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

Item Type:	Article
Title:	Fluorescent labeling of CRISPR/Cas9 RNP for gene knockout in HSPCs and iPSCs reveals an essential role for GADD45b in stress response
Creators Name:	Nasri, M. and Mir, P. and Dannenmann, B. and Amend, D. and Skroblyn, T. and Xu, Y. and Schulze-Osthoff, K. and Klimiankou, M. and Welte, K. and Skokowa, J.
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.
Keywords:	CRISPR-Cas Systems, Cell Line, DNA Damage, Differentiation Antigens, Gene Editing, Gene Knockout Techniques, Gene Targeting, Guide RNA, Hematopoietic Stem Cells, Induced Pluripotent Stem Cells, Macromolecular Substances, Protein Binding, Ribonucleoproteins, Stress, Physiological
Source:	Blood Advances
ISSN:	2473-9529
Publisher:	American Society of Hematology
Volume:	3
Number:	1
Page Range:	63-71
Date:	8 January 2019
Official Publication:	https://doi.org/10.1182/bloodadvances.2017015511
External Fulltext:	View full text on PubMed Central
PubMed:	View item in PubMed

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