Genome Engineering of Hematopoietic Stem Cells Using CRISPR/Cas9 System

Methods Mol Biol. 2022:2429:307-331. doi: 10.1007/978-1-0716-1979-7_20.


Ex vivo genetic manipulation of autologous hematopoietic stem and progenitor cells (HSPCs) is a viable strategy for the treatment of hematologic and primary immune disorders. Targeted genome editing of HSPCs using the CRISPR-Cas9 system provides an effective platform to edit the desired genomic locus for therapeutic purposes with minimal off-target effects. In this chapter, we describe the detailed methodology for the CRISPR-Cas9 mediated gene knockout, deletion, addition, and correction in human HSPCs by viral and nonviral approaches. We also present a comprehensive protocol for the analysis of genome modified HSPCs toward the erythroid and megakaryocyte lineage in vitro and the long-term multilineage reconstitution capacity in the recently developed NBSGW mouse model that supports human erythropoiesis.

Keywords: Base editing; Clonal analysis; Erythroid differentiation; HDR; HSPCs; Hemoglobinopathies; Megakaryopoiesis; NBSGW mouse; NHEJ; Nucleofection; Transduction.

MeSH terms

  • Animals
  • CRISPR-Cas Systems* / genetics
  • Gene Editing / methods
  • Hematopoietic Stem Cell Transplantation*
  • Hematopoietic Stem Cells
  • Mice
  • Transplantation, Autologous