Dynamics and mechanisms of CRISPR-Cas9 through the lens of computational methods

Saha, Aakash; Arantes, Pablo Ricardo; Palermo, Giulia

doi:10.1016/j.sbi.2022.102400

Cited by 14 publications

(21 citation statements)

References 44 publications

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“…Therefore, further structural investigations are needed to clarify such detailed molecular mechanisms. Indeed, using our full-length model it is also possible to carry out long time scale MD simulations to investigate the dynamic process of SaCas9 from the inactive state to the active state, as in previous studies on SpCas9 [ 30 ].…”

Section: Discussionmentioning

confidence: 99%

Full-Length Model of SaCas9-sgRNA-DNA Complex in Cleavage State

Zhu

Qian

et al. 2023

IJMS

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Staphylococcus aureus Cas9 (SaCas9) is a widely used genome editing tool. Understanding its molecular mechanisms of DNA cleavage could effectively guide the engineering optimization of this system. Here, we determined the first cryo-electron microscopy structure of the SaCas9-sgRNA-DNA ternary complex. This structure reveals that the HNH nuclease domain is tightly bound to the cleavage site of the target DNA strand, and is in close contact with the WED and REC domains. Moreover, it captures the complete structure of the sgRNA, including the previously unresolved stem-loop 2. Based on this structure, we build a full-length model for the ternary complex in cleavage state. This model enables identification of the residues for the interactions between the HNH domain and the WED and REC domains. Moreover, we found that the stem-loop 2 of the sgRNA tightly binds to the PI and RuvC domains and may also regulate the position shift of the RuvC domain. Further mutagenesis and molecular dynamics simulations supported the idea that the interactions of the HNH domain with the WED and REC domains play an important role in the DNA cleavage. Thus, this study provides new mechanistic insights into the DNA cleavage of SaCas9 and is also useful for guiding the future engineering of SaCas9-mediated gene editing systems.

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Section: Discussionmentioning

confidence: 99%

Full-Length Model of SaCas9-sgRNA-DNA Complex in Cleavage State

Zhu

Qian

et al. 2023

IJMS

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“…34,35 The TIP3P model was employed for water, 36 and the Li & Merz model was used for Mg 2+ ions. 37 We have extensively employed these force field models in computational studies of CRISPR-Cas systems, 38 showing that they perform well for long timescale simulations on Anton-2. 39 The Li & Merz model also reported a good description of Mg 2+ bound sites, in agreement with quantum/classical simulations.…”

Section: Methodsmentioning

confidence: 99%

An Alpha-helical Lid Guides the Target DNA toward Catalysis in CRISPR-Cas12a

Saha

Ahsan

Arantes

et al. 2022

Preprint

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CRISPR-Cas12a is a powerful RNA-guided genome-editing system, also emerging as a robust diagnostic tool that cleaves double-stranded DNA using only the RuvC domain. This opens an overarching question on how the spatially distant DNA target strand (TS) traverses toward the RuvC catalytic core. Here, continuous tens of microsecond-long molecular dynamics and free- energy simulations reveal that an ⍺-helical lid, located within the RuvC domain, plays a pivotal role in the traversal of the TS by anchoring the crRNA:TS hybrid and elegantly guiding the TS toward the RuvC core, as also corroborated by DNA cleavage experiments. In this mechanism, the REC2 domain pushes the crRNA:TS hybrid toward the core of the enzyme, while the Nuc domain aids the bending and accommodation of the TS within the RuvC core by bending inward. Understanding of this cardinal process in the functioning of Cas12a will enrich fundamental knowledge and facilitate further engineering strategies for genome-editing.

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“…The recently discovered CRISPR-Cas technology is being extensively used for facile genome editing and offers tremendous promise for the treatment of genetic disorders, to development of diagnostic tools, improve genetically modified crops, and a myriad of innovations across life sciences. − In parallel to the crucial contributions from structural and biochemical studies, molecular simulations contributed to understanding the large-scale dynamics and the local fluctuations driving the biophysical function of gene editing complexes. Here, we briefly review the role of state-of-the-art computations in the CRISPR-Cas revolution. , …”

Section: Gene Editing Systemsmentioning

confidence: 99%

“…Here, we briefly review the role of state-of-the-art computations in the CRISPR-Cas revolution. 136,137 The CRISPR-Cas9 genome editing tool is based on the endonuclease enzyme Cas9, which associates with a CRISPR RNA (crRNA), used as a guide for matching DNA sequences. The recognition of a short protospacer adjacent motif (PAM: a specific nucleotide sequence adjacent to the target DNA site) is the primary step for the activation of the Cas9 protein.…”

Section: ■ Gene Editing Systemsmentioning

confidence: 99%

Machines on Genes through the Computational Microscope

Sinha

Pindi

Ahsan

et al. 2023

J. Chem. Theory Comput.

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Macromolecular machines acting on genes are at the core of life’s fundamental processes, including DNA replication and repair, gene transcription and regulation, chromatin packaging, RNA splicing, and genome editing. Here, we report the increasing role of computational biophysics in characterizing the mechanisms of “machines on genes”, focusing on innovative applications of computational methods and their integration with structural and biophysical experiments. We showcase how state-of-the-art computational methods, including classical and ab initio molecular dynamics to enhanced sampling techniques, and coarse-grained approaches are used for understanding and exploring gene machines for real-world applications. As this review unfolds, advanced computational methods describe the biophysical function that is unseen through experimental techniques, accomplishing the power of the “computational microscope”, an expression coined by Klaus Schulten to highlight the extraordinary capability of computer simulations. Pushing the frontiers of computational biophysics toward a pragmatic representation of large multimegadalton biomolecular complexes is instrumental in bridging the gap between experimentally obtained macroscopic observables and the molecular principles playing at the microscopic level. This understanding will help harness molecular machines for medical, pharmaceutical, and biotechnological purposes.

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Dynamics and mechanisms of CRISPR-Cas9 through the lens of computational methods

Cited by 14 publications

References 44 publications

Full-Length Model of SaCas9-sgRNA-DNA Complex in Cleavage State

Full-Length Model of SaCas9-sgRNA-DNA Complex in Cleavage State

An Alpha-helical Lid Guides the Target DNA toward Catalysis in CRISPR-Cas12a

Machines on Genes through the Computational Microscope

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