CRISPR Screen to Identify Neutrophil Regulators of Interferon-Gamma; Signaling in Acute Lung Injury
Abstract
Acute lung injury (ALI) can be caused by infections, trauma, blast injuries, and inhalation of toxic material such as burn pit fumes. Neutrophils constitute the initial response to many injuries and infections and secrete a number of mediators such as interferon gamma (IFN) that contribute to both pathogen clearance and host tissue damage. Neutrophils are an attractive target for host-directed therapies intended to limit organ damage. However, neutrophil signaling is notoriously difficult to study due to their short lifespan, the lack of immortalized cell lines that recapitulate neutrophil biology, and an absence of tools that allow neutrophil-specific gene modification in vivo and in vitro. This project uses CRISPR-Cas9 technology in mice to perform large-scale in vivo screening and identify regulators of neutrophil IFN in the setting of Streptococcus pneumoniae-induced ALI. The overall goal is to identify targetable pathways that may be used to treat ALI and to create a platform for efficient genome-wide studies of neutrophil function. In the initial reporting period, we have been successful in establishing the relevant Cas9-GFP mouse line and optimized antibody panels for fluorescence-activated cell sorting (FACS). We have also optimized murine hematopoietic stem cell (HSPC) purification. We have established, amplified, purified, and tested a lentiviral library for expression of sgRNAs; based on published data from collaborators we have altered our choice of library to assist adequate recovery of guide RNAs. In February 2019 work on this project was largely paused by the COVID-19 pandemic when UNC halted non-essential and non-COVID research projects and reduced animal colonies.
Document Details
- Document Type
- Technical Report
- Publication Date
- May 01, 2021
- Accession Number
- AD1165149
Entities
People
- Robert S Hagan
Organizations
- University of North Carolina at Chapel Hill