Peer review of: AcrIF9 tethers non-sequence specific dsDNA to the CRISPR RNA-guided surveillance complex (2020)

Reviewers' comments: 
Reviewer #1 (Remarks to the Author): 

This short paper reports on the mechanism of the anti-CRISPR AcrIF9 (F9), which inhibits the type 
I-F (Csy) effector. This Acr was one of the first identified but has not been characterised 
structurally or mechanistically. This is achieved here using a combination of cryo-EM and some 
basic biochemical analyses. The Cryo structure reveals that AcrIF9 binds to the type I-F complex 
as two monomers by interacting with the Cas7 subunit in a manner that will block target RNA 
search and binding – a mechanism remarkably similar to AcrIF1. 

As judged by EMSA, AcrIF9 does not bind dsDNA on its own up to a concentration of 3 µM, but 
does inhibit Csy binding to target DNA when present at 20 nM concentration (ten-fold less than the 
Csy concentration) – see point 3 below. However, at higher stoichiometries, F9 no longer prevents 
binding to DNA, which is also difficult to explain. 

A cryo structure was also determined for the complex incubated with dsDNA, revealing binding of 
the DNA to the surface of F9, allowing mapping of the interface as a basic area of F9 with 
conserved basic residues (see point 5). The authors speculate that F9 functions partly by 
sequestering Csy on random DNA as well as blocking target DNA binding. 

Overall there is some decent cryo-EM data here paired with some preliminary, contradictory and 
variable quality EMSA data. This leaves any conclusion beyond “F9 probably functions like F1” to 
be on rather shaky ground. 

Specific points: 
1. AcrIF9 is described as a 5-stranded anti-parallel beta sheet cradling an alpha helix, but no 
structural comparison is offered. Are there any similar folds in PDB? Can the structure be 
compared more fully to AcrIF1, given their striking convergence in binding sites? Can the two 
binding sites be compared, beyond saying they are “similar”. 
2. The “triplicate” EMSAs shown in figure S3 are strikingly different from one another to a 
surprising degree. For example, the third gel in S3b is quite fundamentally different from the first 
gel. In the second and third replicates it looks like there is a significant proportion of shifted 
material half way up the gel over a wide protein range, suggesting an intermediate species, but 
the gel in figure 2 doesn’t conform to this. These experiments should be repeated to give more 
confidence in the data and their interpretation. Ideally, an alternative technique such as 
fluorescence polarization should be considered, as binding or displacement of Csy will give rise to a 
large anisotropy change using a method that measures true equilibria. 
3. Figure 2a suggests that one tenth molar ratio of AcrIF9 almost completely blocks binding of Csy 
to target DNA. Given that at least 90% of the Csy complex should be free and available to bind 
DNA (assuming non-cooperative binding of AcrIF9), what is the authors’ view of the mechanism 
here? Notably, in the replicates, this effect is not observed at 20 nM F9 to nearly the same extent, 
and in the middle gel of S3a there appears to be 50:50 binding to target DNA at a molar ratio of 
1:2, which could make more sense. 
4. It would help to understand how Csy binds to target and non-target DNA in the absence of F9. 
Although this has no doubt been published previously it would be helpful to show this data for the 
DNA species used here. It is important to understand how F9 affects DNA binding for both target 
and non-target DNA. 
5. A binding interface for F9 on dsDNA is observed that does not involve the Csy complex. The text 
states that “we attempted to make structure-guided mutations of these residues, but the F9 
mutants failed to bind the Csy complex”. Leaving aside the observation that there is no data in the 
paper to support this (were all 5 made? Did they purify as for the wild-type protein?), this is 
another puzzling observation as the binding interface between F9 and Csy has not been modified. 


Reviewer #2 (Remarks to the Author): 

Hirschi et al. present the cryo-EM structures of Csy-AcrIF9 complex and Csy-AcrIF9-dsDNA 
complex at ~3.9 Å and 6.9 Å respectively. In combination with biochemical data, the authors show 
AcrIF9 not only sterically blocks the binding of target DNA to crRNA, but also promotes nonspecific 
recruitment of dsDNA, potentially sequestering the complex from target DNA. Even without the 
high-resolution structure of Csy-AcrIF9-dsDNA complex, the biochemical data and the current 
density map support their conclusions. This paper significantly broadens the current view of the 
mechanisms for anti-CRISPR proteins mediated immune suppression, and will be of interest in the 
community given that CRISPR-Cas immunity is of broad interest. 

During the revision session, the structure of Csy-AcrF9 complex has been described in a PNAS 
paper (PNAS first published March 13, 2020 https://doi.org/10.1073/pnas.1922638117). With 
2.57 Å high-resolution Csy-AcrF9 complex structure, the authors of the PNAS paper proposed 
binding of AcrF9 to the Csy complex prevents target DNA binding to crRNA, similar to how AcrF1 
works, also in line with part of Hirschi et al. ‘s conclusions. The author may briefly discuss the 
results in the PNAS paper. Even the high-resolution structure of Csy-AcrF9 complex recently has 
been reported, as far as I see, the non-sequence specific dsDNA bound to Csy-AcrF9 complex 
reported in Hirschi et al. paper significantly advances the understanding of how anti-CRISPR 
proteins work, I still support this paper to be published. 

Minor comments: 

1) Fig. 1b, the color of Cas7.6f thumb label is not in blue, making the label not clear that what it 
represents. 

2) The authors should provide detailed information in the method session about how Csy-AcrIF9 
and Csy-AcrIF9-dsDNA complexes are assembled. 

3) By comparing Supplementary Fig.1a and Supplementary Fig.4a, it’s confusing about the peaks 
they choose. The authors might use different columns to purify the complex, are the two major 
peaks in each file related to each other in these two gel filtration files? 

4) In supplementary Fig, 4a, the label “10, 11, 13, 15” is not clear about what it presents. 



Reviewer #3 (Remarks to the Author): 

The authors present structural and biochemical analyses of the anti-CRISPR protein AcrIF9 that 
targets the type I-F CRISPR-Cas system. They have determined the 3D structure of AcrIF9 bound 
to the RNA-guided surveillance complex (Csy) by cryo-EM at an overall resolution of 3.9 Å. Two 
copies of AcrIF9 bind to the Csy complex at positions reminiscent of the AcrIF1 binding sites. 
AcrIF9, like AcrIF1, blocks target DNA binding in Electrophoretic Mobility Shift Assays with sub-
stoichiometric ratios of AcrIF9 to Csy complex. However, at higher stoichiometric ratios, AcrIF9 
induces target and non-target DNA binding to Csy. The 3D reconstruction of the Csy-AcrIF9-dsDNA 
ternary complex, determined by cryo-EM at an overall resolution of 6.9 Å, shows that AcrIF9 
tethers dsDNA to the Csy complex. 
As acknowledged by the authors, a similar phenomenon of anti-CRISPR-induced non-specific DNA 
binding to CRISPR-Cas surveillance complex has previously been reported for AcrIIA11 that targets 
the type II CRISPR-Cas9 system (https://doi.org/10.7554/eLife.46540). Although the manuscript 
under review presents the first example of a type I anti-CRISPR protein promoting non-specific 
DNA binding to the Csy complex in vitro, major revisions are required to strengthen the authors 
findings and provide an advance in understanding anti-CRISPR proteins. In particular, the role for 
dsDNA binding in AcrIF9’s mode of inhibition remains elusive. What would be the benefit of a 
concentration-dependent two-pronged CRISPR-Cas inhibition mechanism while the inhibition of 
target DNA binding and cleavage is effective at low concentration of anti-CRISPR protein (sub-
stoichiometric to stoichiometric ratios of AcrIF9 to the Csy complex)? Noteworthy, the paper by 
Zhang K et al. entitled “Inhibition mechanisms of AcrF9, AcrF8, and AcrF6 against type I-F 
CRISPR-Cas complex revealed by cryo-EM”, which has been published in the course of reviewing 
this manuscript (www.pnas.org/cgi/doi/10.1073/pnas.1922638117), presents a highly detailed 
cryo-EM structure of AcrIF9 bound to the Csy complex at an overall resolution of 2.6 Å and shows 
that AcrIF9 functions via the competitive binding to DNA binding sites on Cas7f subunits. Overall, 
further data interpretation and additional experiments could greatly improve the manuscript. 
Besides, all methodological details should be provided for a better understanding of the results and 
for the sake of reproducibility. 

Major comments 

1) Page 3: “To achieve this goal, we co-expressed and purified the crRNA-guided surveillance 
complex (Csy) with AcrIF9 and determined the structure […]” 
- Here one understands that AcrIF9 together with the crRNA-guided surveillance complex were co-
expressed. However, there is no paragraph related to this procedure in the Methods. Alternatively, 
if AcrIF9 and the Csy complex have been produced independently, as described in the Methods, 
and then mixed to make the complex, this procedure should be described in the Methods. 
This point should be clarified. 

2) Fig 1, panels d-e; page 4: “The two AcrIF9 binding sites are nearly identical. Residues 
interacting with each of the AcrIF9 molecules superimpose with an r.m.s.d. of 1.4 Å (see 
methods). The contacts include: a hydrogen bonding network involving AcrIF9 residues 11 to 15, a 
ß-sheet on the Cas7f thumb, and a nucleobase from the crRNA (Fig. 1c); interactions between 
AcrIF9 arginine 17 and residues in the Cas7f thumb (Fig. 1d); and the formation of an “aromatic 
clamp” around the Cas7f thumb loop by AcrIF9 residues 39 to 41 (Fig. 1d). AcrIF9.1, but not 
AcrIF9.2, also interacts with Cas8f (Fig 1e).” 
-This description of the intermolecular contacts between AcrIF9 and the Csy complex should be 
revised for a better reading and understanding. 
- The authors mention an “aromatic clamp” formed by AcrIF9 residues, which suggests 
hydrophobic or stacking interactions between AcrIF9 and Cas7f residues. However, panel d shows 
that these aromatic residues hydrogen bond with Cas7f. 
- The authors mention “interactions between AcrIF9 arginine 17 and residues in the Cas7f thumb”. 
The term “interactions” is vague. According to panel d, these interactions are hydrogen bonds, as 
are all intermolecular contacts described in this section. 
- The authors indicate that the two AcrIF9 binding sites are nearly identical. Is the only difference 
the interaction with Cas8f? This interaction between Cas8f and AcrIF9.1, shown as one hydrogen 
bond in panel e, should also be described in the text. 
- The numbering of all interacting residues and nucleobases should be reported in the text and the 
figure panels. 

3) Page 5: “Based on the similarity of the binding sites for AcrIF1 and AcrIF9, we predicted that 
AcrIF9 would sterically block target binding.” 
-The authors have previously shown that AcrIF1 interacts with Cas7f lysine residues (K85, K254, 
K257) that are involved in target DNA binding (http://dx.doi.org/10.1016/j.cell.2017.03.012). 
Based on Fig 1 panel d, the lysine 85, which is located in the thumb of Cas7f, may also interact 
with AcrIF9. Testing the binding of AcrIF9 to wild type Csy and to the K85A mutant used in the 
paper mentioned above, could experimentally prove that AcrIF9 is a steric inhibitor of target DNA 
binding. 
4) Page 6: Fig 2, panel A 
- In the EMSA with target dsDNA, the strong unbound DNA band in the 20 nM lane is puzzling. 
Indeed, it indicates that most of the Csy molecules are inhibited, which cannot be the case with a 
steric inhibitor at sub-stoichiometric concentration. The second EMSA presented in the 
Supplementary Fig 3 panel a would be more appropriate for the main figure. 
- In the EMSA with target and non-target dsDNAs, how do you interpret the DNA migration profiles 
of the 200 nM lanes? At 200 nM of AcrIF9, and with two AcrIF9 binding sites per Csy molecule, not 
all Csy molecules are fully occupied by the inhibitor. 
- Overall, how do you interpret these EMSA in terms of the AcrIF9 molecular mechanism? If a Csy-
mediated DNA cleavage assay in the presence of a stoichiometric ratio of AcrIF9 shows an 
inhibition of the Csy activity (which is not the case for AcrIIA11), what would be the benefit of 
binding dsDNA at high concentrations of AcrIF9? 

5) Page 5: “we next determined the structure of the Csy-AcrIF9 complex bound to dsDNA at a 
nominal resolution of ~6.9 Å (Fig. 2b and Supplementary Fig. 4).” 
- The procedure used to make the Csy-AcrIF9-dsDNA ternary complex is not described in the 
Methods. 

6) Page 5: “The reconstruction reveals two stretches of non-contiguous helical density that are not 
accounted for by Csy or AcrIF9, but are consistent with the size and shape of B-form DNA (Fig. 
2c).” 
-The EMSA with target and non-target dsDNAs show that non-specific DNA binding occurs at 400 
nM of AcrIF9, which is a concentration of AcrIF9 allowing the occupation of all AcrIF9 binding sites 
on the Csy complexes. This indicates that two AcrIF9 per Csy complex are required to bind non-
specific dsDNA. 
Is this compatible with the 3D reconstruction showing two non-contiguous dsDNAs bound to the 
Csy-AcrIF9 complex? 

7) Page 5: “The model reveals that the dsDNA segments are interacting with a positive patch on 
AcrIF9. […] Potential interactions are formed between dsDNA and the Csy complex (Fig. 2 c-d), 
however the resolution does not allow for reliable model building or structure-guided 
mutagenesis.” 
- The authors have showed that AcrIF9 alone does not bind to dsDNA. They have also identified 
potential interactions between the Csy complex and dsDNA in the 3D reconstruction of the ternary 
Csy-AcrIF9-dsDNA complex. These results indicate that AcrIF9 and Csy surfaces both contribute to 
the dsDNA binding interfaces. Even though the resolution of the Csy-AcrIF9-dsDNA 3D 
reconstruction does not allow the modeling of amino acid side chains, the rigid and flexible fitting 
of the Csy-AcrIF9 model within the Csy-AcrIF9-dsDNA 3D reconstruction may enable the mapping 
of Csy positively charged residues and the identification of a positive patch at the Csy-AcrIF9 
interfaces accommodating the dsDNAs. The authors have also identified some AcrIF9 basic 
residues that mediate DNA binding using the Csy-AcrIF9 model. 

8) Page 6: “Collectively, the structures and biochemistry presented here reveal a two-pronged 
mechanism for AcrIF9 mediated immune suppression.” 
- Based on the presented data, “suggest” would be more appropriate than “reveal”. 

9) Page 7: Data availability 
- The authors present a pseudo-atomic model of the AcrIF9-Csy-dsDNA complex in the Fig 2, but 
this model has not been deposited in the PDB. A Cα backbone model of this ternary complex 
should be made available. 

Minor comments 

1) References list 
- References 12 to 16 are not called in the text. 

2) Page 3: “[…] anti-parallel beta sheet cradling […]”, and page 4: “[…] a ß-sheet on […]” 
- Check the spelling (greek versus latine letters). 

3) Page 4: “[…] accounting for ~30% of the total solvent accessible surface area […]” 
- Is this the surface area of AcrIF9 or of the thumb of Cas7f? 

4) Page 4: “The two AcrIF9 binding sites are nearly identical.” 
- “Similar” instead of “nearly identical”. 

5) Page 4: “Residues interacting with each of the AcrIF9 molecules superimpose with an r.m.s.d. 
of 1.4 Å (see methods).” 
- Is this the rmsd for equivalent Cα atoms or all backbone atoms? 

6) Fig 1 
- Add in the legend what the yellow dotted lines represent.