TY  - JOUR
AU  - Nasri, Masoud
AU  - Mir, Perihan
AU  - Dannenmann, Benjamin
AU  - Amend, Diana
AU  - Skroblyn, Tessa
AU  - Xu, Yun
AU  - Schulze-Osthoff, Klaus
AU  - Klimiankou, Maksim
AU  - Welte, Karl
AU  - Skokowa, Julia
TI  - Fluorescent labeling of CRISPR/Cas9 RNP for gene knockout in HSPCs and iPSCs reveals an essential role for GADD45b in stress response.
JO  - Blood advances
VL  - 3
IS  - 1
SN  - 2473-9537
CY  - Washington, DC
PB  - American Society of Hematology
M1  - DKFZ-2020-00155
SP  - 63 - 71
PY  - 2019
AB  - 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.
KW  - Antigens, Differentiation (NLM Chemicals)
KW  - GADD45B protein, human (NLM Chemicals)
KW  - Macromolecular Substances (NLM Chemicals)
KW  - RNA, Guide (NLM Chemicals)
KW  - Ribonucleoproteins (NLM Chemicals)
LB  - PUB:(DE-HGF)16
C6  - pmid:30622144
C2  - pmc:PMC6325296
DO  - DOI:10.1182/bloodadvances.2017015511
UR  - https://inrepo02.dkfz.de/record/153073
ER  -