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Updated: Jan 27, 2026
High-throughput Screening for Broad-spectrum Chemical Inhibitors of RNA Viruses
Published on: May 5, 2014
Broad-spectrum enzymatic inhibition of CRISPR-Cas12a
Gavin J Knott1, Brittney W Thornton1, Marco J Lobba2
1Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA.
Abstract:
Cas12a is a bacterial RNA-guided nuclease used widely for genome editing and, more recently, as a molecular diagnostic. In bacteria, Cas12a enzymes can be inhibited by bacteriophage-derived proteins, anti-CRISPRs (Acrs), to thwart clustered regularly interspaced short palindromic repeat (CRISPR) adaptive immune systems. How these inhibitors disable Cas12a by preventing programmed DNA cleavage is unknown. We show that three such inhibitors (AcrVA1, AcrVA4 and AcrVA5) block Cas12a activity via functionally distinct mechanisms, including a previously unobserved enzymatic strategy. AcrVA4 and AcrVA5 inhibit recognition of double-stranded DNA (dsDNA), with AcrVA4 driving dimerization of Cas12a. In contrast, AcrVA1 is a multiple-turnover inhibitor that triggers cleavage of the target-recognition sequence of the Cas12a-bound guide RNA to irreversibly inactivate the Cas12a complex. These distinct mechanisms equip bacteriophages with tools to evade CRISPR-Cas12a and support biotechnological applications for which multiple-turnover enzymatic inhibition of Cas12a is desirable.
Insights
Bacteriophages use anti-CRISPR proteins (Acrs) to disable Cas12a bacterial immune systems. Three Acrs (AcrVA1, AcrVA4, AcrVA5) use distinct methods, including a novel enzymatic strategy, to block Cas12a DNA cleavage.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- Cas12a is a versatile RNA-guided nuclease employed in genome editing and molecular diagnostics.
- Bacterial CRISPR-Cas12a adaptive immune systems are targeted by bacteriophage-derived anti-CRISPR proteins (Acrs).
- The mechanisms by which Acrs inhibit Cas12a's DNA cleavage activity remain largely uncharacterized.
Purpose of the Study:
- To elucidate the distinct molecular mechanisms employed by three Acrs (AcrVA1, AcrVA4, AcrVA5) to inhibit Cas12a.
- To identify novel strategies used by phages to evade CRISPR-Cas12a immunity.
- To explore potential biotechnological applications of Cas12a inhibitors.
Main Methods:
- Biochemical assays to assess Cas12a nuclease activity in the presence of AcrVA1, AcrVA4, and AcrVA5.
- Analysis of protein-protein interactions to understand Acr-Cas12a complex formation.
- Investigation of guide RNA processing and target DNA recognition.
Main Results:
- AcrVA4 and AcrVA5 inhibit Cas12a by preventing double-stranded DNA (dsDNA) recognition; AcrVA4 induces Cas12a dimerization.
- AcrVA1 acts as a multiple-turnover inhibitor, cleaving the guide RNA's target-recognition sequence to irreversibly inactivate Cas12a.
- This study reveals a previously unknown enzymatic strategy for Cas12a inhibition.
Conclusions:
- Bacteriophages utilize diverse Acr mechanisms to counteract CRISPR-Cas12a immunity.
- The distinct inhibition strategies of AcrVA1, AcrVA4, and AcrVA5 offer new insights into Cas12a regulation.
- The multiple-turnover inhibition by AcrVA1 presents opportunities for developing novel biotechnological tools.
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