Scientists Further Powerful Gene Editing Tech’s Caliber with Rapid, Low Cost COVID-19 Diagnosis

By Sahana Shankar, Ph.D. Candidate

Traditionally, coronavirus infections are identified by either RT-PCR, which amplifies and quantifies the viral genetic material from a patient sample, or neutralizing immunoglobulins in the host (antibodies), or sequencing the patient genome to look for viral nucleic acid sequences (antigen). These tests are sufficiently sensitive and specific. However, they require trained personnel, specific molecular reagents, and infrastructure. With healthcare systems overwhelmed due to the COVID pandemic, researchers have been working on rapid tests that can be applied to diagnose large populations. Despite their low sensitivity, antibody and serology tests have become popular in the past few months to help screen people during the pandemic.

In due course, scientists began to explore ways to combat COVID-19 through the powerful gene-editing technology of CRISPR/Cas9. Even before this pandemic, way back in 2017, Harvard and MIT researchers, including Feng Zhang, developed a version of CRISPR called SHERLOCK to detect viruses like Zika and Dengue. In the following year, Jennifer Doudna’s lab developed DETECTR to detect the HPV virus. Since the onset of COVID-19, these systems were upgraded to combat the spread of SARS-CoV-2. Similarly, companies such as Mammoth Biosciences developed their CRISPR-based SARS-CoV-2 diagnostic tests for which it received the NIH’s Rapid Acceleration of Diagnostics (RADx) program.

Now, a research team from China has developed a new rapid diagnostic assay for COVID-19 using CRISPR to bridge the gap between highly sensitive RT-PCR and rapid, high-throughput antibody tests. This assay can detect the SARS-CoV-2 virus with high specificity and sensitivity without the requirement of thermocyclers and reagents.

CRISPR/Cas9 Gene Editing

CRISPR, which stands for “clusters of regularly interspaced short palindromic repeats,” was discovered in bacteria and archaea as an immune system against viral attacks. It is a specialized region of DNA harboring nucleotide repeats and spacers which are replaced with virus DNA to memorize previous viral encounters. This way, a secondary infection could be precisely identified by comparing sequence similarity with the spacer region. The CRISPR machinery then generates a copy of the spacer that binds complementary to the viral DNA and induces efficient viral clearance through Cas enzymes. This naturally occurring phenomenon was adopted by scientists, and today gene editing is made possible by guiding the Cas enzyme to one’s gene of interest through a complimentary guide RNA.

CRISPR/Cas has emerged as a powerful tool for gene editing and targeting in the past decade. Widely used in research for creating mutant and deletion cells, scientists have recently harnessed CRISPR for in vitro diagnosis of nucleic acids with the Gene-Xpert assay, which can detect mycobacteria in lung fluids for tuberculosis diagnosis. In the present study, published in PLOS Pathogens on August 27, 2020, researchers from Vision Medicals Center for Infectious Diseases, Guangdong, China, have combined polymerase-based amplification of viral DNA and CRISPR/Cas-mediated signal detection. This assay provides high specificity due to the amplification of SARS-COV-2-specific DNA and improved sensitivity due to the enzymatic signal of CRISPR/Cas.

The authors analyzed the SARS-CoV-2 sequences from COVID-19 patients to compare and identify the variability and consensus regions among different SARS-CoV-2 viral strains that could be used to design a CRISPR probe. They identified three regions as potential target sequences – two in the Orf1ab gene and the other in the N gene. These sequences were differentiable from other pathogenic coronaviruses. These regions were used to design RPA primers and guide RNAs for Cas13a enzymes. The Orf1ab region was the most suitable for targeting and amplification.

The CRISPR-COVID system was tested for specificity by challenging it with a wide panel of microbes commonly found in human respiratory disorders such as mycobacterium, adenovirus, influenza virus, and other human coronaviruses. The assay could consistently detect SARS-CoV-2 at near-single copy sensitivity. The authors then confirmed clinical diagnostic potential by analyzing 114 samples, which were obtained from COVID-19 patients, human CoV cases, and healthy subjects, along with appropriate positive and negative controls. The test detected COVID cases at 100% sensitivity with no false positives.

Higher Sensitivity, Lower Cost

Comparing the CRISPR-COVID test with RT-PCR and sequencing-based diagnostic tests showed better turn-around time (40min) than sequencing and higher specificity and sensitivity than PCR, suggesting that CRISPR-based test can be used in cases with low viral loads. The assay can be scaled up to offer diagnostic tests at lower costs.

The authors concede that the CRISPR-COVID test shares similarities with other molecular tests in sample collection and RNA extraction, and viral strains with mutations in the probe region may limit the sensitivity of the assay. This study provides a comparison of various diagnostic platforms available for COVID detection, which can guide resource optimization to improve our response to the current public health crisis due to COVID-19.

Editor: Rajaneesh K. Gopinath, Ph.D.

Related Article: COVID-19: An Improved ELISA Test for Rapid Antibody Detection Provides Edge Over Current Players

References

1. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5556276/
2. https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1008705

Related posts:

COVID-19 Antibody: Vir Biotechnology Partners with Samsung Biologics for Large Scale Manufacturing Human Monoclonal Antibody Breakthroughs Provide New Hope against COVID-19 COVID-19 Vaccine: Novavax Gets Funding from the US Department of Defense A Secondary Route of Viral Entry Could Explain Enhanced Pathogenicity of COVID-19

Author

Sahana Shankar