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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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. microbial composition at two test sites Detection Sensitivity Methods Covid positive samples with RT-PCR Ct values from 16-39 were processed through the CRISRP enhanced mNGS pipeline. Data processing workflow 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. Disclosures All Authors: No reported disclosures.

Background The COVID-19 pandemic has brought awareness to the dangers of emerging pathogens to global human health and welfare. Sensitivity and flexibility are important features for methods used to detect emerging pathogens. While PCR testing is rapid and sensitive, a significant advantage next generation sequencing (NGS) approaches have over PCR-based analyses is the ability to detect previously undiscovered pathogens while also providing genomic information that can detect SARS-CoV-2 genome sequence, identify source of co-infection, and the host transcriptional response in a single workflow. The critical component enabling this approach is Jumpcode CRISPRclean technology which removes abundant human and bacterial ribosomal RNA sequences from NGS libraries. CRISPRclean workflow easily integrates into next generation sequencing projects Schematic of the Jumpcode CRISPRclean protocol Methods CRISPRclean was applied to contrived infected tissue samples including human lung RNA spiked with serially diluted amounts of SARS-CoV-2 RNA and bacterial RNA. NEB RNA libraries were prepared and treated with CRISPRclean protocol, then sequenced on Illumina instruments. Data analysis was performed using Jumpcode proprietary software to measure alignment and depletion rates, the Silva database for rRNA read alignment, and Kraken2 and CosmosID pipelines for k-mer based metagenomic investigation. Fold enrichment of SARS-CoV-2 reads after CRISPRclean depletion of libraries prepared from contrived samples. CRISPRclean treatment of the fully contrived samples increases the fraction of reads that map to the SARS-CoV-2 genome by an average of ~10-fold Results CRISPRclean treatment of the contrived samples increases ~10 fold of reads that map to the SARS-CoV-2 genome. For the 60 viral copies of SARS-CoV-2 sample, the number of reads mapping to the SARSCoV-2 genome increases from ~10,000 reads to ~70,000 reads. A similar increase in reads occurs for S. aureus. The percentage of SARS-CoV-2 genome covered at 1X and 10X also increases. Similar results were achieved even after downsampling the datasets to 5M reads. There is a ~4-fold increase in bacterial species detection in these stool samples after CRISPRclean treatment. Percentage of SARS-CoV-2 genome covered at 1X and 10X increases as a result of rRNA depletion. Coverage of the SARS-CoV-2 genome at 50 million reads. Number of reads aligning to the S. aureus and SARS-CoV-2 genomes increases after CRISPRclean depletion. For the sample containing 0.0001% SARS-CoV-2, (60 viral copies), the number of reads mapping to the SARS-CoV-2 genome increases from ~10,000 reads to ~70,000 reads. CosmosID Shotgun Metagenomics Analysis heat map showing read alignments to viral genomes. The yellow color indicates high read counts. The CosmosID shotgun metagenomic analysis software was used to analyze the sequencing data, classify the sequences and generate the heat map. Conclusion Metatranscriptomics powered by CRISPR-mediated rRNA depletion offers a robust methodology to acquire viral genomic data, microbiome composition, co-infection information, and the transcriptional status of the host immune response in a single workflow. This sequencing-based approach can be available on the first day of the next viral outbreak and should be considered as a first-line test for novel zoonotic virus detection. Bacterial species composition of patient stool samples before and after CRISPRclean depletion. ~4-fold increase in bacterial species detection in these stool samples after CRISPRclean treatment. Sequencing data downsampled to 20 million reads. Disclosures Keith Brown, n/a, Jumpcode Genomics (Board Member, Employee, Shareholder)

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