ArticleNucleic acids research2023
A DNA unwinding equilibrium serves as a checkpoint for CRISPR-Cas12a target discrimination.
Article in Nucleic acids research, 2023. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 10 papers.
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Who cites it
10 citing papers in PubMed.
- Machine Learning for CRISPR-Based Diagnostics.International journal of molecular sciences · 2026Review
- An overview of CRISPR-artificial intelligence theranostics: Current and emerging applications.Biomaterials translational · 2026Review
- Structural integrity and side-chain interaction at the loop region of the bridge helix modulate Cas9 substrate discrimination.Nucleic acids research · 2025Article
- Dynamic basis of supercoiling-dependent DNA interrogation by Cas12a via R-loop intermediates.Nature communications · 2025Article
- PAM-adjacent DNA flexibility tunes CRISPR-Cas12a off-target binding.Scientific reports · 2025Article
- Leveraging CRISPR gene editing technology to optimize the efficacy, safety and accessibility of CAR T-cell therapy.Leukemia · 2024Review
- CRISPR-Cas12a exhibits metal-dependent specificity switching.Nucleic acids research · 2024Article
- Cas12a domain flexibility guides R-loop formation and forces RuvC resetting.Molecular cell · 2024Article
- CRISPR-Cas12a exhibits metal-dependent specificity switching.bioRxiv : the preprint server for biology · 2024Article
- Advancements in genome editing tools for genetic studies and crop improvement.Frontiers in plant science · 2024Review
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5 authors.
Funding
Abstract
CRISPR-associated proteins such as Cas9 and Cas12a are programable RNA-guided nucleases that have emerged as powerful tools for genome manipulation and molecular diagnostics. However, these enzymes are prone to cleaving off-target sequences that contain mismatches between the RNA guide and DNA protospacer. In comparison to Cas9, Cas12a has demonstrated distinct sensitivity to protospacer-adjacent-motif (PAM) distal mismatches, and the molecular basis of Cas12a's enhanced target discrimination is of great interest. In this study, we investigated the mechanism of Cas12a target recognition using a combination of site-directed spin labeling, fluorescent spectroscopy, and enzyme kinetics. With a fully matched RNA guide, the data revealed an inherent equilibrium between a DNA unwound state and a DNA-paired duplex-like state. Experiments with off-target RNA guides and pre-nicked DNA substrates identified the PAM-distal DNA unwinding equilibrium as a mismatch sensing checkpoint prior to the first step of DNA cleavage. The finding sheds light on the distinct targeting mechanism of Cas12a and may better inform CRISPR based biotechnology developments.
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Registered trials
Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.