A CRISPR-enhanced metagenomic NGS test to improve pandemic preparedness

The lack of preparedness for detecting and responding to the SARS-CoV-2 pathogen (i.e. COVID-19) has caused enormous harm to public health and the economy. Testing strategies deployed on a population scale at ‘Day Zero', i.e., the time of the first reported case, would be of significant value. Next Generation Sequencing (NGS) has such capabilities;however, it has limited detection sensitivity for low copy number pathogens. Here we leverage the CRISPR-Cas9 system to effectively remove abundant sequences not contributing to pathogen detection and show that NGS detection sensitivity of SARS-CoV-2 approaches that of RT-qPCR. The resulting sequence data can also be used for variant strain typing, co-infection detection, and individual human host response assessment, all in a single molecular and analysis workflow. This NGS workflow is pathogen agnostic and, therefore, has the potential to transform how large-scale pandemic response and focused clinical infectious disease testing are pursued in the future. Graphical Next generation sequencing could provide ‘Day Zero' testing for pandemic preparedness however, abundant uninformative sequences mask the signal from low level pathogens. Chan et al. establish a method using the CRISPR-Cas system to remove uninformative sequences in vitro to achieve sensitivity and specificity of pathogen detection comparable to RT-qPCR.

A Universal Day Zero Infectious Disease Testing Strategy Leveraging CRISPR-based Sample Depletion and Metagenomic Sequencing (preprint)

The lack of preparedness for detecting the highly infectious SARS-CoV-2 pathogen, the pathogen responsible for the COVID-19 disease, has caused enormous harm to public health and the economy. It took ~60 days for the first reverse transcription quantitative polymerase chain reaction (RT-qPCR) tests for SARS-CoV-2 infection developed by the United States Centers for Disease Control (CDC) to be made publicly available. It then took >270 days to deploy 800,000 of these tests at a time when the estimated actual testing needs required over 6 million tests per day. Testing was therefore limited to individuals with symptoms or in close contact with confirmed positive cases. Testing strategies deployed on a population scale at "Day Zero" i.e., at the time of the first reported case, would be of significant value. Next Generation Sequencing (NGS) has such Day Zero capabilities with the potential for broad and large-scale testing. However, it has limited detection sensitivity for low copy numbers of pathogens which may be present. Here we demonstrate that by using CRISPR-Cas9 to remove abundant sequences that do not contribute to pathogen detection, NGS detection sensitivity of COVID-19 is comparable to RT-qPCR. In addition, we show that this assay can be used for variant strain typing, co-infection detection, and individual human host response assessment, all in a single workflow using existing open-source analysis pipelines. This NGS workflow is pathogen agnostic, and therefore has the potential to transform how both large-scale pandemic response and focused clinical infectious disease testing are pursued in the future.

Conserved Genomic Terminals of SARS-CoV-2 as Co-evolving Functional Elements and Potential Therapeutic Targets

To identify features in the genome of the SARS-CoV-2 pathogen responsible for the COVID-19 pandemic that may contribute to its viral replication, host pathogenicity, and vulnerabilities, we investigated how and to what extent the SARS-CoV-2 genome sequence differs from other well-characterized human and animal coronavirus genomes. Our analyses suggest the presence of unique sequence signatures in the 3'-untranslated region (UTR) of betacoronavirus lineage B, which phylogenetically encompasses SARS-CoV-2, SARS-CoV, as well as multiple groups of bat and animal coronaviruses. In addition, we identified genome-wide patterns of variation across different SARS-CoV-2 strains that likely reflect the effects of selection. Finally, we provide evidence for a possible host microRNA-mediated interaction between the 3'-UTR and human microRNA hsa-miR-1307-3p based on predicted, yet extensive, complementary base-pairings and similar interactions involving the Influenza A H1N1 virus. This interaction also suggests a possible survival mechanism, whereby a mutation in the SARS-CoV-2 3'-UTR leads to a weakened host immune response. The potential roles of host microRNAs in SARS-CoV-2 replication and infection, and the exploitation of conserved features in the 3'-UTR as therapeutic targets warrant further investigation.

The novel Mechanical Ventilator Milano for the COVID-19 pandemic.

This paper presents the Mechanical Ventilator Milano (MVM), a novel intensive therapy mechanical ventilator designed for rapid, large-scale, low-cost production for the COVID-19 pandemic. Free of moving mechanical parts and requiring only a source of compressed oxygen and medical air to operate, the MVM is designed to support the long-term invasive ventilation often required for COVID-19 patients and operates in pressure-regulated ventilation modes, which minimize the risk of furthering lung trauma. The MVM was extensively tested against ISO standards in the laboratory using a breathing simulator, with good agreement between input and measured breathing parameters and performing correctly in response to fault conditions and stability tests. The MVM has obtained Emergency Use Authorization by U.S. Food and Drug Administration (FDA) for use in healthcare settings during the COVID-19 pandemic and Health Canada Medical Device Authorization for Importation or Sale, under Interim Order for Use in Relation to COVID-19. Following these certifications, mass production is ongoing and distribution is under way in several countries. The MVM was designed, tested, prepared for certification, and mass produced in the space of a few months by a unique collaboration of respiratory healthcare professionals and experimental physicists, working with industrial partners, and is an excellent ventilator candidate for this pandemic anywhere in the world.

Conserved Genomic Terminals of SARS-CoV-2 as Coevolving Functional Elements and Potential Therapeutic Targets.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected over 40 million people worldwide, with over 1 million deaths as of October 2020 and with multiple efforts in the development and testing of antiviral drugs and vaccines under way. In order to gain insights into SARS-CoV-2 evolution and drug targets, we investigated how and to what extent the SARS-CoV-2 genome sequence differs from those of other well-characterized human and animal coronavirus genomes, as well as how polymorphic SARS-CoV-2 genomes are generally. We ultimately sought to identify features in the SARS-CoV-2 genome that may contribute to its viral replication, host pathogenicity, and vulnerabilities. Our analyses suggest the presence of unique sequence signatures in the 3' untranslated region (3'-UTR) of betacoronavirus lineage B, which phylogenetically encompasses SARS-CoV-2 and SARS-CoV as well as multiple groups of bat and animal coronaviruses. In addition, we identified genome-wide patterns of variation across different SARS-CoV-2 strains that likely reflect the effects of selection. Finally, we provide evidence for a possible host-microRNA-mediated interaction between the 3'-UTR and human microRNA hsa-miR-1307-3p based on the results of multiple computational target prediction analyses and an assessment of similar interactions involving the influenza A H1N1 virus. This interaction also suggests a possible survival mechanism, whereby a mutation in the SARS-CoV-2 3'-UTR leads to a weakened host immune response. The potential roles of host microRNAs in SARS-CoV-2 replication and infection and the exploitation of conserved features in the 3'-UTR as therapeutic targets warrant further investigation.IMPORTANCE The coronavirus disease 2019 (COVID-19) outbreak is having a dramatic global effect on public health and the economy. As of October 2020, SARS-CoV-2 has been detected in over 189 countries, has infected over 40 million people, and is responsible for more than 1 million deaths. The genome of SARS-CoV-2 is small but complex, and its functions and interactions with human host factors are being studied extensively. The significance of our study is that, using extensive SARS-CoV-2 genome analysis techniques, we identified potential interacting human host microRNA targets that share similarity with those of influenza A virus H1N1. Our study results will allow the development of virus-host interaction models that will enhance our understanding of SARS-CoV-2 pathogenesis and motivate the exploitation of both the interacting viral and host factors as therapeutic targets.

Conserved Genomic Terminals of SARS-CoV-2 as Co-evolving Functional Elements and Potential Therapeutic Targets.

To identify features in the genome of the SARS-CoV-2 pathogen responsible for the COVID-19 pandemic that may contribute to its viral replication, host pathogenicity, and vulnerabilities, we investigated how and to what extent the SARS-CoV-2 genome sequence differs from other well-characterized human and animal coronavirus genomes. Our analyses suggest the presence of unique sequence signatures in the 3'-untranslated region (UTR) of betacoronavirus lineage B, which phylogenetically encompasses SARS-CoV-2, SARS-CoV, as well as multiple groups of bat and animal coronaviruses. In addition, we identified genome-wide patterns of variation across different SARS-CoV-2 strains that likely reflect the effects of selection. Finally, we provide evidence for a possible host microRNA-mediated interaction between the 3'-UTR and human microRNA hsa-miR-1307-3p based on predicted, yet extensive, complementary base-pairings and similar interactions involving the Influenza A H1N1 virus. This interaction also suggests a possible survival mechanism, whereby a mutation in the SARS-CoV-2 3'-UTR leads to a weakened host immune response. The potential roles of host microRNAs in SARS-CoV-2 replication and infection, and the exploitation of conserved features in the 3'-UTR as therapeutic targets warrant further investigation.

Conserved Genomic Terminals of SARS-CoV-2 as Co-evolving Functional Elements and Potential Therapeutic Targets (preprint)

ABSTRACTTo identify features in the genome of the SARS-CoV-2 pathogen responsible for the COVID-19 pandemic that may contribute to its viral replication, host pathogenicity, and vulnerabilities, we investigated how and to what extent the SARS-CoV-2 genome sequence differs from other well-characterized human and animal coronavirus genomes. Our analyses suggest the presence of unique sequence signatures in the 3’-untranslated region (UTR) of betacoronavirus lineage B, which phylogenetically encompasses SARS-CoV-2, SARS-CoV, as well as multiple groups of bat and animal coronaviruses. In addition, we identified genome-wide patterns of variation across different SARS-CoV-2 strains that likely reflect the effects of selection. Finally, we provide evidence for a possible host microRNA-mediated interaction between the 3’-UTR and human microRNA hsa-miR-1307-3p based on predicted, yet extensive, complementary base-pairings and similar interactions involving the Influenza A H1N1 virus. This interaction also suggests a possible survival mechanism, whereby a mutation in the SARS-CoV-2 3’-UTR leads to a weakened host immune response. The potential roles of host microRNAs in SARS-CoV-2 replication and infection, and the exploitation of conserved features in the 3’-UTR as therapeutic targets warrant further investigation.Competing Interest StatementThe authors have declared no competing interest.View Full Text