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

Abstract

Highlights d CRISPR/Cas9 RNPs and AAV-DJ donor vectors lead to efficient HR in mouse HSPCs d No off-target activity was detected when sgRNAs with high specificity were used d Gene-targeted HSPCs fully engrafted and repopulated all immune cell lineages in vivo d Rag2-repaired HSPCs restore B and T cell development in vivo

Protocol for Efficient CRISPR/Cas9/AAV-Mediated Homologous Recombination in Mouse Hematopoietic Stem and Progenitor Cells

“… Mouse strains: We have successfully performed reporter knockins in HSPCs isolated either from C57BL/6 or Cas9 transgenic mice as reported previously ( Chu et al., 2016b ). The targeted HSPCs can be transplanted into sublethally irradiated Rag2 −/− cγ −/− ( Tran et al., 2019 ) or lethally irradiated immunocompetent mice ( Duran-Struuck et al., 2008 ) to assess engraftment efficiency of the targeted HSPCs. Healthy and low passage HEK293T cells are critical for a high yield of AAV.…”

“…Our protocol allows the simultaneous targeting of multiple gene loci in mouse HSPCs. In Tran et al. (2019) , we inserted in-frame coding sequences of T2A-mCherry and T2A-BFP reporters into the last exon of the Lmnb1 and beta-actin ( Actb ) genes, respectively.…”

“…Upon transplantation into irradiated immunodeficient mice, the targeted HSPCs engraft efficiently and fully reconstitute the hematopoietic system with all immune cell lineages detected in the bone marrow and spleen of the transplanted animals at 8- or 16-weeks post reconstitution (see Tran et al., 2019 ).…”

“…This approach enables the functional study of hematopoietic disease mutations in vivo , without requiring germline mutagenesis. For complete details on the use and execution of this protocol, please refer to Tran et al. (2019) .…”

“…For complete details on the use and execution of this protocol, please refer to Tran et al. (2019) .…”