Cited by 4,507CRISPR and Genetic EngineeringAdvanced biosensing and bioanalysis techniquesRNA regulation and disease

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CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity

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Chen, Janice S., Ma, Enbo, Harrington, Lucas B., Da Costa, Maria, Tian, Xinran, Palefsky, Joel M., Doudna, Jennifer A.·2018·Science

DOI 10.1126/science.aar6245PMC PMC6628903

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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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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: Chen, Janice S.
Harrington, Lucas B.
Da Costa, Maria
Tian, Xinran
Palefsky, Joel M.
Doudna, Jennifer A.
Venue: Science
Publisher: American Association for the Advancement of Science (AAAS)
Published: 2018-04-27
Volume: 360
Issue: 6387
Pages: 436-439
ISSN: 0036-8075 (ISSN-L)
1095-9203
DOI: 10.1126/science.aar6245
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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CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity

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