Table 2.
Notable CRISPR–Cas-based diagnostic platforms for SARS-CoV-2 designed in 2020 and 2021
| CRISPR–Cas type | Technique name | Attendant methods | Target genes | Limit of detection (LOD) | Duration time | References | ||
|---|---|---|---|---|---|---|---|---|
| Amplification method | Detection method | |||||||
| Type II (Cas9) | Cas9/D10A with nickase activity |
CRISPR array-mediated primer exchange reaction-based biochemical circuit cascades |
PER | Electrochemical biosensing | Nucleocapsid (N) | 5 nM for synthesized SARS-CoV-2 genome in the human cell lysate | Three steps (CRISPR reactant, PER reactant, and detection) within 110 min | [33] |
| Francisella novicidae Cas9 | CRISPR/Cas9-mediated triple-line lateral flow assay (TL-LFA) | RT-RPA | Lateral flow visual readout | Simultaneous dual genes; envelope (E) and Orf1ab | 100 RNA copies per reaction (25 μl) | Two steps at 37 °C within 40 min | [34] | |
| Campylobacter jejuni NCTC11168 (CjeCas9) | Leveraging engineered tracrRNAs and on-target DNAs for parallel RNA detection (LEOPARD) | RT-PCR or free-amplification | Polyacrylamide gel electrophoresis or Bioanalyzer | Spike (S) | One copy, or 1.7 aM in the original dilution, of RNA compared with 3 × 108 copies, or 0.6 nM in the original dilution, without preamplification | unspecified | [31] | |
| Type V (Cas12) | Cas12a (Cpf1) | DNA endonuclease-targeted CRISPR trans reporter (DETECTR) | RT–LAMP | Lateral flow visual readout | Nucleocapsid (N) and envelope (E) | 10 copies per µl of nasopharyngeal swab samples | Three steps within 30–40 min | [35] |
| CRISPR-based fluorescent diagnosis system for COVID-19 (COVID-19 CRISPR-FDS) | RT-RPA | Fluorescence-based detection in 96-well microtiter plate and adaptable to smartphone-read chip format | Nucleocapsid (N) and envelope (E) | 2 copies per µl of nasal swab samples | Three steps within 50 min | [36, 37] | ||
| In vitro Specific CRISPR-based Assay for Nucleic acid detection (iSCAN) | RT-LAMP | Lateral flow and fluorescence-based detection | Nucleocapsid (N) and envelope (E) | 10 copies per µl of Nasopharyngeal swab samples | One and two steps at 62 °C within 60 min | [38] | ||
| CRISPR–Cas12a naked eye readout (CRISPR–Cas12a-NER) | RT-RAA | Fluorescence-based detection | orf1a, orf1b nucleocapsid (N) and envelope (E) | 10 copies per µl input | Three steps within 60 min | [39] | ||
| Specific enhancer for PCR-amplified Nucleic Acid (SENA) | rRT-PCR | Fluorescence-based detection | Orf1ab (O) and nucleocapsid (N) | 1.6 copies per µl of pharyngeal and nasopharyngeal swabs with 95% confidence | Three steps within rRT-PCR time and CRISPR detection time | [40] | ||
| Manganese-enhanced Cas12a detection (MeCas12a) | RT-RAA | Fluorescence-based detection | Envelope (E) | 5 copies with Mn2+ compared to 10 copies with Mg2+ | Three steps within 45 min | [41] | ||
| ENHANCE system | RT-LAMP | Fluorescence-based assay and lateral flow assay | Nucleocapsid (N) | 3–300 copies per µl | Three steps within 40–60 min | [42] | ||
| One-pot visual SARS-CoV-2 detection system named (opvCRISPR) | RT-LAMP | Fluorescence-based assay | Spike (S) | 5 copies per µl | Three steps within 45 min | [43] | ||
| All-In-One Dual CRISPR–Cas12a (AIOD-CRISPR) | RT-RPA | Fluorescence-based assay | Nucleocapsid (N) | 5 copies per µl | One-step amplification and detection at room temperature (37 °C) within 20 min | [44] | ||
| Microfluidic Isotachophoresis (ITP)-CRISPR-based | RT-LAMP | Fluorescence-based assay | Nucleocapsid (N) and envelope (E) | 10 copies per µl of Nasopharyngeal swab samples | Within 30 min | [45] | ||
|
Digital warm-start CRISPR (DWS-CRISPR) |
RT-DAMP | Fluorescence-based assay | Nucleocapsid (N) | 5 copies/μl RNA in the chip tenfold higher sensitivity than tube-based bulk assay format | One step in QuantStudio 3D digital chip initiating at above 50 °C | [46] | ||
| Cas12b (C2c1) | CRISPR-assisted detection (CASdetec) | RT-RAA | Fluorescence-based assay | RdRp | 10 copies per µl | one step at 42 °C within 60 min | [47] | |
| SHERLOCK Testing in One Pot (STOP) | RT-LAMP | Fluorescence-based assay and lateral flow assay | Nucleocapsid (N) | 100 copies/reaction | One-step amplification and detection at 60 °C within 45 min | [48] | ||
| Type VI (Cas13) | Cas13a (C2c2) | Specific High-sensitivity Enzymatic Reporter unlocking (SHERLOCK) | RT–RPA and T7 transcription | Fluorescence-based assay and lateral flow assay | Spike (S), nucleoprotein (N) replicase polyprotein 1ab (Orf1ab) | 42 RNA copies per reaction of Nasopharyngeal swab | Three steps within 60 min | [26, 49] |
| Combinatorial arrayed reactions for multiplexed evaluation of nucleic acids (CARMEN-Cas13) | PCR or RT-RPA | Fluorescence-based assay using mixes of color-coded amplified sequences | spike (S), nucleoprotein (N) replicase polyprotein 1ab (Orf1ab) | 104 copies per µl for synthetic targets | Three steps | [50, 51] | ||
| Streamlined highlighting of infections to navigate epidemics (SHINE) | RT–RPA and T7 transcription | Fluorescence-based assay and lateral flow assay | ORF1a | 10 copies per µl with 100% specificity | One step | [27] | ||
| Cas13 assisted saliva-based & smartphone integrated testing (CASSPIT) | RT-RPA and T7 transcription | Lateral flow assay integrated with a smartphone application | S and Orf1ab | 200 copies/reaction for the S gene with a corresponding Ct value of 35.4 | Two steps dual-heat inactivation (37 °C for 10 min and 95 °C for 5 min) | [25] | ||
PER primer exchange reaction, LAMP loop-mediated isothermal amplification, rLAMP RNA transcription following LAMP, RAA recombinase-aided amplification, RPA recombinase polymerase amplification, DAMP dual-priming isothermal amplifications