Gene Knockout in Hematopoietic Stem and Progenitor Cells Followed by Granulocytic Differentiation

Methods Mol Biol. 2020:2115:455-469. doi: 10.1007/978-1-0716-0290-4_26.

Abstract

In this chapter, we present an optimized CRISPR/Cas9 RNP nucleofection approach for gene knockout (KO) in hematopoietic stem and progenitor cells (HSPCs). With experimentally proved active locus-specific sgRNAs, we routinely reach over 80% gene KO in HSPCs, thus avoiding the need for cell sorting or enrichment of targeted cell population. Additionally, we provide a protocol for in vitro granulocytic differentiation of HSPCs after gene KO and detailed description of granulocyte function tests which can be applied to study the effects of a particular gene KO.

Keywords: CRISPR/Cas9; Gene knockout; Granulocytic differentiation; Hematopoietic stem and progenitor cells; Nucleofection; Ribonucleoprotein; Single-guide RNA.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • CRISPR-Cas Systems*
  • Cells, Cultured
  • Gene Editing / methods*
  • Gene Knockout Techniques / methods
  • Granulocytes / cytology*
  • Granulocytes / metabolism
  • Hematopoietic Stem Cells / cytology*
  • Hematopoietic Stem Cells / metabolism
  • Humans
  • Leukopoiesis*
  • RNA, Guide, CRISPR-Cas Systems / genetics

Substances

  • RNA, Guide, CRISPR-Cas Systems