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

Forsberg, Kevin J.; Schmidtke, Danica T.; Werther, Rachel; Hausman, Deanna; Stoddard, Barry; Kaiser, Brett K.; Malik, Harmit S.

doi:10.1101/2020.09.28.317578

Cited by 5 publications

(2 citation statements)

References 99 publications

(85 reference statements)

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“…The strongly reduced target recognition efficiency in absence of supercoiling suggests that CRISPR-Cas systems have evolutionary been optimized to support efficient recognition of invaders with supercoiled DNA. This is supported by the discovery of a novel anti-CRISPR protein, which nicks plasmid DNA to release supercoils in order to strongly reduce the targeting efficiency of CRISPR-Cas9 55 as well as the torque-dependent post-cleavage behavior of Cas9 itself 56 . The genomes of eukaryotic cells are however considerably less supercoiled than prokaryotic cells.…”

Section: Discussionmentioning

confidence: 99%

Dynamic interplay between target search and recognition for the Cascade surveillance complex of type I-E CRISPR-Cas systems

Aldag

Rutkauskas

Madariaga-Marcos

et al. 2022

Preprint

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CRISPR-Cas effector complexes enable the defense against foreign nucleic acids and have recently been exploited as molecular tools for precise genome editing at a target locus. To bind and cleave their target, the CRISPR-Cas effectors first have to interrogate the entire genome for the presence of a matching sequence. Matching is achieved by base-pairing between the crRNA of the complexes and the DNA target strand such that an R-loop is formed. R-loop formation starts at a specific PAM motif and progresses reversibly in single base-pair steps until mismatches stop further progression or until the full target is recognized and destroyed. The reversible nature of this process entails that even a fully matching target should only become recognized with a low probability per target encounter. The details of this process, which directly affect the effectiveness of the target search, remain unresolved. Here we dissect the target search process of the Type I CRISPR-Cas complex Cascade by simultaneously monitoring DNA binding and R-loop formation by the complex. We directly quantify the low target recognition probabilities and show that they increase with increasing negative supercoiling. Furthermore, we demonstrate that Cascade uses a combination of three-dimensional and limited one-dimensional diffusion along the DNA contour for its target search. The latter allows for rapidly scanning the PAM sequences in a given region and, importantly, significantly increasing the overall efficiency of the target search by repeatedly revisiting the sites. Overall we show that target search and target recognition are tightly linked and that DNA supercoiling and limited 1D diffusion need to be considered when understanding target recognition and target search by CRISPR-Cas enzymes and engineering more efficient and precise variants.

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Section: Discussionmentioning

confidence: 99%

Dynamic interplay between target search and recognition for the Cascade surveillance complex of type I-E CRISPR-Cas systems

Aldag

Rutkauskas

Madariaga-Marcos

et al. 2022

Preprint

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“…and 5 Acr proteins against the subtype II-C CRISPR-Cas system. [18][19][20] Structural and mechanistic studies revealed that type II Acrs inhibit their host Cas9 proteins through diverse mechanisms. They occupy the protospacer adjacent motif interaction site of Cas9 to compete with target DNA binding, 21,22 block the active site of the Cas9 nuclease domain, 23 induce nonfunctional Cas9 dimerization, 24,25 and cause Cas9 degradation in cell culture.…”

Section: Self-association and Interaction Of Acriic2mentioning

confidence: 99%

Structural Investigation of Self-Assembly and Target Binding of Anti-CRISPR AcrIIC2

et al. 2021

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Anti-CRISPR (Acr) proteins are phage-borne inhibitors of the CRISPR-Cas immune system in archaea and bacteria. AcrIIC2 from prophages of Neisseria meningitidis disables the nuclease activity of type II-C Cas9, such that dimeric AcrIIC2 associates with the bridge helix (BH) region of Cas9 to compete with guide RNA loading. AcrIIC2 in solution readily assembles into oligomers of variable lengths, but the oligomeric states are not clearly understood. In this study, we investigated the dynamic assembly of AcrIIC2 oligomers, and identified key interactions underlying the self-association. We report that AcrIIC2 dimers associate into heterogeneous high-order oligomers with the equilibrium dissociation constant K D *8 lM. Oligomerization is driven by electrostatic interactions between charged residues, and rational mutagenesis produces a stable AcrIIC2 dimer with intact Cas9 binding. Remarkably, the BH peptide of Cas9 is unstructured in solution, and undergoes a coil-to-helix transition upon AcrIIC2 binding, revealing a unique folding-upon-binding mechanism for Acr recognition.

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Anti-CRISPR proteins: a weapon of phage-bacterial arm race for genome editing

Jaiswal,

Singh,

Kumar

et al. 2023

Nucleus

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

Cited by 5 publications

References 99 publications

Dynamic interplay between target search and recognition for the Cascade surveillance complex of type I-E CRISPR-Cas systems

Dynamic interplay between target search and recognition for the Cascade surveillance complex of type I-E CRISPR-Cas systems

Structural Investigation of Self-Assembly and Target Binding of Anti-CRISPR AcrIIC2

Anti-CRISPR proteins: a weapon of phage-bacterial arm race for genome editing

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