Sep 25, 2019

SHERLOCK: nucleic acid detection with CRISPR nucleases

Nature Protocols
Max J KellnerFeng Zhang

Abstract

Rapid detection of nucleic acids is integral to applications in clinical diagnostics and biotechnology. We have recently established a CRISPR-based diagnostic platform that combines nucleic acid pre-amplification with CRISPR-Cas enzymology for specific recognition of desired DNA or RNA sequences. This platform, termed specific high-sensitivity enzymatic reporter unlocking (SHERLOCK), allows multiplexed, portable, and ultra-sensitive detection of RNA or DNA from clinically relevant samples. Here, we provide step-by-step instructions for setting up SHERLOCK assays with recombinase-mediated polymerase pre-amplification of DNA or RNA and subsequent Cas13- or Cas12-mediated detection via fluorescence and colorimetric readouts that provide results in <1 h with a setup time of less than 15 min. We also include guidelines for designing efficient CRISPR RNA (crRNA) and isothermal amplification primers, as well as discuss important considerations for multiplex and quantitative SHERLOCK detection assays.

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Mentioned in this Paper

Gene Amplification
CRISPR-Cas Systems
Workflow
Endonuclease
Proteins, Recombinant DNA
Nucleic Acid Amplification Tests
Recombinase
Alkaline Ribonuclease
Oligonucleotide Primers
Biotechnology

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