A Universal 'Day Zero' Infectious Disease Testing Strategy Exploiting Crispr-Based Sample Depletion And Metagenomic Sequencing

ABSTRACT

Background. The lack of preparedness for detecting the highly infectious SARS-CoV-2 pathogen - the pathogen responsible for the COVID-19 disease - caused enormous harm to the public health, the economy and society as a whole. It took ~60 days for the first RT-PCR tests for SARS-CoV-2 infection developed by the United States Centers for Disease Control (CDC) to be made 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 only individuals with symptoms or individuals in close contact with confirmed positive cases. Testing strategies that can be deployed on a population scale at 'day zero' (i.e., at the time of the first reported case) are needed. Next Generation Sequencing (NGS) has such day zero capabilities with the potential to enable feasible and broad large-scale testing strategies, however it has limited detection sensitivity for low copy numbers of pathogens which may be present. Here we demonstrate that using CRISPR-Cas9 to remove abundant sequences that do not contribute to pathogen detection, NGS detection sensitivity is equivalent to RT-PCR. 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 radically transform how both very large-scale pandemic response and focused clinical infectious disease testing are pursued in the future. Methods. Covid positive samples with RT-PCR Ct values from 16-39 were processed through the CRISRP enhanced mNGS pipeline. Results. Sn/Sp compared to RT-PCR was 97%/100%. Strain calling concordance compared to amplicon sequencing was 100%. Co-infections from Covid positive samples were identified with high confidence. Host response signatures match the published literature. Conclusion. Applying CRISPR enhanced metagenomic NGS at Day Zero of the next pandemic can mitigate the time gap in developing approved diagnostics at population scale and potentially save lives.