Structural basis for RNA-mediated assembly of type V CRISPR-associated transposons
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Abstract
CRISPR systems have been co-opted by Tn7-like elements to direct RNA-guided transposition. Type V-K CRISPR-associated transposons rely on the concerted activities of the pseudonuclease Cas12k, the AAA+ ATPase TnsC, the Zn-finger protein TniQ, and the transposase TnsB. Here we present a cryo-electron microscopic structure of a target DNA-bound Cas12k-transposon recruitment complex comprising RNA-guided Cas12k, TniQ, TnsC and, unexpectedly, the ribosomal protein S15. Complex assembly on target DNA results in complete R-loop formation mediated by critical interactions between TniQ and the trans-activating crRNA, and is coupled with TniQ-dependent nucleation of a TnsC filament. In vivo transposition assays corroborate our structural findings, and biochemical and functional analyses of S15 supports its role as a bona fide component of the type V crRNA-guided transposition machinery. Altogether, our work uncovers key aspects of the mechanisms underpinning RNA-mediated assembly of CRISPR-associated transposons that will guide their development as programmable site-specific gene insertion tools.
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Review coordinated via ASAPbio’s crowd preprint review
This review reflects comments and contributions by Ruchika Bajaj and Michael Robichaux. Review synthesized by Michael Robichaux.
The manuscript presents a cryo-electron microscopy focused study of a recombinant type V-K CRISPR-associated Cas12k transposon recruitment complex from Scytonema hofmanni that is DNA-bound and includes a complete R-loop formation. In addition to mapping the assembly and interactions within this transposon complex, the study also details the discovery of ribosomal protein S15 as an essential component for the transposition activity of the complex. The work presented in this manuscript may contribute to the development of new programmable CRISPR-associated genome-engineering tools in eukaryotic cells.
Major comments
- The figures in the manuscript are …
Review coordinated via ASAPbio’s crowd preprint review
This review reflects comments and contributions by Ruchika Bajaj and Michael Robichaux. Review synthesized by Michael Robichaux.
The manuscript presents a cryo-electron microscopy focused study of a recombinant type V-K CRISPR-associated Cas12k transposon recruitment complex from Scytonema hofmanni that is DNA-bound and includes a complete R-loop formation. In addition to mapping the assembly and interactions within this transposon complex, the study also details the discovery of ribosomal protein S15 as an essential component for the transposition activity of the complex. The work presented in this manuscript may contribute to the development of new programmable CRISPR-associated genome-engineering tools in eukaryotic cells.
Major comments
- The figures in the manuscript are generally well-organized and clear. In particular, the 2D diagram of the Cas12k-TnsC complex in Figure 1A is a useful figure panel; however, please consider refining the diagram for readability by replacing the current nucleotide sequence rearrangement with simpler shapes or graphics.
- For the structural complex models in Figure 2, please consider adding annotations that highlight both the completed R-loop as well as the 122॰ angled confirmation of the PAM distal to proximal DNA, which are both features that are highlighted in the Results section text.
- The title for the “TniQ nucleates TnsC filament formation” Results section and the title for Figure 4 are both possibly overstated since these mechanistic conclusions are based solely on transposition assay results.
- In the discussion, please consider revising the language used to describe the mechanism of transposon complex assembly (the model in Figure 7) to better justify a rationale for proposing a “cooperative” assembly mechanism that is based on the data in this manuscript, which is a structural assessment of the whole complex and its sub-complex interactions.
Minor comments
- In the first section of Results section, consider adding a description of the recombinant system used to purify the protein complex used for cryo-EM as done for the Figure 1 legend (“V-K CRISPR-associated transposon system from Scytonema hofmanni (Strecker et al., 2019)”).
- For Figure S1B, the orientation map is not clear, an adjustment to the color contrast may improve the clarity of this panel.
- For the cryo-EM data in Figures S2, please better define the TnsC oligomer organization (i.e., hexameric, variable). Also for Figure S2, please consider improving the image contrast for the angular distribution images in panel B.
- For Figure S3, both the incomplete R-loop and the missing Cas12k-sgRNA + TsnC contacts described in the text for this non-productive complex structure are not evident or identifiable in the models presented in the figure. Please consider annotations or descriptions in the figure legend.
- For Figure S4, please consider defining all rotations and dispositions that make up the conformational rearrangements in the RuvC domain, as described in the Results section text.
- For Figure 2, please consider adding a 2D diagram of the current complex structure in comparison to previously-reported structural models.
- The organization of Figure 3 is too busy, please consider re-formatting for clarity.
- For Figure S8, please consider including a “zoomed-out” image of the Cas12k+S15 structure.
- In the concluding paragraph of the Discussion section, please elaborate more on how the findings from this work may impact the “genome engineering application of CRISPR-associated transposons”.
Comments on reporting
- As outlined in Figure S1, 75K particles were used for the final cryo-EM reconstruction of the Cas12k-TsnC recruitment complex. Please consider discussing the structural elements or discrepancies of the other classified particles.
- Table S2 and S3 appear to be missing.
- In the “TniQ recognizes tracrRNA and R-loop” Results section, please specify which TniQ and tracrRNA mutations reduced transposition activity.
Suggestions for future studies
Please consider future studies that address the relevance of this transposon complex structure to physiological processes via cell-based assays.
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