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

The current coronavirus disease 2019 (COVID-19) pandemic is caused by the global circulation of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is highly transmissible and contagious. Prompt identification of the virus was required for epidemic prevention, as well as controlling the spread of the virus.

Study: A smartphone-based visual biosensor for CRISPR-Cas powered SARS-CoV-2 diagnostics. Image Credit: CI Photos / Shutterstock.com

Background

Quantitative real-time polymerase chain reaction (qPCR) has been the most popular method for the detection of SARS-CoV-2. To date, this method has been standardized by the World Health Organization (WHO) for the diagnosis of COVID-19.

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

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

Furthermore, the applications of plasmonic gold nanoparticles (AuNPs) in biosensing have gained importance in recent years. Several advantages of smartphones such as their interactivity, portability, and cameras, have also been found to be important in biosensing. The coupling of smartphones with biosensing provides a field-deployable and user-friendly analytical device.

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 findings

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

If the ssDNA is labeled with a fluorophore (F) at 5′ and a quencher (Q) at 3’ ends, the cleavage led to an unquenched state. This resulted in enhanced fluorescent signals that were used for the quantitative analysis of the target dsDNA. Additionally, a linker ssDNA was used to hybridize with pre-made AuNPs-DNA probe pairs via complementary base pairing.

In the absence of target DNA, the linker ssDNA would remain uncut, leading to hybridization-induced aggregation of the AuNPs probes that could undergo “pulldown.” This resulted in a redshift in their absorbance, turning the solutions colorless after centrifugation.

In the presence of target DNA, cleavage took place and there was no aggregation of AuNPs probes. This resulted in the solution being colored after centrifugation.

These color changes could be captured by a smartphone installed with Color Picker App or the naked eye. Thus, the detection of SARS-CoV-2 took place based on the color changes.

Is CRISPR-Cas12a feasible 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 aligned to a few of the related coronaviruses including the severe acute respiratory syndrome (SARS-CoV), Middle East respiratory syndrome (MERS-CoV), and human coronaviruses (HumanCoV) for evaluation of their specificity.

Following this, some preliminary experiments were carried out on the plasmid that contained the N gene fragment of SARS-CoV-2. The results of the experiment indicated that trans-cleavage of Cas12a was triggered only in the presence of the target N gene dsDNA amplicons and crRNA.

Furthermore, a selectivity assay was designed that showed that the fluorescence intensities of only the SARS-CoV-2 samples were increased. This demonstrated that the selectivity was high without cross-reaction of non-SARS-CoV-2 targets.

Moreover, detection of SARS-CoV-2 RNA took place with the help of the Cas12a-crRNA complex. The results indicated that there was a linear relationship between the RNA and fluorescence intensities.

Detection of SARS-CoV-2 was stimulated by the production of lentiviruses harboring 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. Additionally, it was also found that the performance of the CRISPR-Cas12a fluorescent assay was quite comparable with that of the traditional qRT-PCR analysis.

To carry out rapid detection of SARS-CoV-2, a visual detection method was developed that was independent of the microplate reader and could be easily read with the help of a smartphone. Therefore, the proposed biosensor was tested with pseudoviruses that contained the N fragment.

The results indicated that the tube devoid of SARS-CoV-2 specific nucleic acids was colorless after centrifugation. Comparatively, the tube that contained SARS-CoV-2 specific nucleic acids displayed a grade of color changes that were dependent on the concentration of the SARS-CoV-2.

Amplification-free detection of SARS-CoV-2 pseudoviruses indicated the limit of detection (LOD) to be 10⁶ copy/microliter (μL), which was quite higher than the amplification adopted detection. Furthermore, a linear correlation existed between the concentration of the pseudovirus. Lightness values were obtained by the smartphone camera.

Moreover, the CRISPRCas12a fluorescent and visual biosensors were found to have 100% consistency with qPCR. The area under curve value (AUV) was better for this method as compared to qPCR.

Repeatability and reproducibility are considered important for biosensor development. Investigation of the relative standard deviations (RSD) values was found to be less than 6%, which corresponded with acceptable repeatability and reproducibility.

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

The current study involved 50 clinical respiratory samples, out of which 20 were affected by COVID-19, and 30 were obtained from healthy subjects. The RNA samples obtained from all the subjects were reversely transcribed and amplified by PCR.

The PCR products were then tested by CRISPR-Cas12a powered visual biosensor. The results indicated 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 quite effective in determining the potential of the CRISPR-Cas12a visual biosensor in the detection of SARS-CoV-2, it had certain limitations.

Firstly, PCR amplification requires thermal cycles that could be replaced by some isothermal amplification technology. Secondly, the biosensor comprised multistep liquid transfer that could be integrated into a single-pot reaction, which would help minimize instrumentation and simplify the procedure.

Written by

Suchandrima Bhowmik

Suchandrima has a Bachelor of Science (B.Sc.) degree in Microbiology and a Master of Science (M.Sc.) degree in Microbiology from the University of Calcutta, India. The study of health and diseases was always very important to her. In addition to Microbiology, she also gained extensive knowledge in Biochemistry, Immunology, Medical Microbiology, Metabolism, and Biotechnology as part of her master's degree.