Credit: NIH
The first confirmed case of SARS-CoV-2 occurred around December 1, 2019. From there, it took about 42 days for the SARS-CoV-2 viral genome sequence to be publicly released, and another month for the first 90 initial test kits to be given to high-risk individuals. Over the course of the next 6 to 8 months, the U.S. was able to produce approximately 800,000 tests per day—when the estimated need was 6 million per day.
To say the world was not ready for a global pandemic in 2020 would be an understatement. While the world was eventually able to get COVID-19 under control, it was obvious better planning was needed in the case of another—likely—pandemic. In 2021, the White House launched a $65.3 billion plan to transform the way the United States responds to pandemics in part by vastly accelerating vaccine development, testing and production.
And while vaccines are certainly a critical part of the plan, Azeem Siddique, director of research & development at Jumpcode Genomics, says there’s more to it.
“What is necessary to enable the proposed vision of pandemic preparedness is testing and response strategies that are deployable at day zero to combat any future pathogen outbreak before it progresses to a pandemic,” said Siddique. “Such an approach, by necessity, needs to be pathogen agnostic and, ideally, would provide more detailed information about an individual’s condition than the mere presence of a pathogen.”
Siddique believes next-generation sequencing (NGS) can fulfill those requirements—with some adaptation.
In a paper published in Cell Reports Methods, scientists from Jumpcode Genomics and The Translational Genomics Research Institute describe the development of a CRISPR-enhanced metagenomic NGS test to improve pandemic preparedness.
The researchers developed the new protocol to overcome the limitations they saw when trying to apply NGS methods during the COVID-19 pandemic. The foremost hinderance was that traditional NGS-based metagenomic tests for COVID-19 were inefficient because of relatively large sample input requirements and lower sensitivity for the virus due to abundant, uninformative nucleic acid molecules. This new method, however, overcomes that limitation by using a CRISPR system to specifically target and remove abundant host and microbial ribosomal RNA (rRNA) sequences.
“Uninformative material often makes up the vast majority of content of sample and, unfortunately, data from an infectious agent, can easily be swamped by uninformative data. Removal of uninformative sequences before they are sequenced allows one to focus on the pathogen sequences of interest and reduce sequencing costs,” explained Siddique.
Jumpcode’s CRISPR-NGS approach uses CRISPR-Cas technology to cleave and remove sequencing library fragments of little interest to researchers prior to sequencing. The study results show the “CRISPRclean kit” exhibited a 70% and 61% reduction in rRNA-aligned reads at 5 and 50 ng RNA inputs, respectively, and was 15 to 18% more efficient at removing rRNA (eukaryotic and bacterial) than the current gold standard of RiboZero Plus, which reports a 52% and 46% reduction in rRNA-aligned reads. In addition, nearly 2x the number of bacterial species were identified in the CRISPRclean-treated samples compared with RiboZero Plus.
Importantly, the study results also showed that the sensitivity and specificity of the CRISPR-NGS method are comparable with the gold-standard RT-qPCR. According to the findings, the sensitivity was 97% for non-depleted and 100% for depleted samples, while the specificity was 100% for non-depleted and 100% for depleted samples. Thus, the results show the characteristics and clinical relevance of the CRISPRclean NGS assay is equal to that of RT-qPCR. In addition, the gain in sensitivity was primarily realized with low viral load samples, which are challenging for mNGS to resolve without CRISPRclean depletion. For example, genome coverage increased to over 85% in 11 (73%) of 15 low-viral-load samples.
Day Zero application
With sensitivity at least comparable if not better than the current gold standard, the researchers envision the application of CRISPR-NGS technology on “Day Zero” of future infectious disease outbreaks.
“Since NGS provides information on the total nucleic acid content of a sample, it allows researchers to identify pathogens without prior knowledge of sample composition or the pathogen itself. This makes it ideal for deployment on Day Zero of a pandemic when little or nothing is known about the infectious agent,” said Siddique.
Additionally, the single-test CRISPR-NGS approach also provides information on co-infections and the human immune response—both of which make it a highly attractive option for Day Zero deployment.
NGS-based sample testing also already exists in many laboratories and facilities throughout the U.S., so the infrastructure is there—or at least the bones of it. Unfortunately, fewer locations have the capabilities to handle potentially infectious samples, nor have streamlined wet-lab and analysis workflows specifically for pathogen detection. Another hindrance would be transportation logistics. Moving samples from areas of outbreaks, especially rural regions in resource-poor countries, and ensuring that they get to central testing and sequencing facilities in a timely manner without loss or degradation still remains one of the most challenging aspects of fighting a pandemic.
“But, with the aid of government investment, the NGS community is working to enable faster, cheaper NGS workflows with push-button analyses that are also easily scalable so that they can handle large-scale patient screening once an outbreak occurs,” said Siddique. “This means that depending on the location of an outbreak and its accessibility to public health officials, NGS methods today could be deployed within as little as a few days after an infectious disease outbreak.”
Additional pathogens
The study authors say their approach is applicable across a wide range of genomic applications. The study showed the strategy can detect other viral pathogens, emphasizing the pathogen-agnostic nature of the mNGS approach.
For example, several of the non-SARS-CoV-2 viruses identified in the study are associated with respiratory illness. Previous literature has suggested that rhinovirus can block or inhibit SARS-CoV-2 replication in lung epithelial cells by triggering an interferon response, so this information could be useful to predict outcome or severity of disease.
Only NGS enables strain characterization, identification of clinically relevant variants, information on co-infections, and host response expression patterns.
“Although both RT-qPCR and amplicon-based targeted sequencing technologies are important tools for detecting and tracking pathogens as they evolve and will continue to have a vital role for routine detection, clearly neither can meet the Day Xero requirement for a novel zoonotic pathogen. Our approach provides the added benefit of generating whole-genome sequence information, which is of crucial importance when dealing with a novel zoonotic virus,” conclude the study authors.