The CRISPR-Cas9 system from Neisseria meningitidis (NmeCas9) is capable of programmable and site-specific cleavage of single-stranded RNA targets in vitro in a manner guided by CRISPR RNA and independent of any protospacer adjacent motif sequence. NmeCas9 efficiently cleaves RNA substrates containing sequences complementary to the CRISPR RNA, and mutations in the HNH nuclease domain abolish this ribonuclease activity. In contrast to SpyCas9 which requires an additional oligonucleotide for RNA cleavage, NmeCas9 has a natural RNA-guided ribonuclease activity without any co-factors. This expands the targeting capabilities of the CRISPR-

1. Short Article
Programmable RNA Cleavage and Recognition by a
Natural CRISPR-Cas9 System from Neisseria
meningitidis
Graphical Abstract
Highlights
d Neisseria meningitidis Cas9 has a natural, PAM-independent
RNase activity in vitro
d Cleavage of single-stranded RNA by NmeCas9 is CRISPR
RNA-guided
d RNA cleavage can be blocked by type II-C anti-CRISPR
proteins
d Catalytically inactive dNmeCas9 recognizes ssRNA targets
sequence-specifically
Authors
Beth A. Rousseau, Zhonggang Hou,
Max J. Gramelspacher, Yan Zhang
Correspondence
yzhangbc@med.umich.edu
In Brief
Rousseau et al. demonstrate that the
type II-C CRISPR-Cas9 from Neisseria
meningitidis (Nme) has a natural,
programmable RNase activity in vitro.
The NmeCas9 protein can recognize and
cleave RNA target in a CRISPR-RNA-
guided fashion. This activity does not
require any PAM sequence in the target or
PAM-presenting auxiliary cofactor.
Rousseau et al., 2018, Molecular Cell 69, 1–9
March 1, 2018 ª 2018 Elsevier Inc.
https://doi.org/10.1016/j.molcel.2018.01.025

2. Molecular Cell
Short Article
Programmable RNA Cleavage and Recognition
by a Natural CRISPR-Cas9 System
from Neisseria meningitidis
Beth A. Rousseau,1,2 Zhonggang Hou,1,2 Max J. Gramelspacher,1 and Yan Zhang1,3,*
1Department of Biological Chemistry, University of Michigan, Ann Arbor, MI 48109, USA
2These authors contributed equally
3Lead Contact
*Correspondence: yzhangbc@med.umich.edu
https://doi.org/10.1016/j.molcel.2018.01.025
SUMMARY
The microbial CRISPR systems enable adaptive
defense against mobile elements and also provide
formidable tools for genome engineering. The Cas9
proteins are type II CRISPR-associated, RNA-guided
DNA endonucleases that identify double-stranded
DNA targets by sequence complementarity and pro-
tospacer adjacent motif (PAM) recognition. Here we
report that the type II-C CRISPR-Cas9 from Neisseria
meningitidis (Nme) is capable of programmable,
RNA-guided, site-specific cleavage and recognition
of single-stranded RNA targets and that this ribonu-
clease activity is independent of the PAM sequence.
We define the mechanistic feature and specificity
constraint for RNA cleavage by NmeCas9 and also
show that nuclease null dNmeCas9 binds to RNA
target complementary to CRISPR RNA. Finally, we
demonstrate that NmeCas9-catalyzed RNA cleav-
age can be blocked by three families of type II-C
anti-CRISPR proteins. These results fundamentally
expand the targeting capacities of CRISPR-Cas9
and highlight the potential utility of NmeCas9 as a
single platform to target both RNA and DNA.
INTRODUCTION
Clustered, regularly interspaced, short, palindromic repeats
(CRISPR) loci and their associated (cas) genes constitute an
adaptive defense system that is widespread in bacteria and
archea and that limits horizontal genetic transfer (Barrangou
et al., 2007; Marraffini and Sontheimer, 2008). CRISPR spacers,
which are acquired from invaders’ genomes and integrated
between CRISPR repeats, specify the nucleic acid targets for
CRISPR interference (Barrangou et al., 2007; Marraffini, 2015).
The CRISPR locus is transcribed and processed into small
RNAs called CRISPR RNAs (crRNAs), which guide the Cas pro-
tein effectors to destroy complementary targets (Brouns et al.,
2008; Hale et al., 2009; Wright et al., 2016). Based upon cas
gene content, the diverse CRISPR-Cas systems are categorized
into two classes, six major types, and nearly thirty subtypes
(Koonin et al., 2017), and the majority of these systems confer
interference by DNA targeting (Garneau et al., 2010; Marraffini
and Sontheimer, 2008; Wright et al., 2016).
The Cas9 proteins, which are the single protein effectors of
type II CRISPRs, generally function as RNA-guided DNA endo-
nucleases (Gasiunas et al., 2012; Jinek et al., 2012) and provide
revolutionary tools for programmable genome engineering in eu-
karyotes and prokaryotes, with the type II-A SpyCas9 being the
most commonly used (Cho et al., 2013; Cong et al., 2013; Hwang
et al., 2013; Jiang et al., 2013; Jinek et al., 2013; Mali et al., 2013).
When programmed by crRNA and another RNA cofactor called
tracrRNA (Deltcheva et al., 2011), SpyCas9 targets double-
stranded DNA (dsDNA) through the recognition of protospacer
adjacent motif (PAM) sequence (Jinek et al., 2012), DNA unwind-
ing, R-loop formation, and the triggering of DNA scission (Stern-
berg et al., 2014). The HNH and RuvC nuclease domains of Cas9
cleave the crRNA-complementary and non-complementary
target strands, respectively (Gasiunas et al., 2012; Jinek et al.,
2012). Catalytically inactive ‘‘dead’’ Cas9s (dCas9s), which can
bind to DNA targets without inducing any DNA breaks (Jinek
et al., 2012), have also been harnessed as eukaryotic genome-
binding platforms to deliver effector domains for the regulation,
imaging, or modification of the specific chromosomal loci
(Wright et al., 2016). In addition, the newly discovered type II
anti-CRISPR (Acr) proteins, encoded by mobile genetic ele-
ments (MGEs), are potent Cas9 inhibitors and can block Cas9-
mediated DNA cleavage and gene editing (Harrington et al.,
2017; Pawluk et al., 2016; Rauch et al., 2017). These and other
Cas9-based DNA manipulation tools are transforming biomed-
ical research.
There are a few CRISPR systems capable of RNA targeting.
For example, the type VI effector Cas13 is a promiscuous RNase
that, upon activation by crRNA-guided RNA recognition, de-
grades nearby RNAs non-specifically (Abudayyeh et al., 2016;
East-Seletsky et al., 2016). The type II-B Cas9 from Francisella
novicida (Fno) employs the tracrRNA, rather than crRNA, as a
guide to silence an endogenous transcript, yet the protein en-
coding the ribonuclease activity remains unidentified (Sampson
et al., 2013). Type III CRISPRs use large multi-protein complexes
to confer immunity through transcription-dependent co-degra-
dation of the DNA and its transcripts (Elmore et al., 2016; Samai
et al., 2015). A separate type-III-associated RNase, Csm6,
Molecular Cell 69, 1–9, March 1, 2018 ª 2018 Elsevier Inc. 1
Please cite this article in press as: Rousseau et al., Programmable RNA Cleavage and Recognition by a Natural CRISPR-Cas9 System from Neisseria
meningitidis, Molecular Cell (2018), https://doi.org/10.1016/j.molcel.2018.01.025

3. provides signal-activated, non-specific RNA clearance (Jiang
et al., 2016; Kazlauskiene et al., 2017; Niewoehner et al., 2017).
Several reports have indicated that Cas9s lack crRNA-guided
RNA cleavage activity (Gasiunas et al., 2012; Ma et al., 2015).
SpyCas9, however, can be tricked into cleaving RNAs in vitro by
an exogenously supplied PAMmer DNA oligo that hybridizes to
the ssRNA target-flanking region and presents the DNA PAM on
the opposing strand. Specific binding of SpyCas9 to the RNA
target can also be achieved with a longer PAMmer that extends
into the crRNA-complementary region (O’Connell et al., 2014).
These in vitro findings open up doors to develop CRISPR-based
RNA-targeting tools to recognize and manipulate specific RNAs
in vitro and in vivo. Indeed, a PAMmer- and dSpyCas9-based
strategy has enabled the pull-down of RNAs from cell extracts
(O’Connell et al., 2014) and the visualization of stress granule
RNAs in mammalian cells (Nelles et al., 2016). In addition,
dSpyCas9 was repurposed to eliminate disease-associated, toxic
repetitive RNAs (Batra et al., 2017), and more recently, the Cas13
RNase wasadopted for invivoknockdown and editing ofmamma-
lian transcripts (Abudayyeh et al., 2017; Cox et al., 2017).
Despite these advances, it is unclear whether any other Cas9
ortholog has PAMmer-inducible or even intrinsic crRNA-pro-
grammable RNase activity. Here we explore the RNA-targeting
potential of Nme CRISPR-Cas9, a type II-C system that has pre-
viously been shown to limit DNA natural transformation in its
native context N. meningitidis (Zhang et al., 2013) and has
been adopted as an eukaryotic gene-editing platform (Esvelt
et al., 2013; Hou et al., 2013; Lee et al., 2016; Zhang, 2017).
We find that NmeCas9 has a natural PAMmer-oligo-independent
and programmable RNase activity in vitro. We describe the func-
tional determinants, mechanistic features, and specificity con-
straints for RNA cleavage by NmeCas9 and show that this activ-
ity can be blocked by three families of type II-C Acr proteins. We
also find that catalytically inert dNmeCas9 binds to RNA targets
in a sequence-specific manner.
RESULTS
A Natural RNA-Guided Ribonuclease Activity of
NmeCas9 In Vitro
The type II-C CRISPR-cas locus from N. meningitidis strain 8013
consists of three cas genes (cas1, cas2, and cas9), a tracrRNA
locus, and a CRISPR array (Zhang et al., 2013; Figure 1A). We
expressed recombinant, FLAG-tagged NmeCas9 in E. coli and
isolated it by heparin, ion exchange, and size exclusion chroma-
tography (Figure S1A). In vitro RNA cleavage assays were per-
formed using purified protein, in vitro-transcribed tracrRNA
and crRNA, and a fluorescent end-labeled ssRNA oligonucleo-
tide bearing a target region complementary to the spacer of
crRNA-sp25 (Figure 1B). We found that NmeCas9 efficiently cat-
alyzes in vitro cleavage of the RNA substrate, resulting in one
prominent labeled cleaved product, and that this reaction re-
quires the cognate crRNA, the tracrRNA, and Mg2+
or Mn2+
(Fig-
ures 1C and S1B). A DNA guide containing sequences identical
to crRNA-sp25 cannot support RNA cleavage (Figure S1C), indi-
cating that NmeCas9’s RNase activity is strictly RNA guided.
Strikingly, unlike SpyCas9, NmeCas9 catalyzed efficient
RNA cleavage without any PAMmer oligo co-factor (Figure 1C),
reflecting a fundamental distinction between the RNase activities
of these two Cas9 orthologs. We also tested whether a similar
PAMmer strategy would modulate RNA cleavage by NmeCas9
by pre-annealing the same ssRNA target to various top-strand
partners (Figure 1D, lower, and Figure S1D) and analyzing the
resulting substrates. RNA cleavage was minimally enhanced
by a 22-nucleotide (nt) DNA PAMmer but greatly impeded or
completely abrogated on a fully annealed DNA-RNA heterodu-
plex or on a double-stranded RNA substrate (Figure 1D, upper).
These results reveal that NmeCas9-mediated RNA cleavage is
specific for ssRNA target and is independent of any PAMmer
provided in trans.
Next, we tested whether NmeCas9’s ribonuclease activity
requires a PAM equivalent in the target RNA (termed rPAM,
50
-AAUCN4-30
for NmeCas9) by assaying a mutant RNA sub-
strate with 3-nt mutations (AAUC to AUAG) introduced into the
rPAM (Figure 1E). The same triple mutation was sufficient to
abolish dsDNA cleavage by NmeCas9 (Zhang et al., 2015).
This rPAM mutant RNA substrate was cleaved as efficiently as
the wild-type counterpart (Figure 1E), indicating that RNA cleav-
age by NmeCas9 is rPAM-independent. All Cas9 enzymes
described to date employ the HNH and RuvC domains to cut
the crRNA-complementary and non-complementary strands of
the dsDNA targets, respectively (Gasiunas et al., 2012; Jinek
et al., 2012). For NmeCas9, active site residues D16 in the
RuvC domain and H588 in the HNH domain were previously
shown to be essential for dsDNA targeting in vivo and in vitro
(Zhang et al., 2013, 2015). To test the involvement of these two
active sites in RNA cleavage, we purified and analyzed three
mutant NmeCas9 proteins (D16A, H588A, and the double mutant
[dm, D16A+H588A]) (Figures S1A and S2B). RNA cleavage
was abolished for the H588A and dm proteins but not for the
D16A mutant (Figure 1F), suggesting that the HNH domain of
NmeCas9 mediates the ssRNA cleavage and therefore is
capable of both DNA and RNA scission.
In addition, a time-course experiment revealed that RNA
cleavage at 37
C became detectable within one minute and pla-
teaued after 30 minutes and that it occurs more slowly at room
temperature (Figure S1E). RNA cleavage was robust under a
wide range of NmeCas9-RNA ribonucleoprotein (RNP) concen-
trations (Figure S1F) or monovalent salt concentrations (KCl, Fig-
ure S1G). By analyzing serial crRNA mutants with 50
truncations
(in the 24-nt spacer portion) or 30
truncations (in the 24-nt repeat
portion), we found that RNA cleavage is reduced with a 18-nt to
20-nt spacer and completely lost with a 16-nt spacer (Figure S3A)
and that the first 8 nts of the crRNA repeat are sufficient to sup-
port robust RNA cleavage by NmeCas9 (Figure S3B).
RNA-Guided RNA Cleavage by NmeCas9 Is
Programmable
To assess the programmability of the NmeCas9 ribonuclease,
we first attempted to redirect RNA cleavage to different positions
within the same ssRNA target. We created two variants of the
crRNA-sp25, walkÀ2 and walk+3, which match different regions
of the same target (Figure 2A). These two variants were analyzed
alongside the wild-type counterpart in a cleavage site mapping
experiment. Both variants directed in vitro RNA cleavage, and
importantly their cleavage sites moved in concert with the
2 Molecular Cell 69, 1–9, March 1, 2018
Please cite this article in press as: Rousseau et al., Programmable RNA Cleavage and Recognition by a Natural CRISPR-Cas9 System from Neisseria
meningitidis, Molecular Cell (2018), https://doi.org/10.1016/j.molcel.2018.01.025

4. Figure 1. NmeCas9 Possesses a Natural RNA-Guided Ribonuclease Activity In Vitro
(A) Schematics depicting the CRISPR-Cas9 of N. meningitidis strain 8013 and domain organization of NmeCas9. Individual elements are not drawn to scale.
Black rectangles, CRISPR repeats; yellow diamonds, CRISPR spacers; gray boxes, cas1, cas2, cas9, and tracrRNA genes; black arrows, transcription driven by
repeat-embedded promoters. CTD, C-terminal domain; R, arginine-rich motif; REC, recognition domains; HNH, HNH domain; RuvC, RuvC domains.
(B) A schematic for the complex of the crRNA-sp25, tracrRNA, and ssRNA target 25. Yellow, crRNA spacer; gray, crRNA repeat; bold, rPAM; green, FAM label.
(C) NmeCas9 cleaves ssRNA target efficiently in vitro, and this reaction requires the cognate crRNA, the tracrRNA, and divalent metal Mg2+
. Non-cog crRNA,
crRNA-sp23. The uncleaved probe and cleavage products are indicated.
(D) An exogenously supplied PAMmer DNA oligo has a modest effect on NmeCas9-catalyzed ssRNA cleavage. The panel of pre-annealed nucleic acid substrates
(depicted at the bottom) were assayed for in vitro RNA cleavage as in (C). Black, RNA; gray, DNA; red, crRNA-complementary region; yellow, PAM or rPAM; green,
FAM label.
(E) NmeCas9 cleaves ssRNA in an rPAM-independent manner. An rPAM mutant RNA substrate was analyzed for cleavage as in (C). Red, mutated sequences in
the rPAM.
(F) An intact HNH domain is required for RNA cleavage by NmeCas9. Nuclease domain active site mutants of NmeCas9 were tested as in (C). Dm, double mutant
(D16A +H588A).
Molecular Cell 69, 1–9, March 1, 2018 3
Please cite this article in press as: Rousseau et al., Programmable RNA Cleavage and Recognition by a Natural CRISPR-Cas9 System from Neisseria
meningitidis, Molecular Cell (2018), https://doi.org/10.1016/j.molcel.2018.01.025

5. Figure 2. NmeCas9-Catalyzed RNA Cleavage Is Programmable
(A) A schematic depicting the ssRNA target 25 and three matching crRNAs, (crRNA-sp25-wt, walkÀ2, and walk+3). Yellow, crRNA spacer; gray, crRNA repeat;
bold, rPAM; green, FAM label; red arrows, predominant RNA cleavage sites mapped out in (B).
(B) RNA cleavage site mapping experiment. SsRNA target 25 and the three crRNAs used are shown in (A). The NmeCas9 cleavage products and RNase T1 and
hydrolysis ladders were subjected to 30
dephosphorylation by T4 polynucleotide kinase and separated by 15% denaturing PAGE. T1, RNase T1 ladder; OH-
,
hydrolysis ladder. À2, walkÀ2; +3, walk+3; wt, crRNA-sp25. Sites of G cleavage by RNase T1 are indicated; sites of NmeCas9 cleavage (G30, A27, G25 for the
three crRNAs, respectively) are marked by red arrows.
(C) Serial single-nt mutants of crRNA-sp25 were analyzed for NmeCas9-catalyzed RNA cleavage. In M1 through M19, single-nt mutation is introduced into every
other position of the crRNA spacer. The location and sequence of each mutation (in red) are shown at the top. Yellow, crRNA spacer; gray, crRNA repeat; red
arrow, RNA cleavage site. Non-cog, crRNA-sp23.
(D) A schematic depicting the ssRNA target 9 and the two matching crRNAs (crRNA-sp9-wt and walk-1). Yellow, crRNA spacer; gray, crRNA repeat; bold, rPAM;
red arrows, predicted RNA cleavage sites; green, FAM label; magenta, Cy5 label.
(E) NmeCas9’s ribonuclease activity is re-programmable on a different RNA substrate. The two crRNAs shown in (D) were assayed for in vitro cleavage on ssRNA
target 9. The same denaturing gel is subjected to FAM (left) and Cy5 (right) detection.
4 Molecular Cell 69, 1–9, March 1, 2018
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6. guide-target complementarity (Figure 2B). By comparing the
NmeCas9 cleavage products with RNase T1 and hydrolysis
ladders, we found that the wild-type crRNA-sp25 and the two
variants predominantly directed cuts after A27, G25, and G30,
respectively (Figure 2B), indicating that NmeCas9 catalyzes
RNA scission between the 3rd
and the 4th
nts of the crRNA-paired
target region proximal to the 50
end of the target (Figure 2A). This
is consistent with the DNA double-strand breaks (DSBs) be-
tween the 3rd
and the 4th
nts generated by NmeCas9 (Zhang
et al., 2015) and other Cas9 orthologs (Gasiunas et al., 2012;
Jinek et al., 2012). The fact that the two variants of crRNA-
sp25 effectively changed target-flanking sequence in the RNA
substrate corroborated earlier finding in Figure 1E that RNA
cleavage is rPAM-independent.
Next, we sought to investigate the specificity rule governing
NmeCas9’s tolerance for mismatches in the crRNA-RNA target
complementarity. We created and tested serial crRNA mutants
each bearing a single-nt deviation from the wild-type sequence
at every odd position within the spacer (Figure 2C, upper).
Only the two single mismatches at the 3rd
and 5th
nt, which are
close to the cleavage site, abrogated in vitro RNA cleavage;
the other mutations either did not affect cleavage or only caused
modest defects (e.g., at the 9th
or 17th
nt) (Figure 2C, lower). We
also analyzed crRNA mutants with multiple mismatches and
found that RNA cleavage was diminished by short (2–4 nts) mu-
tations clustered around the cleavage site (Figure S3C, lanes 4
and 8), but was only partially reduced by 2–3 nts of mismatches
in regions away from the cleavage site (Figure S3C, lanes 3 and
5–7). CrRNAs with 4 or more nts of mismatches all exhibited
severe cleavage defects (Figure S3C, lanes 8–10). Overall, the
NmeCas9 ribonuclease has certain degree of tolerance for
guide-target mismatches that are not next to the cleavage site.
Programmable RNA cleavage by NmeCas9 was also observed
with a different dual-fluorophore-labeled RNA substrate bearing
the target sequence for spacer 9 of N. meningitis strain 8013
(Figure 2D). RNA cleavage guided by crRNA-sp9 resulted in
one predominant 50
product and one major 30
product (Figure 2E,
Cy5 and FAM labeled, respectively). For a variant of crRNA-sp9,
walk-1, which has the guide-target pairing region shifted by 1 nt,
the cleavage site moved in concert (Figures 2D and 2E). Collec-
tively, these results demonstrate that NmeCas9 ribonuclease is
crRNA-guided and programmable.
Type II-C Anti-CRISPRs Inhibit In Vitro RNA Cleavage by
NmeCas9
In light of the recent discovery of type II Acrs that inhibit Cas9-
mediated genetic interference, DNA cleavage, and genome edit-
ing (Harrington et al., 2017; Hynes et al., 2017; Pawluk et al.,
2016; Rauch et al., 2017), we wondered whether the three
families of type II-C Acrs also inhibit NmeCas9-catalyzed
RNA cleavage. We purified four Acr proteins—AcrIIC1Nme,
AcrIIC2Nme, AcrIIC3Nme and AcrIIA4 (Figure 3A)—and analyzed
them in our RNA cleavage assay. Notably, pre-incubation of
NmeCas9 with increasing amounts of AcrIIC1Nme, AcrIIC2Nme,
or AcrIIC3Nme all resulted in blockage of RNA cleavage dose
dependently, and near-complete inhibition was achieved with
all the three Acr proteins at or above 3-fold molar excess over
NmeCas9 (Figure 3B). In contrast, cleavage was not affected
by increasing amounts of AcrIIA4 (Figure 3B), a control Acr that
specifically blocks DNA cleavage by two type II-A Cas9s,
SpyCas9 and Listeria monocytogenes (Lmo) Cas9 (Rauch
et al., 2017). In a quality-control plasmid cleavage experiment,
the three type II-C Acrs all prevented linearization of a target
plasmid DNA by NmeCas9 (Figure 3C), consistent with previous
Figure 3. Type II-C Anti-CRISPRs Block
RNA Cleavage by NmeCas9
(A) A Coomassie-stained 15% SDS-PAGE of pu-
rified Acr proteins. The predicted molecular weight
of AcrIIC1Nme, AcrIIC2Nme, Flag-AcrIIC3Nme, and
AcrIIA4 (a type II-A control Acr) are about 9.8, 14.4,
14.6, and 10.4 kDa, respectively. Molecular weight
markers are indicated.
(B) Three type II-C anti-CRISPR proteins AcrIIC1,
AcrIIC2, and AcrIIC3, but not AcrIIA4, inhibit
NmeCas9-catalyzed RNA cleavage. NmeCas9
was incubated with increasing amounts of various
Acr proteins for 10 minutes, assembled with the
tracrRNA and crRNA for another 10 minutes, and
then mixed with fluorescently labeled RNA sub-
strate (25 nM) to license in vitro RNA cleavage.
Each Acr protein was used at 0.75-, 1.5-, 3-, and
6-fold molar equivalents relative to NmeCas9 RNP
(500 nM). The cleavage reactions were analyzed
by denaturing PAGE.
(C) All three purified type II-C Acr proteins, but not
AcrIIA4, are potent inhibitors for NmeCas9-medi-
ated plasmid DNA cleavage. N, nicked; L, linear-
ized plasmid, about 3.6 kb; SC: supercoiled. Mo-
lecular size markers are indicated. Reactions were
done as in (B) except that 8 nM of plasmid DNA was
added as the substrate, and the reactions were
analyzed by agarose gel and SYBR Safe staining.
Molecular Cell 69, 1–9, March 1, 2018 5
Please cite this article in press as: Rousseau et al., Programmable RNA Cleavage and Recognition by a Natural CRISPR-Cas9 System from Neisseria
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7. reports (Harrington et al., 2017; Pawluk et al., 2016; Rauch et al.,
2017). In summary, these results showed that, in addition to their
known roles in blocking DNA targeting, AcrIIC1Nme, AcrIIC2Nme,
and AcrIIC3Nme can all block NmeCas9-catalyzed in vitro RNA
cleavage as well.
RNA-Guided, Sequence-Specific Binding of NmeCas9 to
RNA Targets In Vitro
To investigate how NmeCas9 engages ssRNA target, we turned
to electrophoretic mobility shift assays (EMSAs). Divalent metals
were omitted from the reactions to render NmeCas9 catalytically
inactive. We observed robust mobility shifts forming on the fluo-
rescently labeled RNA target only when NmeCas9, a matching
crRNA, and the tracrRNA were included (Figure 4A, lane 2 from
the left, top bands). Importantly, these shifts were greatly
reduced when a non-cognate crRNA was used instead (Fig-
ure 4A, lane 3 from the left), indicative of sequence-specific, sta-
ble binding of NmeCas9 RNP to the ssRNA target. There were a
few other shifts with intermediate mobility in all the binding reac-
tions containing the cognate crRNA-sp25 (Figure 4A, lanes 2, 4,
7, and 8 from the left), likely reflecting binding events mediated
by RNA-RNA interactions only. The same rPAM mutant RNA
substrate used in Figure 1E was also analyzed in EMSA experi-
ments and exhibited no binding defects at all (Figure 4B),
suggesting that NmeCas9 recognizes its RNA target in an
rPAM-independent way. This is in stark contrast to Cas9-medi-
ated dsDNA binding, in which PAM recognition is a prerequisite
(Jinek et al., 2012; Sternberg et al., 2014).
The NmeCas9-mediated retarded migration in EMSAs
occurred in a dose-dependent manner (Figure 4C), and the bind-
ing was not affected by active site mutations in either the HNH or
the RuvC domains (Figure 4D); this suggests that the recognition
of RNA substrate can happen independently of RNA scission. It
is reported that SpyCas9 is a single turnover enzyme that holds
on to all four ends of the products after cleaving the duplexed
DNA target (Richardson et al., 2016; Sternberg et al., 2014).
We investigated whether the RNA cleavage products here are
released from the NmeCas9 RNP. To this end, we performed
Figure 4. dNmeCas9 Binds ssRNA Target
In Vitro in a crRNA-Guided Manner
(A) CrRNA-guided, sequence-specific recognition
of RNA target by NmeCas9. Electrophoretic
mobility shift assays (EMSAs) were performed
using the 50
FAM-labeled ssRNA target 25 (25 nM),
NmeCas9 (500 nM), and various small RNAs
(500 nM) as indicated. Binding was performed in
cleavage reaction buffer (but with Mg2+
omitted)
at 37
C for half an hour. Non cog, non-cognate
crRNA-sp23.
(B) The association of NmeCas9 with RNA target is
rPAM-independent. The same rPAM mutant RNA
substrate used in Figure 1E was analyzed here
using EMSAs. Binding reactions were done as in
(A). Red, mutated sequences in the rPAM; non-
cog, crRNA-sp23
(C) Dose-dependent binding to the RNA target by
NmeCas9-RNP complex. Binding reactions were
done as in (A) except that 10 nM substrate is used.
The concentrations of NmeCas9 RNP used were
indicated.
(D) The wild-type NmeCas9 and its nuclease
domain active site mutants (D16A, H588A, and
dm) were assayed for sequence-specific binding
to the RNA target, as in (A). Dm, double mutant;
non-cog, crRNA-sp23.
(E) The 30
, but not the 50
, product of RNA
cleavage remained bound by the NmeCas9
RNP. NmeCas9-catalyzed RNA cleavage re-
actions were done as in Figure 1C for two sepa-
rate fluorescent RNA substrates, target 25 and
target 9. Half of the reactions were analyzed by
6% native PAGE (upper), and the same native
gel was visualized by FAM (left) and Cy5 (right)
detection. The other half of the reactions were
quenched by proteinase K treatment and form-
amide loading dye, then analyzed by 15% dena-
turing PAGE (lower), and the same denaturing
gel was visualized by FAM (left) or Cy5 (right).
Schematics of RNA substrates used were at the
top. Green, FAM label; magenta, Cy5 label. Note
that the 50
cleavage product dissociates from the
NmeCas9 (upper right).
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8. standard in vitro RNA cleavage assay and analyzed the reactions
using two parallel approaches. Half of reactions were quenched
and analyzed by denaturing PAGE (Figure 4E, lower), and the
other half were analyzed by native PAGE (Figure 4E, upper).
While robust cleavage was observed on both ssRNA target 25
and target 9 (Figure 4E, lower), the 50
cleavage products (FAM-
labeled on target 25, Cy5-labeled on target 9) were released
from the NmeCas9 RNP (Figure 4E, upper). In contrast, the
30
cleavage products (FAM-labeled on target 9), which contain
20 nts of sequence complementary to the cognate crRNA,
were present only in higher molecular-weight shifts on native
PAGE, suggesting that they were largely still bound by the
NmeCas9 RNP (Figure 4E, upper panel, left). We also did similar
analysis using dsDNA substrates for NmeCas9. DNA target-
strand or non-target-strand oligonucleotides that were fluores-
cently labeled at either the 30
or 50
end were annealed with
appropriate partner strands (Figure S2A), and the resulting
DNA duplexes were all robustly cleaved by NmeCas9 (Fig-
ure S2D). Native PAGE analysis revealed that all but one of the
four ends of DNA cleavage products remain largely bound by
NmeCas9 (Figure S2C, left), whereas the 50
product of non-target
DNA strand was released (Figure S2C, right). Taken together,
these results suggest that NmeCas9 is likely a single turnover
enzyme for both ssRNA cleavage and dsDNA cleavage.
DISCUSSION
Expanded Targeting Capacities for CRISPR-Cas9
Systems
Central to the CRISPR genome editing revolution are the Cas9
DNA endonucleases, which can be easily programmed to cut
any dsDNA target of interest through PAM recognition and
guide-target base pairing (Wright et al., 2016). Our work here re-
vealed that the type II-C Cas9 from N. meningitidis is capable of
programmable, RNA-guided, sequence-specific cleavage and
recognition of ssRNA target (Figures 1, 2, and 4). Importantly, un-
like the type II-A SpyCas9 (O’Connell et al., 2014), NmeCas9’s
RNase activity is independent of PAMmer DNA oligo auxiliary
factor (Figures 1C and 1D). While the revision of this paper was
under consideration, Strutt et al. (2018) reported similar natural
RNase activity for the Cas9s of Staphylococcus aureus (Sau)
and Campylobacter jejuni (Cje). Therefore, NmeCas9 represents
one of the first native Cas9 endoribonucleases that expand
the targeting capacities of CRISPR-Cas9. It is also tempting to
speculate that additional natural Cas9 RNase may exist in the
divergent Cas9 family.
The rPAM- and PAMmer-independent nature of this ribonu-
clease activity implies that the selection of ssRNA substrate is
achieved mainly through RNA-RNA pairing between the guide
and the target, without a requirement for rPAM recognition by
NmeCas9. This feature may help enable the manipulation of
cellular messenger RNAs while avoiding collateral cleavage or
binding to the corresponding genomic sites lacking the PAMs.
In fact, our in vitro assay revealed that NmeCas9 cleaves a no-
rPAM RNA target without affecting the corresponding dsDNA
substrate, which contains no correct PAM sequence (Figure S4,
lanes 7–12 from left). Moreover, the same HNH nuclease domain
of NmeCas9 that cleaves the target DNA strand also cleaves the
ssRNA substrate. This is not surprising, given the existence of
HNH motifs in homing endonucleases that can cleave both
DNA and RNA molecules (Pommer et al., 2001). Future structural
studies are needed to understand how the NmeCas9 and its HNH
domain accommodate various kinds of nucleic acid targets.
Acr proteins inhibit DNA targeting by Cas9s through distinct
strategies (Dong et al., 2017; Harrington et al., 2017; Pawluk
et al., 2016; Rauch et al., 2017), and these mechanistic informa-
tion could be illuminating in the context of NmeCas9-catalyzed
RNA cleavage. For example, AcrIIA4 structurally mimics a
PAM for SpyCas9 (Dong et al., 2017), so an analogous Acr for
NmeCas9 would not be expected to interfere with RNA cleavage,
which is an rPAM- and PAMmer-independent process. AcrIIC1 is
a broad-spectrum inhibitor that disarms diverse type II-C Cas9
orthologs by direct binding to the HNH domain (Harrington
et al., 2017); therefore, it is not surprising that AcrIIC1 blocks
RNA cleavage that relies on the HNH domain of NmeCas9.
Potential RNA-Targeting Applications
RNA plays critical and diverse biological roles, and RNA-target-
ing tools have the potential to transform research and medicine.
There are existing platforms such as RNA interference, antisense
oligonucleotides, and designer RNA-binding proteins (e.g.,
Pumilios) that can recognize specific transcripts or exogenous
RNA tags (Nelles et al., 2015). Recently, the CRISPR-Cas13 sys-
tem that targets RNA was repurposed as a new tool to knock
down or edit specific mammalian transcripts (Abudayyeh et al.,
2017; Cox et al., 2017), and dSpyCas9 can also help to remove
repetitive RNAs in human cells (Batra et al., 2017). These pro-
grammable RNA-targeting tools will revolutionize how we modu-
late RNA metabolism in the cells.
NmeCas9 can potentially provide a unique single platform to
achieve both dsDNA targeting and RNA targeting tasks, whereas
Cas13 exclusively targetsRNA.The PAMmer-independent nature
of its RNase activity makes NmeCas9 a desired system to circum-
vent challenges that come with the delivery and toxicity of ssDNA
PAMmer oligos. In addition, the fact that NmeCas9, SpyCas9,
and Cas13s use orthogonal guide RNAs (Abudayyeh et al.,
2016; Esvelt et al., 2013) can be exploited to achieve distinct but
multiplexed RNA-targeting tasks. Moreover, the three families of
type II-C Acr proteins can provide off switches to enable precise
control of NmeCas9-based RNA cleavage applications.
Biological Implications of NmeCas9 RNA Targeting
Species of the genus Neisseria rely on natural transformation for
frequent genetic exchange (Hamilton and Dillard, 2006). In
CRISPR-containing N. meningitidis strains, dsDNA cleavage by
Cas9 provides a barrier to genetic material transfer through nat-
ural transformation (Zhang et al., 2013). NmeCas9, along with
several other type II-C Cas9s, are also found to be capable of
robust ssDNA cleavage in vitro, a feature proposed to play an
evolutionarily ancestral role in restricting the ssDNA genome of
filamentous bacteriophages or transforming ssDNAs (Ma et al.,
2015; Zhang et al., 2015).
As for the RNase activity of NmeCas9, the physiological rele-
vance in Neisseria cells remains to be determined. One possibil-
ity is that RNA targeting plays an auxiliary role in defense by help-
ing the clearance of transcripts derived from transforming DNAs
Molecular Cell 69, 1–9, March 1, 2018 7
Please cite this article in press as: Rousseau et al., Programmable RNA Cleavage and Recognition by a Natural CRISPR-Cas9 System from Neisseria
meningitidis, Molecular Cell (2018), https://doi.org/10.1016/j.molcel.2018.01.025

9. or bacteriophages. Another possibility is that NmeCas9 may
target endogenous Neisseria RNAs in a crRNA-guided fashion.
We bioinformatically examined potential matches between all
the twenty-five spacers from native mature crRNAs and the
self-chromosome of N. meningitidis strain 8013. BLASTN
searches revealed no perfect or near-perfect hits (up to 4 nts
of mismatches allowed), consistent with an earlier finding that
Neisseria CRISPR spacers primarily match to the genomes of
other strains or species (Zhang et al., 2013). The best matches
for all but one crRNA have 7 or more nts of mismatches spread
out in regions both proximal and distal to the presumed
RNA cleavage site. This degree of mispairing, according to
our in vitro mutagenesis study (Figures 2C and S3C), would
largely abolish NmeCas9-catalyzed RNA cleavage. Therefore,
we cautiously speculate that robust cleavage of endogenous
Neisseria transcripts by NmeCas9 and existing CRISPR spacers
is not very likely but could arise. Nonetheless, future work is
needed to determine if crRNA-directed RNA binding or cleavage
of endogenous transcripts by NmeCas9 occurs in Neisseria, and
if so, what biological consequences could result.
STAR+METHODS
Detailed methods are provided in the online version of this paper
and include the following:
d KEY RESOURCES TABLE
d CONTACT FOR REAGENT AND RESOURCE SHARING
d EXPERIMENTAL MODEL AND SUBJECT DETAILS
B Escherichia coli BL21 (DE3)
B Escherichia coli JM109
d METHOD DETAILS
B Plasmid construction
B In vitro transcription (IVT) and purification of
small RNAs
B In vitro cleavage assay
B RNA ladders and cleavage site mapping
B Expression and purification of NmeCas9 protein
B Expression and purification of Acr proteins
B Electrophoretic mobility shift assay (EMSA)
d QUANTIFICATION AND STATISTICAL ANALYSIS
SUPPLEMENTAL INFORMATION
Supplemental Information includes four figures and one table and can be
found with this article online at https://doi.org/10.1016/j.molcel.2018.01.025.
ACKNOWLEDGMENTS
The authors would like to thank Erik Sontheimer, Hank Seifert, Daniel Goldman,
and Michael Sheets for critical reading of the manuscript. We thank Chase Beisel
and Cynthia Sharma for communicating unpublished work, Tianmin Fu (Harvard
Medical School) for advice on protein purification, and Xufei Zhou for graphical
assistance. This work was supported by the National Institutes of Health
(GM117268 to Y.Z.) and University of Michigan institutional funds to Y.Z.
AUTHOR CONTRIBUTIONS
B.A.R, Z.H. and Y.Z. designed and conducted experiments. M.J.G. and Z.H.
purified proteins. Z.H. and Y.Z. wrote the manuscript. All authors edited the
manuscript.
DECLARATION OF INTERESTS
The authors declare no competing interests.
Received: November 20, 2017
Revised: December 18, 2017
Accepted: January 18, 2018
Published: February 15, 2018
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Molecular Cell 69, 1–9, March 1, 2018 9
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meningitidis, Molecular Cell (2018), https://doi.org/10.1016/j.molcel.2018.01.025

11. STAR+METHODS
KEY RESOURCES TABLE
REAGENT or RESOURCE SOURCE IDENTIFIER
Chemicals, Peptides, and Recombinant Proteins
Urea Sigma Aldrich #U5378-1KG
Trizma base Sigma Aldrich #T1503-5KG
Boric acid Sigma Aldrich #B6768-5KG
EDTA Sigma Aldrich #EDS-500G
DTT Thermo Fisher Scientific #BP17225
HEPES Sigma Aldrich #H4034-100G
Proteinase K Thermo Fisher Scientific #FEREO0491
Magnesium chloride hexahydrate Sigma Aldrich #M2670-500G
Potassium chloride Sigma Aldrich #P9541-500G
Sodium chloride Thermo Fisher Scientific #S271-3
Sodium acetate Sigma Aldrich #S2889-250G
Sodium bicarbonate Sigma Aldrich #S5761-500G
Heparin Sigma Aldrich #H4784-250MG
Glycogen Sigma Aldrich #10901393001
Q5 Hot Start High-Fidelity DNA Polymerase New England Biolabs #M0493L
Invitrogen SYBR Safe DNA Gel Stain Thermo Fisher Scientific #S33102
Invitrogen Ambion Gel Loading Buffer II Thermo Fisher Scientific #AM8547
Formamide deionized Sigma Aldrich #F9037-100ML
Formaldehyde solution Sigma Aldrich #F1635-25ML
Orange G Sigma Aldrich #O3756-25G
Glycerol Thermo Fisher Scientific #BP229-4
40% Acrylamide/Bis solution, 19:1 Bio-Rad #1610145
30% Acrylamide/Bis solution, 37.5:1 Bio-Rad #1610158
Ammonium persulfate Bio-Rad #A3678-100G
TEMED Sigma Aldrich #T9281-50ml
Coomassie Brilliant Blue G-250 Amresco #0615-10G
LB Broth (Lennox) Sigma Aldrich #L3022-1KG
Hydrochloric acid Thermo Fisher Scientific #A14-500
Agar Sigma Aldrich #A1296-1KG
IPTG Thermo Fisher Scientific #BP1755-10
FastBreak Cell Lysis Reagent Promega #V8573
Imidazole Sigma Aldrich #I202-500G
Bond-Breaker Neutral pH TCEP Solution Thermo Fisher Scientific #PI77720
Tobacco Etch Virus (TEV) protease Ryan Baldridge lab N/A
T5 Exonuclease New England Biolabs #M0363S
Taq DNA Ligase New England Biolabs #M0208L
b-Nicotinamide adenine dinucleotide (NAD+) New England Biolabs #B9007S
T4 Polynucleotide Kinase New England Biolabs #M0201S
DpnI New England Biolabs #R0176S
T4 DNA Ligase New England Biolabs #M0202L
Yeast tRNA (10 mg/mL) Thermo Fisher Scientific #AM7119
RNase T1 Thermo Fisher Scientific #EN0541
(Continued on next page)
e1 Molecular Cell 69, 1–9.e1–e4, March 1, 2018
Please cite this article in press as: Rousseau et al., Programmable RNA Cleavage and Recognition by a Natural CRISPR-Cas9 System from Neisseria
meningitidis, Molecular Cell (2018), https://doi.org/10.1016/j.molcel.2018.01.025

12. CONTACT FOR REAGENT AND RESOURCE SHARING
Please direct any requests for further information or reagents to the Lead Contact, Yan Zhang (yzhangbc@med.umich.edu).
EXPERIMENTAL MODEL AND SUBJECT DETAILS
Escherichia coli BL21 (DE3)
E. coli BL21 (DE3) cells were used for protein production for in vitro experiments. Cells were grown at 18
C in Lysogeny Broth (LB)
medium supplemented with 50 mg/mL kanamycin.
Escherichia coli JM109
This strain was used for cloning of plasmids. Cells were grown at 37
C in LB medium supplemented with 50 mg/mL kanamycin.
METHOD DETAILS
Plasmid construction
The NmeCas9 gene was amplified from plasmid pGCC2/p-NmeCas9 (Zhang et al., 2013) and cloned via Gibson assembly into
pET28b digested with NcoI and NotI. A flag tag was then inserted at the C terminus of NmeCas9 using Q5 site-directed mutagenesis.
To create expression constructs for NmeCas9 active site mutants, a $2 kb fragment containing the active site regions were amplified
from templates pGCC2/p-NmeCas9 D16A, H588, and dm (Zhang et al., 2013) and subcloned into the pET28/NmeCas9-Flag
backbone using Gibson assembly. To create the target plasmid used in Figure 3C, an annealed oligo pair containing validated target
sequence for spacer 25 was clone into the BamHI and HindIII sites of pCDF-1. See Table S1 for all primers used.
The four anti-CRISPR genes AcrIIC1Nme, AcrIIC2Nme, AcrIIC3Nme, and AcrIIA4 were amplified from pGCC2/p-AcrIIC1Nme, pGCC2/
p-AcrIIC2Nme, pGCC2/p-AcrIIC3Nme (Pawluk et al., 2016) and pCSW21 (Addgene #86836) respectively, and cloned via the NdeI and
Continued
REAGENT or RESOURCE SOURCE IDENTIFIER
Critical Commercial Assays
AmpliScribe T7-Flash Transcription Kit Epicenter #ASF3507
QIAprep Spin Miniprep Kit QIAGEN #27106
QIAquick Gel Extraction Kit QIAGEN #28706
Ni-NTA agarose QIAGEN #30210
HiTrap Heparin HP GE Healthcare #17-0407-03
HiTrap SP HP GE Healthcare #7115201
HiPrep Sephacryl S-200 HR GE Healthcare #17-1166-01
Experimental Models: Organisms/Strains
Escherichia coli JM109 Promega #L2005
Escherichia coli BL21 (DE3) EMD Millipore-Sigma #70235
Oligonucleotides
See Table S1 for sequences of oligonucleotides used in this study. N/A N/A
Recombinant DNA
pET28b EMD Millipore-Sigma #69865-3
pET28/NmeCas9-Flag This paper N/A
pET28/NmeCas9-Flag D16A This paper N/A
pET28/NmeCas9-Flag H588A This paper N/A
pET28/NmeCas9-Flag D16A H588A This paper N/A
pET28/His-TEV-AcrIIC1Nme This paper N/A
pET28/His-TEV-AcrIIC2Nme This paper N/A
pET28/His-TEV-Flag-AcrIIC3Nme This paper N/A
pET28/His-TEV-AcrIIA4 This paper N/A
pCDF-1b Novagen #71330-10UG
pCDF-1b/protospacer 25 This paper N/A
pSTblue/U6-Nme sgRNA(Tdtomato) Hou et al., 2013 #47871, Addgene
Molecular Cell 69, 1–9.e1–e4, March 1, 2018 e2
Please cite this article in press as: Rousseau et al., Programmable RNA Cleavage and Recognition by a Natural CRISPR-Cas9 System from Neisseria
meningitidis, Molecular Cell (2018), https://doi.org/10.1016/j.molcel.2018.01.025

13. HindIII sites into pET28b. A TEV protease site between His tag and the Acr gene was introduced via PCR primers. A flag tag was
also added to the beginning of AcrIIC3Nme to improve solubility, as described before (Pawluk et al., 2016). See Table S1 for all primers
used.
In vitro transcription (IVT) and purification of small RNAs
The crRNAs and tracrRNA were generated by in vitro transcription (AmpliScribe T7-Flash Transcription Kit), and gel purified by 15%
denaturing PAGE. Gel slices were eluted with agitation overnight in 0.3M NaCl-TE (10 mM Tris, 1 mM EDTA, pH7.5), and RNAs were
recovered by isopropanol precipitation. Fluorescent-labeled RNA and DNA oligonucleotides (IDT) were gel purified by 15% dena-
turing PAGE. Transcription templates used in this study were either annealed DNA oligos (annealing buffer is 100 mM NaCl,
10 mM Tris, pH 8.5) or gel-purified PCR products. See Table S1 for all oligonucleotides used.
In vitro cleavage assay
All cleavage reactions were carried out in 1X cleavage buffer [20mM HEPES pH 7.5, 50 mM KCl, 0.1 mM EDTA, 0.5 mM DTT, and
10 mM MgCl2] at 37
C for 30 minutes, unless otherwise indicated. NmeCas9 (500 nM) was mixed with crRNA and tracrRNA
(500 nM each) in buffer, and then fluorescently labeled RNA or DNA substrates were added to a final concentration of 25 or
50 nM. The finished reactions were treated with proteinase K (2 mg/ml) at 37
C for 15 min, quenched with either equal volume of
2x Gel loading buffer II (Invitrogen) or two volumes of 1.5x formaldehyde-formamide loading dye (1.5xTBE, 2.3 M formaldehyde,
53% formamide, 20 mM EDTA pH 8, 2.5 mg/mL Orange G), and then analyzed on 15% denaturing PAGE and visualized on Biorad
Chemidoc MP imager. Plasmid cleavage by NmeCas9 in Figure 3C is carried out similarly, except that plasmid DNA was added to a
final concentration of 5 nM. After proteinase K treatment, the reactions were separated on 1% agarose gel, stained by SYBR Safe
(Invitrogen).
For Acr inhibition experiments, NmeCas9 (500 nM) was pre-mixed with 0.35, 0.75, 1.5, and 3 mM of purified Acr proteins in 1X cleav-
age buffer for 10 minutes at room temperature, tracrRNA and crRNA (500 nM ea) were then added to the incubation for 10 minutes at
room temperature. Next, fluorescently labeled RNA or DNA substrates (or plasmids DNA) were added to a final concentration of 25 or
50 nM (or 8 nM for plasmid), and the reactions were carried out and analyzed as described above.
RNA ladders and cleavage site mapping
RNA ladders in Figure 2B were generated by alkaline hydrolysis and RNase T1 digestion of 8 pmole fluorescently-labeled ssRNA
target 25, followed by 30
de-phosphorylation by T4 polynucleotide kinase (NEB). The reactions were then phenol: chloroform ex-
tracted and ethanol precipitated before 10% of each was analyzed by 15% UREA PAGE. Alkaline hydrolysis ladder was prepared
by incubating the RNA target in 50 mM NaHCO3, pH 9.2, 1mM EDTA and 6.75 ng/mL yeast tRNA (Thermo Fisher Scientific) at
95
C for 20 minutes. RNase T1 ladder was prepared by combing the RNA target with 4U of RNase T1 (Thermo Fisher Scientific)
in a 50 mL reaction (50 mM Tris, pH 7.5, 2 mM EDTA, and 3 mg/ml yeast tRNA) for 5 minutes at 37
C. The NmeCas9-catalyzed
RNA cleavage reactions were treated by T4 polynucleotide kinase, phenol: chloroform extraction and ethanol precipitation, then
analyzed alongside the RNA ladders on a 15% sequencing PAGE gel.
Expression and purification of NmeCas9 protein
For NmeCas9, E. coli BL21 (DE3) cells were grown in LB medium at 37
C to OD600 0.4-0.6. Protein expression was induced with
0.5 mM IPTG at 18
C for 16 hr. Cells were harvested, resuspended in lysis buffer (1xPBS, 350 mM NaCl, 0.5 mM TCEP, 1xFastBreak
lysis reagent) and lysed by sonication. Clarified lysates were loaded onto a heparin column (HiTrap Heparin HP, GE Healthcare), and
eluted with a step gradient of NaCl (1xPBS with 600 mM, 850 mM, and 2M of NaCl). Fractions containing Cas9 were pooled, diluted
10-fold using 1xPBS, loaded onto an ion exchange column (HiTrap SP HP, GE Healthcare), and eluted with a step gradient of NaCl
(1xPBS with 600 mM, 850 mM, and 2M of NaCl). Cas9 containing fractions were pooled, concentrated to 1ml, and further purified
using a size exclusion column (HiPrep Sephacryl S-200). S200 column fractions containing monomeric Cas9 were pooled, concen-
trated and stored at À80
C.
Expression and purification of Acr proteins
Acr proteins were purified using E. coli BL21 (DE3) as described previously (Pawluk et al., 2016) with minor modifications. Briefly,
BL21 (DE3) cells were grown in LB medium at 37
C to OD600 0.4-0.6. Protein expression was induced with 0.5 mM IPTG either at
37
C for 3hr for AcrIIC1, AcrIIC2 and AcrIIA4, or at 18
C for 16hr for Flag-AcrIIC3. Cells were pelleted and resuspended in lysis buffer
(20 mM HEPES pH7.5, 300 mM NaCl, 20 mM imidazole, 0.5 mM TCEP, 1x FastBreak lysis reagent) and lysed by sonication. Clarified
lysate was purified with Ni-NTA resin, and His-tagged ACR proteins were eluted with 20 mM HEPES pH7.5, 300 mM NaCl, and
500 mM imidazole. The elution was mixed with His-tagged TEV protease and dialyzed into 20 mM HEPES pH 7.5, 300 mM NaCl,
and 0.5mM DTT overnight at 4
C. Cleaved, untagged Acr proteins were purified using new Ni-NTA resin by collecting the unbound
fraction with the exception of Flag-ACRIIC3, which was purified after TEV cleavage on sizing column. Purified proteins were concen-
trated and flash frozen in small aliquots and stored at À80
C.
e3 Molecular Cell 69, 1–9.e1–e4, March 1, 2018
Please cite this article in press as: Rousseau et al., Programmable RNA Cleavage and Recognition by a Natural CRISPR-Cas9 System from Neisseria
meningitidis, Molecular Cell (2018), https://doi.org/10.1016/j.molcel.2018.01.025

14. Electrophoretic mobility shift assay (EMSA)
The in vitro binding reactions were assembled the same way as for cleavage reactions, except that 30 mg/ml heparin is included and
MgCl2 is omitted from the buffer. Binding was carried out at 37
C for 30 minutes, then the reactions were supplemented with 10%
glycerol and resolved on 5% native TBE-PAGE, and gel visualized using Biorad Chemidoc MP imager. For the cleavage-gel shift
experiments in Figures 4E and S2, regular cleavage reaction was performed (without heparin, with Mg2+
), then half of the reaction
was supplemented with 10% glycerol and analyzed by 5% native TBE-PAGE, the other half treated with proteinase K and resolved
on 15% denaturing TBE-PAGE.
QUANTIFICATION AND STATISTICAL ANALYSIS
All in vitro experiments were repeated three times, and representative gel images were shown. Quantification for RNA cleavage
experiments in Figures S1E and S3C was done using the Image lab software (Bio-Rad).
Molecular Cell 69, 1–9.e1–e4, March 1, 2018 e4
Please cite this article in press as: Rousseau et al., Programmable RNA Cleavage and Recognition by a Natural CRISPR-Cas9 System from Neisseria
meningitidis, Molecular Cell (2018), https://doi.org/10.1016/j.molcel.2018.01.025