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CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity
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Abstract
CRISPR-Cas12a (Cpf1) proteins are RNA-guided enzymes that bind and cut DNA as components of bacterial adaptive immune systems. Like CRISPR-Cas9, Cas12a has been harnessed for genome editing on the basis of its ability to generate targeted, double-stranded DNA breaks. Here we show that RNA-guided DNA binding unleashes indiscriminate single-stranded DNA (ssDNA) cleavage activity by Cas12a that completely degrades ssDNA molecules. We find that target-activated, nonspecific single-stranded deoxyribonuclease (ssDNase) cleavage is also a property of other type V CRISPR-Cas12 enzymes. By combining Cas12a ssDNase activation with isothermal amplification, we create a method termed DNA endonuclease-targeted CRISPR trans reporter (DETECTR), which achieves attomolar sensitivity for DNA detection. DETECTR enables rapid and specific detection of human papillomavirus in patient samples, thereby providing a simple platform for molecular diagnostics.
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“Cas12a enzymes are single crRNA-guided endonucleases measuring 3.9 kb”
From Viral Delivery Systems for CRISPR (2019) · cites this paper as [78]
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“their distinguishable differences from Cas9 include: a T nucleotide-rich PAM site (5′-TTTV), self-catalysis of crRNA maturation, and a staggered dsDNA cut [76,77,78,79].”
From Viral Delivery Systems for CRISPR (2019) · cites this paper as [78]
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“Additionally, Cas12a′s trans-nuclease activity is utilized to detect sequence-specific nucleic acid (Chen et al., 2018).”
From Emerging applications of gene editing technologies for the development of climate-resilient crops (2025) · cites this paper as Chen et al., 2018
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“their distinguishable differences from Cas9 include: a T nucleotide-rich PAM site (5′-TTTV), self-catalysis of crRNA maturation, and a staggered dsDNA cut [76,77,78,79].”
From Viral Delivery Systems for CRISPR (2019) · cites this paper as [78]
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“their distinguishable differences from Cas9 include: a T nucleotide-rich PAM site (5′-TTTV), self-catalysis of crRNA maturation, and a staggered dsDNA cut [76,77,78,79].”
From Viral Delivery Systems for CRISPR (2019) · cites this paper as [78]
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Metadata
- Authors
- Chen, J. S., Ma, E., Harrington, L. B., Da Costa, M., Tian, X., Palefsky, J. M., & Doudna, J. A.
- Affiliations
- University of California, Berkeley (US)
- Department of Medicine, University of California, San Francisco, San Francisco, CA 94143, USA.
- University of California, San Francisco (US)
- Howard Hughes Medical Institute (US)
- Corresponding author
- Chen, Janice S.
- Ma, Enbo
- Harrington, Lucas B.
- Doudna, Jennifer A.
- ORCID
- Venue
- Science
- Published
- 2018-04-27
- Volume
- 360
- Issue
- 6387
- Pages
- 436-439
- ISSN
- 0036-8075 (ISSN-L)
- 1095-9203
- PMC
- PMC6628903
- OpenAlex
- W2787024379
- Cited by
- 4,507
- References
- 48
- Citations by year
- 2026: 759 · 2025: 816 · 2024: 736 · 2023: 643 · 2022: 624 · 2021: 452 · 2020: 271 · 2019: 135 · 2018: 67
- FWCI
- 82.27
- Type
- journal article
- Language
- en
- Open access
- green
- doi.org
- Locations
- Science (publishedVersion)
- bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion)
- PubMed (publishedVersion)
- Europe PMC (PubMed Central) (acceptedVersion)
- PubMed Central (acceptedVersion)
- eScholarship (California Digital Library) (submittedVersion)
- Funders
- National Science Foundation (MCB-1244557)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Advanced biosensing and bioanalysis techniques
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA regulation and disease
- Keywords
- CRISPR/Cas12a, single-stranded DNA cleavage, isothermal amplification, DETECTR, DNA detection, HPV testing, molecular diagnosis, genome editing
- MeSH terms
- Clostridiales, Eubacteriales, Bacterial Proteins, DNA, Single-Stranded, Endodeoxyribonucleases, Endonucleases, Kinetics, Substrate Specificity, DNA Cleavage, CRISPR-Cas Systems, CRISPR-Associated Proteins
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