CRISPR Diagnostics: SHERLOCK and DETECTR Work as Claimed
“CRISPR-based diagnostic platforms, such as SHERLOCK, utilise Cas13 enables rapid and highly sensitive detection by binding to the specific viral RNA sequence which would be amplified by RT-RPA or RT-LAMP. Activating the Cas-enzyme that cleaves the target RNA and nearby RNA molecules. This then creates a detectable signal(fluorescence). Parallely, DETECTR operates by initially converting the viral RNA to DNA using reverse transcription and amplifying it using RT-LAMP(reverse transcription loop-mediated isothermal amplification), then gets recognised by the Cas12 enzyme, which activates and cleaves the nearby infected DNA molecules, simillary this produces a visual signal in 30-45 minutes as well.”
Summary
SHERLOCK employs Cas13 to recognize amplified viral RNA (using RT‑RPA or RT‑LAMP with T7 transcription); Cas13 activation triggers collateral RNA cleavage that produces a fluorescent signal. DETECTR first reverse‑transcribes viral RNA and amplifies it with RT‑LAMP, then Cas12 binds the DNA amplicon and cleaves nearby reporter DNA, generating a visual readout in roughly 30‑45 minutes.
Sources 59 searched
- SHERLOCK: Nucleic acid detection with CRISPR nucleases - PMC
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 ...
- SHERLOCK and DETECTR: CRISPR-Cas Systems as Potential Rapid Diagnostic Tools for Emerging Infectious Diseases - PMC
To conclude, SHERLOCKv2 is a highly sensitive quantitative diagnostic platform suitable for multiplex signal detection and colorimetric detection on lateral flow strips (45) (Table 2). ... 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).
- Sherlock CRISPR SARS-CoV-2 Kit - Instructions for Use
The SherlockTM CRISPR SARS-CoV-2 kit is designed to detect RNA from upper respiratory · specimens (anterior nasal swabs, nasopharyngeal swabs, oropharyngeal swabs, nasopharyngeal · wash/aspirate or nasal aspirate) and bronchoalveolar lavage specimens from patients suspected · of COVID-19 by their healthcare provider. RNA is extracted from clinical samples using the · PureLink™ Viral ...
- SARS-CoV-2 RNA DETECTR Assay
sequence lacks the PAM required for Cas12 activity. Although some primers have partial homology to the organisms of interest, it is unlikely · for cross-reactivity to occur with these organisms as RT-LAMP requires complementarity
- Clinical validation of a Cas13-based assay for the detection of SARS-CoV-2 RNA | Nature Biomedical Engineering
SHERLOCK detection of SARS-CoV-2 relies on RT–RPA to isothermally amplify viral gene segments of interest, followed by CRISPR–Cas-mediated detection of the amplified genes (in this case, using Cas13a from L. wadei (LwaCas13a), shown previously to confer the highest sensitivity in SHERLOCK-type detection9).
- CRISPR–Cas12-based detection of SARS-CoV-2 | Nature Biotechnology
Conventional RNA extraction can be used as an input to DETECTR (LAMP preamplification and Cas12-based detection for E gene, N gene and RNase P), which is visualized by a fluorescent reader or lateral flow strip. e, Lateral flow strip assay readout. A positive result requires detection of at least one of the two SARS-CoV-2 viral gene targets (N gene or E gene, as indicated in the interpretation matrix).
- Nucleic acid detection with CRISPR-Cas13a/C2c2 | Science
Of the methods explored, recombinase polymerase amplification (RPA) (18) afforded the greatest sensitivity and could be coupled with T7 transcription to convert amplified DNA to RNA for subsequent detection by LwCas13a. We refer to this combination of amplification by RPA, T7 RNA polymerase transcription of amplified DNA to RNA, and detection of target RNA by Cas13a collateral RNA cleavage–mediated release of reporter signal as SHERLOCK.
- Nucleic acid detection with CRISPR-Cas13a/C2c2 - PubMed
The target site is highlighted in blue. (C) Cas13a detection of RNA with RPA amplification (SHERLOCK) can detect ssRNA target at concentrations down to ~2 aM, more sensitive than Cas13a alone.
- RT-LAMP CRISPR-Cas12/13-Based SARS-CoV-2 Detection Methods - PMC
In a study, the CRISPR-mediated detection step was optimized to prevent false-negative results caused by SARS-CoV-2 genome alterations [28]. Cas12a from the Lachnospiraceae bacterium (LbCas12a) was combined with two distinct crRNAs to target the RT-LAMP amplicons. When one of the target sites is mutated, the other crRNA can still function properly, resulting in positive COVID-19 results.
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