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.
Highlights
The CRISPR-Cas9 technology has developed into an essential tool for gene editing and genome manipulation in many eukaryotic cell types [1]
CRISPR-Cas9 efficiently targets the locus of the cell surface protein CD46 in primary human B cells
We aimed at testing the functional knockout of genes in primary human B cells with the CRISPR-Cas9 technology
Summary
The CRISPR-Cas technology has developed into an essential tool for gene editing and genome manipulation in many eukaryotic cell types [1]. This versatile system has been successfully applied to various mammalian cells and cell lines, but its application in certain primary human cells is far from routine. Studies that applied CRISPR-Cas mediated gene editing in primary human T cells resulted in low targeting efficiencies and cellular toxicity [2,3] probably due to the delivery of the Cas encoding plasmid DNA together with the two indispensable RNAs to assemble a functional CRISPR-Cas complex in the cells. The highly efficient knockout of a target gene in primary human T cells proved that the RNP-based CRISPR-Cas technology can be applied to develop T cell based immunotherapies and suggested that it might be adaptable to other primary cell types [6]
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.