SHERLOCK and DETECTR: CRISPR-Cas Systems as Potential Rapid Diagnostic Tools for Emerging Infectious Diseases

Abstract

Infectious diseases are one of the most intimidating threats to human race, responsible for an immense burden of disabilities and deaths. Rapid diagnosis and treatment of infectious diseases offers a better understanding of their pathogenesis. According to the World Health Organization, the ideal approach for detecting foreign pathogens should be rapid, specific, sensitive, instrument-free, and cost-effective. Nucleic acid pathogen detection methods, typically PCR, have numerous limitations, such as highly sophisticated equipment requirements, reagents, and trained personnel relying on well-established laboratories, besides being time-consuming. Thus, there is a crucial need to develop novel nucleic acid detection tools that are rapid, specific, sensitive, and cost-effective, particularly ones that can be used for versatile point-of-care diagnostic applications. Two new methods exploit unpredicted in vitro properties of CRISPR-Cas effectors, turning activated nucleases into basic amplifiers of a specific nucleic acid binding event. These effectors can be attached to a diversity of reporters and utilized in tandem with isothermal amplification approaches to create sensitive identification in multiple deployable field formats. Although still in their beginning, SHERLOCK and DETECTR technologies are potential methods for rapid detection and identification of infectious diseases, with ultrasensitive tests that do not require complicated processing. This review describes SHERLOCK and DETECTR technologies and assesses their properties, functions, and prospective to become the ultimate diagnostic tools for diagnosing infectious diseases and curbing disease outbreaks.

Keywords: CRISPR-Cas diagnostic tools; DETECTR; SHERLOCK; infectious diseases.

FIG 1

CRISPR-Cas technologies for nucleic acid detection in SHERLOCKv2, SHERLOCK, and DETECTR assays. In the absence of its nucleic acid target, the Cas nuclease is inactive. When binding to its guide crRNA to a related target (RNA for Cas13a, ssDNA or dsDNA for Cas12a), the nuclease is activated, leading to catalytic cleavage of off-target nucleic acids (RNA for Cas13a, ssDNA for Cas12a). This collateral nuclease activity is turned into an amplified signal by providing reporter probes with a fluorophore (green) linked to a quencher (white) by a short oligonucleotide (black). (Left) Schematic of SHERLOCKv2, with direct detection of viral infection (for example) in bodily fluids. (Middle) Schematic of the SHERLOCK system. Nucleic acid is extracted from clinical samples (for example), and the target is amplified by recombinase polymerase amplification (RPA) with either RNA or DNA as the input (reverse transcriptase recombinase polymerase amplification [RT-RPA] or RPA, respectively). RPA products are detected in a reaction mixture containing T7 RNA polymerase, Cas13, a target-specific crRNA, and an RNA reporter that fluoresces when cleaved. (Right) Schematic of the DETECTR system.

FIG 2

Comparison of SARS-CoV-2 assay workflows for DETECTR, SHERLOCK, and CDC/WHO.