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Updated: May 27, 2025
Substrate Generation for Endonucleases of CRISPR/Cas Systems
Published on: September 8, 2012
AcrVIB1 inhibits CRISPR-Cas13b immunity by promoting unproductive crRNA binding accessible to RNase attack
Katharina G Wandera1, Stefan Schmelz2, Angela Migur1
1Helmholtz Institute for RNA-based Infection Research (HIRI), Helmholtz Centre for Infection Research (HZI), 97080 Würzburg, Germany.
Abstract:
Anti-CRISPR proteins (Acrs) inhibit CRISPR-Cas immune defenses, with almost all known Acrs acting on the Cas nuclease-CRISPR (cr)RNA ribonucleoprotein (RNP) complex. Here, we show that AcrVIB1 from Riemerella anatipestifer, the only known Acr against Cas13b, principally acts upstream of RNP complex formation by promoting unproductive crRNA binding followed by crRNA degradation. AcrVIB1 tightly binds to Cas13b but not to the Cas13b-crRNA complex, resulting in enhanced rather than blocked crRNA binding. However, the more tightly bound crRNA does not undergo processing and fails to activate collateral RNA cleavage even with target RNA. The bound crRNA is also accessible to RNases, leading to crRNA turnover in vivo even in the presence of Cas13b. Finally, cryoelectron microscopy (cryo-EM) structures reveal that AcrVIB1 binds a helical domain of Cas13b responsible for securing the crRNA, keeping the domain untethered. These findings reveal an Acr that converts an effector nuclease into a crRNA sink to suppress CRISPR-Cas defense.
Insights
Anti-CRISPR proteins (Acrs) inhibit CRISPR-Cas immunity. AcrVIB1, a novel Acr, prevents Cas13b function by promoting crRNA degradation, not by blocking complex formation, revealing a new defense suppression mechanism.
Area of Science:
- Microbiology
- Molecular Biology
- Structural Biology
Background:
- CRISPR-Cas systems provide adaptive immunity in prokaryotes.
- Anti-CRISPR proteins (Acrs) are key regulators that inhibit CRISPR-Cas activity.
- Most known Acrs target the Cas nuclease-CRISPR (cr)RNA ribonucleoprotein (RNP) complex.
Purpose of the Study:
- To elucidate the mechanism of AcrVIB1, an Acr targeting the Cas13b system.
- To investigate how AcrVIB1 inhibits CRISPR-Cas13b immune defense.
Main Methods:
- Biochemical assays to study protein-nucleic acid interactions.
- In vivo experiments to assess crRNA stability and defense activity.
- Cryo-electron microscopy (cryo-EM) to determine structural mechanisms.
Main Results:
- AcrVIB1 binds Cas13b, promoting unproductive crRNA binding and subsequent degradation.
- AcrVIB1 inhibits Cas13b's RNA cleavage activity and collateral damage.
- Cryo-EM reveals AcrVIB1 binding to a Cas13b helical domain, preventing crRNA stabilization.
Conclusions:
- AcrVIB1 functions upstream of RNP formation, acting as a crRNA sink.
- This mechanism represents a novel strategy for suppressing CRISPR-Cas defense.
- AcrVIB1 provides new insights into the regulation of CRISPR-Cas13b systems.
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