Efficient CRISPR/Cas9-Mediated Gene Knockin in Mouse Hematopoietic Stem and Progenitor Cells

Abstract

Summary: Mutations accumulating in hematopoietic stem and progenitor cells (HSPCs) during development can cause severe hematological disorders. Modeling these mutations in mice is essential for understanding their functional consequences. Here, we describe an efficient CRISPR/Cas9-based system to knock in and repair genes in mouse HSPCs. CRISPR/Cas9 ribonucleoproteins, in combination with recombinant adeno-associated virus (rAAV)-DJ donor templates, led to gene knockin efficiencies of up to 30% in the Lmnb1 and Actb loci of mouse HSPCs in vitro. The targeted HSPCs engraft and reconstitute all immune cell lineages in the recipient mice. Using this approach, we corrected a neomycin-disrupted Rag2 gene. The Rag2-corrected HSPCs restore B and T cell development in vivo, confirming the functionality of the approach. Our method provides an efficient strategy to study gene function in the hematopoietic system and model hematological disorders in vivo, without the need for germline mutagenesis. : Using CRISPR/Cas9 and AAV-DJ vectors for donor template delivery, Tran et al. achieve high homologous recombination efficiencies (median of 30%) in mouse hematopoietic stem and progenitor cells (HSPCs). The targeted HSPCs engraft and repopulate all immune cell lineages after primary and secondary reconstitution of immunodeficient recipients. Keywords: CRISPR/Cas9, ribonucleoprotein, RNP, adeno-associated virus, AAV, non-homologous end joining, NHEJ, homologous recombination, HR, hematopoietic stem and progenitor cells, HSPCs, high efficiency, gene knockin, gene repair