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Fluorescent labeling of CRISPR/Cas9 RNP for gene knockout in HSPCs and iPSCs reveals an essential role for GADD45b in stress response

Masoud Nasri, Perihan Mir, Benjamin Dannenmann, Diana Amend, Tessa Skroblyn, Yun Xu, Klaus Schulze-Osthoff, Maksim Klimiankou, Karl Welte and Julia Skokowa

Key Points

  • Fluorescent labeling of CRISPR/Cas9–gRNA RNP enables sorting of edited HSPCs and iPSCs for further applications.

  • GADD45B plays a crucial role in UV stress-induced response of HSPCs and iPSCs.

Abstract

CRISPR/Cas9-mediated gene editing of stem cells and primary cell types has several limitations for clinical applications. The direct delivery of ribonucleoprotein (RNP) complexes consisting of Cas9 nuclease and guide RNA (gRNA) has improved DNA- and virus-free gene modifications, but it does not enable the essential enrichment of the gene-edited cells. Here, we established a protocol for the fluorescent labeling and delivery of CRISPR/Cas9–gRNA RNP in primary human hematopoietic stem and progenitor cells (HSPCs) and induced pluripotent stem cells (iPSCs). As a proof of principle for genes with low-abundance transcripts and context-dependent inducible expression, we successfully deleted growth arrest and DNA-damage-inducible β (GADD45B). We found that GADD45B is indispensable for DNA damage protection and survival in stem cells. Thus, we describe an easy and efficient protocol of DNA-free gene editing of hard-to-target transcripts and enrichment of gene-modified cells that are generally difficult to transfect.

  • Submitted December 24, 2017.
  • Accepted November 28, 2018.
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