ArticleNucleic acids research2021
Decoupling the bridge helix of Cas12a results in a reduced trimming activity, increased mismatch sensitivity and impaired conformational transitions.
Article in Nucleic acids research, 2021. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 17 papers.
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Who cites it
17 citing papers in PubMed.
- Molecular mechanisms and biotechnology applications of CRISPR-Cas12a.Nature reviews. Molecular cell biology · 2026Review
- Helicity of the bridge helix of Cas12a regulates on-target DNA cleavage efficiency and off-target cleavage propensity.Biochemical and biophysical research communications · 2026Article
- Bridge helix of Cas12a is an allosteric regulator of R-loop formation and RuvC activation.Nature communications · 2026Article
- And… cut! - how conformational regulation of CRISPR-Cas effectors directs nuclease activity.The Biochemical journal · 2025Review
- CRISPR-Cas12a REC2-Nuc interactions drive target-strand cleavage and constrain trans cleavage.Nucleic acids research · 2025Article
- Structural and mechanistic insights into the sequential dsDNA cleavage by SpCas12f1.Nucleic acids research · 2025Article
- Recent advances in CRISPR-based single-nucleotide fidelity diagnostics.Communications medicine · 2025Review
- Kinetic analysis and engineering of thermostable Cas12a for nucleic acid detection.Nucleic acids research · 2025Article
- Cas12a domain flexibility guides R-loop formation and forces RuvC resetting.Molecular cell · 2024Article
- An alpha-helical lid guides the target DNA toward catalysis in CRISPR-Cas12a.Nature communications · 2024Article
- Nonspecific interactions between Cas12a and dsDNA located downstream of the PAM mediate target search and assist AsCas12a for DNA cleavage.Chemical science · 2023Article
- Structures of apo Cas12a and its complex with crRNA and DNA reveal the dynamics of ternary complex formation and target DNA cleavage.PLoS biology · 2023Article
- Optimized protocols for the characterization of Cas12a activities.Methods in enzymology · 2023Article
- Improved genome editing by an engineered CRISPR-Cas12a.Nucleic acids research · 2022Article
- Allosteric activation of CRISPR-Cas12a requires the concerted movement of the bridge helix and helix 1 of the RuvC II domain.Nucleic acids research · 2022Article
- CRISPR-Cas12a-mediated DNA clamping triggers target-strand cleavage.Nature chemical biology · 2022Article
- MgProceedings of the National Academy of Sciences of the United States of America · 2021Article
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5 authors.
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No grant is acknowledged in the PubMed record.
Abstract
The widespread and versatile prokaryotic CRISPR-Cas systems (clustered regularly interspaced short palindromic repeats and associated Cas proteins) constitute powerful weapons against foreign nucleic acids. Recently, the single-effector nuclease Cas12a that belongs to the type V CRISPR-Cas system was added to the Cas enzymes repertoire employed for gene editing purposes. Cas12a is a bilobal enzyme composed of the REC and Nuc lobe connected by the wedge, REC1 domain and bridge helix (BH). We generated BH variants and integrated biochemical and single-molecule FRET (smFRET) studies to elucidate the role of the BH for the enzymatic activity and conformational flexibility of Francisella novicida Cas12a. We demonstrate that the BH impacts the trimming activity and mismatch sensitivity of Cas12a resulting in Cas12a variants with improved cleavage accuracy. smFRET measurements reveal the hitherto unknown open and closed state of apo Cas12a. BH variants preferentially adopt the open state. Transition to the closed state of the Cas12a-crRNA complex is inefficient in BH variants but the semi-closed state of the ternary complex can be adopted even if the BH is deleted in its entirety. Taken together, these insights reveal that the BH is a structural element that influences the catalytic activity and impacts conformational transitions of FnCas12a.
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