Single-cell transcriptomics suffers from lapses in coverage of the full transcriptome, providing an incomplete gene expression profile of the cell. Here, we introduce single-cell CRISPRclean (scCLEAN), an in vitro molecular method that can be universally inserted into any single-cell RNA-seq workflow to improve the sensitivity of the assay. Utilizing CRISPR/Cas9, scCLEAN works to selectively remove highly abundant uninformative molecules, redistributing ~50% of reads to enrich for lowly expressed transcripts. Utilizing immune cells, we describe a validation of scCLEAN showing a 2.1-fold enrichment in library complexity with negligible off-target effects. Subsequently, applying scCLEAN to single-cell iso-seq samples results in a 4.6-fold improvement in unique isoform detection. Thus, demonstrating a benefit in short and long read sequencing applications. Finally, we illustrate the ability of scCLEAN to elucidate biological insights by applying it to two participant cohorts of cardiovascular samples, bringing to light novel molecular characteristics including inflammatory signatures.

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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.SIGNIFICANCE STATEMENTThe lack of preparedness for detecting infectious pathogens has had a devastating effect on the global economy and society. Thus, a ‘Day Zero’ testing strategy, that can be deployed at the first reported case and expanded to population scale, is required. Next generation sequencing enables Day Zero capabilities but is inadequate for detecting low levels of pathogen due to abundant sequences of little biological interest. By applying the CRISPR-Cas system to remove these sequences in vitro, we show sensitivity of pathogen detection equivalent to RT-qPCR. The workflow is pathogen agnostic, and enables detection of strain types, co-infections and human host response with a single workflow and open-source analysis tools. These results highlight the potential to transform future large-scale pandemic response.