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

摘要

Mutations accumulating in hematopoietic stem andprogenitor cells (HSPCs) during development cancause severe hematological disorders. Modelingthese mutations in mice is essential for understandingtheir functional consequences. Here, we describe anefficient CRISPR/Cas9-based system to knock inand repair genes in mouse HSPCs. CRISPR/Cas9 ribonucleoproteins,in combination with recombinantadeno-associated virus (rAAV)-DJ donor templates,led to gene knockin efficiencies of up to 30% in theLmnb1 andActbloci of mouse HSPCs in vitro. The targetedHSPCs engraft and reconstitute all immune celllineages in the recipient mice. Using this approach,we corrected a neomycin-disrupted Rag2 gene. TheRag2-corrected HSPCs restore B and T cell developmentin vivo, confirming the functionality of theapproach. Our method provides an efficient strategyto study gene function in the hematopoietic systemand model hematological disorders in vivo, withoutthe need for germline mutagenesis.