P (right). Scale bars, 100 nm. (D) Profile along dashed linesin (C) showing a higher region on origami P.

the expected width of DNA strands and confirming the formationof a nanoscale local environment of PAM.
We next explored how the PAM antenna affected Cas9 binding.
We monitored the binding events of Cy5-labeled sgRNA/Cas9 withthe target DNA localized on origami B and P, respectively. To thisend, we hybridized a 3′-end extended sgRNA with a Cy5-tagged
short ssDNA strand (fig. S4). To avoid cleavage, we used catalyticallyinactive Cas9 (dCas9) in these experiments. Binding dynamics ofdCas9 was characterized by analyzing the variation of the fluorescenceintensity at the target DNA position with time. We found that theoccurrence of binding events on origami P (94 ± 1.8%) was considerably higher than that on origami B (68 ± 3.5%) (Fig. 2A and
fig. S5). The distribution of binding events on individual origami
revealed a considerable enrichment of dCas9 on origami P over thatobserved on origami B with a considerably higher multiple binding(n > 1) probability, suggesting that PAM antennae operate as dCas9-­binding hotspots.
Next, we investigated the binding efficiency of dCas9 on origami
with antennae using AFM. Besides origami P and B, origami harboring
non-PAM antennae (referred to as origami N) were assembled withthe same local spatial environment as origami P. AFM height profiling
was used to study the static binding of sgRNA/dCas9 on origami B,
N, and P. Binding to the origami was manifested as a height increase
Wang et al., Sci. Adv. 2020; 6 : eaay9948

8 May 2020in their center region (Fig. 2B and fig. S6). Height profile analysis
revealed that sgRNA/dCas9 bound to all origami with the target DNA;
however, the presence of the PAM antennae led to an increase of~31% in the binding efficiency, which coincides well with the observations in the kinetic studies described above.
To substantiate the idea that PAM antennae represent binding
hotspots for Cas9, we perform Markov chain simulations to investigate
the movement of the nuclease on antenna-carrying origami (Fig. 2C).
Transient interactions between Cas9 and PAM antenna were modeledby scaling binding and dissociating rates (blank model with nobinding, non-PAM antenna model with high binding rate and highdissociation rate, and PAM antenna model with high binding rateand low dissociation rate). Counting the number of occurrences of
Cas9 in each spatial unit, we obtained a heatmap exhibiting theprobability distribution for Cas9 to appear at each unit. The heatmap
showed ~2-fold higher intensity near the PAM antenna for a 5:1 ratioof binding and dissociation rates, representing a Cas9-binding hotspot
due to the strong enhancement by the surrounding PAM antenna.
PAM antennae enhance target cleavage
Having established that PAM antennae enhance Cas9 binding, wenext explored the effect of PAM microenvironments on target
cleavage at the single-molecule level. AFM imaging of origami B
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DNA origami

CSCIENCE ADVANCES | RESEARCH ARTICLE

Bound B
68 ± 3.5%

sgRNA/dCas9binding eventson B

Bound P
94 ± 1.8%

Percentage

UnboundsgRNA/dCas9binding events
on P

1.0

>4 times

0.8

4 times0.6

0.0

Origami B

Once
B

1

or

0.5

0

P

0

40

80

120
Time (s)160

200

Origami N

Origami P

0.2

0.1

0.0

55.2% (n = 355)

39.9% (n = 228)

-2

0

2
4
6
8 10 12Center height (nm)

-2

0

2
4
6
8 10 12Center height (nm)40.2% (n = 355)

-2

0

2
4
6
8 10 12Center height (nm)
-4
×10
10

C

8
6
4
10 nm
t =0

Cas9

PAM

10 nm

Blank

10 nm
Non-PAM antenna

10 nm
PAM antenna

2

Fig. 2. PAM antenna creates Cas9-binding hotspot on a rectangular DNA origami. (A) Left: The frequency of binding events on origami P is higher than that on origami
B. Middle: Statistics of the number of binding events on each origami during the observation period shows the enrichment of sgRNA/dCas9 on origami P. Right: Sample
intensity–time traces at an individual target position of origami B (blue) and P (red) showing single dCas9 binding event on origami B and two dCas9 binding events onorigami P. a.u., arbitrary units. (B) Center height distribution showing two peaks corresponding to AFM topographic images of bound and unbound DNA origami. State
composition (red bars for the bound state and blue bars for the unbound state) shows a higher bound percentage on origami P (n = 355, P = 55.2%) over origami B (n = 228,
P = 39.9%) and origami N (n = 355, P = 40.2%). (C) Heatmaps showing that the spatial distribution of Cas9 is rearranged with PAM antennae, whereas non-PAM antennae
with high dissociation rate had no obvious effect on Cas9 distribution. For the initial condition, Cas9 molecules were randomly placed in a 60 nm–by–60 nm region.

before and after incubating with sgRNA/Cas9 showed that mostorigami (>85%) were occupied by sgRNA/Cas9 (fig. S7), suggestingthat Cas9 remained tightly bound to both cleaved ends of the target
strand (TS; Fig. 3A and fig. S8) and therefore could not be used tomonitor the cleavage event. To monitor cleavage, we used instead
total internal reflection fluorescence microscopy (TIRFM) to observe
the fluorescence variation of Cy3-labeled of either the PAM-proximalor PAM-distal ends on the non-TS (NTS; Fig. 3B). A large fractionof Cy3 fluorescence rapidly disappeared when Cy3 was labeled on
the PAM-proximal end (Fig. 3B), indicating cleavage and release of
the Cy3-labeled fragments. Control experiments were conducted byinjecting buffer without sgRNA/Cas9, which confirmed that the fluorescence disappearance was caused by target cleavage and release(fig. S9). By contrast, when Cy3 was labeled on the PAM-distal end(Fig. 3C), the release process was notably slower. In the following
studies, the PAM-proximal end of the NTS was labeled for accuratecharacterization of target cleavage.
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Next, we quantitatively studied the kinetics of the cleavage processby analyzing the time-dependent intensity traces derived from real-­time TIRFM imaging. The lifetime of individual target DNA molecules was determined by the time point at which the fluorescenceintensity dropped to baseline (Fig. 3B). We next monitored the target
cleavage on origami B, N, and P, respectively, to explore the effect of
PAM antennae. The individual lifetimes yielded the accumulated
cleavage fraction over time. We performed three repeated experiments to obtain averaged cleavage fraction with error bars (Fig. 3D).
We found that the cleaved fraction for targets on origami P harboring
PAM antennae was significantly higher (87 ± 4%) than that onorigami B (77 ± 1%) and N (74 ± 3%), suggesting that the presenceof PAM antennae significantly enhanced target cleavage efficiency.
Several studies showed that CRISPR-Cas9 can edit genome lociin different spatial contexts (29). To investigate whether CRISPR
cleavage is affected by the structure of the local environment, we
compared the cleavage of targets surrounded by non-PAM–containing
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Normalized counts

Twice

0.2

UnboundB

3 times

0.4

Fluorescence intensity (a.u.)ASCIENCE ADVANCES | RESEARCH ARTICLE
A

Cy3 onB (i)Cy3 off

2

ReleaseBinding

1

NTS

ReleaseBeforeBinding

(ii)

0
(iii)

1.0
PAM-distal labeled

0.8
0.6

PAM-proximal labeled

0.4
0.2
0.0

Cy3 fluorescence

Fraction of unreleased Cy3
Cleaved fraction

DNA origami

4

2

0.9
B

0.8

2

4

6

8

10

N

0.7

P

0.6
20

Non-PAM dsDNA
in solution

F

Target on
DNA origami
PAM-rich dsDNA
in solution

0.4

15

1
2

12

14

16

Time (min)0.810Time (min)16 min14tLifetime
0

**

5

12

Release

E

0

10

300

1.0

0.2

8

400

4
6
8 10 12
Time (min)0.66sgRNA/Cas9

200
0

1.0

0.0

2

500

0.6
0.4
0.2
0.0

Bl
B

N

P

N

on

M
PA

d

N
sD

M
PA

an

A

-ri

k

in

ch

l
so

ut

D
ds

io

n

NA

i

o
ns

lu

tio

n

Fig. 3. Enhanced target cleavage induced by PAM antenna. (A) Schematic illustration of the state of the Cy3 label corresponding to the binding of sgRNA/Cas9 and therelease of cleaved fragment. NTS, non-TS. (B) Target cleavage and release kinetics imaged using TIRFM. (i) A representative imaging field under TIRFM before (left) and15 min after injection of sgRNA/Cas9 (right) when Cy3 is labeled on the PAM-proximal end of NTS. (ii) Snapshots of two targets in the imaging field at the indicated time
points. (iii) Cleavage kinetics showing the lifetime of sgRNA/Cas9 for the two targets in (ii). (C) Fraction of unreleased Cy3 decays with time for Cy3 labeled on the PAM-­
proximal end (red) and PAM-distal end (blue) of NTS. (D) Curve of cleaved fraction calculated from single-molecule assay of origami N, P, and B. The zoomed-in view showscleaved fraction within 20 min after injecting sgRNA/Cas9 to origami N, P, and B. Data are mean ± SD (n = 3 independent experiments). **P = 0.01 (unpaired, two-sided ttest). (E) Schematics depicting the binding of sgRNA/Cas9 to target on origami B with non-PAM dsDNA and PAM-rich dsDNA in solution. (F) Cleaved fraction within 20 min
after injecting sgRNA/Cas9 to origami B with blank, non-PAM dsDNA, and PAM-rich dsDNA in solution. Error bars represent SD for three independent experiments.

antennae (origami N) with that of targets in antenna-free origami(origami B). Cleavage fraction measurements showed no significant
difference between origami N and B (Fig. 3D), suggesting that Cas9binding and cleavage were not significantly affected by the spatialorganization of non-PAM sequences. This independence was also
shown in dCas9-binding measurements (Fig. 2B).
To determine whether the formation of a structured PAM environment is required for enhanced target cleavage, we tested whether
freely diffusive antenna strands affect target cleavage (Fig. 3E). The
presence of 1 M non-PAM strands in the reaction buffer showedno obvious influence on target cleavage (Fig. 3F), which is consistent
with the effect of non-PAM antennae. However, the presence ofWang et al., Sci. Adv. 2020; 6 : eaay9948

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1 M PAM strands in the reaction buffer remarkably decreased thecleaved fraction of target by 30%, which is consistent with previous
studies that show that PAM-containing dsDNA added in trans act
as competitors to the specific target (18, 30). Compared to theenhancing effect of PAM antennae on DNA origami, the inhibiting
effect of freely diffusive PAM antenna strands in the reaction buffer
indicates that the confinement of PAM antennae into a structured
environment was necessary for cleavage efficiency enhancement.
Localized diffusion of Cas9 within PAM antennae
To investigate the mechanism by which PAM antennae improve thetarget cleavage efficiency, we tracked the movement of dCas9 within
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D

1
2

Cy3

C

After

Cleaved fraction

TS

SCIENCE ADVANCES | RESEARCH ARTICLE
the PAM antennae at the single-molecule level. Specifically, we
localized the position of fluorescently labeled sgRNA/dCas9 after
their binding to the origami. Analysis of time trajectories of individual
dCas9 reveals a remarkably larger range of displacement on origami
P than on origami B (Fig. 4B and fig. S10). Statistical analysis ofdCas9 displacement from its initial position revealed that the maximum
displacement range of dCas9 in 200-ms time interval on origami B
was within 40 nm, reflecting the range of displacement of tightly
bound dCas9 (Fig. 4A). In contrast, on origami P, this range ofdisplacement increases to nearly ~80 nm, suggesting the localized
diffusion of dCas9 among the PAM antennae (Fig. 4B). An origami
on which PAM-rich antennae were placed remotely from the target(origami O; fig. S11A) was used to study the dependence of localized
diffusion on target-PAM antenna distance. Both binding and cleavage results showed that remotely located PAM-rich regions do notfavor the transfer of Cas9 to the target as efficiently as when they areplaced in the proximal environment of the target (figs. S11 and S12).
To further study how the localized diffusion mechanism of Cas9
within the PAM microenvironments results in enhanced target
cleavage, we numerically simulated the spatial distribution of unbound Cas9 using a random walk model (Fig. 4C). In this model,
Cas9 performs localized diffusion with a short step length inside
PAM antenna and free diffusion with a long step length outside. To
qualitatively study the effect of localized diffusion on the spatial
distribution of Cas9, we simplified the PAM microenvironment into
a 10.5 nm–by–10.5 nm area with uniformly distributed 49 PAMs,
and the target site was set at the center of this area. Initially, Cas9s
were uniformly distributed in a 60 nm–by–60 nm region (fig. S13A).
By counting the number of occurrences of unbound Cas9 in eachspatial unit, we obtained a heatmap exhibiting the probability dis-

B
P

0.20
0.15
0.10
0.05
0.00

0

20
40
60
80
Displacement (nm)

100

40

Y displacement (nm)Y displacement (nm)Normalized counts

B

0.25

Programming of PAM antennae to modulate the target
microenvironment
Because the facilitated targeting of Cas9 by the antennae is distancedependent (fig. S11), we investigated whether the effects of the PAM
microenvironment could be modulated through the programmingof PAM antennae. We first synthesized two-layered PAM antennae
on DNA origami (Fig. 5A, middle). AFM images showed a PAM
island larger than that observed with regular antennae (Fig. 5B). We
then assessed the effect of the outer-layered PAM antennae on target
cleavage. Real-time cleavage assays showed no significant differenceon either two-layered antenna origami or origami P (Fig. 5C),
indicating that the outer-layered PAM antenna did not contributeto enhanced target cleavage. This result is consistent with ourobservation that remote PAM does not contribute to Cas9’s binding

20
0
−20
−40

−20

0

20

20
0
−20
−40

40

X displacement (nm)

C

40

−20

0

20

40

X displacement (nm)

D
−3
×10
3

sgRNA/Cas9

PAM

Target2.5

Free diffusion

2
1.5
1

Localized diffusion

0.5
10 nm

10 nm

Fig. 4. Localized diffusion mode target search increases target binding. (A) Displacement distribution of dCas9 obtained from trajectories on origami B and P.
(B) Difference in trajectories of sgRNA/dCas9 on origami B (left) and P (right) suggests diffusion of sgRNA/dCas9 among PAMs. (C) Possible modes for target searching of
sgRNA/Cas9 within PAM antenna including localized diffusion inside PAM antennae and free diffusion in solution. (D) Left: Localized diffusion inside PAM microenvironment
increases the probability of finding unbound Cas9 at the target location (center position). Right: The probability distribution of unbound Cas9 shows no change when the
nuclease is allowed to display only free diffusion inside PAM antennae.
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A

tribution for unbound Cas9 to appear at each location. The heatmap
showed an increased probability of finding Cas9 inside PAM antennae
and reached a maximum near the center (Fig. 4D). The increased
probability for the unbound Cas9 to appear at the target site
concurs with our experimental results of binding and cleavage assay
on origami P, indicating that localized targeting diffusion has apositive effect on target binding. In contrast, a model of the freely
diffusing Cas9 inside and outside PAM antennae predicted a uniform spatial distribution of unbound Cas9 that showed no increased
distribution at the center, suggesting that localized diffusion modeis essential for target binding enhancement. These observations,combined with the single-molecule tracking results, indicate that
the PAM-rich origami microenvironment enhances target cleavage
through a localized search diffusional mechanism, which is distinctly
different from that operating in solution.

SCIENCE ADVANCES | RESEARCH ARTICLE
A

Original staple

PAM-richer dsDNA

PAM-rich dsDNA

Target

PAM-richer

Two-layer

One-layer

6 PAM

10 PAM

6 PAM

Cy3

21.76 nm10.88 nm

3

C

2
1
0

Height (nm)

E

50
100
Distance (nm)5

PAM-richer
PAM-rich

4
3
2
1
0
0

50
100
Distance (nm)

F

3

0.8
0.6

2
Blank
One-layer
Two-layer

0.4
0.2
0.0

Cleaved fraction

0

D ×10

1.0
Cleaved fraction

Two-layer
One-layer

0

5

10
15
Time (min)

1

20

10 nm

0

1

2

3

0

1

2

3

−3

G ×10

1.0

−3

×10

3

0.8
0.6

2
Blank
Two-layer
PAM-richer

0.4
0.2
0.0

0

5

10
15
Time (min)

20

1

10 nm

−3

×10

Fig. 5. Programming PAM antenna for microenvironment modulation. (A) Schematic design and AFM characterization of one-layered PAM antenna (left; origami P),
two-layered PAM antenna (middle), and PAM-richer antenna (right) on DNA origami. (B) Profiles of one- and two-layered PAM antenna on DNA origami. (C) Curve ofcleaved fraction showing that the outer layer of PAM antenna does not contribute to cleavage enhancement. Error bars represent SD for three independent experiments.
(D) A larger spatial range of PAM antennae does not increase the appearance probability of unbound Cas9 inside the PAM antennae region. Error bars represent SD forfive independent simulations. (E) Profiles of two-layered PAM and PAM-richer antenna on DNA origami. (F) Curve of cleaved fraction showing that PAM-richer antenna
induces higher target cleavage efficiency. Error bars represent SD for three independent experiments. (G) Numerical simulations suggest that higher PAM density leads
to an increase in appearance probability of unbound Cas9 inside the antennae. Error bars represent SD for five independent simulations.

to target. To better understand this result, we simulated the spatial
distribution of unbound Cas9 appearance probability in the presenceof a larger PAM area (18 nm by 18 nm) using the localized diffusion
model. We found that the appearance probability at the center was
identical to that of the 10.5-nm PAM antenna model (Fig. 5D). It is
possible that the increased capture, associated with the use of more
PAMs, is offset by the concomitant larger diffusion range. The
ineffectual contribution of the outer-layered PAM antenna is consistent with the idea that the effect of PAM antennae is distancedependent, further confirming the importance for PAM sequences
to form a local microenvironment around the target to enhancecleavage.
We next investigated how the density of PAM in the microenvironment affects target cleavage. To this end, we constructed
PAM-richer origami harboring more PAMs on every antenna strand(Fig. 5A, right). Topographic profiles of AFM images of PAM-richer
Wang et al., Sci. Adv. 2020; 6 : eaay9948

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antenna revealed a central area of identical width to that of the two-­layered PAM-rich origami, but with a considerably increased height(Fig. 5E). Single-molecule cleavage kinetics of PAM-richer origami
led to increased cleavage efficiency when compared to that observedwith two-layered PAM origami (Fig. 5F). Simulations were conductedto model the effect of PAM density within the same PAM spatial
range (18 nm by 18 nm). We found that the appearance probabilityof unbounded Cas9 was further increased in areas near the center of
PAM antenna when the PAM density was increased (Fig. 5G),
consistent with the observed enhanced target cleavage. Together,
these results showed that higher density of PAM within the effective
distance could further enhance target cleavage efficiency, because
more PAMs increase the ability to recruit Cas9 molecules that could
diffuse to the target site. The positive correlation between PAM
density and target cleavage efficiency indicates that it is possible toregulate the microenvironment through arrangement of PAMs.
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Height (nm)

B 4

21.76 nm−3

SCIENCE ADVANCES | RESEARCH ARTICLE
Diffusion

D

SA-T1+ sgRNA/Cas9

T1
Target

PAM
Diffusion

Non-PAM

T3

PAM

Non-PAM

C

1.0

1.00

T1
T2

0.4

T3
0.2
0.0

0.95

1

10
Time (min)

100

2.0

−0.5

0.90
0.85

1.5

−1.0

0.80
0.75

Enhancement ratio
T1
T3

0.0

T1 T2 T3

−1.5

1.0
0.5

1.0

1.5

2.0

Ratio (sgRNA/Cas9:DNA)

Fig. 6. Target cleavage efficiency enhancement with linear adjacent PAM antenna. (A) Schematics show sgRNA/Cas9 binding process on target flanking by linear
PAM antenna containing eight tandem PAMs (T1), target separated with PAM antenna by 6-bp spacer (T2), and target flanking by non-PAM sequence (T3). (B) Averaged
cleavage fraction of target from independently repeated experiments confirms cleavage efficiency enhancement with adjacent linear PAM antenna. Error bars represent
SD for three independent experiments. (C) Final cleaved fraction of T1, T2, and T3 from six repeated experiments. Data from different experiments are indicated by different colors. The P values were calculated by paired two-sided t test (n = 6), where ns means P > 0.05 and **P < 0.01. (D) AFM images of T1 and T3 at 2 nM incubated with 20 nM
sgRNA/dCas9 showing that the binding position varies on T1, while dCas9 occupies a specific position on T3. (E) Cleavage fraction of T1 and T3 with 0×, 0.5×, 1×, and 2×
sgRNA/Cas9 (red and black; left y axis) and cleavage enhancement ratio with PAM antenna (green; right y axis). Error bars represent SD for three independent experiments.

Programming PAM antenna for bulk CRISPR-Cas9 editing
Having substantiated the target cleavage enhancement of PAM
microenvironments at the single-molecule level, we next tested
whether the PAM microenvironment could be programmed for more
efficient gene editing in bulk. We designed linear PAM antennae (inthe PAMs are present in cis with the target) either adjacent to thetarget site (T1; Fig. 6A) or separated from the target site by a 6-bp
non-PAM sequence (T2). For comparison, we synthesized a target
with non-PAM adjacent sequence (T3). These targets were designed to
mimic targets flanked by PAM-containing and non-PAM–containing
sequences within the genome.
To explore whether a linear PAM microenvironment can enhancetarget cleavage in bulk, we performed cleavage assays using polyacrylamide gel electrophoresis by incubating T1, T2, and T3 withthe same concentration of sgRNA/Cas9. Gel analysis showed thatboth T1 and T2 were thoroughly cleaved after incubating with sgRNA/
Cas9 at a 1:2 ratio for 1 hour, while a retained uncleaved band wasobserved for T3 (fig. S14B). We further monitored the cleavage
kinetics by interrupting the incubation of targets and sgRNA/Cas9
at different time points. Quantification of cleaved fraction fromrepeated experiments showed that targets next to PAM antennae T1
and T2 were cleaved faster than T3 (Fig. 6B and fig. S14A). Finalcleavage efficiency from repeated experiments of T1 (97 ± 1%) was
higher than that of T3 (88 ± 5%). We note that this improvement
effect is statistically significant (P < 0.01; Fig. 6C).
Wang et al., Sci. Adv. 2020; 6 : eaay9948

8 May 2020

To better understand the enhanced sgRNA/Cas9 functionality,
we investigated the effects of linear (in cis) PAM antennae on target
search process. We performed Markov chain simulations to study
the distribution of Cas9. Heatmaps of Cas9 distribution showedenhanced intensity near the antennae, suggesting that a binding
hotspot was formed by the linear PAM antennae (fig. S15). We next
conducted binding observations using T1 and T3 to investigate
whether the linear antennae adjacent to target could increase binding
efficiency. To add polarity to the dsDNA target, we attached a
streptavidin at one end (fig. S16). We found that a higher fraction of
T1 was bound to dCas9 than of T3 (fig. S17), which is consistent
with the hotspot effect of PAM antennae suggested by the simulations. Detailed investigation of the binding of Cas9 to these two typesof targets showed that the binding positions varied on T1, while
sgRNA/dCas9 occupied a fixed position on T3 (Fig. 6D). This observation indicates that linear PAM antennae can recruit Cas9 moleculesthat can be further translocated to the specific target site, contributing
to increased cleavage efficiency. Comparing cleavage kinetics of T1
and T2, we found that both the cleavage speed and final cleaved
fraction were indistinguishable for these two targets (Fig. 6B). The
maintained enhancing effect of cleavage observed with T2 suggeststhat the short non-PAM spacer sequence between target site and the
PAM antenna did not hinder the translocation of Cas9 (Fig. 6A,
middle). The ability of Cas9 to cross non-PAM regions provides awider target site selection range for more efficient gene editing.
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0.6

Cleaved fraction

0.8

ns

E

**

Log10 (cleaved fraction)

B

Cleaved fraction

SA-T3+ sgRNA/Cas9

T2

Cleaved T1/cleaved T3

ASCIENCE ADVANCES | RESEARCH ARTICLEDISCUSSION

In this work, we designed PAM antennae in spatial proximity witha Cas9 target site on a DNA origami platform. We studied the effectof PAM antennae on target binding and cleaving. We find that
PAM antennae anchored spatially close to the target enhance thelocal Cas9 distribution by recruiting more Cas9 molecules aroundthe target, which, in turn, results in higher target cleavage efficiency.
On the basis of the antenna effect on DNA origami, we designed alinear PAM antenna adjacent to target sequence and achievedenhancement of target cleavage in bulk.
Structured microenvironments have been shown to be criticallyimportant in many biochemical reactions both in vitro and in vivo.
However, the effect of PAM microenvironments on CRISPR-Cas9
gene editing has hitherto largely been neglected. Extensive efforts
have been focused on the sequence and structure optimization ofCas9, guide RNA, and the recognition site to achieve higher gene-­
editing efficiency. Progress has also been made through chemical
modulation or DNA replacement of guide RNA. However, gene-editing
efficiency needs to be further enhanced to improve in vivo applications of CRISPR. Here, we find that fine-tuning the PAM microenvironment affords a new route to improve the efficiency of gene
editing. By programming PAM antennae, we achieved improvementin binding and cleavage efficiency, providing a new paradigm forthe optimization of CRISPR-Cas9 targeting sites. Moreover, thisapproach can be combined with other strategies, including Cas9
optimization and guide RNA modification, to achieve more efficientgenome editing in future work.
Our data also reveal that Cas9 bound to a PAM antenna cancross short non-PAM spacer sequences (perhaps by linear diffusion),allowing a much wider selection range of target sites that are notdirectly flanked by PAM antennae. As each chromosome in thehuman genome has an uneven distribution of PAMs, the nanoscale
local PAM environment surrounding each targeting site could affectWang et al., Sci. Adv. 2020; 6 : eaay9948

8 May 2020

the cleavage efficiency differently. A detailed inspection of chromosome 1 showed that PAM intensity varies from 0 to 40 per 100 bp(fig. S20), the range of which is consistent with the number of PAMs
in our experiments, suggesting that programming PAM antennae
has potential for improving genome editing efficiency and specificity.
Taking advantage of DNA origami, DNA editing process was studied
with different PAM antennae mimicking the three-dimensional
microenvironment of chromatin. Three-dimensional characterizationof genome structures showed that chromosomes are folded into
highly packed topological-associated domains and long-range loops(31). A previous study reported that a 5 kbp locus could form ahighly condensed structure less than 35 nm in diameter (32). The
effects of PAM antennae found here suggest that PAM microenvironments surrounding the target in the context of chromatinshould play an important role for in vivo gene editing. Using theantenna effects of PAM-rich microenvironment may facilitate
efficient gene editing with a lower concentration of sgRNA/Cas9,
which may further reduce the burden of delivery and the impact oncell activities, and advance the utility of the CRISPR-Cas9 genome
editing system for practical purposes.
MATERIALS AND METHODS

Preparation of sgRNAAn optimized version of sgRNA proposed by Huang et al. (33) wasused with customized DNA matching region. To prepare sgRNA, we
prepared a dsDNA template containing the T7 promoter by polymerase chain reaction (PCR) amplification of a pair of forward andreverse primers using KOD polymerase (Toyobo; table S1). The product was purified using an agarose gel DNA extraction kit (TaKaRa,
9762). The obtained template was used for further transcription
using an in vitro transcription T7 kit (TaKaRa, 6140). The sgRNA
was purified using TRIzol (Invitrogen, 15596018) according to the
manufacturer’s instructions. The purified sgRNA was stored at −80°C.
Preparation of DNA origami with designed target site
and antennae
Staples purchased from Sangon Biotech (Shanghai) were dissolved
in deionized water, and stored in 100 M at −20°C. Extended staples(table S2) for target and antennae were premixed to 1 M. Original
staples were premixed to 500 nM, and edge staples were removed to
avoid stacking force while annealing. The origami folding mixture(100 l) was prepared by mixing M13mp18 (to 2 nM; New England
Biolabs, N4040S), extended staples (to 10 nM), and original staples(to 10 nM) in 1× TAE-Mg2+ buffer (40 mM tris, 2 mM EDTA,
and 12.5 mM MgAc2). The mixture was annealed using PCR Thermal
Cycler (Applied Biosystems) with the following procedure: 95°C
for 3 min, 95° to 4°C at a rate of 0.1°C/10 s in 0.1°C steps, 4°C
forever. The annealed mixture was incubated with 50× hybridization
strands of the extended staples for 4 hours under room temperature.
DNA origami was purified three times at 3000g for 10 min using
100K centrifugal filters (Millipore) in an Eppendorf centrifuge andstored at 4°C for further use.
Gel electrophoresis for cleavage kinetics
For cleavage kinetics analysis of T1, T2, and T3, mixtures containing
160 nM target and 320 nM sgRNA/Cas9 with 1× reaction buffer
were prepared on ice and divided into 10-l parts. Mixtures wereincubated at 37°C, and one sample for each of the three targets was
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To explore the effects of sgRNA/Cas9 concentration on target
cleavage with and without PAM antennae, we incubated T1 and T3
with increasing levels of sgRNA/Cas9 and quantified the cleavage
fractions from gel assays (Fig. 6E and fig. S14C). The higher cleavage efficiency observed for T1 than for T3 under each concentration
condition suggests that gene editing could be achieved using lesssgRNA/Cas9 through programming of PAM antennae. The ratio ofcleaved fraction of T1 to T3 showed a higher cleavage enhancement
ratio using PAM antennae at low sgRNA/Cas9 concentration (Fig. 6E),
thereby potentially enabling more efficient gene editing when thedelivery efficiency of CRISPR system is limited.
We further studied whether PAM antennae could influence off-­target DNA cleavage. A mismatch 8-bp upstream PAM was introducedon T1 and T3 (M8-PAM and M8; fig. S18A). We found that thecleavage efficiency of M8-PAM was higher than that of M8 (fig. S18),
suggesting that the presence of PAM antennae could also increase
the cleavage of off-target sites, which may potentially reduce thespecificity of gene editing. However, with the coexistence of target
and off-target sites, the cleavage of target with M8-PAM did not
differ from that with M8 (fig. S19A). In contrast, the cleavage of an
off-target site (M8) was significantly decreased with T1 than withT3 (fig. S19B), suggesting that higher specificity could be achieved
with PAM antennae at target site and avoiding PAM antennae atpotentially off-target sites.

SCIENCE ADVANCES | RESEARCH ARTICLE
taken at indicated time points: 0, 1, 2, 4, 6, 8, 10, 30, and 60 min.
Samples taken out were put into a 70°C thermostat water bathimmediately and incubated for 10 min to inactivate Cas9. After
samples were all treated in 70°C, they were incubated with ribonuclease at 37°C for 30 min. Products were mixed with 2 l of 6× loading
buffer before loading to 10% polyacrylamide gel. The gel was run
with a constant voltage of 120 V for 80 min. At the end of the run,the gel was stained with GelRed and imaged using G:Box Chemi-XL(Syngene). Cleavage efficiency analysis at each time point was
performed using the Gels tool in the ImageJ software.

TIRFM experiments
TIRFM imaging was performed using a Nikon N-STORM microscope endowed with an electron-multiplying charge-coupled device
camera (Andor iXon Ultra 897) and a solid-state 561-nm excitation
laser. Multi–band-pass filters (422 to 478 nm, 502 to 549 nm, 581 to
625 nm, and 674 to 686 nm) were used for collecting fluorescenceemission. TIRFM imaging experiments were performed in imaging
buffer (40 mM tris, 2.1 mM EDTA, 20 mM Hepes, 100 mM NaCl,
5 mM MgCl2, and 12.5 mM MgAc2) at 20°C. For target cleavage
experiments, glass-bottom dishes (D-29-10-1-N; Cellvis) were treatedusing plasma cleaner to make it hydrophilic. DNA origami (10 l,
0.2 nM) was placed on the glass surface and incubated for 5 min to
attach on the surface. The excitation intensity was adjusted to guaranteethat Cy3 labels could survive more than 100 frames. To monitor thecleavage kinetics, we set an automated acquisition procedure for
15 or 20 min with 20-s intervals. Survival time distributions andaccumulated cleaved fractions were calculated by custom-written
MATLAB (MathWorks) programs. Polyethylene glycol (PEG)–
passivated coverslips (34) were used for sgRNA/dCas9 tracking
experiments (see Supplementary Methods for biotin-PEG coverslip
preparation). Movies were obtained to record sgRNA/dCas9 binding
events on origami B and P. Binding events and single-particle tracking were performed using custom-written MATLAB programs (seeSupplementary Methods for details).
SUPPLEMENTARY MATERIALSSupplementary material for this article is available at http://advances.sciencemag.org/cgi/
content/full/6/19/eaay9948/DC1
View/request a protocol for this paper from Bio-protocol.

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Acknowledgments
Funding: This work was financially supported by the Ministry of Science and Technology ofChina (2016YFA0201200), the NSFC (grant nos. 21834007, 21722502, 21505045, and21705048), the Shanghai Municipal Education Commission–Gaofeng Clinical Medicine Grant
Support (20171913), and the Nanomachines program (KC1203) funded by the Office of Basic
Energy Sciences of the U.S. Department of Energy (DOE) contract no. DE-AC02-05CH11231 (toC.B.). Author contributions: C.F. and C.B. directed the research. F.W., Y.H., and Q.L. performed
the experiments. F.W., Y.H., Q.L., J.L., H.Z., X.Z., and L.W. analyzed the data. C.F., F.W., and C.B.
conceived the experiment and wrote the manuscript. All authors read the manuscript andprovided comments. Competing interests: The authors declare that they have no competinginterests. Data and materials availability: All data needed to evaluate the conclusions in
the paper are present in the paper and/or the Supplementary Materials. Additional data
related to this paper may be requested from the authors.
Submitted 4 August 2019
Accepted 19 February 2020
Published 8 May 2020
10.1126/sciadv.aay9948
Citation: F. Wang, Y. Hao, Q. Li, J. Li, H. Zhang, X. Zhang, L. Wang, C. Bustamante, C. Fan,
Programming PAM antennae for efficient CRISPR-Cas9 DNA editing. Sci. Adv. 6, eaay9948 (2020).

Downloaded from http://advances.sciencemag.org/ on May 12, 2020

Wang et al., Sci. Adv. 2020; 6 : eaay9948

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Programming PAM antennae for efficient CRISPR-Cas9 DNA editing
Fei Wang, Yaya Hao, Qian Li, Jiang Li, Honglu Zhang, Xueli Zhang, Lihua Wang, Carlos Bustamante and Chunhai Fan

Sci Adv 6 (19), eaay9948.
DOI: 10.1126/sciadv.aay9948

http://advances.sciencemag.org/content/6/19/eaay9948

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