AcrIIA7 hijacks tracrRNA to block CRISPR-Cas system

Domain: biology
Published: 2026-03-14
Editorial outcome: major_revision

Abstract
The CRISPR–Cas9 system provides adaptive immunity against invading genetic elements through a
dual-RNA-guided DNA cleavage mechanism. This system relies on the precise assembly of a
ribonucleoprotein (RNP) complex composed of the Cas9 endonuclease, a CRISPR-derived RNA
(crRNA), and a trans-activating CRISPR RNA (tracrRNA). Around 100 anti-CRISPR proteins that
inhibit CRISPR–Cas systems have been identified, and the mechanisms by which they act are
increasingly being elucidated. However, the inhibitory mechanisms of many Acrs, including AcrIIA7,
remain poorly understood. Here, we present the structure of AcrIIA7 and uncover a previously
unrecognized mechanism by which it inhibits Cas9 function. Structural and biochemical analyses reveal
that AcrIIA7 specifically binds to tracrRNA, preventing its association with crRNA and thereby
blocking formation of the active Cas9 RNP complex. This tracrRNA hijacking mechanism represents a
unique strategy for CRISPR inhibition, in which an anti-CRISPR protein targets an RNA scaffold
essential for Cas9 activation rather than interacting directly with the Cas9 protein. Our findings provide
the first structural insight into tracrRNA-targeted anti-CRISPR activity and highlight RNA–RNA
interaction interfaces as vulnerable nodes in CRISPR–Cas immunity.

Introduction
CRISPR–Cas systems provide adaptive immunity in bacteria and archaea by recognizing and destroying
invading genetic elements such as bacteriophages and plasmids 1-4. CRISPR-Cas systems are
categorized into two main classes, Class1 and Class2, based on their locus composition and effector
protein organization, and further divided into six types (I-VI)5,6. Class 1 CRISPR–Cas systems, which
include types I, III, and IV, are composed of multi-subunit RNA-guided surveillance complexes, while
Class2 CRISPR-Cas systems, exemplified by type II systems, utilize a single effector protein, such as
Cas9 7-9. Among these, the Type II-A CRISPR–Cas9 system from Streptococcus pyogenes (SpyCas9)
has been most extensively characterized and widely repurposed for genome engineering 10,11. The Cas9
endonuclease is guided to its DNA target by a dual-RNA complex composed of a CRISPR-derived
RNA (crRNA) and a trans-activating CRISPR RNA (tracrRNA), which together form a stable RNA
duplex that enables specific recognition and cleavage of target DNA 10,12. The assembly of this
ribonucleoprotein (RNP) complex is a critical step in the activation of Cas9 function8,13-15.

To counteract CRISPR-mediated immunity, bacteriophages and mobile genetic elements have evolved
anti-CRISPR (Acr) proteins that inhibit various stages of the CRISPR–Cas immune pathway 16-19. Since
the initial discovery of Acr proteins in 2013 18, approximately 100 distinct variants have been identified
through a combination of functional screening and bioinformatic analyses 20-22. Due to the frequent lack
of sequence homology and shared structural features among Acrs, their classification is primarily based
on the specific CRISPR–Cas systems they inhibit 18,23,24.

These proteins employ diverse mechanisms to disrupt CRISPR-Cas activity, including inhibition of
DNA binding, interference with nuclease activity, or sequestration of essential cofactors 17,22,25-28. Most
recently, AcrIF25 was reported to inhibit CRISPR function by binding to Cas7 and preventing the
formation of a DNA-cleaving complex 28. Among these, AcrIIA proteins are known to inhibit Cas9, yet
for many members of this family, the molecular mechanisms of inhibition remain poorly defined.
AcrIIA7 is one such anti-CRISPR protein that has been previously shown to inhibit SpyCas9, but its
mechanism of action remained elusive 29. Recent study also showed that AcrIIA7 might be a phage-
encoded anti-CBASS protein that inhibits host cyclic-oligonucleotide-based immunity by sequestering
cyclic dinucleotide (CDN) molecules 30. Our current study was designed to elucidate the molecular
mechanism underlying the inhibition of Cas9 by AcrIIA7. In this study, we determined the high-
resolution crystal structure of AcrIIA7 from Phocaeicola dorei (hereafter called pdAcrIIA7) and
conducted structure-based functional analyses to elucidate its inhibitory mechanism. The pdAcrIIA7
gene originates from a bacteriophage, but as is the case for many Acr genes, it is incorporated into the
bacterial chromosome through phage infection. Strikingly, we found that AcrIIA7 does not interact with
Cas9 directly, but instead binds to tracrRNA with high specificity and affinity. This interaction prevents
tracrRNA from forming a duplex with crRNA, thereby blocking the formation of the functional Cas9
RNP complex and ultimately inhibiting Cas9 activity. This mechanism represents a previously
uncharacterized mode of anti-CRISPR action that we refer to as tracrRNA hijacking.

Our findings provide the first structural and mechanistic insight into how an anti-CRISPR protein
targets the RNA component of the CRISPR–Cas9 system rather than the Cas protein itself. This
discovery not only expands our understanding of anti-CRISPR diversity but also highlights RNA–RNA
interactions as a underexplored axis of vulnerability in CRISPR-mediated immunity. The tracrRNA
hijacking strategy employed by AcrIIA7 may represent a broader class of anti-CRISPR mechanisms and
opens avenues for the design of synthetic CRISPR inhibitors that operate via RNA targeting.

Results
The structural studies revealed that AcrIIA7 from Phocaeicola dorei is composed of three
domains, and among them, the head domain is flexible

The investigation into the inhibition mechanism of AcrIIA7 commenced with the overexpression and
purification of the full-length pdAcrIIA7, paving the way for subsequent structural analysis. The
purification process involved Ni-NTA affinity chromatography followed by size exclusion
chromatography (SEC), where distinct peak was observed. SDS-PAGE analysis confirmed the purity of
the pdAcrIIA7 protein in the main peak eluted around 14mL (Fig. 1A). Crystallizations trials using
protein samples eluted from the main peak were successful, and diffraction data from a single crystal
were collected at the PAL synchrotron. Initial molecular replacement (MR) phasing, using a predicted
structural model from Alphafold2, was unsuccessful. To resolve the phasing issue, we performed
SelenoMet labelling for the purpose of Se-SAD phasing, which ultimately determined the 2.16 Å
structure of pdAcrIIA7. The final structural model was refined to R = 20.12 % and R = 25.14 %.
work free
The diffraction data and refinement statistics are summarized in Supplementary Table 1.

The crystal belonged to space group P2 2 2 and contained four molecules (Molecule A ~D) in the
1 1 1
asymmetric unit (ASU) (Fig. 1B). The final structural model showed that molecules B and C of
pdAcrIIA7 covered most of pdAcrIIA7 (residues M1 to E150), while molecules A and D excluded 22
and 28 N-terminal amino acids, respectively, due to unclear electron density. The structure of
pdAcrIIA7 consisted of seven α-helices (α1–α7) and seven 𝛽–sheets (𝛽1–𝛽7) (Fig. 1C and D).
Following the two long helices of head domain (α1 and α2), the core domain is formed by five 𝛽-
sheets(𝛽1,𝛽2 and 𝛽5-𝛽7) and three α-helices (α3,α4 an d α7), extending further two α-helices (α5–α6)
and two 𝛽-sheets (𝛽3–𝛽4) as extended domain (Fig. 1C and D). Four molecules in the same ASU
exhibited slight structural differences in their head domains (α1–α2) and in the α7 regions of their core
domains (Fig. 1E~G). RMSD analysis indicated that molecules B and A differed by 1.8Å, whereas
molecules B and C, as well as molecules B and D, showed relatively larger disparities with RMSD
values of 2.6Å and 2.2Å, respectively (Fig. 1E). Furthermore, the α1 and α2 helices in the head domain
of molecules B and C were found to be inclined at different angels (Fig. 1F). In the core domain, the
alpha helix was well conserved in molecules A and B, while in molecules C and D, these regions
formed a loop configuration rather than alpha helix (Fig. 1G).

An analysis of the electrostatic potential on the molecular surface revealed a distinct distribution of
charges; negative charges were predominantly localized in the head and the extended domains, whereas
positive charges were primarily concentrated in the core domain (Fig. 1H). B-factor analysis highlighted
notable rigidity in the core and head domains, evidenced by their low B-factor values (average 48.12Å).
In contrast, the extended domain exhibited high B-factor values (average 68.98Å), indicating greater
structural flexibility (Fig. 1I).

The utilization of the DALI server to explore proteins structurally akin to pdAcrIIA7 revealed the
sulfate transporter protein as the most structurally similar (Fig. 1J). Despite the sulfate transporter
emerging as the closest match, the modest Z-score (6.0) and sequence identity (5%) underscored a
discernible lack of structural congruence. In addition, the other selected proteins exhibited similarly low
Z-scores (ranging from around 5.7-4.6) and sequence identities (2~10%), collectively signifying the
novelty of the AcrIIA7 structure. Indeed, the fold of pdAcrIIA7 appears to be unprecedented within the
existing structural repertoire. While we were preparing our manuscript, the structure of Tad2 (AcrIIA7
from human gut metagenomic libraries) was published, which represented a form of AcrIIA7 lacking
the head domain and anti-CRISPR activity 30.

Since the MR phasing method using the predicted structural model by Alphafold2, failed, we were
intrigued to compare the predicted model with the experimental structure. To achieve this, we aligned
two structures. The structural alignment of pdAcrIIA7 with the Alphafold2 predicted model revealed
notable differences. Although the overall fold was the consistent between the two models, significant
structural discrepancies were observed in the head domain. The α1 and α2 helices in the predicted
model were notably distorted, and several regions did not match when compared to the crystal structure
of pdAcrIIA7 (Fig. 1K). These structural discrepancies could explain the unsuccessful MR phasing.
Overall, based on the structural studies of pdAcrIIA7, we concluded that pdAcrIIA7 is composed of
three domains, and among them, the head domain is flexible.

AcrIIA7 forms a distinct and robust homo-tetrameric structure in solution
Indeed, although numerous Acrs inhibit Cas9 activity as monomers, previous research has
demonstrated that some Acrs operate in a dimeric configuration 31,32. In the case of pdAcrIIA7, the SEC
results indicated that pdAcrIIA7 eluted between the molecular sizes of 158kDa and 44kDa, producing a
prominent peak in the chromatogram (Fig. 1A). However, SEC alone was insufficient to determine the
stoichiometry of pdAcrIIA7. To confirm its stoichiometry, we employed multi-angle light scattering
(MALS) analysis. MALS yielded an experimental molecular weight of 74.86 kDa, with a fitting error of
1.12 % (Fig. 2A). Given the theoretically calculated molecular weight of monomeric pdAcrIIA7 with a
C-terminal 6X histidine tag is 17.82 kDa, the MALS data conclusively indicated that pdAcrIIA7 exists
as a tetramer in solution. This tetrameric structure of pdAcrIIA7 provides further insights into the
diverse mechanisms of Acr function, revealing that higher-order oligomerization may play a crucial role
in their ability to inhibit Cas9 activity.

Further crystallographic packing analysis revealed three types of putative dimers: Mol B(D)/Mol A(C),
Mol B(A)/Mol C(D), Mol B(A)/Mol D(C) (Fig. 2B and C). To ascertain the biologically relevant form,
we conducted protein-protein interface (PPI) analysis using the PDBePISA server 33. The PPI analysis
showed complexation significance score (CSS) for each dimer: Mol B(D)/Mol A(C) (0.000), Mol
B(A)/Mol C(D) (0.100), and Mol B(A)/Mol D(C) (1.000). Notably, the Mol B(A)/Mol D(C) dimer
exhibited the greatest biological relevance, as indicated by its superior CSS compared to other dimers
with lower scores (Fig. 2B).

Upon closer examination of each dimer’s structural details, it was observed that the Mol B/Mol A dimer
formed through a loop connecting 𝛽5 to α7 in the core domain, forming 2 hydrogen bonds between
G133 and Q138 of each molecule. (Fig. 2D) The Mol B(A)/Mol C(D) dimer predominantly interacted
through 5 hydrogen bonds in the core domain, with an additional hydrogen bond in the head domain
(Fig. 2E). In the case of Mol B/ Mol D dimer, which scored the highest in CSS, displayed a more
intricate interaction pattern with 14 hydrogen bonds and 6 salt bridges between two molecules,
primarily in both the core domain and the extended domain (Fig. 2F).

Anti-CRISPR activity of AcrIIA7 relies on the absence of its head domain
To evaluate the inhibitory effect of AcrIIA7 on the enzymatic activity of Cas9, we established an in
vitro target DNA cleavage assay. We selected Streptococcus pyogenes Cas9 (SpyCas9) for this assay
due to its known anti-CRISPR activity against SpyCas9 and its widespread use in genome-editing
applications. SpyCas9 is a large nuclease composed of 1368 amino acid residues. The assay employed
an 1100bp double-stranded DNA (dsDNA) substrate containing the target region with the PAM
sequence (5’-NGG-3’), along with a specifically designed single guide RNA (sgRNA) to direct
SpyCas9 to the target site. We tested varying concentrations of AcrIIA7 to quantitatively assess its
inhibitory potential. As expected, when SpyCas9 was incubated with sgRNA to form the
ribonucleoprotein (RNP) complex, the enzyme successfully cleaved the target DNA (Fig. 3A). The
inhibitory capacity of AcrIIA7 was further evaluated by varying the incubation order of the components.
Despite using a final AcrIIA7 concentration of 10 μM, which is much higher than the effective
concentrations of other AcrIIA family members, AcrIIA7 did not inhibit DNA cleavage of SpyCas9
under any tested condition (Fig. 3A). Given that previous studies have identified AcrIIA7 from human
gut metagenome (meAcrIIA7) as an inhibitor of SpyCas9, we decided to align the sequences of
pdAcrIIA7 and meAcrIIA7 to identify potential differences. Sequence alignment revealed that
pdAcrIIA7 possesses an additional 48 residues at the N-terminus (Fig. 3B). Apart from this notable
difference, the sequences were largely conserved between the two proteins (Fig. 3B). Structurally, these
extra 48 residues found in pdAcrIIA7 correspond to the head domain (Fig. 3C and D). Leveraging the
experimental structure of the complete AcrIIA7, we constructed a model depicting the tetrameric
arrangement of the meAcrIIA7 (Fig. 3E). To ascertain the functionality of pdAcrIIA7 in the absence of
the head domain, we engineered a head domain-deleted pdAcrIIA7 (ΔN48AcrIIA7)), subsequently
conducting a comparative analysis with the intact pdAcrIIA7, ΔN48AcrIIA7, and the meAcrIIA7.

The stoichiometries of ΔN48AcrIIA7 and meAcrIIA7 were analyzed through MALS. The results
revealed an experimental molecular weight of 52.46 kDa for the ΔN48 AcrIIA7 and 52.79 kDa for the
meAcrIIA7. Considering the theoretically calculated molecular weight of both monomeric
ΔN48AcrIIA7 and meAcrIIA7 to be around 12.7 kDa, the MALS data demonstrated that the
ΔN48AcrIIA7 and the meAcrIIA7 exist as a tetramer in solution. This observation indicated that the
absence of the head domain does not impede tetrameric formation (Fig. 3F and G).

In the in vitro cleavage-inhibition assay using SpyCas9, both the meAcrIIA7 and the ΔN48AcrIIA7
were subjected in the same experimental conditions as the full-length pdAcrIIA7. In contrast to the full
length pdAcrIIA7, which did not exhibit inhibitory function in this assay, the meAcrIIA7 and
ΔN48AcrIIA7 variants displayed significant inhibition of DNA cleavage. Notably, a distinct non-
cleaved band was observed, particularly when the ANcrIIA7 was pre-incubated with sgRNA (Fig. 3H and
I). Further assays examining concentration and order dependency with ΔN48AcrIIA7 demonstrated that
inhibition increased with higher concentrations , but only when AcrIIA7 was added prior to Cas9 RNP
complex formation (Fig. 3J). These findings suggest that the head domain of pdAcrIIA7 interferes with
its inhibitory functions, rendering the full length pdAcrIIA7 unable to impede DNA cleavage.
Additionally, DNA recovery was observed only when AcrIIA7 was introduced before the assembly of
the SpyCas9-sgRNA complex, indicating that AcrIIA7 likely inhibits Cas9 activity by preventing proper
RNP formation and thereby obstructing target DNA recognition.

AcrIIA7 inhibits the DNA cleavage activity of Cas9 without directly binding to the protein
Acr proteins are known to employ diverse strategies to inhibit Cas9, with many acting directly on the
Cas9 protein to suppress its DNA cleavage activity 31,34-36. Previous studies have demonstrated that
numerous Acrs achieve inhibition by physically blocking key functional domains of Cas9, such as the
HNH or RuvC nuclease domains 35, REC3 domain34, BH domain37, and WED-PI domain 38. Building
upon these findings, we further investigated the inhibition mechanism of AcrIIA7 through binding
assays conducted with full-length Cas9 proteins from multiple species, including Streptococcus
pyogenes (SpyCas9), Neisseria meningitidis (NmeCas9), Haemophilus parainfluenzae (HpaCas9), and
Simonsiella muelleri (SmuCas9). These experiments were designed to identify potential cross-species
interactions and to determine whether AcrIIA7 exhibits a conserved inhibitory mechanism across
different Cas9 variants. When purified AcrIIA7 was mixed with various Cas9 proteins, no co-migrations
were detected with any of the Cas9 variants upon the SEC experiments, suggesting that AcrIIA7 does
not directly bind to Cas9 to exert its inhibitory activity (Fig. 4A and Supplementary Fig. 1A~C). Given
that the full-length AcrIIA7 was confirmed to be inactive, we conducted the identical binding assay
using meAcrIIA7 and the ΔN48AcrIIA7 to determine if the presence of the head domain hindered
interactions between AcrIIA7 and Cas9. The assay also showed no binding results, indicating that,
regardless of the presence of the head domain, AcrIIA7 inhibits DNA cleavage independently of
directly binding to Cas9 (Fig. 4B and C, and Supplementary Fig. 2A~C and 3A~C). We also performed
pull-down assays to examine whether AcrIIA7 can interact with SpyCas9 in a cellular context, which
would allow detection of weak or transient interactions that may not be captured by SEC co-elution
analysis. Pull-down experiments were performed using both full length pdAcrIIA7 and the ΔN48
variant. In all cases, we did not observe detectable interaction between SpyCas9 and either AcrIIA7
construct under the tested conditions (Supplementary Fig. 4). These results support the conclusion that
AcrIIA7 does not directly bind SpyCas9 as part of its inhibitory mechanism, consistent with our SEC
analysis.

Due to the potential ambiguity of analyzing co-migration tendency of AcrIIA7 with full length Cas9s
using SEC based experiments, we additionally performed binding assays with distinct functional
domains of SpyCas9, including RuvC, REC, HNH, WED, and PI. Each domain was expressed and
purified (REC1-2, REC3, HNH and WED), except for the PI domain, which was insoluble in our
experiments (Supplementary Fig. 5A). We conducted the SEC binding tests between each domain of
SpyCas9 and both full length pdAcrIIA7 and ΔN48AcrIIA7. These experiments demonstrated that
neither full-length pdAcrIIA7 nor ΔN48AcrIIA7 exhibited obvious co-migration with various domains
of Cas9, suggesting that the inhibitory mechanism of AcrIIA7 operates independently of direct binding
to Cas9 (Supplementary Fig. 5B~I).

AcrIIA7 lacking head domain directly binds RNA, specifically tracrRNA
Despite the inhibitory activity of AcrIIA7 has been validated through in vitro assays, its precise
mechanism of action remains elusive due to limitations of previous binding assays with Cas9 proteins.
To gain further insight into its functional properties, we analyzed the surface electrostatic potential of
tetrameric pdAcrIIA7, as the electrostatic characteristics of a protein surface are often closely linked to
its biological function. This analysis revealed that a deep groove is formed toward the core domain of
the AcrIIA7 tetramer, and that this groove is lined with numerous positively charged residues (Fig. 5A).
Based on this observation, we hypothesized that the protein may be capable of binding to nucleotides.
To explore whether AcrIIA7 directly binds to nucleotides, we initially performed EMSA using sgRNA
that has been used for the formation of active SpyCas9. The result showed a clear RNA band super-shift
when RNA was incubated with both ΔN48AcrIIA7 and meAcrIIA7, but no shifts were observed with
full-length pdAcrIIA7, indicating that only AcrIIA7 without the head domain directly binds to sgRNA
(Fig. 5B). This finding aligns with previous experimental results of in vitro assay, which also revealed
that only AcrIIA7 without the head domain inhibited DNA cleavage, demonstrating that the head
domain-deleted AcrIIA7 directly binds to sgRNA to inhibit the Cas9-mediated DNA cleavage.
Interestingly, in the case of sgRNA, an upper shift of the band was observed even in the absence of
protein, when only buffer was added (Fig. 5B). This shift was presumed to result from structural
changes in the sgRNA under the buffer conditions. However, the protein-induced super-shift appeared
at a distinctly higher position, allowing clear differentiation between nonspecific buffer effects and
specific binding of the protein (Fig. 5B). Because the C-terminal His-tag of AcrIIA7 could potentially
affect its interaction with RNA, we generated an N-terminally tagged version of AcrIIA7 and examined
its binding to sgRNA. The N-terminally tagged protein exhibited the same binding behavior
(Supplementary Fig. 6), indicating that the presence and position of the tag does not influence RNA
interaction.

We next asked whether AcrIIA7 also binds to other types of nucleic acids. EMSAs with ssDNA and
dsDNA showed no band shifts in the presence of AcrIIA7, indicating its specific interaction with RNA,
but not DNA (Fig. 5C and D).

The guide RNA (gRNA) in bacteria and generated sgRNA used for our assay consists of two
components: CRISPR-derived RNA (crRNA), which specifically binds to target DNA with its
complementary sequence, and trans-activating CRISPR RNA (tracrRNA), which binds to Cas9 to form
Cas9-RNP complex (Fig. 5E). To comprehensively understand the binding mode of AcrIIA7 to gRNA,
we performed additional EMSA with crRNA and tracrRNA, respectively. AcrIIA7 did not exhibit band
shifts with crRNA (Fig. 5F), while super-shifts were detected with tracrRNA (Fig. 5G). Interestingly,
although active AcrIIA7, without head domain; ΔN48AcrIIA7 and meAcrIIA7 showed super-shifts with
tracrRNA, full-length pdAcrIIA7 also showed distinct band shifts with tracrRNA (Fig. 5G). These
results suggest that the head domain of pdAcrIIA7 does not significantly affect binding to shorter RNA
molecules like tracrRNA but may impose steric hindrance when interacting with longer RNAs such as
sgRNA. One additional noteworthy observation is that pdAcrIIA7 constructs containing the head
domain show two distinct bands in the EMSA with tracrRNA (Fig. 5G). The presence of two bands
might be due to structural heterogeneity resulting from the flexible head domain, leading to different
conformational states of AcrIIA7.

AcrIIA7 binds nucleic acids in a structure-dependent fashion
After confirming that AcrIIA7 binds tracrRNA, we wondered whether this interaction is RNA-specific,
sequence-specific, or instead driven by recognition of a particular stem–loop structure within the
tracrRNA. To address this question, we synthesized the tracrRNA from an Haemophilus parainfluenzae
(Hpa) strain belonging to the type II-C system, which contains a completely different stem–loop
architecture (Fig. 6A), and examined its binding to AcrIIA7 using EMSA. The results showed that
neither pdAcrIIA7 nor the head domain–deficient AcrIIA7 bound to the type II-C tracrRNA with the
distinct stem–loop structure (Fig. 6A). This finding indicates that AcrIIA7 recognizes and interacts with
RNA in a structure-dependent manner, binding only to tracrRNAs that possess a specific structural
configuration. These results indicate that AcrIIA7, which has been reported to inhibit type II-A systems,
indeed acts in a type II-A–specific manner, and that this specificity arises from recognition of a
particular tracrRNA structural feature rather than from RNA sequence alone.

We next asked whether AcrIIA7 binds specifically to RNA, and what would happen if a DNA molecule
possessed a stem–loop structure similar to that of tracrRNA. To address this question, we synthesized an
ssDNA containing the same stem–loop architecture as the type II-A tracrRNA (Supplementary Fig 7).
We refer to this molecule as structured ssDNA (SssDNA) and examined its interaction with AcrIIA7 by
EMSA. Interestingly, both meAcrIIA7 and Δ48AcrIIA7, which lack the head domain, were able to bind
SssDNA, whereas pdAcrIIA7 did not show detectable binding (Supplementary Fig 7). Although the
presence of the head domain in pdAcrIIA7 may slightly interfere with DNA binding, the observation
that AcrIIA7 variants bind to a stem–loop–containing DNA clearly demonstrates that AcrIIA7 does not
interact with nucleic acids in an RNA-specific or sequence-specific manner. Rather, AcrIIA7 recognizes
and binds nucleotides in a structure-dependent fashion.

If AcrIIA7 binds nucleotides containing three stem–loop structures, an important next question is which
stem loops (SL) are specifically recognized and what constitutes the minimal binding motif within
tracrRNA. To address this question, we synthesized a series of gRNA variants (SL12, SL1, SL0) in
which individual stem–loop elements were selectively removed (Fig. 6B) and examined their
interactions with AcrIIA7 using EMSA (Fig. 6C and D). Removal of SL3 did not abolish binding,
although the affinity for AcrIIA7 was noticeably reduced (Fig, 6C and D). In contrast, gRNA variants
lacking both SL2 and SL3, or lacking all three stem–loops (SL1–SL3), showed little to no detectable
binding to AcrIIA7 (Fig. 6C and D). These results indicate that SL3 is not strictly required for binding,
whereas SL1 and SL2 are essential. We therefore conclude that SL1 and SL2 constitute the minimal
structural motif necessary for AcrIIA7 recognition.

tracrRNA binds within the deep groove formed by the AcrIIA7 tetramer core, which is lined with
positively charged residues
To investigate how AcrIIA7 recognizes and binds to tracrRNA, we first identified functionally
important and evolutionarily conserved residues within AcrIIA7 using the Consurf server 39. This
analysis revealed that conserved residues are primarily located in the core domain, with a few also
present in the head domain, including E6 and E10 (Fig. 7A~C). Among these, we focused on conserved
positively charged residues positioned within the deep groove formed by the core domains upon
tetramerization, as they may play a critical role in RNA binding (Fig. 7D). Based on this reasoning, we
selected five conserved, positively charged residues—K58, K66, K71, K93, and R101—as candidates
likely involved in RNA interaction. Supporting this hypothesis, an AlphaFold3 model of the AcrIIA7–
tracrRNA complex showed that tracrRNA fits into the deep groove of the core domain, forming
interactions with these positively charged residues, further reinforcing our prediction (Fig. 7E).

To experimentally validate these predictions, we introduced charge- or size-disrupting mutations by
substituting each residue with aspartic acid or tryptophan, generating five mutants: K58D, K66W,
K71W, K93W, and R101W. Since the N-terminally truncated form (ΔN48) of AcrIIA7 shows the most
distinct RNA binding behavior (Fig. 5B and G), all mutations were introduced into the ΔN48
background. We then examined the RNA-binding ability of these mutants using EMSA with sgRNA.
As expected, the K58D, K66W, and K71W mutants failed to form any detectable complex with sgRNA,
showing complete loss of RNA binding (Fig. 7F). In contrast, K93W and R101W showed an upper-
shifted band indicative of complex formation, but the shifts differed from that of the wild-type,
suggesting altered or weakened binding (Fig. 7F).

To further corroborate these findings, an in vitro cleavage-inhibition assay was performed using the
mutant variants. The results were consistent with those of the sgRNA binding assays: the K58D and
K71W mutants displayed significantly reduced Cas9 inhibitory activity due to their diminished affinity
for sgRNA (Fig. 7G). For the K93W and R101 W mutants, which showed slightly weaker band shifts in
the sgRNA EMSA, a corresponding reduction in cleavage-inhibition activity was also observed
compared to the wild type (Fig. 7G).

Although the K66W mutant, previously shown to have completely lost sgRNA-binding affinity,
exhibited instability at 37℃, preventing target DNA detection in the in vitro assay, results from other
mutants collectively confirmed that sgRNA binding by pdAcrIIA7 is essential for its Cas9 cleavage-
inhibition mechanism. These findings underscore the critical role of conserved, surface-exposed
positively charged residues within the deep groove of AcrIIA7 tetramer in mediating tracrRNA
interactions and inhibiting Cas9 activity (Fig. 7F and G).

The proposed scenario of AcrIIA7-mediated Cas9 inhibition
Based on our series of experimental results, we propose a previously uncharacterized inhibitory
mechanism for AcrIIA7, in which it hijacks tracrRNA to prevent gRNA formation, thereby inhibiting
Cas9 activity. This mode of inhibition represents a previously uncharacterized strategy among known
anti-CRISPR proteins. To validate that tracrRNA hijacking by AcrIIA7 directly impairs gRNA
formation, we synthesized crRNA_repeat and anti-repeat_tracrRNA, and incubated them in annealing
buffer at 37℃ for 2 hours to mimic gRNA formation. The gRNA, formed via annealing of the
complementary repeat and anti-repeat regions, was visualized as an upper band on native gel. Despite
testing multiple annealing conditions, optimal gRNA formation remained challenging. Nevertheless, a
distinct band corresponding to gRNA was detected, allowing us to assess AcrIIA7-mediated inhibition
under these conditions (Supplementary Fig. 8A~C).

Both full length pdAcrIIA7 and meAcrIIA7 were tested for their ability to inhibit gRNA formation.
As protein concentrations increased, the intensity of the gRNA decreased, indicating that AcrIIA7 inhibits
gRNA assembly in a concentration-dependent manner through tracrRNA sequestration (Fig. 8A and B,
Supplementary Fig. 9A and B). Notably, pdAcrIIA7 inhibited gRNA formation completely at 20μM,
whereas meAcrIIA7 achieved the same effect at just 2μM, demonstrating a tenfold greater potency. This
supports the idea that while the head domain of pdAcrIIA7 does not eliminate its ability to bind
tracrRNA, it reduces binding efficiency, particularly in the Phocaeicola dorei-derived AcrIIA7 variant.

Order of addition experiments further confirmed the role of AcrIIA7 in gRNA formation. For full length
pdAcrIIA7, when crRNA and tracrRNA were pre-incubated before AcrIIA7 addition, gRNA formation
proceeded effectively (Fig. 8C and D, Supplementary Fig. 9C and D). However, when AcrIIA7 was
added prior to or simultaneously with both RNAs, gRNA formation was significantly impaired (Fig. 8C
and Supplementary Fig. 9C). In contrast, meAcrIIA7 exhibited potent inhibition regardless of the
incubation order, consistent with its higher affinity for tracrRNA (Fig. 8D and Supplementary Fig. 9D).
These findings collectively demonstrate that AcrIIA7 directly disrupts gRNA formation through
tracrRNA hijacking, thereby blocking CRISPR-Cas activity.

Although our model predicted that hijacking of tracrRNA would prevent proper gRNA assembly and
subsequently block its loading onto Cas9, we sought to obtain more explicit experimental evidence
supporting this mechanism. To this end, we separately synthesized crRNA and tracrRNA and performed
experiments in which their order of addition with Cas9 and AcrIIA7 was systematically varied.
Although activation of Cas9 using separately prepared crRNA and tracrRNA produced lower activity
than using a pre-assembled sgRNA, Cas9 activity was still detectable (Supplementary Fig.10). When
tracrRNA or gRNA encountered AcrIIA7 before interacting with Cas9, Cas9 activity was completely
inhibited in all cases. However, when the RNAs first interacted with Cas9 to form a fully assembled
RNP complex, AcrIIA7 was no longer able to inhibit Cas9 activity (Supplementary Fig. 10). These
results clearly demonstrate that AcrIIA7 cannot inhibit Cas9 once the RNP complex has been formed
and that AcrIIA7 exerts its inhibitory effect by binding to tracrRNA or gRNA prior to RNP assembly.

In conclusion, inhibitory strategy of AcrIIA7 centers on tracrRNA hijacking, which serves as the
foundation for its two inhibitory effects: (1) halting gRNA maturation through tracrRNA sequestration
and (2) preventing the assembly of the Cas9-RNP complex by blocking gRNA loading onto Cas9 (Fig.
9).

Methods
Cloning, overexpression, and purification of AcrIIA7s, various Cas9 from different species, and
various Cas9 domains for structural and biochemical studies
The gene encoding AcrIIA7 (residues 1–150) from Phocaeicola dorei (accession number:
WP_038611802) was synthesized by Bionics (Daejeon, Republic of Korea) and subsequently inserted
into a pET21a plasmid vector (Novagen, Madison, WI, USA), equipped with a C-terminal hexa-
histidine tag for affinity chromatography. Cloning was executed utilizing NdeI and XhoI restriction
sites. Following transformation into Escherichia coli BL21(DE3) competent cells, a single colony was
selected and cultured in lysogeny broth (LB) medium for protein expression. For preparation of
selenomethionine-labeled protein used in crystallographic studies, cells were alternatively grown in 1L
SelenoMet Medium base supplemented with 50mL SelenoMet Nutrient mix and 4 mL SelenoMet
solution (Molecular Dimension). For generation of the N-terminally histidine-tagged AcrIIA7, the
AcrIIA7 coding sequence was additionally cloned into the pET28a vector using the same NdeI and XhoI
restriction sites, with a stop codon introduced at the C-terminus to prevent C-terminal tagging.
Following transformation into E. coli BL21(DE3) competent cells, protein expression was carried out in
LB medium. For all expression cultures, cells were grown at 37°C with 50 μg/mL ampicillin or 25
μg/mL kanamycin, until the optical density at 600 nm reached 0.7–0.8, upon which 0.25 mM isopropyl-
β-D-1-thiogalactopyranoside (IPTG) was added. The subsequent incubation, at 20°C for 18 hours in a
shaking incubator, facilitated protein expression.

Following culture harvest via centrifugation at 2350 × g and 20°C for 15 minutes, the cells were
resuspended in 20 mL lysis buffer (20 mM Tris–HCl pH 8.0 and 500 mM NaCl) and lysed via
ultrasonication at 4°C. The resulting lysate and supernatant were separated by centrifugation at 27,000 ×
g and 4°C for 30 minutes. The supernatants were collected, combined with Ni-nitrilotriacetic acid
(NTA) affinity resins, and incubated at 4°C for 2 hours. Subsequently, the mixture was loaded onto a
gravity-flow column, and after elution, the resin-bound AcrIIA7 was eluted using elution buffer (20 mM
Tris–HCl pH 8.0, 500 mM NaCl, and 250 mM imidazole).

Further purification of target protein samples was performed by size exclusion chromatography (SEC)
on a Superdex 200 Increase 10/300 GL column connected to an ÄKTA Explorer system. Eluted
fractions were pooled, concentrated to 10 mg/mL, and subjected to structural and biochemical analyses.
Protein sample purity was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE).

Additionally, expression constructs for Streptococcus pyogenes Cas9 (#62731), Neisseria meningitides
Cas9 (#71474), Haemophilus parainfluenzae Cas9 (#121540), and Simonsiella muelleri Cas9 (#121541)
were procured from Addgene. The SpyREC12, SpyREC3, SpyHNH, and SpyWED domain expression
constructs were generated via cloning assays utilizing S. pyogenes Cas9 plasmids as templates. All PCR
products were subsequently cloned into the pET28a vector using NdeI and XhoI restriction sites. The
Cas9 proteins and domains were cultured in lysogeny broth (sigma-lennox), expressed, and purified
using the same methodology as for AcrIIA7.

Oligonucleotide sequences and plasmid information are provided in Supplementary Data 1. Plasmids
generated in this study are available from the corresponding author upon reasonable request.

Crystallization and X-ray diffraction data collection
Crystallization of pdAcrIIA7 was executed in a controlled environment at 20°C employing the sitting
drop vapor diffusion technique. Initial crystals were achieved by combining 1 μL of protein solution
(10.23 mg/mL protein in SEC buffer) with 1 μL of reservoir solution (comprising 20%(w/v) PEG 3000
and 0.1M acetate pH 4.5) and equilibrating the mixture using 0.5 mL of reservoir solution. The
definitive crystals utilized for X-ray diffraction analysis were cultivated on plates, with a solution
comprising 1 μL of protein solution and 1 μL of reservoir solution containing 15% (w/v) PEG 3000 and
0.1M acetate pH 4.7 over three days.

Following crystal growth, a single crystal was carefully chosen and immersed in a reservoir solution
augmented with 23% (v/v) glycerol for cryoprotection. X-ray diffraction data acquisition was conducted
at -178°C utilizing the BL-5C beamline at the Pohang Accelerator Laboratory (Pohang, Korea).
Subsequent data processing, encompassing indexing, integration, and scaling, was performed using
HKL2000 software 42.

Structure determination and refinement.
The initial structure of pdAcrIIA7 was determined using AutoSol, SAD phasing, an automated
experimental phasing with model building software, in the package of PHENIX 43. Subsequent model
building with refinement was performed using phenix.autobuild, phenix.refine, and Coot 44,45. The
structure quality and stereochemistry were validated using the MolProbity46. All structural figures were
generated using the PyMOL program 47. Representative electron density map was provided at
Supplementary Fig. 15

Multi-angle light scattering analysis (MALS)
SEC-coupled multi-angle light scattering (SEC-MALS) experiments were carried out to determine the
absolute molecular mass of AcrIIA7 in solution. Purified AcrIIA7 protein in SEC buffer was injected
into a Superdex 200 Increase 10/300 GL column pre-equilibrated with SEC buffer. SEC-MALS was
performed at 20 °C with a 0.5 mL/min buffer flow rate. The ÄKTA explorer system (GE Healthcare)
was coupled with a DAWN-Treos MALS detector (Wyatt Technology, Santa Barbara, USA). A
reference molecular mass value was established using bovine serum albumin for calibration. Data were
processed and assessed using ASTRA software (Wyatt Technology).

In vitro anti-CRISPR activity assay
To assess the inhibitory potential of AcrIIA7 and its mutants on Cas9-mediated target cleavage, a series
of in vitro target DNA cleavage assays were conducted. SpyCas9 enzyme, acquired from NEB, a single
guide RNA (sgRNA), generated using the EnGen® sgRNA Synthesis Kit, S. pyogenes (NEB), and each
AcrIIA7 wild-type and its mutant variants were added and combined in different order. For the first
binding, the mixture was incubated at 25°C for an hour. In the subsequent components were added to
the mixture, then incubated at 25°C for 30 minutes. Finally, the target DNA was added to the reactions
and incubated in NEBuffer r3.1(NEB) at 37°C for 25minutes. Proteinase K was added and incubated for
10 minutes to terminate the reaction.

All reaction products were subjected to loading and separation through electrophoresis on 4%
polyacrylamide gels. The visualization of DNA cleavage products was accomplished by staining the
gels with SYBR GOLD. The resulting gel images were analyzed to evaluate the inhibitory impact of the
wild-type AcrIIA7 and the various mutant forms on Cas9-mediated target cleavage.

Size exclusion chromatography assay for complex formation
Size exclusion chromatography (SEC) was employed to investigate whether AcrIIA7 forming
complexes between various Cas9s from different species, or SpyCas9 domains. Each wild-type and N-
term deleted AcrIIA7 from Phocaeicola dorei and AcrIIA7 from metagenome were mixed with various
Cas9s and each Cas9 domain, followed by incubation at 4 °C for 1 hour. Then, the resulting mixture
was loaded onto a Superdex 200 Increase 10/300 GL column (GE Healthcare) pre-equilibrated with
SEC buffer (20 mM Tris–HCl pH 8.0, 150 mM NaCl). The eluted fractions corresponding to the peaks
of interest were collected, and the samples were subsequently analyzed by SDS-PAGE stained with
Coomassie Brilliant Blue. To assess the complex formation between AcrIIA7 and the Cas9 proteins or
each domain, the migration patterns, and the co-migration of protein bands within the gel were
examined and analyzed.

Pull down assay
6x His-tagged SpyCas9 cloned into the pET21a vector and untagged AcrIIA7 cloned into the pET24a
vector were co-transformed into E. coli BL21(DE3) competent cells. A single co-transformed colony
was selected and cultured under the same conditions used for AcrIIA7 expression. Cells were harvested
by centrifugation and resuspended in lysis buffer containing 20 mM Tris-HCl pH 8.0,150 mM NaCl.
The cell suspension was lysed by sonication, and the clarified lysate was incubated with Ni-NTA
agarose beads for 2 hours to capture 6x His-tagged SpyCas9. After incubation, the bead-protein
complexes were loaded onto a gravity flow column, washed with buffer containing 20 mM Tris-HCl pH
8.0,150mM NaCl, 80 mM Imidazole. Bound proteins were eluted with buffer containing 20 mM Tris-
HCl pH 8.0,150 mM NaCl, 250 mM Imidazole. Eluted samples were analyzed by SDS-PAGE and
visualized by Coomassie Brilliant Blue staining.

Mutagenesis
Site-directed mutagenesis was performed utilizing a QuickChange kit obtained from Stratagene (San
Diego, CA, USA), following the manufacturer's instructions. Primer sequences used for mutagenesis are
listed in Supplementary Data 1. The resulting mutations were subjected to sequencing analysis to verify
the precision of the introduced alterations. This step ensured the fidelity of the modifications. Following
successful validation, all mutant protein variants were produced and purified by applying the identical
methodology previously described for the wild-type protein 48.

Electrophoretic mobility shift assay with polyacrylamide gel (EMSA-P)
Each purified target DNA (20 nM), sgRNAs (1 50 nM), crRNA (150 nM), type II-A tracrRNA (150
nM), type II-C tracrRNA (150 nM), structured single strand (Sss) RNA (20 nM), tracrRNA variants
(150 nM) were incubated with AcrIIA7 proteins at room temperature (25°C) for 1 hour. The samples
were then separated by gel electrophoresis at 100 V on a 10 % native 0.5 × TBE (Tris-Borate EDTA)
polyacrylamide gel. After electrophoresis, gels were stained with SYBR Gold (Invitrogen, Waltham,
MA, USA) and visualized according to the manufacturer’s instructions.

For the gRNA formation, synthesized crRNA_repeat (300 nM), and anti-repeat_tracrRNA(300 nM)
were incubated in a buffer containing 10 mM Tris pH 8.0, 50 mM NaCl, 1 mM EDTA at 37°C for 2
hours. The formed gRNA complexes were also analyzed by electrophoresis on a 10 % native
polyacrylamide gel in 0.5X TBE buffer and visualized using SYBR Gold dye.

In vitro anti-CRISPR activity assay using separately synthesized tracrRNA and crRNA
To further investigate the order-dependent inhibitory activity of AcrIIA7, additional in vitro target DNA
cleavage assays were performed using crRNA and tracrRNA supplied as separate components. crRNA,
tracrRNA, SpyCas9, and AcrIIA7 were combined in various orders, as specified for each experimental
condition. Depending on the reaction sequence, RNA–AcrIIA7 binding reactions were incubated at 25
°C for 1 hour, whereas crRNA–tracrRNA annealing reactions were incubated for 2 hours in buffer
containing 30 mM Tris–HCl (pH 7.5) and 100 mM potassium acetate. Where indicated, Cas9 was
subsequently added and incubated at 25 °C for 30 minutes. For all conditions, target DNA was added as
the final step in the presence of NEBuffer r3.1 (NEB), followed by incubation at 37 °C for 40 minutes.
Reactions were terminated by the addition of Proteinase K and incubation for 10 minutes.

All reaction products were subjected to loading and separation through electrophoresis on 4%
polyacrylamide gels. The visualization of DNA cleavage products was accomplished by staining the
gels with SYBR GOLD. The resulting gel images were analyzed to evaluate the inhibitory impact of the
wild-type AcrIIA7 and the various mutant forms on Cas9-mediated target cleavage.

Native Polyacrylamide Gel Electrophoresis (Native-PAGE) assay
The binding of AcrIIA7 to cyclic nucleotides was analyzed using Native-PAGE assay. The cyclic
nucleotides used in this study were cGG(c-di-2’-dGMP) and 1’’-2’-gcADPR(2’-cADPR). These cyclic
nucleotides (5, 10, 20, and 50μM) were incubated with 20μM AcrIIA7 at 18°C for 30min. The reaction
mixtures were then loaded onto 5% native polyacrylamide gels and electrophoresed at 120V. After
electrophoresis, the gels were visualized by Coomassie Brilliant Blue staining.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This study elucidates a novel mechanism by which AcrIIA7 inhibits Cas9 through tracrRNA sequestration, supported by\nrigorous structural and biochemical data. The discovery of RNA-targeted anti-CRISPR activity represents a significant\nadvance in the field. While the conclusions are generally well-supported, key issues require resolution to strengthen the\nmanuscript’s impact and address the inconsistency with published literature.\nMajor Concerns：\n1. Reconciliation with Li et al. (Nature 2024): The recent study reporting that an AcrIIA7 homolog (HgmTad2) lacks anti-\nCRISPR activity against SpyCas9 directly challenges this work. The authors should provide a detailed structural\ncomparison between pdAcrIIA7 and HgmTad2 (e.g., RMSD analysis, oligomerization states) and discuss potential reasons\nfor functional discrepancies. Possible explanations include species-specific variations, the role of the head domain in\nautoinhibition, or methodological differences. Testing HgmTad2 in parallel assays or proposing testable hypotheses to\nresolve this conflict is essential. Besides, it is interesting to test whether pdAcrIIA7 could sequester cyclic signaling\nmolecules like HgmTad2 to inhibit other bacterial immune systems.\n3. Non-specific EMSA bands (Fig. 5B): The sgRNA band shift in buffer-only controls complicates data interpretation. The\nauthors should optimize buffer conditions (e.g., salt concentration, divalent cations) to minimize non-specific effects.\n4. Mechanistic specificity: The study does not explore whether AcrIIA7 selectively targets Type II-A tracrRNAs. Testing\ntracrRNAs from other subtypes (II-B/C) or correlating inhibition with RNA structural features (e.g., conserved stem-loops or\nlength) would strengthen mechanistic claims. The Discussion should address this open question.\n5. DNA-binding controls (Fig. 5C–D): The conclusion that AcrIIA7 \"does not bind DNA\" is inadequately supported. The DNA\nsequences/lengths used should be specified, and structured DNAs (e.g., stem-loops) with difference lengths shall be tested\nto rule out motif-specific binding. The Results section must clarify DNA parameters.\n6. Weak gRNA signal (Fig. 7): The faint gRNA bands undermine conclusions about AcrIIA7-mediated inhibition. The authors\nshould optimize annealing conditions or use RNAs with other labels. Quantifying band intensity via densitometry is\nrecommended if optimization fails.\nMinor points:\n1. Introduction Reorganization: The Introduction lacks logical flow, with disjointed paragraphs covering CRISPR basics, Acr\nclassification, and AcrIIA7 without clear narrative progression. It should be restructured to: (i) introduce CRISPR-Cas as\nadaptive immunity, (ii) summarize Acr diversity and unresolved mechanisms, (iii) focus on AcrIIA7’s knowledge gap, and (iv)\nconcisely state the study’s aims.\n2. Protein sequences: The authors should provide the protein as well as DNA sequences of the AcrIIA7 homologs they are\nusing in this study, such as pdAcrIIA7, meAcrIIA7, et al.", "reviewer_2": "Lee et al. significantly advances the understanding of anti-CRISPR mechanisms by elucidating how AcrIIA7, a broadly\ndistributed yet poorly understood protein, inhibits Cas9: the study unveils a novel tracrRNA hijacking strategy, where AcrIIA7\ndirectly binds to tracrRNA, preventing its association with crRNA and effectively disrupting both gRNA formation and the\nsubsequent assembly of the functional Cas9-RNP complex, thereby halting DNA cleavage; the determination of AcrIIA7's\nunique tetrameric structure reveals a positively charged groove crucial for tracrRNA binding, while functional assays\ndemonstrate that the head domain regulates AcrIIA7's activity, sterically hindering its interaction with mature gRNA; these\nfindings not only expand our knowledge of anti-CRISPR diversity and highlight RNA-RNA interactions as a key vulnerability\nwithin CRISPR-Cas systems, but also pave the way for the development of innovative genome-editing tools and strategies.\nAlthough the experiments have been well-performed and the manuscript is written somewhat technically but overall very\nclear, details in some experimental schemes and data interpretation should be revised to reach the scope in Nature\nCommunications. Thus, the publication of this work should bce considered only after clarifying our concerns considerably as\na major revision.\nMajor concerns:\n1. Although the data show that AcrIIA7 predominantly exists as a tetramer in solution, it is not definitively demonstrated that it\nbinds to tracrRNA in this specific oligomeric state and whether the tetramer is indeed the sole biologically active unit.\n2. It would substantially strengthen the proposed hijacking mechanism to determine whether AcrIIA7 functions as a\nmonomer, dimer, or tetramer upon RNA binding. Binding assays with tracrRNA/sgRNA and Cas9 inhibition assays using\nmonomeric or dimeric AcrIIA7 variants should be performed to confirm that only the tetramer can serve as the functional\nentity.\n3. While a direct interaction between ΔN48AcrIIA7 and tracrRNA has been established in vitro, the study currently lacks in\nvivo evidence. The authors need to confirm this interaction in a cellular context, for instance, by performing RNA\nImmunoprecipitation (RIP) or CLIP-seq.\n4. The evidence for gRNA loading inhibition remains indirect. Order-of-addition cleavage assays imply a pre-assembly effect,\nbut do not directly monitor RNP formation.\n5. Additional experiments using separate crRNA and tracrRNA with Cas9 and target DNA, varying Acr concentration and\naddition order, or pull-down assays of Cas9-RNA complexes, would more directly demonstrate inhibition at the loading step.\n6. Physiological relevance of gRNA formation assays: The authors should discuss whether the faint bands in Figure 7 are\nphysiologically relevant, and clarify if their findings reflect physiological inhibition potential or an artifact of the RNA-only\nannealing assay.\n7. The data suggest that AcrIIA7 recognizes structural features of tracrRNA rather than sequence. Experiments with\ncompensatory mutations disrupting/restoring stem-loop pairing, or truncated RNAs containing minimal motifs, would provide\ndirect evidence for structure-dependence in RNA recognition.\n8. Have the authors performed any assays, such as CLIP-seq or in vitro footprinting, to identify the precise binding sequence\nbetween AcrIIA7 and tracrRNA? Determining the exact binding region would greatly help in understanding the proposed\ntracrRNA hijacking mechanism.\n9. Given that the head domain is located near the predicted RNA-binding groove, it is important to rule out any influence of\nthe affinity tag on RNA binding or Cas9 inhibition. A comparison with tag-cleaved or N-terminally tagged constructs would\nbe helpful and may be necessary.\n10. The authors conclude, based on SEC co-elution, that AcrIIA7 does not interact with Cas9. The authors are encouraged\nto temper their interpretation accordingly and, if possible, to perform complementary experiments capable of detecting\ntransient or weak interactions in a cellular context.\n11. In Figure 5, the RNA-only band intensity remains similar across protein concentrations, while in the tracrRNA assays, the\nfree RNA band is nearly absent in the sgRNA EMSA. (i) Have lower protein concentrations been tested to resolve shifts\nmore clearly? (ii) Quantifying band intensities to estimate binding affinities (e.g., Kd values) would strengthen the\nconclusions.\n12. In Figure 5b, the band shift appears to change depending on protein concentration. Could this be due to conformational\nchanges of AcrIIA7 or tracrRNA, or are there other possible reasons?\nMinor concerns:\n1. In Figures 2E and 2F, the specific residues are not clearly distinguishable. To improve clarity, the authors should use\ndistinct colors or labeling to unambiguously highlight each residue being discussed.\n2. In Figures 3D and 3E, additional labeling would help clarify the specific regions being highlighted and allow readers to\nbetter distinguish between domains or datasets.\n3. In Figures 3, 7C, and 7D, the notation for incubation order could be improved. Please specify explicitly that the numbers\nshown correspond to the order of incubation.\n4. In Figure 4, the SDS-PAGE and SEC profiles of SpyCas9 show heterogeneity and possible aggregation.\n5. The legends for Figure 5B and 5G are incomplete. They should explicitly state that the lane labeled 'B' represents the\n'buffer only' control.\n6. In Figure 5G, the two bands observed in the EMSA experiment with full-length pdAcrIIA7 and tracrRNA are insufficiently\nexplained. Clear annotation of these bands is recommended. Similarly, in Figures 5C, 5D, 5F, and 5G, the identity of specific\nbands should be clarified by additional labeling, as was done in Figure 5B.\n7. The abbreviations used in Figure 6F (DW, Pd, Me) are not defined. Please specify their meanings in the figure legend for\nclarity.\n8. In Figure 6G, the labeling of AcrIIA7 mutants currently includes a concentration symbol, which is misleading in this context\nand should be corrected.\n9. In Figures 3, 7C, and 7D, the notation for incubation order could be improved. Please specify explicitly that the numbers\nshown correspond to the order of incubation.\n10. To improve the narrative flow, consider combining the first and second sections of the Results, merging them could\ncreate a more cohesive and logical presentation. (from 241 -332)\n11. Throughout the figures, hyphens (-) have been used where minus signs () are appropriate.", "reviewer_3": "The research field of bacterial immunity driven by the discovery of the CRISPR-Cas system is highly active; many\nresearchers explore diverse defense mechanisms, predict novel systems through genomic analysis, and investigate the\ncomplex interplay between bacterial defense and viruses (phages). Compared to the great advances in our understanding of\nthe CRISPR-Cas system, the inhibitory mechanisms by viruses have been relatively poorly understood. In this manuscript,\nthe authors report the structure and function of a viral inhibitor of CRISPR-cas9. The main finding in this manuscript includes\ni) the atomic structure of AcrIIA7, ii) Characterization of RNA-binding site, iii) the specific interaction with tracrRNA. Based on\nthe structural and biochemical studies, the authors propose a new inhibitory mechanism of tracrRNA hijacking. This finding\nsignificantly advances our knowledge in inhibitory mechanisms of the major bacterial defence mechanism. In addition,\nspecific detection of tracrRNA by a small protein is of a potential medical interest. However, the RNA binding data in the\ncurrent manuscript is not sufficient to claim that AcrIIA7 is tracrRNA-specific.\nMajor points\n1. The mutational studies identify the tracrRNA binding site in the protein, but what motif in tracrRNA is responsible for the\nbinding is not clarified. the minimal sequence with the major binding affinity for AcrIIA7 needs to be investigated.\n2. The authors claim AcrIIA7 shows tracrRNA-specific binding, but the RNA-binding experiments were conducted only for\ncrRNA and tracrRNA. Since AcrIIA7 work in bacterial cells including numerous cellular RNA species, the authors need to\naddress whether AcrIIA7 specifically binds tracrRNA than other cellular RNAs. A reliable method may be a pull-down assay\nusing AcrIIA7 as a bait followed by sequencing of bound RNAs.\nMinor points\n1. In the proposed functionl mechanism, as far as I understand, AcrIIA7 should be a viral protein, not a bacterial protein.\nDoes AcrIIA7 from Phocaeicola dorei mean that the gene is identified from sequencing from total bacterial DNA, infected by\nvirus and thus including viral DNA? If so, this needs to be clearly explaind.\n2. The indicaton of the full name of pdAcrIIA7 is missing.\n3. AcrIIA7 seems to strongly bind both free tracrRNA or gRNA? Would the protein be able to dissociate crRNR and\ntracrRNA? If so, this could be added in the inhibitory mechanism.\n4. In Fig 5G, tracrRNA shows a single band, which is completely shifted by 2.5 uM pdAcrIIA7. But in Fig 7A, tracrRNA is in\ntwo bands, which is not much shifted at the same protein concentration. This conflict needs to be addressed."}, "author_response": "Dear Editor,\nWe have revised our manuscript based on the reviewers’ suggestions by performing\nsubstantial experiments.\nEspecially,\n1. We synthesized the tracrRNA from a type II-C CRISPR system, which represents a\ndifferent subtype with a structurally distinct tracrRNA, and examined its interaction with\nAcrIIA7 (Fig. 6A).\n2. To further test whether AcrIIA7 binding is dependent on the stem–loop structure of\ntracrRNA, we synthesized an ssDNA engineered to adopt the same structure as tracrRNA and\nanalyzed its binding to AcrIIA7 (Fig. 6B).\n3. To determine which stem–loop(s) of tracrRNA are recognized by AcrIIA7, we generated\nthree tracrRNA variants lacking specific stem–loop elements and examined their interactions\nwith AcrIIA7. Through these analyses, we were able to identify the minimal tracrRNA motif\nrequired for AcrIIA7 binding (Fig. 6C, D, E).\n4. As a complementary approach to the SEC-based analysis of the interaction between Cas9\nand AcrIIA7, we generated a tag-free AcrIIA7 construct and performed pull-down assays\nwith Cas9 to further validate their interaction (Supplementary Fig. 4).\n5. To exclude the possibility that the His tag influences the interaction with tracrRNA, we\ngenerated AcrIIA7 constructs bearing either a C-terminal or an N-terminal tag and analyzed\ntheir binding to tracrRNA (Supplementary Fig. 6).\n6. Instead of using sgRNA, we separately synthesized tracrRNA and crRNA and performed\norder-dependent assays to examine Cas9 activity and its inhibition by AcrIIA7\n(Supplementary Fig. 9)\nThrough these additional experiments, we confirmed that AcrIIA7 is a protein that recognizes\nnucleotides in a structure-dependent manner and specifically binds to stem–loops 1 and 2 of\ntype II-A tracrRNA. For this reason, AcrIIA7 selectively inhibits type II-A CRISPR systems\nand does not interact with other cellular RNAs. Instead, it binds to and hijacks tracrRNA,\nthereby enabling evasion of the bacterial immune system, further supporting the model we\nproposed.\nPlease find our detailed responses to the reviewers’ comments on the following pages.\nThank you very much for your time and effort in editing our manuscript, and we hope that it\nis now suitable for publication in “Nature Communications”.\nSincerely,\nHyun Ho Park, Ph.D.\nProfessor of Pharmacy\nReviewers' Comments to Author:\nReviewer: 1\nWe thank the reviewer for his/her constructive comments on our work.\n<Major points>\nComments:\n1. Reconciliation with Li et al. (Nature 2024): The recent study reporting that an AcrIIA7\nhomolog (HgmTad2) lacks anti-CRISPR activity against SpyCas9 directly challenges this\nwork. The authors should provide a detailed structural comparison between pdAcrIIA7 and\nHgmTad2 (e.g., RMSD analysis, oligomerization states) and discuss potential reasons for\nfunctional discrepancies. Possible explanations include species-specific variations, the role of\nthe head domain in autoinhibition, or methodological differences. Testing HgmTad2 in\nparallel assays or proposing testable hypotheses to resolve this conflict is essential. Besides, it\nis interesting to test whether pdAcrIIA7 could sequester cyclic signaling molecules like\nHgmTad2 to inhibit other bacterial immune systems.\nResponse: We first thank the reviewer for raising this important point and appreciate the\nopportunity to clarify these details. The sequences of HgmTad2 and the meAcrIIA7 used in\nour experiments are identical, while pdAcrIIA7 contains an additional 48 residues at the N-\nterminus The overall structures and stoichiometries of these proteins are indeed the same\n(New Supplementary Fig. 12). The only difference is that pdAcrIIA7 contains an additional\nhead domain; nevertheless, both proteins function as tetramers.\nTo examine whether pdAcrIIA7 and meAcrIIA7, like HgmTad2, could bind cyclic signaling\nmolecules and thereby inhibit the CBASS immune pathway, we tested their binding to cyclic\nsignaling molecules. Both proteins bound these molecules efficiently, regardless of the\npresence or absence of the head domain (New Supplementary Fig. 13). This indicates that\npdAcrIIA7 is also capable of sequestering cyclic signaling molecules and may therefore play\na role similar to that of HgmTad2.\nFinally, regarding why HgmTad2 failed to display Acr activity, we believe the experimental\ndesign used in the previous Nature paper study provides a clear explanation. In that work,\nHgmTad2 was added only after Cas9 and gRNA had already been combined, meaning that\nCas9 RNP complexes were pre-formed. Under such conditions, HgmTad2 would no longer\nbe able to hijack tracrRNA, which we propose is essential for its inhibitory function.\nConsistent with this interpretation, our own results show that neither pdAcrIIA7 nor\nmeAcrIIA7 can inhibit Cas9 once the RNP complex has been assembled.\nWe have included these comparative analyses in Supplementary Figures 12 and 13 and\nfurther elaborated on them in the Discussion section. We sincerely thank the reviewer once\nagain for the constructive comments, which have helped to strengthen the manuscript.\n2. Non-specific EMSA bands (Fig. 5B): The sgRNA band shift in buffer-only controls\ncomplicates data interpretation. The authors should optimize buffer conditions (e.g., salt\nconcentration, divalent cations) to minimize non-specific effects.\nResponse: We fully agree with the reviewer’s comment and recognize the importance of\nminimizing such non-specific bands. Indeed, we attempted many of the conditions suggested\nby the reviewer, as well as additional variations; however, this band (arising not from specific\nprotein–RNA binding but rather from intrinsic conformational changes of the RNA itself)\ncould not be eliminated. For this reason, we included a water-only condition as a control to\ndemonstrate that this non-specific band is not produced under conditions lacking RNA\nstructural rearrangement.\nIn fact, such non-specific bands are commonly observed in RNA EMSA experiments in\nwhich the RNA undergoes secondary-structure transitions. As an example, we have attached\na published result showing a similar phenomenon.\n3. Mechanistic specificity: The study does not explore whether AcrIIA7 selectively targets\nType II-A tracrRNAs. Testing tracrRNAs from other subtypes (II-B/C) or correlating\ninhibition with RNA structural features (e.g., conserved stem-loops or length) would\nstrengthen mechanistic claims. The Discussion should address this open question.\nResponse: We thank the reviewer for raising this highly interesting and important point that\nclearly warrants careful evaluation. As suggested by the reviewer, we examined whether\nAcrIIA7 is capable of binding tracrRNAs from other CRISPR–Cas subtypes. To address this\nquestion, we synthesized the tracrRNA from an Haemophilus parainfluenzae (Hpa) strain\nbelonging to the type II-C system, which contains a completely different stem–loop\narchitecture (Fig. 6A), and examined its binding to AcrIIA7 using EMSA. The results showed\nthat neither pdAcrIIA7 nor the head domain–deficient AcrIIA7 bound to the type II-C\ntracrRNA with the distinct stem–loop structure (Fig. 6A). This finding indicates that AcrIIA7\nrecognizes and interacts with RNA in a structure-dependent manner, binding only to\ntracrRNAs that possess a specific structural configuration. These results indicate that AcrIIA7,\nwhich has been reported to inhibit type II-A systems, indeed acts in a type II-A–specific\nmanner, and that this specificity arises from recognition of a particular tracrRNA structural\nfeature rather than from RNA sequence alone.\nWe have incorporated these new experimental results into the manuscript as the newly added\nsection in the Results, together with the new Figure 6A.\n4. DNA-binding controls (Fig. 5C–D): The conclusion that AcrIIA7 \"does not bind DNA\" is\ninadequately supported. The DNA sequences/lengths used should be specified, and structured\nDNAs (e.g., stem-loops) with difference lengths shall be tested to rule out motif-specific\nbinding. The Results section must clarify DNA parameters.\nResponse: We thank this reviewer for this constructive suggestion. To address the reviewer’s\ncomment, we generated structured ssDNA containing a designed stem-loop motif. The\noligonucleotide was synthesized as a ssDNA and subsequently folded into a secondary\nstructure by heating at 98℃ for 5min, followed by gradual cooling to room temperature.\nWhen this structured ssDNA was used in the binding assay, AcrIIA7 exhibited concentration\ndependent band shifts, indicating direct interaction between AcrIIA7 and structured ssDNA.\nThe binding of AcrIIA7 to ssDNA containing a stem–loop structure highly similar to that of\ntracrRNA suggests that AcrIIA7 does not distinguish between DNA and RNA, but instead\nrecognizes and binds nucleic acids in a structure-dependent manner.\nWe have incorporated this important new finding into the manuscript as a newly added\nsection in the Results, together with the new Figure 6B.\n5. Weak gRNA signal (Fig. 7): The faint gRNA bands undermine conclusions about\nAcrIIA7-mediated inhibition. The authors should optimize annealing conditions or use RNAs\nwith other labels. Quantifying band intensity via densitometry is recommended if\noptimization fails.\nResponse: We attempted several rounds of gRNA band optimization by modifying RNA\nannealing conditions and testing FAM-labeled RNAs, however, the gRNA band signal could\nnot be further improved. As recommended by the reviewer, we therefore quantified gRNA\nformation by densitometric analysis using Image J. gRNA levels were determined based on\nthe relative band intensities of gRNA and free crRNA, with background subtraction. The\nfraction of gRNA formation was calculated as : gRNA formation (%) =\nIgRNA/(IgRNA+IcrRNA)*100. The values were subsequently normalized to the no AcrIIA7\ncontrol condition. This quantification approach enables accurate assessment of AcrIIA7\ndependent inhibition of gRNA formation despite the inherently weak gRNA band signal. The\nresults were included in the new Supplementary Fig. 8.\n<Minor points>\n1. Introduction Reorganization: The Introduction lacks logical flow, with disjointed\nparagraphs covering CRISPR basics, Acr classification, and AcrIIA7 without clear narrative\nprogression. It should be restructured to: (i) introduce CRISPR-Cas as adaptive immunity,\n(ii) summarize Acr diversity and unresolved mechanisms, (iii) focus on AcrIIA7’s knowledge\ngap, and (iv) concisely state the study’s aims.\nResponse: In response to the reviewer’s suggestion, we have revised the “Introduction”\nsection to improve the clarity and coherence of the research narrative by including the most\nrecent relevant publications.\n2. Protein sequences: The authors should provide the protein as well as DNA sequences of\nthe AcrIIA7 homologs they are using in this study, such as pdAcrIIA7, meAcrIIA7, et al.\nResponse: In accordance with the reviewer’s request, we have added the complete protein\nsequences used in this study, including pdAcrIIA7, meAcrIIA7, and pdAcrIIA7 delN48, in\nthe Supplementary table 1.\nReviewer: 2\nWe thank the reviewer for his/her constructive comments on our work.\n<Major points>\nComments:\n1. Although the data show that AcrIIA7 predominantly exists as a tetramer in solution, it is\nnot definitively demonstrated that it binds to tracrRNA in this specific oligomeric state and\nwhether the tetramer is indeed the sole biologically active unit.\n2. It would substantially strengthen the proposed hijacking mechanism to determine whether\nAcrIIA7 functions as a monomer, dimer, or tetramer upon RNA binding. Binding assays with\ntracrRNA/sgRNA and Cas9 inhibition assays using monomeric or dimeric AcrIIA7 variants\nshould be performed to confirm that only the tetramer can serve as the functional entity.\nResponse: We sincerely thank the reviewer for raising this thought-provoking question.\nBecause AcrIIA7 consistently appears as a tetramer, we considered the tetramer to be the\nfunctional unit and conducted all mechanistic analyses using this native oligomeric state. To\nexplore the reviewer’s intriguing suggestion regarding potential functions of alternative\noligomeric states, we attempted to generate monomeric or dimeric variants through structure-\nguided point mutations. However, none of the mutants disrupted tetramer formation.\nTo overcome this limitation, we also designed and tested multiple double and triple mutants,\nyet the tetrameric assembly remained intact. Finally, we attempted to interfere with\noligomerization by fusing large tags (Trx or MBP) to either the N- or C-terminus, but for\nreasons that remain unclear, these tagged constructs also failed to dissociate the tetramer.\nAs suggested by the extensive intermolecular interfaces observed in the crystal structure,\nAcrIIA7 tetramerization appears to be driven by exceptionally strong and widespread\nprotein–protein interactions, which likely explains why the tetrameric form is highly resistant\nto dissociation. We share the reviewer’s enthusiasm for this question, and although we were\nunable to obtain variants with separable oligomeric states, we will continue efforts to\ngenerate such constructs in future studies.\nNevertheless, given the remarkable stability of the tetramer and its consistent presence across\nall variants we tested, we infer that the tetrameric assembly is intrinsically linked to the\nbiological activity of AcrIIA7. Thus, although we cannot experimentally exclude the\npossibility of other functional states, our data strongly support the tetramer as the biologically\nrelevant and mechanistically active unit of AcrIIA7.\n3. While a direct interaction between ΔN48AcrIIA7 and tracrRNA has been established in\nvitro, the study currently lacks in vivo evidence. The authors need to confirm this interaction\nin a cellular context, for instance, by performing RNA Immunoprecipitation (RIP) or CLIP-\nseq.\nResponse: We understand the reviewer’s concern. As suggested, we attempted RNA\nimmunoprecipitation (RIP) experiments; however, due to the numerous variables and\ncomplexities inherent to the cellular environment, we were unable to obtain sufficiently clear\nand reproducible results to include in this revision.\nNevertheless, throughout the revision period, we consistently observed robust binding\nbetween AcrIIA7 and tracrRNA in multiple additional experiments. Moreover, we repeatedly\ndetected a reduction in gRNA formation mediated by AcrIIA7 in cells (Figure 8), and this\ndecrease in gRNA levels was directly correlated with reduced Cas9 activity (new\nSupplementary Fig. 9). Taken together, these independent and converging lines of evidence\nstrongly support the validity of our model in which AcrIIA7 binds to and hijacks tracrRNA,\nthereby preventing efficient gRNA formation and subsequent Cas9 activation.\n4. The evidence for gRNA loading inhibition remains indirect. Order-of-addition cleavage\nassays imply a pre-assembly effect, but do not directly monitor RNP formation.\n5. Additional experiments using separate crRNA and tracrRNA with Cas9 and target DNA,\nvarying Acr concentration and addition order, or pull-down assays of Cas9-RNA complexes,\nwould more directly demonstrate inhibition at the loading step.\nResponse: We agree with the reviewer’s concern regarding the lack of direct evidence for\ngRNA-loading inhibition, and we appreciate the reviewer’s suggestion to address this\nexperimentally. Although our model predicted that hijacking of tracrRNA would prevent\nproper gRNA assembly and subsequently block its loading onto Cas9, we sought to obtain\nmore explicit experimental evidence supporting this mechanism.\nTo this end, we separately synthesized crRNA and tracrRNA and performed experiments in\nwhich their order of addition with Cas9 and AcrIIA7 was systematically varied. Although\nactivation of Cas9 using separately prepared crRNA and tracrRNA produced lower activity\nthan using a pre-assembled sgRNA, Cas9 activity was still detectable (Supplementary Fig. 9).\nWhen tracrRNA or gRNA encountered AcrIIA7 before interacting with Cas9, Cas9 activity\nwas completely inhibited in all cases. However, when the RNAs first interacted with Cas9 to\nform a fully assembled RNP complex, AcrIIA7 was no longer able to inhibit Cas9 activity.\nThese results clearly demonstrate that AcrIIA7 cannot inhibit Cas9 once the RNP complex\nhas been formed and that AcrIIA7 exerts its inhibitory effect by binding to tracrRNA or\ngRNA prior to RNP assembly. This new evidence is presented in Supplementary Fig. 9 and\nincorporated into the main text.\nIf the reviewer still finds the evidence for gRNA-loading inhibition insufficient despite these\nadditional experiments, we are willing to focus our interpretation solely on tracrRNA\nhijacking and remove the statement that AcrIIA7 blocks gRNA loading onto Cas9 after\nhijacking.\n6. Physiological relevance of gRNA formation assays: The authors should discuss whether\nthe faint bands in Figure 7 are physiologically relevant, and clarify if their findings reflect\nphysiological inhibition potential or an artifact of the RNA-only annealing assay.\nResponse: We appreciate the reviewer’s insightful comment regarding the physiological\nrelevance of the faint gRNA bands. Based on our additional analyses, we would like to\nclarify the following points:\n(1) Correlation between gRNA formation and AcrIIA7-mediated inhibition (Figure 7 and\nFigure 3).\nIn the presence of AcrIIA7, gRNA formation decreases in a concentration-dependent manner,\nand the extent of gRNA assembly varies depending on whether AcrIIA7 encounters crRNA\nor tracrRNA first. These trends correlate well with our in vitro order-of-addition inhibition\nassays, in which AcrIIA7 effectively inhibits Cas9 only when it binds the RNA components\nprior to RNP formation. Thus, the faint gRNA bands observed in Figure 7 reflect biologically\nmeaningful RNA intermediates whose formation is directly linked to the inhibitory\nmechanism.\n(2) Physiological relevance demonstrated by additional revision experiments (Supplementary\nFig. 9).\nIn response to another reviewer2’s comment (comments 4&5), we performed further\nexperiments using separately synthesized crRNA and tracrRNA instead of sgRNA to more\nprecisely determine the stage at which AcrIIA7 inhibits Cas9. As expected from the faint\ngRNA formation in vitro, Cas9 activity was substantially reduced compared to reactions\nusing sgRNA. Importantly, however, these faint gRNA were sufficient to activate Cas9, and\nAcrIIA7 fully inhibited Cas9 activity whenever it encountered tracrRNA before Cas9.\nConversely, when crRNA and tracrRNA met Cas9 first to form a complete RNP, AcrIIA7\nfailed to inhibit Cas9.\nTaken together, these results demonstrate that although gRNA formation is inefficient under\nRNA-only in vitro conditions, the faintly assembled gRNA species are biologically\nmeaningful, as they directly correlate with Cas9 activity and AcrIIA7-mediated inhibition.\nWe have incorporated this interpretation into the Discussion section as suggested by the\nreviewer.\n7. The data suggest that AcrIIA7 recognizes structural features of tracrRNA rather than\nsequence. Experiments with compensatory mutations disrupting/restoring stem-loop pairing,\nor truncated RNAs containing minimal motifs, would provide direct evidence for structure-\ndependence in RNA recognition.\n8. Have the authors performed any assays, such as CLIP-seq or in vitro footprinting, to\nidentify the precise binding sequence between AcrIIA7 and tracrRNA? Determining the exact\nbinding region would greatly help in understanding the proposed tracrRNA hijacking\nmechanism.\nResponse: We first thank the reviewer for raising this important and previously overlooked\nquestion. If AcrIIA7 binds tracrRNA, identifying which region of tracrRNA mediates this\ninteraction is indeed a critical issue. While structural analysis of the AcrIIA7–tracrRNA\ncomplex would provide the most definitive insight—and such studies are currently underway\nin our laboratory with promising preliminary progress—we agree that defining a minimal\nbinding motif represents an important and complementary line of investigation.\nGuided by this reviewer’s suggestion, we synthesized a series of gRNA variants in which\nindividual stem–loop elements were selectively removed and examined their interactions with\nAcrIIA7 using EMSA. Removal of stem–loop 3 (SL3) did not abolish binding, although the\naffinity for AcrIIA7 was noticeably reduced. In contrast, gRNA variants lacking both SL2\nand SL3, or lacking all three stem–loops (SL1–SL3), showed little to no detectable binding to\nAcrIIA7. These results indicate that SL3 is not strictly required for binding, whereas SL1 and\nSL2 are essential. We therefore conclude that SL1 and SL2 constitute the minimal structural\nmotif necessary for AcrIIA7 recognition.\nThese new findings identifying the minimal tracrRNA-binding motif have been incorporated\ninto the Results section as a newly added subsection and are presented in Figures 6C, 6D,\nand 6E.\n9. Given that the head domain is located near the predicted RNA-binding groove, it is\nimportant to rule out any influence of the affinity tag on RNA binding or Cas9 inhibition. A\ncomparison with tag-cleaved or N-terminally tagged constructs would be helpful and may be\nnecessary.\nResponse: We thank this reviewer for raising this important point. Our AcrIIA7 constructs\nwere originally expressed with a C-terminal 6x His tag, and we agree that the presence of the\naffinity tag could potentially influence RNA binding or Cas9 inhibition. To directly address\nthis concern, as requested by the reviewer, we generated an additional N-terminally tagged\nversion of the pdAcrIIA7 N48 construct and compared its RNA-binding with that of the C-\nterminally tagged protein. Both constructs exhibited comparable binding affinity toward\nsgRNA in our EMSAs, indicating that the presence or position of the affinity tag does not\ninterfere with the RNA binding mechanism of AcrIIA7. These results support that the\nfunctional conclusions presented in this study are not affected by tag.\nThis result has been described in the main text and is now presented together with the new\nSupplementary Fig. 6.\n10. The authors conclude, based on SEC co-elution, that AcrIIA7 does not interact with Cas9.\nThe authors are encouraged to temper their interpretation accordingly and, if possible, to\nperform complementary experiments capable of detecting transient or weak interactions in a\ncellular context.\nResponse: We appreciate for the reviewer’s insightful suggestion. In agreement with this\nrecommendation, we performed pull-down assays to examine whether AcrIIA7 can interact\nwith SpyCas9 in a cellular context, which would allow detection of weak or transient\ninteractions that may not be captured by SEC co-elution analysis. These experiments were\nperformed using both full length pdAcrIIA7 and the ΔN48 variant. In all cases, we did not\nobserve detectable interaction between SpyCas9 and either AcrIIA7 construct under the\ntested conditions. These results support the conclusion that AcrIIA7 does not directly bind\nSpyCas9 as part of its inhibitory mechanism, consistent with our SEC analysis. The results of\nthis pull-down experiment have been compiled in the new Supplementary Fig. 4 and have\nalso been incorporated into the main text.\n11. In Figure 5, the RNA-only band intensity remains similar across protein concentrations,\nwhile in the tracrRNA assays, the free RNA band is nearly absent in the sgRNA EMSA. (i)\nHave lower protein concentrations been tested to resolve shifts more clearly? (ii) Quantifying\nband intensities to estimate binding affinities (e.g., Kd values) would strengthen the\nconclusions.\nResponse: As suggested by the reviewer, we attempted to determine the dissociation constant\n(Kd) through repeated experiments. However, we found that accurately estimating Kd values\nbased on band intensities was technically challenging, and the resulting values were not\nsufficiently reliable. For this reason, we did not include these data in the current revision.\nWe are currently conducting structural studies of the AcrIIA7–tracrRNA complex and are\nobtaining promising results. In future follow-up studies, we plan to determine the binding\naffinity between AcrIIA7 and tracrRNA using more quantitative and definitive approaches,\nsuch as surface plasmon resonance (SPR) or isothermal titration calorimetry (ITC). We\nsincerely thank the reviewer for this valuable suggestion.\n12. In Figure 5b, the band shift appears to change depending on protein concentration. Could\nthis be due to conformational changes of AcrIIA7 or tracrRNA, or are there other possible\nreasons?\nResponse: We sincerely thank the reviewer for carefully observing this subtle change, which\nwe had not previously considered. Based on the reviewer’s comment, we have reflected on\nthis point, and our interpretation is as follows. When the protein binds to specific RNA, the\nRNA may bend or become compacted, altering not only its molecular mass but also its shape\nand net charge, which leads to nonlinear changes in electrophoretic mobility. Therefore, as\nthe protein concentration increases and more protein molecules bind to the more RNA,\nnumerous RNA species with subtle structural changes are formed, which likely explains the\nslight differences observed in the band positions.\n<Minor points>\n1. In Figures 2E and 2F, the specific residues are not clearly distinguishable. To improve\nclarity, the authors should use distinct colors or labeling to unambiguously highlight each\nresidue being discussed.\nResponse: We have revised the figure according to the reviewer’s suggestion to make the\nresidues more clearly visible.\n2. In Figures 3D and 3E, additional labeling would help clarify the specific regions being\nhighlighted and allow readers to better distinguish between domains or datasets.\nResponse: We have revised the figure according to the reviewer’s suggestion.\n3. In Figures 3, 7C, and 7D, the notation for incubation order could be improved. Please\nspecify explicitly that the numbers shown correspond to the order of incubation.\nResponse: We thank the reviewer for this helpful comment. We have revised Figures 3, 7C,\nand 7D to explicitly indicate the order of incubation. The numbers shown in each panel now\nclearly represent the sequential incubation steps, as described in the revised figures.\n4. In Figure 4, the SDS-PAGE and SEC profiles of SpyCas9 show heterogeneity and possible\naggregation.\nResponse: We thank the reviewer for pointing this out. The SEC and SDS-PAGE profiles of\nSpyCas9 indeed showed slight heterogeneity, which is likely due to partial aggregation\ncommonly observed for Cas9 proteins at high concentrations. We have now included a note\nin the revised figure legend to clarify this observation.\n5. The legends for Figure 5B and 5G are incomplete. They should explicitly state that the lane\nlabeled 'B' represents the 'buffer only' control.\nResponse: We thank the reviewer for pointing this out. We have revised the legends for\nFigures 5B, 5F, and 5G to explicitly indicate that the lane labeled “B” represents the sgRNA\nin buffer control.\n6. In Figure 5G, the two bands observed in the EMSA experiment with full-length pdAcrIIA7\nand tracrRNA are insufficiently explained. Clear annotation of these bands is recommended.\nSimilarly, in Figures 5C, 5D, 5F, and 5G, the identity of specific bands should be clarified by\nadditional labeling, as was done in Figure 5B.\nResponse: We thank the reviewer for this valuable suggestion. We have revised Figures 5C,\n5D, 5F, and 5G to clearly annotate the identity of each band, following the labeling format\nused in Figure 5B.\nThe appearance of two bands in Figure 5G is somewhat ambiguous. This phenomenon was\nobserved only in pdAcrIIA7 constructs that contain the head domain. Structural analysis\nrevealed that this head domain is not essential for tracrRNA binding and is highly flexible.\nTherefore, it is plausible that the presence of two bands is due to structural heterogeneity\nresulting from the flexible head domain, leading to different conformational states of\nAcrIIA7. We have included this interpretation in the revised manuscript.\n7. The abbreviations used in Figure 6F (DW, Pd, Me) are not defined. Please specify their\nmeanings in the figure legend for clarity.\nResponse: We thank the reviewer for this helpful comment. We have revised the legend of\nFigure 6F to define all abbreviations (DW, Pd, and Me) for clarity.\nwhich is misleading in this context and should be corrected.\nResponse: We corrected it.\n9. n Figures 3, 7C, and 7D, the notation for incubation order could be improved. Please\nspecify explicitly that the numbers shown correspond to the order of incubation.\nResponse: Following the reviewer’s recommendation, we improved the notation describing\nthe incubation order in Figures 3, 7C, and 7D. In the revised figures, the numbers explicitly\nindicate the sequential order of component addition, where ‘1’ represents the components that\nwere incubated first, followed by ‘2’ and ‘3’ for subsequent additions. Thus, two components\nlabeled with '1' indicate that they were co-incubated during the initial step. This clarified\nnotation is also described in the updated figure legends to avoid any ambiguity.\n10. To improve the narrative flow, consider combining the first and second sections of the\nResults, merging them could create a more cohesive and logical presentation. (from 241 -\n332)\nResponse: We agree with the reviewer’s point and find it valid. However, in the context of a\ntypical structure–function study, we believe that both structural analysis and subsequent\ninvestigation of the functional unit are equally important components for elucidating the\nmechanism. Therefore, we consider it a reasonable and strategic approach to present these\ntwo aspects as separate sections within the Results, each contributing distinct and essential\ninsights into the protein’s function.\n11. Throughout the figures, hyphens (-) have been used where minus signs () are appropriate.\nResponse: We replaced them to minus signs.\nReviewer: 3\nWe thank the reviewer for his/her constructive comments on our work.\n<Major points>\nComments:\n1. The mutational studies identify the tracrRNA binding site in the protein, but what motif in\ntracrRNA is responsible for the binding is not clarified. the minimal sequence with the major\nbinding affinity for AcrIIA7 needs to be investigated.\nResponse: We sincerely thank the reviewer for raising this important question and providing\nvaluable comments. Reviewer 2 raised a similar point, underscoring the significance of this\nissue. Our response is as follows.\nIf AcrIIA7 binds tracrRNA, identifying which region of tracrRNA mediates this interaction is\nindeed a critical issue. While structural analysis of the AcrIIA7–tracrRNA complex would\nprovide the most definitive insight—and such studies are currently underway in our\nlaboratory with promising preliminary progress—we agree that defining a minimal binding\nmotif represents an important and complementary line of investigation.\nGuided by this reviewer’s suggestion, we synthesized a series of tracrRNA variants in which\nindividual stem–loop elements were selectively removed and examined their interactions with\nAcrIIA7 using EMSA. Removal of stem–loop 3 (SL3) did not abolish binding, although the\naffinity for AcrIIA7 was noticeably reduced. In contrast, tracrRNA variants lacking both SL2\nand SL3, or lacking all three stem–loops (SL1–SL3), showed little to no detectable binding to\nAcrIIA7. These results indicate that SL3 is not strictly required for binding, whereas SL1 and\nSL2 are essential. We therefore conclude that SL1 and SL2 constitute the minimal structural\nmotif necessary for AcrIIA7 recognition.\nThese new findings identifying the minimal tracrRNA-binding motif have been incorporated\ninto the Results section as a newly added subsection and are presented in Figures 6C, 6D,\nand 6E.\n2. The authors claim AcrIIA7 shows tracrRNA-specific binding, but the RNA-binding\nexperiments were conducted only for crRNA and tracrRNA. Since AcrIIA7 work in bacterial\ncells including numerous cellular RNA species, the authors need to address whether AcrIIA7\nspecifically binds tracrRNA than other cellular RNAs. A reliable method may be a pull-down\nassay using AcrIIA7 as a bait followed by sequencing of bound RNAs.\nResponse: We understand the reviewer’s concern. As suggested, we attempted pull-down\nassay using AcrIIA7 as a bait; however, due to the numerous variables and complexities\ninherent to the cellular environment, we were unable to obtain sufficiently clear and\nreproducible results to include in this revision.\nNevertheless, throughout the revision period, we consistently observed robust binding\nbetween AcrIIA7 and tracrRNA in multiple additional experiments. Moreover, we repeatedly\ndetected a reduction in gRNA formation mediated by AcrIIA7 in cells (Figure 8), and this\ndecrease in gRNA levels was directly correlated with reduced Cas9 activity (new\nSupplementary Fig. 9).\nIn addition, through several experiments performed during the revision period—including\nbinding assays with type II-C tracrRNA (Figure 6A), binding assays using ssRNA engineered\nto adopt the same structure as tracrRNA (Figure 6B), and experiments aimed at identifying\nthe minimal binding motif (Figures 6C, 6D, and 6E)—we demonstrated that AcrIIA7 binds\ntracrRNA in a structure-specific manner. These results suggest that AcrIIA7 discriminates\ntracrRNA from other cellular RNAs based on its unique structural features. We have\nincorporated these findings into both the Results and Discussion sections of the manuscript.\n<Minor points>\n1. In the proposed functionl mechanism, as far as I understand, AcrIIA7 should be a viral\nprotein, not a bacterial protein. Does AcrIIA7 from Phocaeicola dorei mean that the gene is\nidentified from sequencing from total bacterial DNA, infected by virus and thus including\nviral DNA? If so, this needs to be clearly explaind.\nResponse: Yes, that is correct. This gene originates from a bacteriophage and was transferred\ninto the bacterial chromosome through phage infection. As the reviewer suggested, we have\nclarified the origin of this gene in the Introduction section, where we explicitly describe this\npoint as follows;\n“The pdAcrIIA7 gene originates from a bacteriophage, but as is the case for many Acr genes,\nit is incorporated into the bacterial chromosome through phage infection”\n2. The indicaton of the full name of pdAcrIIA7 is missing.\nResponse: We indicated the meaning of pdAcrIIA7 in introduction.\n3. AcrIIA7 seems to strongly bind both free tracrRNA or gRNA? Would the protein be able\nto dissociate crRNR and tracrRNA? If so, this could be added in the inhibitory mechanism.\nResponse: We thank the reviewer for raising this highly intriguing topic. Whether AcrIIA7 is\ncapable of dissociating pre-assembled crRNA-tracrRNA duplexes is indeed an important\nmechanistic consideration. Based on our current data, however, AcrIIA7 does not appear to\ndisrupt the crRNA-tracrRNA complex once it is formed. In our gRNA formation assays,\npdAcrIIA7 did not reduce the gRNA band when added after crRNA-tracrRNA duplex\nformation (Fig. 8C), indicating that it does not dissociate the pre-assembled gRNA. In\ncontrast, meAcrIIA7, which has an affinity for gRNA, produced band shifts even in the\npresence of gRNA, but this likely reflects its binding to the stem-loop region of tracrRNA\nwithin the gRNA structure, not dissociation of the duplex itself (Fig. 8D). Furthermore, our\nmapping experiments show that AcrIIA7 binds to the stem-loop region of tracrRNA, which is\ndistinct from the repeat-anti repeat base pairing interface between crRNA and tracrRNA.\nBecause the AcrIIA7 binding site does not overlap with the crRNA-tracrRNA region, it is\nunlikely that AcrIIA7 actively separates the two RNAs. Instead, AcrIIA7 appears to bind the\nstructured region of tracrRNA or gRNA without disrupting the crRNA-tracrRNA pairing.\nTogether, these results suggest that AcrIIA7 inhibits Cas9 not by dissociating crRNA and\ntracrRNA, but rather by binding to the stem-loop region of tracrRNA and preventing proper\ngRNA formation and loading onto Cas9.\nWe have incorporated this important discussion into the “Discussion section” of the\nmanuscript.\n4. In Fig 5G, tracrRNA shows a single band, which is completely shifted by 2.5 uM\npdAcrIIA7. But in Fig 7A, tracrRNA is in two bands, which is not much shifted at the same\nprotein concentration. This conflict needs to be addressed.\nResponse: We appreciate the reviewer’s careful observation. The difference in tracrRNA\nband patterns between Figures 5G and 7A arises from the distinct buffer conditions used in\neach experiment. In Figure 5G, tracrRNA was incubated with pdAcrIIA7 in a buffer\ncontaining 10 mM Tris-HCl (pH 8.0) and 75 mM NaCl, which produces a single, well-\ndefined RNA band that is fully shifted at 2.5μM pdAcrIIA7.\nIn contrast, the assay in Figure 7A essentially required inclusion of the RNA annealing buffer\nfor crRNA–tracrRNA duplex formation, resulting in a different final composition (10 mM\nTris-HCl pH 8.0, 50 mM NaCl, 1 mM EDTA). This altered ionic strength and the presence of\nEDTA affect the folding state and electrophoretic mobility of tracrRNA, producing two\nbands, and also influence the binding affinity and shift behavior of pdAcrIIA7.\nTo avoid misinterpretation, each gel includes buffer-matched controls, ensuring that\ncomparisons are made only within identical experimental conditions. Therefore, although\nFigures 5G and 7A show different migration patterns and shift efficiencies, these differences\nreflect buffer-dependent RNA behavior, not inconsistencies in AcrIIA7 function."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have addressed my concerns.", "reviewer_2": "From the side of Reviewer 2 –\nResponse Comments to Major Comments\n  Major Comments 1 & 2: Regarding whether the Tetramer is the sole functional unit - The authors repeatedly observed that\nAcrIIA7 exists as a stable tetramer in solution. Despite various attempts to induce monomer/dimer states—such as structure-\nbased mutations, multiple mutations, and bulky tag fusion—they failed to disassemble the tetramer. This explanation is\nconsistent with the structural characteristic that the tetramer possesses a very strong intermolecular interface. Although non-\ntetrameric states could not be completely experimentally excluded, it is judged that sufficiently diligent attempts were made\nwithin the currently accessible scope. However, the potential functionality of states other than the tetramer remains a subject\nfor future research.\n  Major Comment 3: Lack of in vivo evidence (ΔN48AcrIIA7–tracrRNA interaction) - RIP experiments in a cellular context\nwere attempted but failed to yield reproducible results; thus, direct in vivo binding evidence has not yet been presented.\nHowever, the authors provided additional data showing a direct correlation between the reduction of gRNA and the\ninhibition of Cas9 activity as AcrIIA7 concentration increases in a cell-based system. This indirectly supports the possibility\nthat the proposed mechanism operates in vivo. While it cannot be seen as complete substitute evidence, it is judged that\nreasonable supplementation achievable at the revision stage has been accomplished.\n  Major Comments 4, 5, 6: Lack of directness regarding gRNA loading inhibition - The authors faithfully performed the\n\"crRNA and tracrRNA separated system + order-of-addition\" experiments suggested by the reviewer. As a result, they\nclearly demonstrated that Cas9 cleavage activity is inhibited only when AcrIIA7 encounters tracrRNA (or gRNA) prior to\nCas9, and that inhibition does not occur if the RNP is formed first. This aligns logically with the hijacking model, which posits\naction prior to the gRNA loading step, and is judged to be data that adequately answers the reviewer's core question\nregarding the mechanistic order.\n  Major Comments 7 & 8: Recognition of tracrRNA structure and binding region - Through EMSA using stem-loop variants,\ntype II-C tracrRNA, and ssDNA mimicking the structure, the authors convincingly showed that AcrIIA7 recognizes structure—\nspecifically SL1 and SL2—rather than sequence. Although high-resolution methods like CLIP-seq or footprinting were not\npresented, it is judged that sufficient indirect and direct evidence regarding structural recognition has been provided within\nthe scope of the current paper.\n  Major Comment 11: Quantitative issues with EMSA (Kd) - The authors explained that they attempted to calculate Kd but\ncould not obtain reliable values due to technical limitations, and they clearly stated plans for follow-up research using\nSPR/ITC. Considering that EMSA is not inherently a precise quantitative experiment, the current data is judged sufficient to\nsupport the core claim of this paper (the tracrRNA hijacking mechanism). However, the absence of quantitative values is\nregrettable, and it is expected to be supplemented in future research.\nResponse Comments to Minor Comments\nThe suggestions regarding figure labeling, legend clarification, notation of incubation order, supplementation of band\ninterpretation, and improvement of narrative structure proposed in the minor comments have been faithfully reflected overall.\nIt is judged that the readability and logical clarity of the manuscript have been improved.\nComprehensive Opinion (Final Assessment)\nI judge that the authors' efforts regarding the function of AcrIIA7 have succeeded in proposing a novel anti-CRISPR\nmechanism that inhibits RNP formation by recognizing and hijacking the structure of tracrRNA. In particular, the structure of\nthe paper—starting from the identification of the tetramer structure, leading to analytic SEC, tracrRNA EMSA, Cas9 cleavage\ninhibition, and the order-dependent gRNA formation assay—possesses an appropriate flow to explain the proposed\nmechanism.\nThe manuscript prior to review was limited to in vitro data and lacked consideration for biochemical controls and reaction\norder. However, through revision, these key weaknesses have been significantly supplemented. Although direct binding\nevidence at the in vivo level or information regarding the conformational state during AcrIIA7 action is still lacking, it is\njudged that sufficient in vitro grounds supporting the hijacking model have been secured through the order-dependent assay\nand various tracrRNA/structure variant experiments.\nTherefore, Reviewer 2’s final opinion is that the currently revised manuscript possesses a logically consistent structure and\nsufficient experimental grounds to carry through its core theme, making it suitable for publication.", "reviewer_3": "Since dsRNA and dsDNA is in A and B form, respectively, the tertiary structure may be different between tracrRNA and\nSssDNA. In addition, the presence and absence of 2'-OH is an important structural element. Therefore, it would be more\nprecise that the tracrRNA binding is dependent on the secondary structure. To be more precise, another experiment would\nbe needed with RNA or DNA with the identical secondary structure and a different sequence.\nAlthough it is interesting that AcrIIA7 binds to both tracrRNA and the corresponding DNA, the data is still preliminary to draw\nany definite conclusion. This data and interpretation are recommended to go to a supplementary figure and discussion.\nProvided that this minor issue is addressed, I recommend this manuscript for publication in Nature Communications."}, "author_response": "Reviewers' Comments to Author:\nReviewer: 1\nWe thank the reviewer for his/her constructive comments on our work.\nComments:\n1. The authors have addressed my concerns.\nResponse: We sincerely appreciate the reviewer’s final approval and thoughtful evaluation of\nour manuscript.\nReviewer: 2\nWe thank the reviewer for his/her constructive comments on our work.\nComments:\nMajor Comments 1 & 2: Regarding whether the Tetramer is the sole functional unit - The\nauthors repeatedly observed that AcrIIA7 exists as a stable tetramer in solution. Despite\nvarious attempts to induce monomer/dimer states—such as structure-based mutations,\nmultiple mutations, and bulky tag fusion—they failed to disassemble the tetramer. This\nexplanation is consistent with the structural characteristic that the tetramer possesses a very\nstrong intermolecular interface. Although non-tetrameric states could not be completely\nexperimentally excluded, it is judged that sufficiently diligent attempts were made within the\ncurrently accessible scope. However, the potential functionality of states other than the\ntetramer remains a subject for future research.\nResponse: We agree with the reviewer that a more detailed analysis of the relationship\nbetween stoichiometry and functional activity will be important, and we intend to address this\nin future work.\nMajor Comment 3: Lack of in vivo evidence (ΔN48AcrIIA7–tracrRNA interaction) - RIP\nexperiments in a cellular context were attempted but failed to yield reproducible results; thus,\ndirect in vivo binding evidence has not yet been presented. However, the authors provided\nadditional data showing a direct correlation between the reduction of gRNA and the\ninhibition of Cas9 activity as AcrIIA7 concentration increases in a cell-based system. This\nindirectly supports the possibility that the proposed mechanism operates in vivo. While it\ncannot be seen as complete substitute evidence, it is judged that reasonable supplementation\nachievable at the revision stage has been accomplished.\nResponse: We sincerely thank the reviewer for the generous evaluation of our work.\nMajor Comments 4, 5, 6: Lack of directness regarding gRNA loading inhibition - The authors\nfaithfully performed the \"crRNA and tracrRNA separated system + order-of-addition\"\nexperiments suggested by the reviewer. As a result, they clearly demonstrated that Cas9\ncleavage activity is inhibited only when AcrIIA7 encounters tracrRNA (or gRNA) prior to\nCas9, and that inhibition does not occur if the RNP is formed first. This aligns logically with\nthe hijacking model, which posits action prior to the gRNA loading step, and is judged to be\ndata that adequately answers the reviewer's core question regarding the mechanistic order.\nResponse: Yes, this was a very important question, and we sincerely thank the reviewer for\nproposing the additional experiment. We believe that these new data have further\nstrengthened and solidified our hypothesis, and we once again express our gratitude to the\nreviewer for this valuable suggestion.\nMajor Comments 7 & 8: Recognition of tracrRNA structure and binding region - Through\nEMSA using stem-loop variants, type II-C tracrRNA, and ssDNA mimicking the structure,\nthe authors convincingly showed that AcrIIA7 recognizes structure—specifically SL1 and\nSL2—rather than sequence. Although high-resolution methods like CLIP-seq or footprinting\nwere not presented, it is judged that sufficient indirect and direct evidence regarding\nstructural recognition has been provided within the scope of the current paper.\nResponse: This was also a necessary and important analysis, and we sincerely thank the\nreviewer for suggesting it.\nMajor Comment 11: Quantitative issues with EMSA (Kd) - The authors explained that they\nattempted to calculate Kd but could not obtain reliable values due to technical limitations,\nand they clearly stated plans for follow-up research using SPR/ITC. Considering that EMSA\nis not inherently a precise quantitative experiment, the current data is judged sufficient to\nsupport the core claim of this paper (the tracrRNA hijacking mechanism). However, the\nabsence of quantitative values is regrettable, and it is expected to be supplemented in future\nresearch.\nResponse: We fully recognize the importance of quantitative analysis. As suggested by the\nreviewer, we will aim to perform more rigorous quantitative measurements in the near future.\nWe sincerely thank the reviewer once again for the detailed and thoughtful comments.\nResponse Comments to Minor Comments: The suggestions regarding figure labeling, legend\nclarification, notation of incubation order, supplementation of band interpretation, and\nimprovement of narrative structure proposed in the minor comments have been faithfully\nreflected overall. It is judged that the readability and logical clarity of the manuscript have\nbeen improved.\nResponse: We sincerely thank the reviewer once again for the many important comments\nand for the careful, detailed suggestions throughout the manuscript. We truly appreciate the\ntime and effort invested in thoroughly reading and improving our work.\nComprehensive Opinion (Final Assessment)\nI judge that the authors' efforts regarding the function of AcrIIA7 have succeeded in\nproposing a novel anti-CRISPR mechanism that inhibits RNP formation by recognizing and\nhijacking the structure of tracrRNA. In particular, the structure of the paper—starting from\nthe identification of the tetramer structure, leading to analytic SEC, tracrRNA EMSA, Cas9\ncleavage inhibition, and the order-dependent gRNA formation assay—possesses an\nappropriate flow to explain the proposed mechanism.\nThe manuscript prior to review was limited to in vitro data and lacked consideration for\nbiochemical controls and reaction order. However, through revision, these key weaknesses\nhave been significantly supplemented. Although direct binding evidence at the in vivo level\nor information regarding the conformational state during AcrIIA7 action is still lacking, it is\njudged that sufficient in vitro grounds supporting the hijacking model have been secured\nthrough the order-dependent assay and various tracrRNA/structure variant experiments.\nTherefore, Reviewer 2’s final opinion is that the currently revised manuscript possesses a\nlogically consistent structure and sufficient experimental grounds to carry through its core\ntheme, making it suitable for publication.\nResponse: We sincerely thank the reviewer once again for the many important comments\nand for the careful, detailed suggestions throughout the manuscript. We truly appreciate the\ntime and effort invested in thoroughly reading and improving our work. We also sincerely\nthank the reviewer for the final approval for publication.\nReviewer: 3\nWe thank the reviewer for his/her constructive comments on our work.\nComments:\n1. Since dsRNA and dsDNA is in A and B form, respectively, the tertiary structure may be\ndifferent between tracrRNA and SssDNA. In addition, the presence and absence of 2'-OH is\nan important structural element. Therefore, it would be more precise that the tracrRNA\nbinding is dependent on the secondary structure. To be more precise, another experiment\nwould be needed with RNA or DNA with the identical secondary structure and a different\nsequence.\nAlthough it is interesting that AcrIIA7 binds to both tracrRNA and the corresponding DNA,\nthe data is still preliminary to draw any definite conclusion. This data and interpretation are\nrecommended to go to a supplementary figure and discussion.\nProvided that this minor issue is addressed, I recommend this manuscript for publication in\nNature Communications.\nResponse: We sincerely thank the reviewer for the careful and thorough reading of our\nmanuscript and for the constructive suggestions that have significantly improved our work. In\naccordance with the reviewer’s recommendation, we have moved the incomplete data related\nto Figure 6b to the Supplementary Information and have moderated our interpretation\naccordingly. We are also grateful to the reviewer for granting final approval for publication."}]

Decision letter