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

Authors

Chen, H.-R., Kadzioch, N. P., Gapp, M., Yang, H.-H., Ruhle, A., Villamizar Cujar, J., Fuchs, T., Schmacke, N. A., Hornung, V., Speck, R. F., Keppler, O. T., Albanese, M.

Abstract

Defining how human host factors shape HIV-1 infection in vivo remains essential for both cure-directed research and the development of genetically engineered cell therapies. CD34+ hematopoietic stem and progenitor cells (HSPCs) offer a tractable entry point into this problem because they can be edited ex vivo and used to generate multilineage human immune systems after xenotransplantation. Here, we establish a CRISPR-Cas9 editing workflow in primary human CD34+ HSPCs to generate knockout human immune systems for functional analysis of HIV-1 dependency and restriction factors. Edited HSPCs retained viability, proliferative capacity, primitive immunophenotypes, and multilineage differentiation potential after Cas9 ribonucleoprotein delivery. Following myeloid differentiation, knockout (KO) of the restriction factor Sterile Alpha Motif and Histidine-Aspartate Domain-Containing Protein 1 (SAMHD1) increased susceptibility of HSPC-derived macrophages to HIV-1 infection, demonstrating that edited HSPC progeny can be used for ex vivo functional interrogation of antiviral host factors. We then extended this platform in vivo by transplanting CCR5-, SAMHD1-, or non-targeting-control-edited HSPCs into immunodeficient mice and challenging reconstituted humanized animals with R5-tropic HIV-1. CCR5 knockout prevented detectable productive viral spread, validating the sensitivity of the model using a clinically relevant HIV dependency factor. In contrast, SAMHD1 knockout accelerated HIV-1 dissemination, with an earlier rise in plasma viremia and increased viral spread over the longitudinal infection time course compared with non-targeting controls, despite limited differences at endpoint. These findings establish CRISPR-edited human HSPC xenotransplantation as a modular platform to dissect HIV-1 host-factor function in an organismal setting. These data support the use of engineered human HSPC-derived immune systems as a preclinical platform to investigate HIV-1 host-factor biology and guide the development of durable cell-based strategies for HIV cure.

Preprint server: bioRxiv
The authors list and abstract were imported from bioRxiv on 06 Aug 2026.

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