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Authors: Ding et al.

Link to paper: https://www.biorxiv.org/content/10.1101/2020.03.19.998724v1

Journal/ Pre-Print: biorxiv

Tags: Diagnostics, Molecular biology

Research Highlights 

1. A rapid, sensitive, single-reaction CRISPR-Cas12a detection system termed AIOD-CRISPR.

2. Assay utilized to detect SARS-CoV2-2 cDNA and HIV RNA and DNA with high sensitivity.

3. Assay achieved comparable sensitivity to RT-PCR method using HIV-1 RNA from human plasma samples.

Summary 

Ding et al. present a “All-In-One Dual CRISPR-Cas12a” (AIOD-CRISPR) assay for effective detection of viral nucleic acids. Dual crRNAs detect viral nucleic acids in isolated DNA/RNA and activate Cas12a to cleave nearby ssDNA-FQ fluorescence reporters. Whole assay run in one pot at 37oC in 40mins with a visual output. The assay shows it can successfully detect DNA and RNA of HIV and cDNA of SARS-CoV-2 at high sensitivity. The assay has comparable sensitivity and speed to RT-PCR methods for detection of HIV-1 RNA extracted from human plasma samples. Assay requires DNA/RNA isolation and is probably fairly expensive to implement.

Impact for SARS-CoV2/COVID19 research efforts

Develop diagnostic tools for SARS-CoV2/COVID19

Study Type

· In vitro study

Strengths and limitations of the paper

Novelty:

Individual components of the reaction are not novel; however, this assay uses a single reaction system compared to previous CRISPR-based nucleic acid detection.

Standing in the field:

RNA-guided CRISPR-Cas nuclease-based nucleic acid detection has shown significant promise in other studies. Use of collateral cleavage of Cas12 and Cas13 has been described in several methods – SHERLOCK and DETECTR – for highly sensitive nucleic acid detection.

Appropriate statistics:

Unpaired two-tailed t test used on technical triplicates, a non-parametric test would have been more appropriate. Some experiments do not use statistics as appears to be only 1 repeat.

Viral model used:

HIV-1 and SARS-CoV-2 nucleic acids/cDNA plasmids

Translatability:

Effective diagnostic method in vitro but no in vivo or clinical studies yet so presently far from translatable.

Main limitations:

· Seems to be predominately a qualitative not quantitative method.

· No negative or positive control for the assay.

· Repeats of some experiments vary dramatically, giving very large errors.

· Some experiments do not have error bars/statistics.

· Different cps’ used between different experiments.

· No SARS-CoV-2 patient or clinical work carried out so no indication of how this works from cell extracts or different stages of diseases.

· No information about specificity of assay with plasma samples.

· Plasma sample test showed it is no more sensitive or quicker than RT-PCR method so may not hold much advantage.

· No information regarding cost in comparison to other methods.