, 960 (2008);
et al.Stan J. J. Brouns,
Small CRISPR RNAs Guide Antiviral Defense in
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per junction. Although 29,689 junctions in HEK
and 24,848 in B cells had only one read, those
were considered highly notable, as we expect
at most 23 reads hitting a junction by chance
in the entire data set (16). Splice junctions were
associated with 81% of the expressed genes. We
also observed splice junctions for ~260 genes
in each cell line that were not classified as ex-
pressed (Tables 1 and 2). Of those, 70% had
between 1 and 4 reads and 30% were silent, sug-
gesting a very low activity. The fact that 2275
expressed genes in HEK and 2013 in B cells
had no splice-junction reads correlated with the
fact that those genes contained fewer exons and
a lower activity than the average, reducing the
probability to hit a splice junction.
We observed 95% of the splicing events ex-
pected in this data set, given the current se-
quencing depth (Table 1) (16). We identified 4096
previously unknown splice junctions in 3106
genes, mostly called by single reads and unique
to one cell type (Table 1). Many of these junc-
tions were associated with actively transcribed
genes exhibiting more exons than average, point-
ing to rare splicing events. Approximately 6%
of all splice-junction reads identified AS events
(6416 junctions in 3916 genes HEK and 5195
junctions in 3262 genes in B cells) (table S9). In
a parallel study surveying the mouse transcriptome,
AS forms were observed for 3462 genes in three
tissues (28), but no attempts were made to search
for previously unrecognized junctions. Within a
cell type, junction reads identify AS in 30% of
the expressed genes, where exon skipping was
largely overrepresented (Fig. 3A). Skipping events
affected mostly one or two exons, with a sharp
decline between one and five exons (Fig. 3B). An
illustrative example of AS is given for PKM2,
also showing that the read density reflects the
exon usage (Fig. 3C). Very complex patterns of
AS could be detected. For instance, with the use
of EIF4G1 coding for the eukaryotic translation
initiation factor 4 gamma 1, we showed 12 AS
junctions in B cells, of which five have not yet
been identified (fig. S3). Although AS is known
to regulate the expression of EIF4G1 (29, 30),
such a complex pattern had never been de-
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31. The Gene Expression Omnibus accession number for the
microarrays and sequence data is GSE11892. Data are
displayed in a public version of browser interfaces
developed by the Max Planck Institute for Molecular
gbrowse/Hs.Solexa) and Genomatix (www.genomatix.de/
MPI). This work was supported in part by the Max Planck
Society, the European Union [AnEUploidy (LSHG-CT-2006-
037627) and BioSapiens (LHSG-CT-2003-503265)], the
National Genome Research Network, and the Federal
Ministry for Education and Research of Germany
[BioChancePLUS-3 (0313724A) to A.K. and M.S.].
Supporting Online Material
Materials and Methods
Figs. S1 to S3
Tables S1 to S9
12 May 2008; accepted 27 June 2008
Published online 3 July 2008;
Include this information when citing this paper.
Small CRISPR RNAs Guide
Antiviral Defense in Prokaryotes
Stan J. J. Brouns,1* Matthijs M. Jore,1* Magnus Lundgren,1Edze R. Westra,1
Rik J. H. Slijkhuis,1Ambrosius P. L. Snijders,2Mark J. Dickman,2Kira S. Makarova,3
Eugene V. Koonin,3John van der Oost1†
Prokaryotes acquire virus resistance by integrating short fragments of viral nucleic acid into clusters
of regularly interspaced short palindromic repeats (CRISPRs). Here we show how virus-derived
sequences contained in CRISPRs are used by CRISPR-associated (Cas) proteins from the host to
mediate an antiviral response that counteracts infection. After transcription of the CRISPR, a complex
of Cas proteins termed Cascade cleaves a CRISPR RNA precursor in each repeat and retains the
cleavage products containing the virus-derived sequence. Assisted by the helicase Cas3, these
mature CRISPR RNAs then serve as small guide RNAs that enable Cascade to interfere with virus
proliferation. Our results demonstrate that the formation of mature guide RNAs by the CRISPR RNA
endonuclease subunit of Cascade is a mechanistic requirement for antiviral defense.
archaea against invading conjugative plasmids,
is acquired by incorporating short stretches of
invading DNA sequences in genomic CRISPR
he clusters of regularly interspaced short
palindromic repeat (CRISPR)–based de-
fense system protects many bacteria and
loci (1, 9, 10). These integrated sequences are
thought to function as a genetic memory that
prevents the host from being infected by viruses
containing this recognition sequence. A num-
ber of CRISPR-associated (cas) genes (11–13)
has been reported to be essential for the phage-
resistant phenotype (1). However, the molec-
ular mechanism of this adaptive and inheritable
defense system in prokaryotes has remained
The Escherichia coli K12 CRISPR/cas sys-
tem comprises eight cas genes: cas3 (predicted
HD-nuclease fused to a DEAD-box helicase),
five genes designated casABCDE, cas1 (predicted
integrase) (13), and the endoribonuclease gene
cas2 (14) (Fig. 1A and table S1). In separate
experiments, each Cas protein was tagged at
both the N and C terminus and produced along
with the complete set of untagged Cas proteins
(15). Affinity purification of the tagged com-
ponent enabled the identification of a protein
complex composed of five Cas proteins: CasA,
CasB, CasC, CasD, and CasE (Fig. 1B). The
1Laboratory of Microbiology, Department of Agrotechnology
and Food Sciences, Wageningen University, Dreijenplein
10, 6703 HB Wageningen, Netherlands.2Biological and
Environmental Systems, Department of Chemical and Pro-
cess Engineering, University of Sheffield, Mappin Street,
Sheffield S1 3JD, UK.3National Center for Biotechnology
Information, National Library of Medicine, NIH, Bethesda,
MD 20894, USA.
*These authors contributed equally to this work.
†To whom correspondence should be addressed. E-mail:
15 AUGUST 2008 VOL 321
on January 9, 2009
complex, denoted Cascade (CRISPR-associated
complex for antiviral defense), could be isolated
from E. coli lysates using any of the tagged sub-
units of the complex as bait, except for CasA.
The function of Cascade was studied by an-
alyzing the effect of in-frame cas gene knock-
outs (16) on the formation of transcripts of the
CRISPR region in E. coli K12 (Fig. 1A). North-
ern analysis of total RNA with single-stranded
spacer sequences as a probe showed transcrip-
tion of the CRISPR region in the direction down-
stream of the cas2 gene (Figs. 1A and 2A) and
no transcription in the opposite direction. Anal-
ysis of control strains (wild type and a non-cas
gene knockout) revealed a small CRISPR-RNA
(crRNA) product of ~57 nucleotides (Fig. 2A).
The same product was present in much higher
amounts in the casA, casB, and casC knockout
strains but absent from strains lacking the over-
lapping genes casD and casE (Fig. 2A). The
small crRNAs seem to be cleaved from a multi-
unit crRNA precursor (pre-crRNA) (7, 17, 18),
as is evident from the presence of two and three
repeat-spacer units (~120 and ~180 nucleotides)
that show up in the DcasA, DcasB, and DcasC
strains (Fig. 2A). The DcasE strain contained a
large pre-crRNA, suggesting that the disruption
of this gene prevents pre-crRNA cleavage.
To study the accumulation and cleavage pat-
terns of crRNAs in the E. coli K12 knockout
strains in more detail and to rule out any effects
of the gene disruptions on the expression of
downstream or upstream cas genes, the five sub-
units of Cascade and the K12-type pre-crRNA
were expressed in E. coli BL21(DE3), which
lacks endogenous cas genes (19). Northern anal-
ysis showed that crRNAs of ~57 nucleotides
were only produced in strains containing the
Cascade complex (Fig. 2B). By omitting the in-
dividual subunits one by one, it became apparent
that the small crRNA was absent only in the
strain that lacked casE (Fig. 2B), indicating that
this is the only Cascade subunit essential for
Activity assays with purified Cascade showed
that the complex is capable of cleaving the E. coli
K12 pre-crRNA into fragments of ~57 nucleo-
tides in vitro (Fig. 2C). However, no cleavage
was observed with either pre-crRNA from E.
coli UTI89, which contains repeats with a dif-
ferent sequence (20), or a non-crRNA template
(Fig. 2C). The RNA cleavage reaction pro-
ceeded in the absence of divalent metal ions and
adenosine triphosphate and reached saturation
level within 5 min. To investigate whether the
CasE subunit is sufficient for pre-crRNA
cleavage activity, it was overproduced as a fu-
sion with the E. coli maltose binding protein
(MalE). Like the complete Cascade, the CasE
fusion protein cleaved only the K12-type pre-
crRNA (Fig. 2D), showing that CasE is an un-
usual endoribonuclease that does not require the
other Cascade subunits. We cannot rule out the
possibility that pre-crRNA cleavage is an auto-
catalytic, ribozyme-like reaction, in which CasE
is an essential RNA chaperone.
CasE belongs to one of the numerous fam-
ilies of repeat-associated mysterious proteins,
the largest and most diverse class of Cas pro-
teins (12, 13). The crystal structure of a CasE
homolog from Thermus thermophilus HB8 shows
that the protein contains two domains with a
ferredoxin-like fold, and displays overall struc-
tural similarity to a variety of RNA-binding
proteins (13, 21). On the basis of structure and
amino acid conservation analysis of this protein
family (fig. S1), the invariant residue His20was
mutated to Ala to analyze the effect on pre-
crRNA cleavage. Northern blots indicated that
crRNAs of ~57 nucleotides were no longer
formed in the strain containing Cascade-
CasEH20A(Fig. 2E). Moreover, although the
mutated CasE was still incorporated into Cas-
cade, the pre-crRNA cleaving ability of purified
Cascade was abolished (Fig. 2F), providing
further support for the essential role of CasE in
pre-crRNA cleavage and suggesting that the
conserved His residue is involved in catalysis.
The crRNA cleavage sites were examined by
simultaneous expression of K12-type pre-crRNA
and Cascade. Under these conditions, the purifi-
cation of Cascade yielded substantial amounts
of copurified RNAs of ~57 nucleotides (Fig. 3A).
Cloning and sequencing of this Cascade-bound
RNA revealed that 85% of the clones [67 out
of 79 clones (67/79)] were derived from crRNAs,
of which 78% (52/67) started with the last eight
bases of the repeat sequence (AUAAACCG)
(Fig. 3B and fig. S2). This well-defined 5′ end
Fig. 1. The composition
of the Cascade complex.
(A) Schematic diagram
of the CRISPR/cas gene
cluster of E. coli K12
W3110. Repeats and
spacers are indicated
by diamonds and rect-
angles, respectively. A palindrome in the repeat is marked by con-
vergently pointing arrows. Protein family nomenclature is as described
in (11, 12). (B) Coomassie blue–stained SDS-polyacrylamide gel of
the affinity purified protein complex using either the N-terminal
StrepII-tag (S) or C-terminal His-tag (H) of each of the subunits
CasB, CasC, CasD, or CasE as bait. Asterisks indicate the 5.5 kD
larger double-tagged subunits. Marker sizes in kilodaltons on the
left; location of untagged subunits on the right.
VOL 32115 AUGUST 2008
on January 9, 2009
Fig. 2. Cascade cleaves
CRISPR RNA precursors
into small RNAs of ~57
nucleotides (marked by
ysis of total RNA of WT
E. coli K12 (WT), a non-
cas gene knockout (Du,
and Cascade gene knock-
outs using the single-
stranded spacer sequence
BG2349 (table S2) as a
probe. (B) Northern blot
E. coli BL21 (DE3) express-
ing the E. coli K12 pre-
crRNA and either the
complete or incomplete
Cascade complex. (C) Ac-
tivity assays with purified
Cascade using in vitro
crRNA from E. coli K12
(repeat sequence: GAGU-
UAAACCG), E. coli UTI89
(repeat sequence: GUUCA-
UAGAAA), and non-crRNA
as substrates. (D) Activity
assays as shown in (C) for
15 min with purified MalE-
LacZa and MalE-CasE fu-
sion proteins. (E) North-
ern blot as shown in (B)
with Cascade or Cascade-
CasEH20A. (F) Activity as-
says as shown in (C) for 30 min with purified Cascade or Cascade-CasEH20A.
Fig. 3. Cleaved crRNAs
(A) Denaturing polyacryl-
amide gel showing the
crRNA (marked by the
arrow) isolated from pu-
rified Cascade in the ab-
sence and presence of
(B) Secondary structure
of pre-crRNA repeats
and example sequences
of cloned crRNAs indicat-
ing the PCS and crRNA
15 AUGUST 2008VOL 321
on January 9, 2009
was followed by a complete spacer sequence
and a less well-defined 3′ sequence ending in
the next repeat region. A transcript of a single
palindromic repeat can fold as a stable stem-
loop of seven base pairs, which may facilitate
recognition by RNA-binding Cas proteins (8, 20),
such as CasE. The pre-crRNA cleavage site
(PCS) appeared to be located immediately up-
stream of the 3′ terminal base of the stem-loop
formed by the repeat (Fig. 3B). The clone li-
brary did not contain crRNAs of 61 nucleotides,
which would be the result of a single endonuclease
cleavage event in each repeat, given the size of a
repeat (29 nucleotides) and most spacers (32
nucleotides). Instead, in agreement with exper-
imental observations (Figs. 2 and 3A), the crRNAs
were truncated at the 3′ end by at least two guano-
sine bases from the endonuclease cleavage site,
removing several stem-forming bases.
To test whether crRNA-loaded Cascade gives
rise to phage resistance, two artificial CRISPRs
were designed against phage Lambda (l). Each
of these CRISPRs targeted four essential l genes
(fig. S3). The coding CRISPR (C1–4) produced
crRNAs complementary to both the mRNA and
the coding strand of these four genes, whereas
the template CRISPR (T1–4) targeted only the
template strand of the same proto-spacer regions
(fig. S3). A nontargeting CRISPR containing
wild-type (WT) spacers with no similarity to the
phage genome served as a control. Plaque as-
says with E. coli showed that the introduction of
either one of these anti-l phage CRISPRs in a
strain expressing only Cascade did not result
in reduced sensitivity of the host to a virulent
Lambda phage (lvir) (Fig. 4A). However, strains
that expressed Cascade and Cas3 were much
less sensitive to phage infection. The template
CRISPR rendered the strain insensitive to the
phage at the highest phage titer tested (>107-fold
less sensitive than the control strain), whereas
the coding CRISPR reduced the sensitivity 102-
fold (Fig. 4A) and produced plaques with a di-
ameter~1/10of the standard l plaque. The phage
omitted(Fig.4A),proving thatbothCascade and
Cas3 are required in this process. Moreover,
strains containing Cas3 and Cascade-CasEH20A
displayed a sensitive phenotype, which shows
that pre-crRNA cleavage is mechanistically re-
quired forphageresistance.The co-expression of
Cas1 and Cas2 had no effect on the sensitivity
proteins are involved in other stages of the
CRISPR/cas mechanism. Plaque assays with
single anti-l spacers (fig. S3) showed that the
total reduction of sensitivity observed with the
four anti-l spacers (C1–4and T1–4) (Fig. 4A)
results from a synergistic effect of the individual
spacers (C1to T4) (Fig. 4B).
Our results demonstrate that a complex of
five Cas proteins is responsible for the matura-
tion of pre-crRNA to small crRNAs that are
critical for mediating an antiviral response. These
mature crRNAs contain the antiviral spacer
unit flanked by short RNA sequences derived
from the repeat on either side termed the 5′ and
3′ handle, which may serve as conserved bind-
ing sites for Cascade subunits, as has been
suggested previously (20). The Cascade-bound
crRNA serves as a guide to direct the complex
to viral nucleic acids to mediate an antiviral
response. We hypothesize that crRNAs target
virus DNA, because anti-l CRISPRs of both
polarities lead to a reduction of sensitivity to the
phage. The model is supported by previous ob-
servations that virus-derived sequences are in-
tegrated into CRISPR loci, irrespective of their
orientation in the virus genome (1–4, 7, 9, 10, 13).
We conclude that the transcription of CRISPR
regions—and the cleavage of pre-crRNA to
mature crRNAs by Cas proteins—is the molec-
ular basis of the antiviral defense stage of the
CRISPR/cas system, which enables prokaryotes
to effectively prevent phage predation.
References and Notes
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material on Science Online.
16. T. Baba, H. Mori, Methods Mol. Biol. 416, 171 (2008).
17. T. H. Tang et al., Proc. Natl. Acad. Sci. U.S.A. 99, 7536
18. T. H. Tang et al., Mol. Microbiol. 55, 469 (2005).
19. J. F. Kim, H. Jeong, R. E. Lenski, personal communication.
20. V. Kunin, R. Sorek, P. Hugenholtz, Genome Biol. 8, R61
21. A. Ebihara et al., Protein Sci. 15, 1494 (2006).
22. We thank T.Verweij, C.G.J.vanHoute, andM.R. Beijerfor
experimental contributions and T. Goosen (Hogeschool
van Arnhem en Nijmegen BioCentre), M. J. Young
(Montana State University), T. Bisseling, and W. M. de Vos
Fig. 4. Engineered CRISPRs confer resistance to l in the presence of Cascade and Cas3. (A) Effect of the
presence of different sets of cas genes on the sensitivity of E. coli to phage lvir. Cells were equipped with
one of two engineered CRISPRs containing four anti-l spacers each (fig. S3). The C1–4CRISPR produces
crRNA complementary to the coding strand and mRNA of lvir, and the T1–4CRISPR targets only the
plaquing, which is the plaque count ratio of the anti-l CRISPR to that of the nontargeting control CRISPR.
(B) Effect of single anti-l spacers (fig. S3) on the sensitivity of E. coli to lvir. Error bars indicate 1 SD.
VOL 32115 AUGUST 2008
on January 9, 2009
(Wageningen University) for helpful discussions. We are Download full-text
grateful for receiving strains from the KEIO collection
distributed by National BioResource Project (National
Institute of Genetics, Japan). We thank U. Dobrindt
(University of Würzburg) for sending genomic material of
E. coli UTI89. This work was financially supported by a Vici
grant from the Dutch Organization for Scientific Research
(Nederlandse Organisatie voor Wetenschappelijk
Onderzoek) and a Marie Curie grant from the European
Union.M.L.was supportedbythe Wenner-Gren Foundations.
Supporting Online Material
Materials and Methods
Figs. S1 to S4
Tables S1 to S3
28 April 2008; accepted 1 July 2008
Suppression of the MicroRNA Pathway
by Bacterial Effector Proteins
Lionel Navarro,1Florence Jay,1Kinya Nomura,2Sheng Yang He,2Olivier Voinnet1*
Plants and animals sense pathogen-associated molecular patterns (PAMPs) and in turn differentially
regulate a subset of microRNAs (miRNAs). However, the extent to which the miRNA pathway
contributes to innate immunity remains unknown. Here, we show that miRNA-deficient mutants of
Arabidopsis partly restore growth of a type III secretion-defective mutant of Pseudomonas syringae.
These mutants also sustained growth of nonpathogenic Pseudomonas fluorescens and Escherichia
coli strains, implicating miRNAs as key components of plant basal defense. Accordingly, we have
identified P. syringae effectors that suppress transcriptional activation of some PAMP-responsive
miRNAs or miRNA biogenesis, stability, or activity. These results provide evidence that, like viruses,
bacteria have evolved to suppress RNA silencing to cause disease.
like Dicer enzymes. The sRNAs guide Argonaute
(AGO)–containing RNA-induced silencing com-
plexes (RISCs) to inhibit gene expression at the
transcriptional or posttranscriptional levels (1). In
the Arabidopsis thaliana microRNA (miRNA)
pathway, miRNA precursors (pre-miRNAs) are
excised from noncoding primary transcripts (pri-
n RNA silencing, double-stranded RNA
(dsRNA) is processed into small RNAs
(sRNAs) through the action of RNase-III–
miRNAs) and processed into mature miRNA
duplexes by Dicer-like 1 (DCL1). Upon HEN1-
catalyzed 2′-O-methylation (2), one miRNA
strand incorporates an AGO1-containing RISC
to direct endonucleolytic cleavage or translational
repression of target transcripts (1). DCL4 and
DCL2 perform major defensive functions by
processing viral-derived dsRNA into small inter-
fering RNAs (siRNAs), which, like miRNAs, are
loaded into AGO1-RISC. As a counterdefensive
strategy, viruses deploy viral suppressors of RNA
silencing, or VSRs (3). RNA silencing also con-
tributes to resistance against bacterial pathogens
(4–7), which elicit an innate immune response
upon perception of pathogen-associated molecu-
lar patterns (PAMPs) by host-encoded pattern
recognition receptors (PRRs). For example, the
Arabidopsis miR393 is PAMP-responsive (4, 8)
and contributes to resistance against virulent
Pseudomonas syringae pv. tomato strain DC3000
(Pto DC3000) (4). Nonetheless, the full extent
to which cellular sRNAs, including miRNAs,
participate in PAMP-triggered immunity (PTI)
in plants remains unknown.
To address this issue, Arabidopsis mutants
defective for siRNA or miRNA accumulation
were challenged with Pto DC3000 hrcC–, a mu-
tant that lacks a functional type III secretion sys-
PTI and, consequently, multiplies poorly on wild-
type Col-0– and La-er–inoculated leaves(Fig. 1A
and fig. S1). However, Pto DC3000 hrcC–
growth was specifically enhanced in the miRNA-
deficient dcl1-9 and hen1-1 mutants (Fig. 1A), in
which induction of the basal defense marker gene
WRKY30 was also compromised (fig. S2A) (10).
Because PTI is also a major component
of nonhost resistance (10, 11), we challenged
dcl1-9 and hen1-1 mutants with P. syringae
pv. phaseolicola (Psp) strain NPS3121, which
infects beans but not Arabidopsis. Both dcl1-9
and hen1-1 mutants sustained Psp NPS3121
growth (Fig. 1B) and displayed compromised
WRKY30 induction (fig. S2B). Enhanced bacte-
rial growth was also observed with the non-
pathogenic Pseudomonas fluorescens Pf-5 and
Escherichia coli W3110 strains (Fig. 1, C and
D). Furthermore, the above nonvirulent bacteria
all induced chlorosis and necrosis on miRNA-
deficient mutants, resembling bacterial disease
1Institut de Biologie Moléculaire des Plantes, CNRS UPR
2353–Université Louis Pasteur, 12 Rue du Général Zimmer,
67084 Strasbourg Cedex, France.
Plant Research Laboratory, Michigan State University, East
Lansing, MI 48824, USA.
*To whom correspondence should be addressed. E-mail:
2Department of Energy
Fig. 1. The Arabidopsis miRNA pathway promotes basal and nonhost resistances
to bacteria. (A) Six-week-old plants were inoculated by syringe infiltration using
a Pto DC3000 hrcC–concentration of 106colony-forming units (CFUs) per
milliliter. Error bars indicate SE of log-transformed data from five independent samples. Similar results were obtained in three independent experiments.
(B to D) Plants were inoculated as in (A) but with Psp NPS3121 (B), P. fluorescens Pf-5 (C), or E. coli W3110 (D). Similar results were obtained in two
independent experiments. (E) Plants were inoculated as in (A) and pictures were taken at 6 days after inoculation.
15 AUGUST 2008 VOL 321
on January 9, 2009