Conformational regulation of CRISPR-
associated nucleases
Authors: Ryan N. Jackson, Paul P.G. van Erp, 
Samuel H. Sternberg, and Blake Wiedenheft
NOTICE: this is the author’s version of a work that was accepted for publication in Current 
Opinion in Microbiology. Changes resulting from the publishing process, such as peer review, 
editing, corrections, structural formatting, and other quality control mechanisms may not be 
reflected in this document. Changes may have been made to this work since it was submitted for 
publication. A definitive version was subsequently published in Current Opinion in Microbiology, 
[VOL#, ISSUE#, (DATE)] DOI# 10.1016/j.mib.2017.05.010.
Jackson, Ryan N. , Paul Bg van Erp, Samuel H. Sternberg, and Blake Wiedenheft. 
"Conformational regulation of CRISPR-associated nucleases." Current Opinion in Microbiology 37 
(June 2017): 110-119. DOI: 10.1016/j.mib.2017.05.010.
Made available through Montana State University’s ScholarWorks 
scholarworks.montana.edu 
Conformational regulation of CRISPR-associated
nucleases
Ryan N Jackson1, Paul BG van Erp2, Samuel H Sternberg3 and
Blake Wiedenheft2Adaptive immune systems in bacteria and archaea rely on small
CRISPR-derived RNAs (crRNAs) to guide specialized
nucleases to foreign nucleic acids. The activation of these
nucleases is controlled by a series of molecular checkpoints
that ensure precise cleavage of nucleic acid targets, while
minimizing toxic off-target cleavage events. In this review, we
highlight recent advances in understanding regulatory
mechanisms responsible for controlling the activation of these
nucleases and identify emerging regulatory themes conserved
across diverse CRISPR systems.
Introduction
Nucleases that degrade DNA and RNA are indispensable
for diverse biological functions [1]. To avoid toxicity
associated with aberrant activity, nucleases are often
controlled by substrate binding at (orthosteric), or away
from (allosteric) the active site. Recent evidence suggests
that CRISPR (Clustered Regularly Interspaced Short
Palindromic Repeat)-associated (Cas) nucleases, which
are essential components of adaptive immune systems
that protect bacteria and archaea from infection by viruses
and plasmids, rely on orthosteric and allosteric control
[2,3]. Presumably these regulatory mechanisms evolved
to efficiently eliminate foreign DNA or RNA, while
avoiding autoimmune reactions associated with destruc-
tion of the bacterial or archaeal genome. Theprogrammable nature of CRISPR-associated nucleases
(e.g., Cas9) has given rise to a powerful new method for
genome engineering, and a comprehensive understand-
ing of how these nucleases function is necessary for safe
implementation [4–7].
CRISPR RNA-guided adaptive immune systems are
structurally and functionally diverse, consisting of two
Classes (1 and 2), six Types (I–VI), and more than
nineteen subtypes distinguished by CRISPR repeat
sequence and cas genes [2,8–10] (Figure 1). Despite this
diversity, all CRISPR systems rely on specialized
nucleases to execute three stages of adaptive immunity;
acquisition, CRISPR RNA biogenesis, and interference
[2,3]. During acquisition, a nuclease active integrase
complex comprised of Cas1 and Cas2 proteins inserts
foreign nucleic acid targets (about 20–40 nt in length)
called ‘protospacers’ into the spacer-repeat array at the
leader end of the CRISPR locus. CRISPR loci are tran-
scribed, and Cas or RNAse III enzymes process these
transcripts into libraries of small CRISPR-derived RNAs
(crRNA). Each crRNA assembles with Cas proteins into
surveillance complexes that use the crRNA to bind com-
plementary DNA (Type I, II, V) or RNA (Type III, VI)
(Figure 1). Target binding induces conformational rear-
rangements within the surveillance complex that activate
either cis-acting nuclease domains located within the
complex, or a trans-acting nuclease that is recruited for
destruction of bound targets.
Structures of Cas nucleases involved in spacer acquisition
(e.g., Cas1) and crRNA biogenesis (e.g., Cas6), suggest
substrate binding at the enzymatic active site (i.e., orthos-
teric) induces conformational rearrangements that regu-
late the nuclease [11–13]. In contrast, Cas nucleases
involved in interference rely on a combination of orthos-
teric and allosteric activation mechanisms. Here we
review the regulatory mechanisms that control Cas
nucleases involved in interference.
Regulation of Class 2 Cas nucleases
Class 2 systems consist of three different Types (i.e.,
Type II, V, and VI) that encode single-subunit Cas
nucleases [2,8,9] (Figure 2). The Type II (Cas9) systems
gained considerable notoriety in 2012 and early 2013 when
the programmable nature of these RNA-guided nucleases
was exploited for precise cleavage of DNA in human cells
(for a review of the primary literature, see [4–6]). In the
Figure 1
Class 2 - Single-subunit
Class 1 - Multi-subunit
CR
IS
PR
 S
ys
te
m
 P
hy
lo
ge
ny
3'5'
spacer
crRNAcas3
cascade genes
 Type I
bind: dsDNA, cut: dsDNA
3'5'
spacer
crRNA
tracrRNA
cas9
 Type II
bind: dsDNA, cut: dsDNA
3'5'
spacer
crRNA
 III-A csm / III-B cmr genes
 Type III
bind: ssRNA, cut: ssRNA & ssDNA
cas10 cas7
csf genes
 Type IV
bind: ?, cut: ?
dinG
Type V
bind: dsDNA, cut: dsDNA
cas12a
3'
3'
5'
5'
spacercrRNA
cas12b
cas12c
tracrRNA
or
(Cas12b)
or
3'5'
spacer
crRNACas13
(C2c2)
 Type VI
bind: ssRNA, cut: ssRNA
3' 5'
tracrRNA
PAM
DNA-Phage
Cas3
PAM
5'
3'
Type V
Cas12a / Cas12b / Cas12c
PAM
rPAM
Cas10
5’
Cas7
Cas7
Cas7
3'
5'
nascent RNA
RNP
transcription
bubble
processive
 DNA cleavage
RNA cleavage
6nt intervals
?
3'
5'
 Type I
Cascade
 Type III
Csm / Cmr 
complex
 Type IV
 Type II
Cas9
blunt-end 
dsDNA cleavage
3'
5'
RNP
staggered 
dsDNA cleavage
non-specific
 ssDNA cleavage 
transcription
bubble
PFS
5'
5' nascent RNA
RNA-Phage
5'
Cas7
Cas7
Cas7
3'
rPA
M
non-specific RNA cleavage 
outside the crRNA:RNA duplex
PFS
5
'
 Type VI
Cas13
(a) (b)
?
 Type IV
 Type III
Csm / Cmr 
complex
(Cpf1) (C2c1) (C2c3)
Current Opinion in Microbiology
Diverse CRISPR systems defend against DNA- and RNA-based invaders.
(a) Phylogenetic tree of CRISPR system Types with nucleic acid substrates that are bound and cleaved. DNA targeting nucleases are labeled red
and RNA targeting nucleases are labeled blue. Genes coding for multi-subunit complexes are indicated with the brackets. (b) Cartoon
representations of each CRISPR system targeting DNA phage, RNA phage, or both.
Figure 2
5'
180°
L1
L2
RuvC
Rec lobe
HNH
Nuc lobe
L1
L2
Cas9
single-guide RNA
5'
3'
repeatspacer
trans-activating
RNA
5'
3'
protospacer
PAM
PA
M
 &
 s
ee
d
 
re
co
gn
iti
on
5'
3'
co
n
fo
rm
at
io
na
l
re
ar
ra
n
ge
m
en
t
R
N
A
-g
ui
de
 lo
ad
in
g
di
re
ct
io
na
l
u
n
w
in
di
ng
complementary
strand
non-complementary 
strand
RuvC
HNH
5' 3’
PAM
protospacer
dsDNA
5' 3'
RuvC
Nuc
repeat spacer
crRNA
Nuc lobe
Rec lobe
?
Type II Type V
Cas12a/Cas12b/Cas12c
5'
5'
3'
3'
5'
3'
concerted
blunt-end dsDNA cleavage
5'
RuvC
Nuc
staggered dsDNA cleavage
5'
RuvC
or
Type VI
Cas13
5'
HEPN HEPN
5'
3'
U
U
3'- seed
1st
2nd
5'- seed
dsDNA
??
5'
HEPN HEPN
crRNA
Rec lobe
Nuc lobe
RNA cleavage near U nucleotides
outside the crRNA:RNA duplex
3'
?
3'
5'
3'
5'
3'
ssRNA
5'PFS
central-seedorder?
RuvC and Nuc RuvC cleaves both strands
rearrangement ?
5'
3'
5'
PFS
G deactivating
or
non-G activating 
 
repeat spacer
3'
5'
3'
?
rearrangement ?
?
bidirectional ?
class-2 single-subunit
(a) (b) (c)
order?
(Cpf1) (C2c1) (C2c3) (C2c2)
non-complementary 
strand
non-complementary 
strand
Current Opinion in Microbiology
Class 2 Nuclease activation mechanisms.
(a) Activation of the Type II Cas9 nuclease (grey with red outline) relies on several regulatory checkpoints. Cas9 adopts a bi-lobed structure that
consists of a nuclease-lobe (Nuc lobe) and an alpha-helical recognition lobe (Rec lobe). Single-guide-RNA loading causes a conformational
rearrangement in the Rec lobe. To bind duplex DNA, a PAM motif (dark red) is recognized followed by 30 seed complementation. Directional
unwinding of the duplex to the PAM-distal side of the guide induces a conformational rearrangement of two peptide linkers (L1 and L2 colored
blue and orange) that stabilize the active conformation of the HNH and RuvC nuclease domains (colored red) to make a blunt-end cut in duplex
DNA. (b) Type V nucleases (Cas12a, Cas12b, and Cas12c; also known as Cpf1, C2c1, and C2c3) also adopt a bi-lobed architecture, are loaded
with an RNA-guide and use PAM and seed recognition to initiate target binding with DNA. However, the DNA cleavage mechanisms may involve
the RuvC domain and Nuc domain, or just the RuvC domain. (c) Like Type II and V, the Type VI (Cas13, also known as C2c2) proteins adopt a bi-
lobed architecture and are programmed with an RNA-guide. Type VI nucleases bind RNA with a central seed within the RNA-guide, and an
adjacent sequence called the PFS (Protospacer Flanking Site) plays a role in nuclease activation.last five years, these nucleases have ushered in a new era
of genome editing technologies that have considerable
potential for applications in molecular medicine, indus-
trial biotechnology, and agriculture.Type II systems
The implementation of Type II systems for genome
editing applications requires a fundamental understand-
ing of how these RNA-guided nucleases assemble, bind
to double-stranded DNA (dsDNA), and then cleave both
strands of the dsDNA target. Initial biochemical work on
the Cas9 ribonucleoprotein from Streptococcus pyogenes
showed that the crRNA base-paired to a trans-activating
tracrRNA, and that these two RNAs could be fused into a
single-guide RNA (sgRNA) capable of directing the Cas9
nuclease to a complementary DNA [14,15]. Subsequent
structural and biochemical studies demonstrated that
Cas9 is a dynamic enzyme, and that target cleavage
requires sequential completion of multiple regulatory
checkpoints.
Cas9 adopts a bi-lobed architecture in which two adjacent
nuclease domains (RuvC and HNH) reside within the
nuclease lobe (NUC lobe), and an alpha-helical lobe (also
known as the recognition or REC lobe) interacts with the
guide RNA–target DNA heteroduplex
[16,17,18,19,20,21]. In the absence of RNA, Cas9
adopts a nuclease-inactive conformation, which under-
goes a dramatic rearrangement upon binding the guide
RNA [16] (Figure 2a). Cas9 relies on base-pairing
between the crRNA guide and DNA targets, but sin-
gle-molecule experiments performed using Cas9 from
Streptococcus pyogenes showed that Cas9 must first recog-
nize a tri-nucleotide motif called a PAM (Protospacer
Adjacent Motif) located next to the DNA target sequence
[22]. In addition to PAM recognition, high-affinity bind-
ing requires precise complementarity between the DNA
target and a PAM-proximal ‘seed sequence’ followed by
directional unwinding of the DNA from the seed to the
PAM-distal end of the target [23] (Figure 2a). Chroma-
tin immunoprecipitation followed by sequencing (ChIP-
seq), revealed that as many as 15 mismatches are allowed
outside of the seed sequence for stable binding, but
incomplete RNA–DNA hybrids are often not cleaved
by Cas9 [24–26].
Initial structures of DNA-bound Cas9 could not explain
why stable binding and incomplete R-loop formation fail
to activate Cas9 cleavage. In these structures, the active
site of the HNH nuclease domain is 30 A˚ away from the
scissile phosphate of the target DNA strand [17,18,27]
(Figure 2a), suggesting that a major conformational rear-
rangement was necessary to activate Cas9-mediated
DNA cleavage. To test this hypothesis and clarify the
substrate features required for Cas9 activation, Fo ̈rster
Resonance Energy Transfer (FRET)-based assays were
used to measure conformational changes in the HNH
domain upon binding to perfectly complementary and
mismatched DNA targets [28]. This work revealed that
the HNH nuclease domain adopts an inactive conforma-
tion on mismatched DNA substrates that are stably bound
by Cas9, explaining why off-target DNA binding events
do not always lead to off-target DNA cleavage. Full
complementarity between target DNA and the crRNA
guide drives conformational changes in the HNH domain
that trigger target-strand cleavage, and allostericallyactivates cleavage of the non-complementary strand by
the RuvC domain, ensuring concerted production of
double-strand breaks [28].
A recent structure of Cas9 bound to a long dsDNA
substrate provides a structural basis for the concerted
activation of the Cas9 nuclease domains [29] (Figure 2).
As observed in all other Cas9 structures, the HNH
domain is inserted between motifs II and III in the RuvC
domain, and is connected to these motifs by two peptide
linkers, L1 and L2. Comparison with previous Cas9
structures reveals that L1 and L2 undergo extensive
rearrangement after binding a dsDNA-target. L1 interacts
with the PAM-distal side of the RNA–DNA hybrid, while
a conserved phenylalanine on L2 stacks with the fourth
nucleobase of the protospacer on the non-complementary
strand. These interactions stabilize a 180 rotation of
the HNH domain and position the active site in proximity
to the scissile phosphate of the complementary strand.
Additionally, these rearrangements position the scissile
phosphate of the non-complementary strand into the
active site of the RuvC nuclease. To avoid steric clashes,
these large conformational rearrangements can only be
achieved if both L1 and L2 move in concert, which
explains why HNH and RuvC activation is coupled.
Cas9 nucleases from organisms other than S. pyogenes
share a similar HNH and RuvC domain linkage, suggest-
ing this concerted activation mechanism may be con-
served across all Cas9 enzymes [16,19,20,21].
Type V systems
Like Cas9, the Type V enzymes – Cas12a, Cas12b, and
Cas12c; also known as Cpf1, C2c1, and C2c3 – contain a
putative nuclease domain (Nuc) inserted between motifs
II and III of a RuvC domain [9,30–35]. Point mutations in
the Nuc domain of Cas12a (Cpf1) inhibited cleavage of
the complementary strand, while mutations in the RuvC
active site disrupted cleavage of both strands [31]. These
data suggested RuvC-mediated cleavage of the non-com-
plementary strand is a pre-requisite for cleavage of the
complementary strand by the Nuc domain. However, a
structure of Cas12b (C2c1) bound to a long single-
stranded DNA suggests the RuvC nuclease may be
responsible for cleaving both strands of the DNA in
sequential order [34]. Indeed, a recent study of Cas12a
provides biochemical evidence that a single active site
within the RuvC domain cleaves both DNA strands using
the same catalytic mechanism, and that activation of this
nuclease function requires substantial conformational
rearrangements to unblock the previously occluded active
site [36]. While it appears that Cas12 enzymes recognize
DNA targets in a distinct way from Cas9, both enzyme
families have evolved mechanisms to sequester the active
sites in their nuclease domains until RNA–DNA hetero-
duplex formation triggers activation.
Type VI systems
Unlike most Class 2 systems, which target dsDNA, the
Type VI Cas nucleases (Cas13, also known as C2c2) bind
and cleave ssRNA using two HEPN (Higher Eukaryote
and Prokaryote Nucleotide-binding) domains [9,37–39]
(Figure 2b). The HEPN RNase domains are activated by
ssRNA base-pairing with the crRNA guide, but nuclease
activity is suppressed if complementarity extends beyond
the guide and into the repeat-derived portion of the
crRNA [37]. Complementarity to the repeat-derived
sequence of the crRNA may explain the inhibition of
Cas13 (C2c2) nuclease activity, similar to regulation of
DNase activity in Type III systems (see below). Notably,
unlike other Class 2 systems, RNA binding by Cas13
activates a non-sequence specific and trans-acting RNase
activity [37,38]; a property which has recently been har-
nessed for sensitive RNA and DNA diagnostics [40].
Thus, target-induced conformational rearrangements
are required to activate the promiscuous HEPN nuclease
domains. While recent structures have shed light on
Cas13 conformational changes that occur upon crRNA
binding [39], further structural and biophysical studies are
necessary to fully understand the conformational rearran-
gements that occur upon target RNA binding and the
mechanism of RNA cleavage in trans.
Regulation of Class 1 nucleases that are
essential for interference.
Class 1 CRISPR systems are phylogenetically and func-
tionally diverse, but a unifying feature of these systems is
that they all rely on multi-subunit crRNA-guided surveil-
lance complexes for detection of invading nucleic acids
[8,41]. Class 1 systems consist of two well-studied Types
(Type I and III), and a recently identified Type IV system
that has not been experimentally tested and is beyond the
scope of this review.
Type I systems
Phylogenetic studies have divided Type I systems into
seven sub-types, I-A to I-F and I-U [8]. All Type I
CRISPR systems rely on multi-subunit surveillance com-
plexes for crRNA-guided detection of dsDNA targets,
and a Cas3 nuclease/helicase that is responsible for target
degradation.
Foreign DNA detection by the Type I-E system in
Escherichia coli relies on a large crRNA-guided complex
called Cascade (CRISPR-associated complex for antiviral
defense) [42,43]. Cascade searches for targets by first
localizing to PAM sequences, which are recognized by
the Cas8e subunit (also called Cse1) [23,44,45,46].
PAM detection destabilizes the target duplex and facil-
itates crRNA-guided sampling of the PAM-proximal
DNA seed sequence [47]. Complementarity between
the crRNA-guide and the seed leads to directional
unwinding of the DNA duplex that proceeds away from
the PAM and triggers a conformational change in Cascadethat is necessary for recruitment of the trans-acting Cas3
nuclease/helicase, which degrades the DNA target
[23,48–50,51]. However, there is an accumulating
body of work showing that Cascade may adopt distinct
conformational states based on interactions with specific
PAM and protospacer combinations, and that these dis-
tinct conformations may regulate the activity of Cas3
[44,49,51,52,53,54–56].
Cascade elicits two distinct immune responses upon
target binding: interference or priming (Figure 3). During
interference, the Cas8e subunit adopts a ‘closed’ confor-
mation capable of recruiting a nuclease active Cas3,
which processively degrades the target DNA
[44,50,51,57–59]. However, strict sequence require-
ments present a potential weakness in the immune sys-
tem, because mutations in the PAM, seed, or specific
positions of the protospacer, allow viruses to escape
CRISPR-Cas immunity [47,60,61]. Bacteria with Type
I immune systems can restore immunity against PAM or
protospacer ‘escape’ mutants using a positive feedback
loop that rapidly updates the CRISPR locus with new
spacers derived from the phage or plasmid genome
[61–64]. This process of rapid acquisition, called
‘priming’, requires a target that is partially complemen-
tary to the crRNA-guide, Cas3, and two proteins that are
essential for adaptation (i.e., Cas1 and Cas2) [62,65].
Mutated targets that elicit a priming response corre-
spond to an ‘open’ conformation of the Cas8e subunit,
which does not effectively recruit nuclease active Cas3
[44,51,52] (Figure 3). However, in the presence of
Cas1 and Cas2, Cascade bound to a priming target
efficiently recruits nuclease-repressed Cas3 [44].
Recent structural and biochemical studies performed
using the Type I-F immune system from Pseudomonas
aeruginosa, in which Cas2 and Cas3 are fused into a single
polypeptide (Cas2/3), indicated that Cas1 and Cas2/3
form a complex, and that Cas1 represses Cas2/3 nuclease
activity until activated by the target bound surveillance
complex [66].
Collectively, these results suggest that the conformational
state of Cascade recruits a nuclease active Cas3 for direct
degradation (interference) or a nuclease repressed Cas1-
2/3 complex for rapid adaptation (priming) [44]. How-
ever, in both instances Cas3 cleavage products are pre-
cursors for CRISPR adaptation [67], suggesting that
priming targets do elicit some level of Cas3 nuclease
activity and that primed adaptation also takes place
during the interference response. It is clear Cas1, 2,
and 3 work together to generate and integrate new
sequences into CRISPR loci, but the mechanism of
allosteric regulation of Cas3 activity by the conforma-
tional state of Cascade remains poorly understood.

Type III systems
Like Type I systems, the Type III systems also rely on
large multi-subunit crRNA-guided surveillance com-
plexes that are divided into sub-types (i.e., Type III-A–
III-D) [8]. Initially these subtypes were functionally
divided into either DNA or RNA targeting immune
systems. However, a series of recent experiments
revealed a sophisticated mechanism that explains how
crRNA-guided binding to complementary RNA alloste-
rically activates a non-sequence specific DNase activity
(Figure 3) [68]. This model reconciles earlier distinctions
between RNA or DNA targeting and reveals that Type
III systems cleave both RNA and DNA targets.
The RNase and DNase active sites in Type III com-
plexes are spatially separated, functionally integrated,
and temporally controlled. RNA binding drives a confor-
mational rearrangement that allosterically activates non-
sequence-specific DNase activity in the Cas10 subunit
[69,70,71,72,73,74,75]. The RNA target is cleaved
in regular 6-nt intervals and structures of Type III com-
plexes have identified residues in the backbone (Cas7-
family) and belly subunits that are necessary for RNA
cleavage [41,68,76]. According to the current model,
periodic cleavage of the RNA target, leads to dissociation
of the RNA fragments, which restores the complex to a
DNase inactive state [68,70,71,72].
In addition to complementarity between the crRNA-
guide and the RNA protospacer, sequence 30 of the
protospacer regulates DNase activity. Complementarity
between the crRNA and the RNA target that extends
beyond the guide and into the 50-repeat of the crRNA
inhibits DNase activation in all Type III systems
[70,71,72,73,74,75,77], but requirements for DNase
activation seem to vary between systems. In Thermotoga
maritima, the DNase function is activated by protospacers
lacking flanking sequence, suggesting complementation
between the crRNA-guide and RNA target is the only
signal necessary for DNase activation and base pairing
with the 50-repeat is inhibitory [71]. However, in several
other systems, hybridization with the protospacer
sequence alone is not sufficient for DNase activation.
In these systems, the DNase remains inactive unless a
non-complementary sequence extends from the proto-
spacer into the 50-repeat [72,73,75], and in some sys-
tems (e.g., Type III-B Cmr system in P. furiosus) this 30
non-complementary RNA sequence must meet specific
sequence requirements [70]. The mechanistic distinc-
tions between these systems and the precise role of the 30
RNA in controlling DNase activation awaits structural
studies of Type III complexes bound to RNAs with 30-
flanking sequence.
Perspective
CRISPR-associated nucleases are phylogenetically and
functionally diverse, yet common regulatory themes arebeginning to emerge. All CRISPR systems that target
DNA rely on protein mediated PAM recognition and
crRNA-guided recognition of the protospacer. However,
binding does not necessarily result in target cleavage.
Recent data indicates that different PAM and protospacer
combinations result in distinct conformational states that
regulate nuclease activity. These new insights are reveal-
ing additional layers of sophistication in prokaryotic adap-
tive immune responses, and understanding how target
selection impacts the activity of these nucleases will have
important implications for applications in genome
editing.
Conflict of interest statement
B.W. is the founder of SurGene LLC, and an inventor on
patent applications related to CRISPR-Cas systems and
applications thereof. S.H.S. is an employee of Caribou
Biosciences, Inc. and an inventor on patents and patent
applications related to CRISPR-Cas systems and applica-
tions thereof.
Acknowledgements
Research in the Wiedenheft lab is supported by the National Institutes of
Health (P20GM103500, P30GM110732-03, R01GM110270, and
R01GM108888), the National Science Foundation EPSCoR (EPS-110134),
the M.J. Murdock Charitable Trust, a young investigator award from
Amgen, Gordon and Betty Moore Foundation, and the Montana State
University Agricultural Experimental Station and Montana University
System Research Initiative (51040-MUSRI2015-03). Research in the
Jackson Lab is supported by Utah State University New Faculty Start-up
funding from the Department of Chemistry and Biochemistry, the Research
and Graduate Studies Office, and the College of Science.
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