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

Janice S Chen1, Enbo Ma1, Lucas B Harrington1

  • 1Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA.

Science (New York, N.Y.)
|February 17, 2018
PubMed

Insights

CRISPR-Cas12a enzymes, beyond genome editing, possess a novel single-stranded DNA (ssDNA) cleavage activity. This discovery led to the development of DETECTR for highly sensitive DNA detection.

Area of Science:

  • Molecular Biology
  • Biochemistry
  • Genetics

Background:

  • CRISPR-Cas12a (Cpf1) proteins are RNA-guided enzymes integral to bacterial adaptive immunity.
  • Cas12a, similar to Cas9, is utilized for targeted DNA cleavage in genome editing applications.

Purpose of the Study:

  • To investigate the DNA cleavage activities of CRISPR-Cas12a beyond targeted double-stranded breaks.
  • To explore the potential of Cas12a's ssDNA cleavage activity for novel detection methods.

Main Methods:

  • Characterization of CRISPR-Cas12a's RNA-guided DNA binding and subsequent cleavage activities.
  • Development of the DNA endonuclease-targeted CRISPR trans reporter (DETECTR) assay by combining Cas12a ssDNase activation with isothermal amplification.
  • Application of DETECTR for the detection of human papillomavirus (HPV) in clinical samples.

Main Results:

  • RNA-guided DNA binding by Cas12a activates an indiscriminate single-stranded DNA (ssDNA) cleavage function, degrading ssDNA molecules.
  • This target-activated, non-specific ssDNase activity is a conserved property among type V CRISPR-Cas12 enzymes.
  • The DETECTR method demonstrated attomolar sensitivity for DNA detection.
  • DETECTR enabled rapid and specific detection of human papillomavirus in patient samples.

Conclusions:

  • CRISPR-Cas12a exhibits a previously unrecognized ssDNA cleavage activity upon target recognition.
  • This ssDNase activity can be harnessed to develop highly sensitive diagnostic tools like DETECTR.
  • DETECTR presents a promising platform for molecular diagnostics, exemplified by HPV detection.

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