CRISPR-Cas9-mediated gene editing in primary human B cells identifies critical cellular factors for Epstein-Barr virus infection

Akidil, Ezgi (2023): CRISPR-Cas9-mediated gene editing in primary human B cells identifies critical cellular factors for Epstein-Barr virus infection. Dissertation, LMU München: Fakultät für Biologie

Abstract

In humans, Epstein-Barr virus (EBV) infection can cause Infectious Mononucleosis and is associated with various malignancies, e.g. Burkitt’s lymphoma, Hodgkin’s lymphoma, and several lymphoproliferative diseases. Upon infection in vitro, EBV activates resting human B cells and transforms them into indefinitely proliferating lymphoblastoid B cell lines (LCL), in which the virus establishes a stable latent infection. While EBV genetics is feasible with EBV mutant viruses, studies into genetic variants of human B cells have not been possible. In my PhD thesis, I succeeded in performing very efficient genetic engineering in primary resting human B cells using Cas9 ribonucleoprotein complexes, followed by EBV infection or culturing the cells on CD40 ligand feeder cells supplemented with IL-4 to drive their cell survival. I demonstrated gene editing of the CD46 locus with very high efficiencies and provided studies into the kinetics of double-strand breaks by Cas9 within hours after nucleofection. Next generation sequencing of CD46 mRNAs metabolically labeled with 4-thiouridine (4sU) documented locus repair and active transcription of the edited gene locus within 24 hours. For a functional proof-of-principle, I targeted an EBV relevant cellular gene, CDKN2A, encoding the cell cycle regulator p16INK4a. Infection of CDKN2A knockout B cells with wild-type EBV or an EBNA3 oncoprotein mutant strain of EBV allowed me to conclude that p16INK4a is the only cellular barrier to EBV-induced B cell proliferation. The efficient targeting of a specific gene loci in primary human B cells is a major achievement in the B cell field. EBV DNA is epigenetically naïve when it is delivered upon infection but is maintained as fully chromatinized extrachromosomal plasmid DNA with mostly repressive marks in latently infected B cells. In my PhDwork, I studied the role of replication–dependent and –independent histone chaperones which supposedly can load cellular histones onto the incoming viral DNA. Using novel CRISPR technology, I investigated the consequences of single or multiple knockouts of histone chaperone genes in primary human B cells. EBV infected CHAF1B depleted B cells were severely compromised and did not survive infection. My results indicated that CHAF1B is a critical factor that regulates replication of EBV DNA and prevents the genotoxic stress in the first days of infec tion with EBV while other histone chaperones such as HIRA, DAXX or ATRX seemed to be less critical during early viral infection.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Themengebiete: 500 Naturwissenschaften und Mathematik ; 500 Naturwissenschaften und Mathematik > 570 Biowissenschaften, Biologie
• Fakultäten: Fakultät für Biologie
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 1. Februar 2023
• 1. Bericht­erstatter:in: Kempkes, Bettina