CRISPR-based tools for Coronavirus Disease 2019 (COVID-19) test
Diagnostic antibodies and antigens for Companion Animal disease testing
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Diagnostic antibodies and antigens for Swine disease testing
Diagnostic antibodies and antigens for Avian disease testing
Diagnostic antibodies and antigens for Multiple animal disease testing
Diagnostic antibodies and antigens for Ruminant disease testing
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Diagnostic antibodies and antigens for infectious and non-infectious Equine/Horse disease testing
SOCAIL MEDIA
Author:
Huajun Bai1 Xiaolong Cai1, 2* Xiaoyan Zhang1
1. R&D Center, GeneMedi Co.Ltd., Shanghai, P.R. China (www.genemedi.com)
2. Hanbio Research Center, Hanbio Tech Co. Ltd., Shanghai, P.R. China (www.hanbio.net)
Abstract:
The outbreak of COVID-19, caused by 2019 novel coronavirus (2019-nCoV), has been a global public health threat and caught the worldwide concern. Scientists throughout the world are sparing all efforts to explore strategies for the determination of the 2019-nCoV virus and diagnosis of COVID-19 rapidly. Several assays are developed for COVID-19 test , including RT-PCR, coronavirus antigens-based immunoassays, and CRISPR-based strategies (Cas13a or Cas12a), etc. Different assays have their advantages and drawbacks, and people should choose the most suitable assay according to their demands. Here, we make a brief introduction about these assays and give a simple overview of them, hoping to help doctors and researchers to select the most suitable assay for the Coronavirus Disease 2019 test (COVID-19 test) .
CRISPR-based tools for Coronavirus Disease 2019 (COVID-19) test
1) Principles for Diagnostics
As RT-PCR assay for COVID-19 requires several reagents, specific instruments and complicated processes, CRISPR-based tools have been developed to test COVID-19 within 30 minutes, which is really easy and simple to operated and saves a lot of time.
A. CRISPR-Cas13a (SHERLOCK)
Based on CRISPR (clustered regularly interspaced short palindromic repeats) technology, single-effector RNA-guided ribonucleases (RNases), such as Cas13a (previously known as C2c2) [12, 13], can be activated to engage in “collateral” cleavage of nearby nontargeted RNAs once recognizing its RNA targets. Recombinase polymerase amplification (RPA) can efficiently amplify DNA from single-molecule to 1012 level at a low isothermal state with no need to break the double-stranded structure of DNA [14]. Combined with RPA and T7 RNA polymerase, the CRISPR-Cas13a system can detect RNA rapidly with attomolar (10-18) sensitivity. This technology can be applied for real-time detection of the presence of target RNAs in vitro with signal amplification by non-specific collateral cleavage of nearby nontargeted reporter RNA (Fig. 4) [15]. Therefore, this system is called Specific High-Sensitivity Enzymatic Reporter UnLOCKing (SHERLOCK) based on the amplification of nucleic acid and Cas13a-mediated collateral cleavage of a reporter RNA.
For COVID-19 determination, two specific gRNAs targeting Orf1ab and S gene are used and the sequences are as follows. The SHERLOCK COVID-19 detection protocol only requires 3 steps: ① 25min incubation for isothermal amplification of the extracted nucleic acid sample with RPA kit; ② 30 min incubation for the determination of pre-amplified viral RNA sequence using Cas13 protein; ③ 2 min incubation for visual read out of the detection result with a commercially-available paper dipstick.
| S gene crRNA | 5’-GAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACGCAGCACCAGCUGUCCAACCUGAAGAAG-3’ |
| Orf1ab-crRNA | 5’-GAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACCCAACCUCUUCUGUAAUUUUUAAACUAU-3’ |
| Reporter RNA | 5’-/56-FAM/mArArUrGrGrCmAmArArUrGrGrCmA/3Bio/-3’ |
Table 2. crRNA for SHERLOCK COVID-19 detection
B. CRISPR-based DETECT Lateral Flow Assay
Similarly, another diagnostic tool based on CRISPR is DETECTR (DNA endonuclease-targeted CRISPR trans reporter) system [16, 17]. DETECTR system carry out reverse transcription as well as DNA amplification simultaneously with loop-mediated amplification (RT-LAMP) [18]. The gRNAs are designed to target E gene and N gene of SARS-CoV-2 with the protospacer adjacent motif (PAM) sequence for Cas12 (also known as Cpf1) enzyme [19]. When Cas12 specifically recognizes and binds to the target single-stranded DNA (ssDNA), it will be activated to completely degrade ssDNA molecules non-specifically. DETECTR system is based on the isothermal amplification of target DNA and Cas12-mediated to collateral cleavage the ssDNA probe. The detailed workflow of DETECTR system-based COVID-19 test is shown in Fig. 6 [20], similarly to SHERLOCK system.
| N gene crRNA | 5′-CACAATTTGCCCCCAGCGCTTCAGCGTTCTTCGG-3′ |
| E gene crRNA | 5′-CTTGCTTTCGTGGTATTCTTGCTAGTTACACTAG-3′ |
| Reporter | 5’-/56-FAM/TTATTATT/3Bio/-3’, IDT |
Table 3. crRNA for DETECTR system COVID-19 detection
2) Advantages and disadvantages
Advantages:
① Easy to use and operate in a large scale.
② No requirement of additional equipment.
③ High specificity and sensitivity. Both of the two molecular diagnostic technologies, SHERLOCK and DETECTR, can be used to detect specific RNA/DNA at the attomolar level [16, 17].
④ Cost little time, within 1h.
⑤ Can detect early infection of SARS-CoV-2.
Disadvantages:
There may exist some off-target effects.
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