citations

                    Highly efficient CRISPR-Cas9-mediated geneknockout in primary human B cells forfunctional genetic studies of Epstein-Barrvirus infection
                

Authors:

Akidil E, Albanese M, Buschle A, Ruhle A, Pich D, Keppler OT, Hammerschmidt W.

In:

Source: PLoS Pathog
Publication Date: (2021)
Issue: 17(4): e1009117

Research Area:

Immunotherapy / Hematology

Gene Expression

Basic Research

Cells used in publication:

B cell, human: Species: human; Tissue Origin: blood

Platform:

4D-Nucleofector® X-Unit

Experiment

2×10^6 freshly isolated primary B cells were washed in PBS and resuspended in 20 µl P3 Primary Cell Nucleofector Solution buffer prepared with Supplement 1 buffer (Lonza) according to the manufacturer’s instructions (P3 Primary Cell 4D-Nucleofector X Kit S). Cells were mixed with 5 µl of the RNP mixture by gently pipetting and were transferred to pre-cooled (4°C) 16 well Nucleocuvette Strips (Lonza). Primary human B cells were nucleofected using the EH-100 program of Lonza´s protocol. 100 µl prewarmed non-supplemented RPMI1640 medium was added to the cells, which were incubated for 15 min at 37°C. The cells were transferred to a single well of a 24-well cluster plate and complete prewarmed cell culture medium containing 20% FCS was added to a final volume of 220 µl to allow cell recovery.

Abstract

Gene editing is now routine in all prokaryotic and metazoan cells but has not received much attention in immune cells when the CRISPR-Cas9 technology was introduced in the field of mammalian cell biology less than ten years ago. This versatile technology has been successfully adapted for gene modifications in human myeloid cells and T cells, among others, but applications to human primary B cells have been scarce and limited to activated B cells. This limitation has precluded conclusive studies into cell activation, differentiation or cell cycle control in this cell type. We report on highly efficient, simple and rapid genome engineering in primary resting human B cells using nucleofection of Cas9 ribonucleoprotein complexes, followed by EBV infection or culture on CD40 ligand feeder cells to drive in vitro B cell survival. We provide proof-of-principle of gene editing in quiescent human B cells using two model genes: CD46 and CDKN2A. The latter encodes the cell cycle regulator p16INK4a which is an important target of Epstein-Barr virus (EBV). Infection of B cells carrying a knockout of CDKN2A with wildtype and EBNA3 oncoprotein mutant strains of EBV allowed us to conclude that EBNA3C controls CDKN2A, the only barrier to B cell proliferation in EBV infected cells. Together, this approach enables efficient targeting of specific gene loci in quiescent human B cells supporting basic research as well as immunotherapeutic strategies.

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