Humanized mouse models include engineered immunodeficient mice that have been transplanted with human hematopoietic stem and progenitor cells (HSPCs; CD34+ cells) to create a functional human immune system. In the past two decades these models have become powerful tools for studying human immune responses to infection and cancer. Efforts are now underway to combine these models with other technologies such as CRISPR gene editing to interrogate specific gene function in the immune system.

A new study reports an efficient method for knocking out genes in human CD34+ cells for transplantation into immunocompromised mice, offering a valuable platform to study the function of immune genes in vivo.

First, the investigators optimized gene knock-out (KO) efficiency in human CD34+ HSPCs by identifying the electroporation conditions and the doses of the Cas9 protein and sgRNAs that achieved maximal gene KO in vitro. The team also determined the optimal number of electroporated cells (30,000) that need to be engrafted into immunocompromised mice to improve engraftment rates in the blood, spleen and bone marrow.

Next, the researchers validated their methodology by knocking out well-characterized immune genes. They notably introduced TCF7-KO or RAG2-KO human CD34+ cells into immunocompromised mice, which allowed them to confirm the role of TCF7 and RAG2 in T-cell development and the generation of mature B and T cells, respectively.

Finally, the team used their humanized mouse reverse genetics system to knock out CCR5 — a gene encoding a co-receptor for HIV entry — in human HSPCs before introducing the cells into immunocompromised mice. They showed that mice reconstituted with the CCR5-KO immune system were protected from HIV-1 infection compared to the control mice.

These findings indicate that the platform will be useful to study the contribution of various genes in controlling human pathogens.

Original reference: Pal, P. et. al. Sci. Adv. 11, eadu1561 (2025)