Document Type

Dissertation - Open Access

Award Date

2024

Degree Name

Doctor of Philosophy (PhD)

Department / School

Chemistry and Biochemistry

First Advisor

Adam D. Hoppe

Abstract

CRISPR/Cas9 is a revolutionary technique for genome editing where Cas9 nucleases can be targeted to specific genomic sites by single-guide RNAs (sgRNAs) containing roughly 20-nucleotide sequences that are complementary to the target region. Double strand breaks created by Cas9 are repaired through non-homologous end-joining (NHEJ) in most cells, resulting in insertions and deletions leading to frame shift mutations in coding regions. Thus, the CRISPR-Cas9 system can be exploited for systematic evaluation of gene function relationships by whole genome screening. We also developed a CRISPR whole‐genome screen and counter‐screen strategy based on a pseudoviral platform that allowed identification of genes specific to SARS‐CoV‐2 spike and vesicular stomatitis virus glycoprotein (VSV‐G) mediated entry. Screening of SARS‐CoV‐2 spike and VSV‐G on the same lentiviral pseudovirus allowed the identification of entry‐specific genes relative to genes associated with retro‐transcription, integration, and reporter expression. Second, a Cre‐Gag fusion protein packaged into the pseudovirus was used to bypass retrotranscription and integration by directly activating a floxed fluorescent protein reporter, thereby increasing specificity for viral entry. Macrophages are important immune cells that engulf pathogens, debris, and apoptotic cells, playing a crucial role in both innate and adaptive immunity. Antibody-dependent cellular phagocytosis (ADCP) is a process where macrophages engulf and destroy pathogens and target cells opsonized by antibodies. We have shown that a Th2 cytokines IL-4 inhibits ADCP of target cells by bone marrow derived macrophages (BMDM). Here, CRISPR-Cas9 whole genome screening on BMDM was used to identify genes that are responsible for IL-4 mediated suppression of ADCP. In this screen, we identified genes contributing to N-linked glycosylation as inhibitors of IL- 4 mediated inhibition of ADCP. Our findings suggest that IL-4 enhances overall glycocalyx abundance, higher negative charge on the cell surface due to the presence of sialic acid at the end of sugar residues, consequently inhibiting target cell phagocytosis through electrostatic repulsion as target cells are also negatively charged. These findings suggest that modulation of glycosylation may be a strategy for enhancing antibody-FcgR interactions and promoting effective immunotherapies for cancer, viral and rheumatological diseases.

Publisher

South Dakota State University

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Rights Statement

In Copyright