Unveiling Cas8 Dynamics and Regulation within a transposon-encoded Cascade-TniQ Complex
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Abstract
Cascade is a class 1, type 1 CRISPR-Cas system with a variety of roles in prokaryote defense, specifically against DNA-based viruses. The Vibrio Cholerae transposon, Tn6677, encodes a variant of the type 1F Cascade known as type 1F-3. This Cascade variant complexes with a homodimer of the transposition protein TniQ and leverages the sequence specificity of Cascade to direct the integration activity of the heteromeric transposase tnsA/B, resulting in site-specific transposition of Tn6677. We desire to uncover the molecular details behind R Loop formation of ‘Cascade-TniQ.’ Due to the lack of a complete model of Cascade-TniQ available at atom-level resolution, we first build a complete model using AlphaFold V2.1. We then simulate this model via classical molecular dynamics and umbrella sampling to study an important regulatory component within Cascade-TniQ, known as the Cas8 ‘bundle.’ Particularly, we show that this alpha helical bundle experiences a free energy barrier to its large-scale translatory motions and relative free energies of its states primarily dependent on a loop within a Cas7 subunit in Cascade-TniQ. Further, we comment on additional structural and dynamical regulatory points of Cascade-TniQ during R Loop formation, such as Cascade-TniQ backbone rigidity, and the potential role TniQ plays in regulating bundle dynamics. In summary, our outcomes provide the first all-atom dynamic representation of one of the largest CRISPR systems, with information that can contribute to understanding the mechanism of nucleic acid binding and, eventually, to transposase recruitment itself. Such information may prove informative to advance genome engineering efforts.
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This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/13625755.
CRISPR-associated transposons (CASTs), such as Cascade-TNiQ, are transposon systems that have co-opted CRISPR-guided surveillance for transposition at specific locations in the genome. The precision and potential programmability of such systems has sparked interest in their practical application. However, the mechanism of R-loop formation (a crucial step in proper function) for Cascade-TNiQ remains unclear. Previously solved experimental structures of Cascade and Cascade-TNiQ have revealed different states of the complex, but what structural features mediate the transition between these states is unknown. In this paper, the authors use molecular dynamics simulations to pinpoint the …
This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/13625755.
CRISPR-associated transposons (CASTs), such as Cascade-TNiQ, are transposon systems that have co-opted CRISPR-guided surveillance for transposition at specific locations in the genome. The precision and potential programmability of such systems has sparked interest in their practical application. However, the mechanism of R-loop formation (a crucial step in proper function) for Cascade-TNiQ remains unclear. Previously solved experimental structures of Cascade and Cascade-TNiQ have revealed different states of the complex, but what structural features mediate the transition between these states is unknown. In this paper, the authors use molecular dynamics simulations to pinpoint the structural features of Cascade-TNiQ that allow it to bind to target DNA and form a complete R loop, and characterize the energetic barriers that could interfere with R loop formation. Our lab's expertise is not in molecular dynamics, but we were intrigued by this paper and attempted a journal club discussion on this. We ran into difficulties due to some of the questions outlined below. Furthermore, we believe the findings in this paper could be valuable to a general scientific audience interested in CASTs, and have some suggestions about how these ideas could be communicated more clearly:
Many details about model building are in the supplementary information section that is currently unavailable. Readers would benefit from a fuller explanation of such methods in the main text instead of the supplement, especially given the complexity of combining Alphafold and cryo-EM models to create the models used for MD simulations.
For example, we are curious how the secondary structure differences noted in the TniQ dimer varied across Alphafold predictions – perhaps this could have influenced the electrostatic interaction energies that were investigated before the discovery of the Cas7 gate.
It is unclear to us how the reaction coordinates are defined for the simulations in this paper, and whether different sets of reaction coordinates were tested to find the set that allowed the most thorough and efficient sampling.
For clarity it would be helpful for readers to have a diagram summarizing the relationships between the DNA- and RNA-HH and DNA- and RNA-J models and which sections of the models were predicted using Alphafold vs experimentally determined.
A summary cartoon/schematic illustrating the reaction progression/energy landscape of the transition from closed to open state, and how the findings of this paper contribute to our understanding of said landscape, would allow for easier understanding of all the findings in this paper.
Competing interests
The authors declare that they have no competing interests.
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