What question did this study set out to answer?

To determine the molecular mechanism by which the phage-derived inhibitor AcrIIA4 disables CRISPR-Cas9 and evaluate its utility in regulating gene editing in human cells.

July 7, 2017Science AdvancesOpen Access

Disabling Cas9 by an anti-CRISPR DNA mimic

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Authors

JSJiyung ShinETH Zurich FJFuguo JiangNational Institute of Clean and Low-Carbon Energy JLJunjie LiuTsinghua University

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Implication

Structural analysis reveals that anti-CRISPR protein AcrIIA4 mimics DNA to block Cas9 recognition in human cells, suggesting a strategy to reduce off-target gene editing.

Key Points

To determine the molecular mechanism by which the phage-derived inhibitor AcrIIA4 disables CRISPR-Cas9 and evaluate its utility in regulating gene editing in human cells.
Determined the 3.9 Å resolution cryo-electron microscopy structure of the Cas9-sgRNA-AcrIIA4 complex.
Performed biochemical order-of-addition binding assays to identify how AcrIIA4 interacts with Cas9-sgRNA and target DNA.
Delivered AcrIIA4 as a purified protein or plasmid vector into human cells with timed administration to measure on-target and off-target Cas9 editing activity.
AcrIIA4 binds with a 1:1 stoichiometry exclusively to assembled Cas9-sgRNA complexes by using an acidic surface to mimic DNA and occupy the protospacer adjacent motif site.
AcrIIA4 prevents target DNA binding when added prior to DNA recognition, but does not disassemble preformed Cas9-sgRNA-DNA complexes.
Timed delivery of AcrIIA4 into human cells maintains on-target Cas9 genome cleavage while substantially suppressing off-target mutations.

Cite This Study

Shin et al. (2017) studied this question.

synapsesocial.com/papers/69fd98197f24f003e827142e https://doi.org/10.1126/sciadv.1701620

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Implication

Structural analysis reveals that anti-CRISPR protein AcrIIA4 mimics DNA to block Cas9 recognition in human cells, suggesting a strategy to reduce off-target gene editing.

Disabling Cas9 by an anti-CRISPR DNA mimic

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Implication

Key Points

Cite This Study

Disabling Cas9 by an anti-CRISPR DNA mimic

Discussion

Member takes

Implication

Key Points

Cite This Study

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