SHERLOCK: Nucleic acid detection with CRISPR nucleases
By M. Kellner, Jeremy Koob, Jonathan Gootenberg, Omar Abudayyeh, Feng Zhang. Nature Protocols, 2019
1,355 citations recorded by the source corpus.
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. Specific high-sensitivity enzymatic reporter unlocking (SHERLOCK) allows multiplexed, portable, and ultra-sensitive detection of RNA or DNA from clinically relevant samples.
Subfields: Biomedical Engineering, Molecular Biology
View Paper: https://www.semanticscholar.org/paper/20b2993c9c81c10957e486af911e81299d4f7044
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Citations in the corpus
- Towards the Optimization of eDNA/eRNA Sampling Technologies for Marine Biosecurity Surveillance (2021, methodology): “Among the most promising approaches are loop-mediated isothermal amplification (LAMP [185]) and recombinase polymerase amplification (RPA), both of which involve the rapid amplification of DNA/RNA using an isothermal amplification reaction with similar sensitivities and specificities to laboratory-b”