Статья

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

Janice S. ChenDepartment of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USAEnbo MaDepartment of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USALucas B. HarringtonDepartment of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USAMaria Da CostaDepartment of Medicine, University of California, San Francisco, San Francisco, CA 94143, USAXinran TianDepartment of Chemistry, University of California, Berkeley, Berkeley, CA 94720, USAJoel M. PalefskyDepartment of Medicine, University of California, San Francisco, San Francisco, CA 94143, USAJennifer A. DoudnaDepartment of Chemistry, University of California, Berkeley, Berkeley, CA 94720, USA

2018en

ABI

Аннотация

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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Идентификаторы

DOI: 10.1126/science.aar6245

Цитирования и источники

Цитирований: 5Использованных источников: 0

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