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We, the students of MICI5029/5049, a Graduate Level Molecular Pathogenesis Journal Club at Dalhousie University in Halifax, NS, Canada, hereby submit a review of the following BioRxiv preprint:

The novel anti-CRISPR AcrIIA22 relieves DNA torsion in target plasmids and impairs SpyCas9 activity.

Kevin J. Forsberg, Danica T. Schmidtke, Rachel Werther, Ruben V. Uribe, Deanna Hausman, Morten O. A. Sommer, Barry L. Stoddard, Brett K. Kaiser, Harmit S. Malik bioRxiv 2021.09.28.317578; doi: https://www.biorxiv.org/content/10.1101/2020.09.28.317578v2

We will adhere to the Universal Principled (UP) Review guidelines proposed in:

Universal Principled Review: A Community-Driven Method to Improve Peer Review. Krummel M, Blish C, Kuhns M, Cadwell K, Oberst A, Goldrath A, Ansel KM, Chi H, O'Connell R, Wherry EJ, Pepper M; Future Immunology Consortium. Cell. 2019 Dec 12;179(7):1441-1445. doi: 10.1016/j.cell.2019.11.029.

SUMMARY: A functional screen for SpyCas9 antagonism led to identification of AcrIIA22 as an anti-CRISPR protein. AcrIIA22 antagonizes SpyCas9 through a unique mechanism - instead of binding to the Cas protein, AcrIIA22 ORF1 functions as a nickase that cleaves supercoiled plasmids. This cleavage causes a release of the torsional stress held by a supercoiled plasmid, which could render these plasmids less susceptible to SpyCas9 targeting. In a trial using phage Mu, AcrIIA22 partially protected against SpyCas9-mediated restriction, in addition to completely protecting plasmids from SpyCas9 targeting and cleavage. AcrIIA22 has no known sequence homology to proteins with known functions, but AcrIIA22 homologs were identified in prophage genomes and small hypervariable regions in bacterial genomes. AcrIIA22 homologs were located near the end of prophage genomes, close to the junction with host genomes, providing insight into gene ancestry. There was evidence that AcrIIA22 promotes recombination in hypervariable bacterial “genomic islands” in the absence of other gene products that typically induce recombination. Most AcrIIA22 homologs were found in bacteria in the CAG-217 genus, and upon further analysis these homologs also inhibited plasmid targeting by Cas9 systems. Thorough analysis revealed that AcrIIA22 did not function via previously described anti-CRISPR mechanisms. The crystal structure of AcrIIA22 was solved, which revealed structural similarity to PC4-like proteins. AcrIIA22 oligomerization was necessary for protection against SpyCas9. Like PC4-like proteins, AcrIIA22 altered DNA topology through the observation of a change in a target plasmid’s migrating form during gel electrophoresis from supercoiled to open-circle. Further in vitro analysis of purified AcrIIA22 protein revealed that AcrIIA22 nicks supercoiled plasmids to protect them from SpyCas9 activity. This function could be inhibited via the mutagenesis of key amino acid D14 to alanine, consistent with observations of a AcrIIA22 homolog with diminished nicking activity (AcrIIA22a).  

OVERALL ASSESSMENT: This pre-print (now published in PLOS Biology) describes a novel ACR mechanism and represents a significant advance in the field. Data was clearly presented in figures and text and the writing was engaging and carried the reader along. Authors used appropriate methodology and data analysis and overall data quality was viewed as a strength. Final conclusions about phage protection by this novel ACR protein could have been more strongly supported by studies of additional phage that feature a supercoiled genome topology that could be a good substrate for AcrIIA22. 

STRENGTHS:

- Clearly presented data, easy to understand. 

- Strong writing, accessible to non-expert reader. 

- Identification of a novel ACR mechanism that is not so easily defeated by rapid microbial evolution. 

WEAKNESSES:   

-  Conclusions about phage protection by this novel ACR protein would have been more strongly supported by studies of additional phage with supercoiled genomes.

-  The Discussion could benefit from some speculation about mechanisms of plasmid nicking. Is there a specific sequence that AcrIIA22 recognizes on the target DNA, or is the nicking carried out with no discrimination other than the target must be supercoiled? Does AcrIIA22 cause single-stranded breaks or are double-strand breaks possible? If so, what is the consequence for the phage/plasmid?  

DETAILED U.P. ASSESSMENT:

OBJECTIVE CRITERIA (QUALITY)

1.  Quality: Experiments (1–3 scale) SCORE = 1 (by the way, 1 is high quality; 3 is low quality)

● Figure by figure, do experiments, as performed, have the proper controls? [note: we use this ‘figure-by-figure' section for broader detailed critiques, rather than only focusing on controls.]

Figure 1: Data proving SpyCas9 expression is not affected by ORF_1 is important enough to move from the Supplement into Figure 1. Minor point: Graph axis labels need to be kept consistent. 

Figure 2: Good. We thought that the graphical representation of the data aided understanding of the conclusions. 

Figure 3: Good. We appreciated the data showing the ability of AcrIIA22 to inhibit other Cas9 systems and the location of the AcrIIA22 homologs in the CAG-217 genus. 

Figure 4: Good. We valued the visualization of the structure of AcrIIA22 and how that revealed the similarities to PC4-like proteins. 

Figure 5: Good. One reader had a minor comment about the differences between the 0.6 uM and 0.3 uM treatment groups, as there appears to be some variation in the starting plasmids compared to the other groups. 

Figure 6: Good. We noticed that in 6C there was still a slight shift of the plasmid into the OC state in the D14A mutant samples. We were curious if there was any speculation as to what may have caused this shift. An additional minor note is that some readers commented that the subtle greyscale colour scheme made the interpretation of the data more challenging. 

Figure 7: Good. We thought that the graphical data representation aided our understanding of the results. Additionally, we thought the data clearly outlined that SpyCas9 is more likely to target DNA that hasn’t already been nicked by AcrIIA22. 

● Are specific analyses performed using methods that are consistent with answering the specific question?

We thought the experimental approaches were sound and were used appropriately to address research questions. 

● Is there appropriate technical expertise in the collection and analysis of data presented?

Yes, appropriate technical expertise was demonstrated. 

● Do analyses use the best-possible (most unambiguous) available methods quantified via appropriate statistical comparisons?

We had no issues with the statistics used throughout this paper. 

● Are controls or experimental foundations consistent with established findings in the field? A review that raises concerns regarding inconsistency with widely reproduced observations should list at least two examples in the literature of such results. Addressing this question may occasionally require a supplemental figure that, for example, re-graphs multi-axis data from the primary figure using established axes or gating strategies to demonstrate how results in this paper line up with established understandings. It should not be necessary to defend exactly why these may be different from established truths, although doing so may increase the impact of the study and discussion of discrepancies is an important aspect of scholarship.

While this paper was an extension of a previous functional screen – we thought that there was sufficient literature cited throughout the paper, and there was enough support and evidence on the comparison between PC4-like proteins and AcrIIA22 function to justify the authors’ conclusions. 

2. Quality: Completeness (1–3 scale) SCORE = 1.5

● Does the collection of experiments and associated analysis of data support the proposed title- and abstract-level conclusions? Typically, the major (title- or abstract-level) conclusions are expected to be supported by at least two experimental systems.

We thought the data supported the abstract-level conclusions. It was clear that AcrIIA22 is able to relieve DNA torsion (changing topology from supercoiled to open circle) and this can impair SpyCas9 targeting against plasmids.  

● Are there experiments or analyses that have not been performed but if ‘‘true’’ would disprove the conclusion (sometimes considered a fatal flaw in the study)? In some cases, a reviewer may propose an alternative conclusion and abstract that is clearly defensible with the experiments as presented, and one solution to ‘‘completeness’’ here should always be to temper an abstract or remove a conclusion and to discuss this alternative in the discussion section.

While this research is complete, we thought that it would have been supported more strongly by determining whether AcrIIA22 could protect other phage that feature supercoiled viral DNA as part of their replication cycle. We also thought that the study would benefit from an exploration of the nicking mechanism. 

3. Quality: Reproducibility (1–3 scale) SCORE = 

● Figure by figure, were experiments repeated per a standard of 3 repeats or 5 mice per cohort, etc.?

Yes, there were sufficient replicates and experimental repeats. 

● Is there sufficient raw data presented to assess rigor of the analysis?

Yes, the supplemental information displayed good support to their figures. We especially appreciated the analytical gels and activity confirmation for the protein purification steps.

Are methods for experimentation and analysis adequately outlined to permit reproducibility?

   Yes, there were no issues with how the methods were described. 

● If a ‘‘discovery’’ dataset is used, has a ‘‘validation’’ cohort been assessed and/or has the issue of false discovery been addressed?

Use of the 2-plasmid kanamycin selection screen was used in a previous research project to identify AcrIIA22 as an antagonist for SpyCas9. This was further confirmed in this research through identification of ORF1 encoding the anti-CRISPR protein that functions through a unique mechanism to previously known ACRs. 

4. Quality: Scholarship (1–4 scale but generally not the basis for acceptance or rejection) SCORE = 

● Has the author cited and discussed the merits of the relevant data that would argue against their conclusion?

Yes, the discussion was thorough and provided the proper context for the research. There was good speculation and discussion of alternative models that were not addressed in the dataset that could be used for future studies.

● Has the author cited and/or discussed the important works that are consistent with their conclusion and that a reader should be especially familiar when considering the work?

The citations were thorough and included many relevant research papers. We appreciated the use of citations for many of the minor points in the Introduction, as they allowed the reader to further investigate any area to gain a fuller understanding. 

● Specific (helpful) comments on grammar, diction, paper structure, or data presentation (e.g., change a graph style or color scheme) go in this section, but scores in this area should not to be significant bases for decisions.

We appreciated the well-written and concise nature of the introduction. We also thought that the Introduction would have benefited from additional details on specific examples of other ACR’s and their mechanisms of action (are there other Acrs that have mechanisms besides Cas binding?). Additionally, adding in a biochemical section discussing protein structure and how it relates to suspected functions, like nicking, would have also aided understanding. 

MORE SUBJECTIVE CRITERIA (IMPACT)

1.Impact: Novelty/Fundamental and Broad Interest (1–4 scale) SCORE = 

● A score here should be accompanied by a statement delineating the most interesting and/or important conceptual finding(s), as they stand right now with the current scope of the paper. A ‘‘1’’ would be expected to be understood for the importance by a layperson but would also be of top interest (have lasting impact) on the field.

The work presented in this paper was nicely put into the proper context of the field in terms of describing the known CRISPR-Cas evasion mechanisms and the novel understanding of Acrs and their functions. The screening approach and the methodology of determining the exact antagonism mechanism were easy to understand. AcrIIA22’s evasion mechanism of relieving supercoiling topology was very well described. One of the most significant points that we discussed was that the research here shows that there are a wide variety of mechanisms of CRISPR immunity evasion that have yet to be discovered. 

● How big of an advance would you consider the findings to be if fully supported but not extended? It would be appropriate to cite literature to provide context for evaluating the advance. However, great care must be taken to avoid exaggerating what is known comparing these findings to the current dogma (see Box 2). Citations (figure by figure) are essential here.

The research shows the strong inhibition of CRISPR-Cas targeting of supercoiled plasmids, and then the less potent protection of the phage Mu by AcrIIA22. While there is a well-reasoned argument for the advantages of this moderate CRISPR-Cas inhibition included in the Discussion, we thought that extending the analysis to other phage infection systems would strengthen this point.  The point was made that the life cycles of native CAG-217 phages are not well understood at this time, and that there are many dsDNA phage genomes which undergo circular topologically restrained stages of their replication cycle, but the implication is that these native phages are better target mobile genetic elements for AcrIIA22 and including those in a protection assay may add considerable strength to the argument for the anti-CRISPR function of AcrIIA22. 

2.Impact: Extensibility (1–4 or N/A scale) SCORE = 

● Has an initial result (e.g., of a paradigm in a cell line) been extended to be shown (or implicated) to be important in a bigger scheme (e.g., in animals or in a human cohort)?

● This criterion is only valuable as a scoring parameter if it is present, indicated by the N/A option if it simply doesn’t apply. The extent to which this is necessary for a result to be considered of value is important. It should be explicitly discussed by a reviewer why it would be required. What work (scope and expected time) and/or discussion would improve this score, and what would this improvement add to the conclusions of the study? Care should be taken to avoid casually suggesting experiments of great cost (e.g., ‘‘repeat a mouse-based experiment in humans’’) and difficulty that merely confirm but do not extend (see Bad Behaviors, Box 2).

N/A, but we are very excited and interested to hear if there will be additional studies to gauge the effects of AcrIIA22 on defense against other phage infections – particularly if there was a test to examine a phage that has an important supercoiled form during some part of its lifecycle.