A CRISPR-enhanced metagenomic NGS test to improve pandemic preparedness

The lack of preparedness for detecting and responding to the severe acute respiratory syndrome coronavirus 2 (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 work flow 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. [Display omitted] •Abundant sequences can be removed from sequencing libraries using CRISPR-Cas9•Sensitivity and specificity of SARS-CoV-2 detection are comparable with RT-qPCR•Data can also be used for strain typing, co-infection detection, and human host response•The NGS work flow can potentially transform infectious disease testing and response RT-qPCR is the gold-standard method for detection of pathogens such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, it lacks day-zero capabilities where the detection method can be deployed at the first reported case and expanded to population scale if needed. Such assays can be developed by incorporating viral genetic information using methods such as next-generation sequencing. However, next-generation sequencing is inadequate for detecting low levels of pathogen due to abundant sequences of little biological interest. We present a work flow where the CRISPR-Cas system is used to remove uninformative sequences in vitro to achieve sensitivity of pathogen detection comparable with RT-qPCR while providing day-zero capabilities. 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 pathog