Efficient CRISPR-Cas9-mediated mutagenesis in primary human B cells for identifying plasma cell regulators

Tuan Anh Le; Van Trung Chu; Andreia C Lino; Eva Schrezenmeier; Christopher Kressler; Dania Hamo; Klaus Rajewsky; Thomas Dörner; Van Duc Dang

doi:10.1016/j.omtn.2022.11.016

Efficient CRISPR-Cas9-mediated mutagenesis in primary human B cells for identifying plasma cell regulators

Mol Ther Nucleic Acids. 2022 Nov 21:30:621-632. doi: 10.1016/j.omtn.2022.11.016. eCollection 2022 Dec 13.

Authors

Tuan Anh Le^{1

2}, Van Trung Chu³, Andreia C Lino^{1

2}, Eva Schrezenmeier^{4

5}, Christopher Kressler^{1

6}, Dania Hamo^{1

6}, Klaus Rajewsky³, Thomas Dörner^{1

2}, Van Duc Dang^{1

2

7}

Affiliations

¹ Deutsches Rheuma-Forschungszentrum, A Leibniz Institute, Charitéplatz 1, 10117 Berlin, Germany.
² Department of Rheumatology and Clinical Immunology, Charité-Universitätsmedizin Berlin, Berlin, Germany.
³ Max-Delbrück-Center for Molecular Medicine in the Helmholtz Association (MDC), Immune Regulation and Cancer, 13125 Berlin, Germany.
⁴ Department of Nephrology and Medical Intensive Care, Charité-Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, and Humboldt-Universität zu Berlin, Berlin, Germany.
⁵ Berlin Institute of Health (BIH), Berlin, Germany.
⁶ Berlin Institute of Health Center for Regenerative Therapies (BCRT), Charité - Universitätsmedizin Berlin, Berlin, Germany.
⁷ Faculty of Biology, VNU University of Science, Vietnam National University, Hanoi, Vietnam.

Abstract

Human B lymphocytes are attractive targets for immunotherapies in autoantibody-mediated diseases. Gene editing technologies could provide a powerful tool to determine gene regulatory networks regulating B cell differentiation into plasma cells, and identify novel therapeutic targets for prevention and treatment of autoimmune disorders. Here, we describe a new approach that uses CRISPR-Cas9 technology to target genes in primary human B cells in vitro for identifying plasma cell regulators. We found that sgRNA and Cas9 components can be efficiently delivered into primary human B cells through RD114-pseudotyped retroviral vectors. Using this system, we achieved approximately 80% of gene knockout efficiency. We disrupted expression of a triad of transcription factors, IRF4, PRDM1, and XBP1, and showed that human B cell survival and plasma cell differentiation are severely impaired. Specifically, that IRF4, PRDM1, and XBP1 were expressed at different stages during plasma cell differentiation, IRF4, PRDM1, and XBP1-targeted B cells failed to progress to the pre-plasmablast, plasma cell state, and plasma cell survival, respectively. Our method opens a new avenue to study gene functions in primary human B cells and identify novel plasma cell regulators for therapeutic applications.

Keywords: B cell differentiation; CRISPR-Cas9; IRF4; MT: RNA/DNA Editing; PRDM1; XBP1; knockout; plasma cell regulators; plasma cells; plasmablasts; primary human B cells.