Structural basis for Cas9 off-target activity

Lin, Chi‐Hui; Cléry, Antoine; Saha, Aakash; Arantes, Pablo; Bargsten, Katja; Irby, Matthew J.; Allain, Frédéric; Palermo, Giulia; Cameron, P. Simmons; Donohoue, Paul D.; Jinek, Martin

doi:10.1016/j.cell.2022.09.026

Cited by 62 publications

(61 citation statements)

References 95 publications

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“…Depending on their position and nature, DNA mismatches can induce an opening of the RNA:DNA hybrid, and lock the catalytic HNH domain in an inactive state (Ricci et al, 2019;Mitchell et al, 2020). These findings were corroborated by X-ray structures of Cas9 bound to off-target substrates, providing a structural rationale for the off-target activity of Cas9 and contributing to the design of guide RNAs and off-target prediction algorithms (Pacesa et al, 2021).…”

Section: Domain Rotation Mechanisms As the Means For Allosteric Regul...mentioning

confidence: 75%

Twisting and swiveling domain motions in Cas9 to recognize target DNA duplexes, make double-strand breaks, and release cleaved duplexes

Wang

Arantes

Ahsan

et al. 2023

Front. Mol. Biosci.

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The CRISPR-associated protein 9 (Cas9) has been engineered as a precise gene editing tool to make double-strand breaks. CRISPR-associated protein 9 binds the folded guide RNA (gRNA) that serves as a binding scaffold to guide it to the target DNA duplex via a RecA-like strand-displacement mechanism but without ATP binding or hydrolysis. The target search begins with the protospacer adjacent motif or PAM-interacting domain, recognizing it at the major groove of the duplex and melting its downstream duplex where an RNA-DNA heteroduplex is formed at nanomolar affinity. The rate-limiting step is the formation of an R-loop structure where the HNH domain inserts between the target heteroduplex and the displaced non-target DNA strand. Once the R-loop structure is formed, the non-target strand is rapidly cleaved by RuvC and ejected from the active site. This event is immediately followed by cleavage of the target DNA strand by the HNH domain and product release. Within CRISPR-associated protein 9, the HNH domain is inserted into the RuvC domain near the RuvC active site via two linker loops that provide allosteric communication between the two active sites. Due to the high flexibility of these loops and active sites, biophysical techniques have been instrumental in characterizing the dynamics and mechanism of the CRISPR-associated protein 9 nucleases, aiding structural studies in the visualization of the complete active sites and relevant linker structures. Here, we review biochemical, structural, and biophysical studies on the underlying mechanism with emphasis on how CRISPR-associated protein 9 selects the target DNA duplex and rejects non-target sequences.

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Section: Domain Rotation Mechanisms As the Means For Allosteric Regul...mentioning

confidence: 75%

Twisting and swiveling domain motions in Cas9 to recognize target DNA duplexes, make double-strand breaks, and release cleaved duplexes

Wang

Arantes

Ahsan

et al. 2023

Front. Mol. Biosci.

Self Cite

View full text Add to dashboard Cite

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“…The reliability of the simulations depends on adequate sampling, convergence, force-field accuracy, and the quality of the experimentally characterized structures. The recent structural studies by Pacesa et al 124,125 have been an added boon to the experimental and computational researchers to obtain a deeper understanding of the molecular mechanism of important events in Cas9 such as off-target effects and catalytic activation. A series of cryo-EM structures captured different stages of R-loop formation and highlighted various checkpoint structures.…”

Section: Conclusion and Future Prospectsmentioning

confidence: 99%

“… 125 These structures give significant insights into the off-target mechanism and provide an excellent template for future computational analysis. 125 …”

Section: Conclusion and Future Prospectsmentioning

confidence: 99%

“…Another recent breakthrough in the structural study provided 19 crystal structures depicting different types of off-target effects in Cas9 genome editing. 125 These structures give significant insights into the off-target mechanism and provide an excellent template for future computational analysis. 125 Future structural characterization (X-ray, cryo-EM, etc.)…”

Section: Conclusion and Future Prospectsmentioning

confidence: 99%

“…125 These structures give significant insights into the off-target mechanism and provide an excellent template for future computational analysis. 125 Future structural characterization (X-ray, cryo-EM, etc.) and conformational dynamics (single-molecule fluorescence resonance energy transfer measurements, nuclear magnetic Resonance) of various uncharacterized Cas9 mutants at various stages of editing pathways (apo, nucleotide-bound, pre-or postcatalytic states, etc.…”

Section: Conclusion and Future Prospectsmentioning

confidence: 99%

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Insights into the Mechanism of CRISPR/Cas9-Based Genome Editing from Molecular Dynamics Simulations

Bhattacharya

Satpati

2022

ACS Omega

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The CRISPR/Cas9 system is a popular genome-editing tool with immense therapeutic potential. It is a simple two-component system (Cas9 protein and RNA) that recognizes the DNA sequence on the basis of RNA:DNA complementarity, and the Cas9 protein catalyzes the double-stranded break in the DNA. In the past decade, near-atomic resolution structures at various stages of the CRISPR/Cas9 DNA editing pathway have been reported along with numerous experimental and computational studies. Such studies have boosted knowledge of the genome-editing mechanism. Despite such advancements, the application of CRISPR/Cas9 in therapeutics is still limited, primarily due to off-target effects. Several studies aim at engineering high-fidelity Cas9 to minimize the off-target effects. Molecular Dynamics (MD) simulations have been an excellent complement to the experimental studies for investigating the mechanism of CRISPR/Cas9 editing in terms of structure, thermodynamics, and kinetics. MD-based studies have uncovered several important molecular aspects of Cas9, such as nucleotide binding, catalytic mechanism, and off-target effects. In this Review, the contribution of MD simulation to understand the CRISPR/Cas9 mechanism has been discussed, preceded by an overview of the history, mechanism, and structural aspects of the CRISPR/Cas9 system. These studies are important for the rational design of highly specific Cas9 and will also be extremely promising for achieving more accurate genome editing in the future.

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Genome Editing Tool CRISPR-Cas: Legal and Ethical Considerations for Life Science

Pandey,

Arora,

Kumar

2024

Gene Editing in Plants

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Structural basis for Cas9 off-target activity

Cited by 62 publications

References 95 publications

Twisting and swiveling domain motions in Cas9 to recognize target DNA duplexes, make double-strand breaks, and release cleaved duplexes

Twisting and swiveling domain motions in Cas9 to recognize target DNA duplexes, make double-strand breaks, and release cleaved duplexes

Insights into the Mechanism of CRISPR/Cas9-Based Genome Editing from Molecular Dynamics Simulations

Genome Editing Tool CRISPR-Cas: Legal and Ethical Considerations for Life Science

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