SHERLOCK: nucleic acid detection with CRISPR nucleases

Abstract

Rapid detection of nucleic acids is integral for 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 for 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 fluorescent and colorimetric readouts that provide results in less than 1 hour. We also include guidelines for designing efficient CRISPR RNA and isothermal amplification primers, and discuss important considerations for multiplex and quantitative SHERLOCK detection assays.

Recent progress in understanding 2019 novel coronavirus (SARS-CoV-2) associated with human respiratory disease: detection, mechanisms and treatment

“…The CRISPR-based SHERLOCK (Specific Highsensitivity Enzymatic Reporter UnLOCKing) technique allows portable, multiplexed and ultrasensitive detection of RNA or DNA from clinically relevant samples. SHERLOCK assays are set up with recombinase-mediated polymerase pre-amplification of DNA or RNA and subsequent Cas13-or Cas12-mediated detection via colorimetric read-outs and fluorescence that provide results in < 1 h with a setup time of < 15 min [28] . Based on the RNA sequence of the novel coronavirus, researchers carefully designed two guide RNAs, one recognising the S gene of the new coronavirus and the other recognising the Orf1ab gene.…”

Integrated Micropillar Polydimethylsiloxane Accurate CRISPR Detection (IMPACT) System for Rapid Viral DNA Sensing

“…17,39 An advantage of the IMPACT chip compared to other detection apparatuses such as the SHERLOCK test strip is that it is a fully enclosed system without extra packaging need. 40,41 This is advantageous as the treated micropillars are sealed before molecular diagnostics. The reporter probes are immobilized within the channel, avoiding degradation issues from outside contaminants.…”

Magnetic Bead-Quantum Dot (MB-Qdot) CRISPR Assay for Instrument-Free Viral DNA Detection

“…Currently, our approach can achieve a detection limit of ~0.5 nM without any target amplification. To improve the sensitivity, our assay can be easily integrated with established isothermal nucleic acids amplification methods such as recombinase polymerase amplification (RPA) 31,15 or Loop-mediated isothermal amplification (LAMP) 32,33 to extend the detection limit to attomolar level. The amplification reagents can be mixed with CRISPR Cas12a protein and crRNA as a "one-pot" assay.…”

“…An ideal POC device that can combat modern era pandemic crises should be simple, inexpensive, accurate, and must be easily operated and understood by people without special training. A recently discovered technology called clustered regularly interspaced short palindromic repeats (CRISPR) in conjunction with CRISPR-associated proteins (Cas) has shown great promise as an alternative to RT-PCR detection 14,15 . This method is significantly simpler than RT-PCR since it does not require complex instrumentation to perform.…”