ArticleNucleic acids research2022
PAM-independent ultra-specific activation of CRISPR-Cas12a via sticky-end dsDNA.
Article in Nucleic acids research, 2022. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 19 papers.
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Who cites it
19 citing papers in PubMed, 64 citations in OpenAlex.
- Cas-regulation-targeting chimera enables selective and tunable control of CRISPR/Cas12a.Nucleic acids research · 2026Article
- A Comprehensive Review on CRISPR-Based Screening and Its Applications.Molecular biotechnology · 2026Review
- CRISPR diagnostics: from trans-nuclease activity to cancer diagnosis.Cell & bioscience · 2026Review
- A Single-Enzyme Activated CRISPR-Cas12a Nano System via Subtly Balanced dsDNA for Kinetic-Gated UDG Detection and Spatiotemporal Cellular Imaging.Advanced science (Weinheim, Baden-Wurttemberg, Germany) · 2026Article
- High-sensitivity, protein-independent detection of dsDNA sequences.Proceedings of the National Academy of Sciences of the United States of America · 2026Article
- Enhanced SfaTnpB enables single-base-specific, one-pot nucleic acid detection for high-sensitivity diagnostics.Nucleic acids research · 2026Article
- Coloring target and off-target effects of genetically modified nucleases by blue & white colony assays.Biotechnology letters · 2025Article
- TdT/Cas12a-based biosensor for sensitive detection of DNA breakpoints in sperm cryopreservation.Scientific reports · 2025Article
- Cas12a Cis-cleavage mediated lateral flow assay enables multiplex and ultra-specific nucleic acid detection.Nature communications · 2025Article
- PAM-free hairpin target binding activates trans-cleavage activity of Cas12a.Nucleic acids research · 2025Article
- Ultrasensitive detection of clinical pathogens through a target-amplification-free collateral-cleavage-enhancing CRISPR-CasΦ tool.Nature communications · 2025Article
- PAM-Independent CRISPR-Cas12a System for Specific Assays of Single Nucleotide Variants.JACS Au · 2025Article
- Topologically constrained DNA-mediated one-pot CRISPR assay for rapid detection of viral RNA with single nucleotide resolution.EBioMedicine · 2025Article
- Detachable DNA Assembly Module to Dissect Tumor Cells Heterogeneity via RNA Pinpoint Screening.Advanced science (Weinheim, Baden-Wurttemberg, Germany) · 2024Article
- Iterative crRNA design and a PAM-free strategy enabled an ultra-specific RPA-CRISPR/Cas12a detection platform.Communications biology · 2024Article
- TracrRNA reprogramming enables direct PAM-independent detection of RNA with diverse DNA-targeting Cas12 nucleases.Nature communications · 2024Article
- Bridging biological samples to functional nucleic acid biosensor applications: current enzymatic-based strategies for single-stranded DNA generation.Analytical sciences : the international journal of the Japan Society for Analytical Chemistry · 2024Review
- Atom-Modified gDNA Enhances Cleavage Activity of TtAgo Enabling Ultra-Sensitive Nucleic Acid Testing.Advanced science (Weinheim, Baden-Wurttemberg, Germany) · 2024Article
- Controllable and reusable seesaw circuit based on nicking endonucleases.Journal of nanobiotechnology · 2024Article
Corrections and comments
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Authors and funding
16 authors at 5 institutions in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
Although CRISPR-Cas12a [clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 12a] combining pre-amplification technology has the advantage of high sensitivity in biosensing, its generality and specificity are insufficient, which greatly restrains its application range. Here, we discovered a new targeting substrate for LbaCas12a (Lachnospiraceae bacterium Cas12a), namely double-stranded DNA (dsDNA) with a sticky-end region (PAM-SE+ dsDNA). We discovered that CRISPR-Cas12a had special enzymatic properties for this substrate DNA, including the ability to recognize and cleave it without needing a protospacer adjacent motif (PAM) sequence and a high sensitivity to single-base mismatches in that substrate. Further mechanism studies revealed that guide RNA (gRNA) formed a triple-stranded flap structure with the substrate dsDNA. We also discovered the property of low-temperature activation of CRISPR-Cas12a and, by coupling with the unique DNA hybridization kinetics at low temperature, we constructed a complete workflow for low-abundance point mutation detection in real samples, which was fast, convenient and free of single-stranded DNA (ssDNA) transformation. The detection limits were 0.005-0.01% for synthesized strands and 0.01-0.05% for plasmid genomic DNA, and the mutation abundances provided by our system for 28 clinical samples were in accordance with next-generation sequencing results. We believe that our work not only reveals novel information about the target recognition mechanism of the CRISPR-Cas12a system, but also greatly broadens its application scenarios.
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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 Socratic graph.