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

Science. 2018 Apr 27;360(6387):436-439. doi: 10.1126/science.aar6245. Epub 2018 Feb 15.

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Bacterial Proteins / chemistry*
  • CRISPR-Associated Proteins / chemistry*
  • CRISPR-Cas Systems*
  • Clostridiales / enzymology*
  • DNA Cleavage*
  • DNA, Single-Stranded / chemistry*
  • Endonucleases / chemistry*
  • Kinetics
  • Substrate Specificity

Substances

  • Bacterial Proteins
  • CRISPR-Associated Proteins
  • DNA, Single-Stranded
  • Cpf1 nuclease, Acidaminococcus
  • Endonucleases