Role of the CRISPR-Cas12a visual biosensor in the detection of SARS-CoV-2

The current 2019 coronavirus disease (COVID-19) pandemic is caused by the worldwide spread of the highly communicable and contagious severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2). Rapid identification of the virus was required to prevent the epidemic and control the spread of the virus.

To learn: A smartphone-based visual biosensor for CRISPR-Cas-based SARS-CoV-2 diagnosis. Image source: CI Photos / Shutterstock.com

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The real-time quantitative polymerase chain reaction (qPCR) is the most popular method for detecting SARS-CoV-2. This method has so far been standardized by the World Health Organization (WHO) for the diagnosis of COVID-19.

Recently, the clustered, regularly arranged short palindromic repeats (CRISPR) together with CRISPR-associated genes (Cas) have shown enormous potential in biosensors. Certain CRISPR-Cas systems, such as Cas13a and Cas12a, have been found to perform non-specific nucleic acid cutting activities upon detection of a target sequence. This activity is also known as trans-cleavage activity, which led to the detection of nucleic acids with high sensitivity and selectivity.

Recent advances have been made in the detection of SARS-CoV-2 using CRISPR-Cas13a or CRISPR-Cas12a, coupled with either fluorescent signal readings or colorimetric signal readings in paper lateral flow assays. However, the sensitivity and selectivity of these methods require further improvement in order to provide satisfactory performance with clinical samples.

In addition, the applications of plasmonic gold nanoparticles (AuNPs) in biosensors have gained in importance in recent years. Several advantages of smartphones such as interactivity, portability, and cameras have also proven important for biosensors. The coupling of smartphones with biosensors offers a user-friendly analysis device that can be used in the field.

A new study published in the journal Biosensors and Bioelectronics aimed to develop a novel, CRISPR-Cas12a-powered visual biosensor for the detection of SARS-CoV-2.

Study results

Viral ribonucleic acid (RNA) was extracted, reverse transcribed and amplified with the aid of SARS-CoV-2 N gene-specific primers in order to obtain double-stranded deoxyribonucleic acid (dsDNA) amplicons. The Cas12a-crRNA complex recognized the dsDNA amplicons, whereupon the trans cleavage of the ssDNA was initiated.

If the ssDNA was labeled with a fluorophore (F) at the 5 ‘and a quencher (Q) at the 3’ ends, the cleavage resulted in an undeleted state. This resulted in increased fluorescence signals which were used for quantitative analysis of the target dsDNA. In addition, a linker ssDNA was used to hybridize with prefabricated AuNPs-DNA probe pairs via complementary base pairing.

Without target DNA, the ssDNA of the linker would remain uncut, which leads to a hybridization-induced aggregation of the AuNPs probes, which could undergo a “pulldown”. This led to a red shift in their absorbance, as a result of which the solutions became colorless after centrifugation.

In the presence of target DNA, cleavage occurred and there was no aggregation of AuNPs probes. As a result, the solution was colored after centrifugation.

These color changes could be detected by a smartphone with the Color Picker app installed or by the naked eye. SARS-CoV-2 was thus detected on the basis of the color changes.

Is CRISPR-Cas12a suitable for the detection of SARS-CoV-2?

To determine the feasibility of CRISPR-Cas12a-based detection, cRNA was designed to correspond to part of the nucleocapsid (N) gene. The designed cRNA and PCR primers were highly conserved and targeted to some of the related coronaviruses, including Severe Acute Respiratory Syndrome (SARS-CoV), Middle East Respiratory Syndrome (MERS-CoV), and Human Coronavirus (HumanCoV) to assess theirs Specificity.

Then some preliminary tests were carried out with the plasmid which contained the N gene fragment of SARS-CoV-2. The results of the experiment showed that the trans cleavage of Cas12a was only triggered in the presence of the dsDNA amplicons and the crRNA of the target N gene.

In addition, a selectivity assay was developed which showed that the fluorescence intensities were only increased in the SARS-CoV-2 samples. This showed that the selectivity with no cross-reactivity from non-SARS-CoV-2 targets was high.

In addition, SARS-CoV-2 RNA was detected using the Cas12a-crRNA complex. The results showed that there was a linear relationship between the RNA and the fluorescence intensities.

The detection of SARS-CoV-2 was stimulated by the production of lentiviruses that harbor genomic fragments (N-gene) of SARS-CoV-2. The results of the assay indicated that the trans cleavage of CRISPR-Cas12a could be useful for the detection of SARS-CoV-2. In addition, it was also found that the performance of the CRISPR-Cas12a fluorescence assay was quite comparable to that of traditional qRT-PCR analysis.

For the quick detection of SARS-CoV-2, a visual detection method was developed that is independent of the microplate reader and can be easily read with the help of a smartphone. Therefore, the proposed biosensor was tested with pseudoviruses containing the N fragment.

The results showed that the tube without SARS-CoV-2-specific nucleic acids was colorless after centrifugation. In comparison, the tube containing SARS-CoV-2 specific nucleic acids showed a degree of color change that was dependent on the concentration of the SARS-CoV-2.

The amplification-free detection of SARS-CoV-2 pseudoviruses resulted in a detection limit (LOD) of 106 copies / microliter (μl), which was significantly higher than the amplified detection. There was also a linear correlation between the concentration of the pseudovirus. Brightness values ​​were determined by the smartphone camera.

In addition, the CRISPRCas12a fluorescent and visual biosensors were found to be 100% consistent with qPCR. The area under the curve (AUV) was better with this method than with qPCR.

Repeatability and reproducibility are considered important for biosensor development. The relative standard deviation (RSD) study found less than 6%, which was acceptable repeatability and reproducibility.

Two different smartphones with different cameras were used to determine if the difference in light and camera could cause discrepancies in the results. Although the two cameras produced slight deviations, they were not statistically significant. This indicated that the proposed biosensor could be used with different smartphones and different lighting conditions.

The current study included 50 clinical airway samples, 20 of which were affected by COVID-19 and 30 were from healthy subjects. The RNA samples obtained from all subjects were reverse transcribed and amplified by PCR.

The PCR products were then tested with a CRISPR-Cas12a powered visual biosensor. The results showed that the CRISPR-Cas12a visual biosensor was able to correctly identify and differentiate the 50 positive and negative samples just like the traditional qPCR results.

limitations

Although the current study was able to determine the potential of the visual biosensor CRISPR-Cas12a in the detection of SARS-CoV-2 quite effectively, it had certain limitations.

First, PCR amplification requires thermal cycling, which could be replaced by isothermal amplification technology. Second, the biosensor included a multi-stage fluid transfer that could be incorporated into a one-pot reaction, which would help minimize instrumentation and simplify the process.

Journal reference:

  • Ma, L., Yin, L., Li, X. et al. (2021). A smartphone-based visual biosensor for CRISPR-Cas-based SARS-CoV-2 diagnosis. Biosensors and Bioelectronics 195. doi: 10.1016 / j.bios.2021.113646.

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