| dc.description.abstract | Recent and current outbreaks of Ebola Virus Disease in West Africa and the Democratic Republic of Congo highlight the need to understand the disease and its pathophysiology. Single-Cell RNA sequencing (scRNA-seq) is a new development that can deeply characterize how the host’s different cell-types modulate their gene expression in response to infection. Recently, our lab used scRNA-seq to study how Ebola virus disease attacks host cell machinery, the first such study for a Risk Group 4 (RG-4) pathogen. ScRNA-seq was tested on blood samples from nonhuman primates infected with Ebola virus, but an adapter artifact from the scRNA library preparation over-amplified in many libraries, and reduced the quantity and quality of meaningful reads. In this study, I use CRISPR-Cas9 to target and degrade this artifact, leaving cDNAs from host genes intact. The CRISPR-Cas9 assays reduced the presence of adapter multimers 10-fold on average, with reductions reaching 37-fold, allowing us to sequence over 15 libraries that failed previously and generate thousands of additional transcriptomes. I then developed a “cell specific PCR,” a method aimed at deeply sequencing the transcriptome of a specific single cell. This method selectively amplifies transcriptomes of interest by employing PCR primers corresponding to a cell’s DNA barcode assigned during scRNA-seq preparation. Finally, I began developing models to study other viral hemorrhagic fevers in a lower containment setting. In all, this study plays a direct role in improving scRNA-seq data to study RG-4 pathogens. By recovering scRNA-libraries and amplifying transcriptomes of interest, we help uncover mechanisms used by different cell-types to respond to viral infection, paving the way for more effective vaccines, diagnostics, and antiviral medication to prevent and treat tragic outbreaks around the world. | |
