ArticleNucleic acids research2025
Boosting CRISPR/Cas12a intrinsic RNA detection capability through pseudo hybrid DNA-RNA substrate design.
Article in Nucleic acids research, 2025. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 9 papers.
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Who cites it
9 citing papers in PubMed.
- Yeast species resource exploration and application in cell factories.Synthetic and systems biotechnology · 2026Review
- RAPID: evaluation of Cas12a protospacer nicking and chimeric reporters for PAM-independent RNA and DNA diagnostics.Nucleic acids research · 2026Article
- Molecular engineering of CRISPR/Cas12a: from activity enhancement to exponential signal amplification.Chemical science · 2026Review
- Flexible regulation of CRISPR/Cas12a activity by spatial confinement effect.Nucleic acids research · 2026Article
- crRNA scaffold remodeling controls CRISPR-Cas12a activity for enhanced performance.Nucleic acids research · 2026Article
- Development of a Sensitive and Specific RPA-CRISPR/Cas12a Assay for Intrahepatic Quantification of HBV cccDNA.International journal of molecular sciences · 2026Article
- An overview of CRISPR-artificial intelligence theranostics: Current and emerging applications.Biomaterials translational · 2026Review
- CRISPR-based platforms for detecting tumor-associated genetic materials in clinical samples.Bioanalysis · 2025Review
- CRISPR/Cas12a DTR system: a topology-guided Cas12a assay for specific dual detection of RNA and DNA targets.Nucleic acids research · 2025Article
Corrections and comments
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Authors and funding
7 authors.
Funding
Abstract
The CRISPR/Cas12a [clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 12a] system is known for its intrinsic RNA-guided trans-cleavage activity; however, its RNA detection sensitivity is limited, with conventional methods typically achieving detection limits in the nanomolar range. Here, we report the development of a "pseudo hybrid DNA-RNA" (PHD) assay that significantly enhances the RNA detection capability of Cas12a. The PHD assay achieves a striking detection limit of 7.7 pM using single CRISPR RNA (crRNA) and 33.8 fM using pooled crRNAs. Importantly, this assay exhibits ultra-high specificity, capable of distinguishing mutated RNA target sequences at the protospacer adjacent motif (PAM)-distal region. It can also detect ultrashort RNA sequences as short as 6-8 nt and long RNAs with complex secondary structures. Additionally, the PHD assay enables PAM-free attomolar-level DNA detection. We further demonstrate the practical utility of the PHD assay by successfully detecting miR-155 biomarkers and human pappilloma virus 16 DNA in clinical samples. We anticipate that the design principles established in this study can be extended to other CRISPR/Cas enzymes, thereby accelerating the development of powerful nucleic acid testing tools for various applications.
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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.