4.2. CRISPR Based COVID-19 Detection
This protocol is extracted from research article:
Nucleic Acid Testing of SARS-CoV-2
Int J Mol Sci, Jun 7, 2021; DOI: 10.3390/ijms22116150

The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas technique has been repurposed for diagnostics and is one of the widely used nucleic acid detection methods [185,186]. Many types of Cas proteins have been developed to create highly accurate and sensitive diagnostic methods [187]. Cas9 has been widely used for genome editing while DNA-targeting Cas12 (also known as Cpf1 or C2c1) effectors and RNA-targeting Cas 13a are more suitable for disease diagnosis [188].

Compared to conventional diagnostic methods such as RT-qPCR, CRISPR-based approaches can quickly provide rapid, visual, highly sensitive, and specific detection due to the collateral cleavage of a reporter dye in the presence of a target [189].

Numerous CRISPR-Cas detection systems have been developed. For example, techniques of a CRISPR–Cas12-based assay have been developed for the detection of SARS-CoV-2 from patient sample RNA, called SARS-CoV-2 DNA Endonuclease-Targeted CRISPR Trans Reporter (DETECTR) [190]. This assay includes simultaneous reverse transcription and isothermal amplification using loop-mediated amplification (RT–LAMP) [191]. An RNA targeting Cas13a dependent platform [156], the SHERLOCK (Specific High Sensitivity Enzymatic Reporter UnLOCKing) technique, offers a simplified test and has a limit of detection of 100 copies of the viral genome [192,193].

Recently, a lyophilized CRISPR-Cas12 assay for SARS-CoV-2 detection (Lyo-CRISPR SARS-CoV-2 kit) has been developed based on reverse transcription (RT), isothermal amplification, and CRISPR-Cas12 reaction [194]. The schematic procedure of CRISPR detection systems is shown in Figure 1D.

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