Boosting CRISPR/Cas12a intrinsic RNA detection capability through pseudo hybrid DNA–RNA substrate design Article Swipe
Jie Qiao
,
Junqi Zhang
,
Qingyuan Jiang
,
Shuqi Jin
,
Ruyi He
,
Bin Qiao
,
Yi Liu
·
YOU?
·
· 2025
· Open Access
·
· DOI: https://doi.org/10.1093/nar/gkaf510
YOU?
·
· 2025
· Open Access
·
· DOI: https://doi.org/10.1093/nar/gkaf510
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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Metadata
- Type
- article
- Language
- en
- Landing Page
- https://doi.org/10.1093/nar/gkaf510
- https://academic.oup.com/nar/article-pdf/53/11/gkaf510/63469941/gkaf510.pdf
- OA Status
- gold
- Cited By
- 4
- References
- 37
- Related Works
- 10
- OpenAlex ID
- https://openalex.org/W4411210004
All OpenAlex metadata
Raw OpenAlex JSON
- OpenAlex ID
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https://openalex.org/W4411210004Canonical identifier for this work in OpenAlex
- DOI
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https://doi.org/10.1093/nar/gkaf510Digital Object Identifier
- Title
-
Boosting CRISPR/Cas12a intrinsic RNA detection capability through pseudo hybrid DNA–RNA substrate designWork title
- Type
-
articleOpenAlex work type
- Language
-
enPrimary language
- Publication year
-
2025Year of publication
- Publication date
-
2025-06-06Full publication date if available
- Authors
-
Jie Qiao, Junqi Zhang, Qingyuan Jiang, Shuqi Jin, Ruyi He, Bin Qiao, Yi LiuList of authors in order
- Landing page
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https://doi.org/10.1093/nar/gkaf510Publisher landing page
- PDF URL
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https://academic.oup.com/nar/article-pdf/53/11/gkaf510/63469941/gkaf510.pdfDirect link to full text PDF
- Open access
-
YesWhether a free full text is available
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-
goldOpen access status per OpenAlex
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https://academic.oup.com/nar/article-pdf/53/11/gkaf510/63469941/gkaf510.pdfDirect OA link when available
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Biology, CRISPR, RNA, DNA, Computational biology, Genetics, Boosting (machine learning), Molecular biology, Gene, Computer science, Machine learningTop concepts (fields/topics) attached by OpenAlex
- Cited by
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4Total citation count in OpenAlex
- Citations by year (recent)
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2025: 4Per-year citation counts (last 5 years)
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37Number of works referenced by this work
- Related works (count)
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10Other works algorithmically related by OpenAlex
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