Ruhle, A. | Search bioRxiv

bioRxiv2026

Optimizing CRISPR/Cas9 genome editing in primary human hematopoietic cells to advance studies into HIV biology

Defining how human host factors shape HIV-1 infection in vivo remains essential for the development of genetically engineered cell therapies. Here, we established a non-viral CRISPR/Cas9 ribonucleoprotein-based platform for efficient single and multiplex gene editing in primary human CD34+ hematopoietic stem and progenitor cells (HSPCs). Edited HSPCs retained viability, proliferative capacity, primitive immunophenotypes, and multilineage differentiation potential and supported targeted reporter knock-in independently of the cell source, cord blood (CB), bone marrow (BM), and mobilized from peripheral blood (MPB). Following differentiation, SAMHD1 knockout increased HIV-1 susceptibility of HSPC-derived macrophages, MX2 knockout also increased HIV infection in cells pre-stimulated with IFN-2a, and CXCR4 knockout blocked X4-tropic HIV-1 infection of HSPC-derived megakaryocytes. We then transplanted CCR5-, SAMHD1-, or non-targeting control-edited HSPCs into immunodeficient mice and challenged reconstituted animals with R5-tropic HIV-1. CCR5 knockout prevented detectable viral spread, validating the model using a clinically relevant dependency factor. In contrast, SAMHD1 knockout accelerated viral dissemination, with earlier plasma viremia and 3.6-fold higher cumulative viremia, although endpoint viral burden was not significantly different from controls. These findings establish transplantation of CRISPR/Cas9-edited human HSPCs as a modular platform for dissecting HIV-1 host-factor function across hematopoietic lineages ex vivo and in vivo and for evaluating engineered cell-based strategies.

bioengineering↗

bioRxiv2020

Highly efficient CRISPR-Cas9-mediated gene knockout in primary human B cells for functional genetic studies of Epstein-Barr virus infection

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. 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. Author summaryHuman hematopoietic stem cells and their derivatives of the myeloid and lymphoid lineages are important targets for gene correction or modifications using the CRISPR-Cas9 technology. Among others, this approach can support site-specific insertion of chimeric antigen receptors (CARs) or T cell receptors (TCRs) into primary T cells. Their subsequent adoptive transfer to patient donors is a promising immunotherapeutic concept that may control chronic infection or certain types of cancer. Human B cells have a similar potential but, in contrast to T cells, they are very sensitive, difficult to handle, and short-lived ex vivo precluding their genetic modification. Here, we provide means to manipulate primary human B cells genetically using in vitro assembled Cas9 ribonucleoprotein complexes and electroporation for their delivery. Our study demonstrates near-to-complete loss of a model target gene and provides examples to evaluate a cellular gene with a critical role during infection with Epstein-Barr virus (EBV).

immunology↗