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000153073 0247_ $$2doi$$a10.1182/bloodadvances.2017015511
000153073 0247_ $$2pmid$$apmid:30622144
000153073 0247_ $$2pmc$$apmc:PMC6325296
000153073 0247_ $$2ISSN$$a2473-9529
000153073 0247_ $$2ISSN$$a2473-9537
000153073 0247_ $$2ISSN$$a2476-9537
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000153073 037__ $$aDKFZ-2020-00155
000153073 041__ $$aeng
000153073 082__ $$a610
000153073 1001_ $$aNasri, Masoud$$b0
000153073 245__ $$aFluorescent labeling of CRISPR/Cas9 RNP for gene knockout in HSPCs and iPSCs reveals an essential role for GADD45b in stress response.
000153073 260__ $$aWashington, DC$$bAmerican Society of Hematology$$c2019
000153073 3367_ $$2DRIVER$$aarticle
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000153073 520__ $$aCRISPR/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.
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000153073 650_7 $$2NLM Chemicals$$aAntigens, Differentiation
000153073 650_7 $$2NLM Chemicals$$aGADD45B protein, human
000153073 650_7 $$2NLM Chemicals$$aMacromolecular Substances
000153073 650_7 $$2NLM Chemicals$$aRNA, Guide
000153073 650_7 $$2NLM Chemicals$$aRibonucleoproteins
000153073 7001_ $$0P:(DE-He78)bc2e8b2ac93b02432d1816f80e26e7e8$$aMir, Perihan$$b1
000153073 7001_ $$aDannenmann, Benjamin$$b2
000153073 7001_ $$aAmend, Diana$$b3
000153073 7001_ $$aSkroblyn, Tessa$$b4
000153073 7001_ $$aXu, Yun$$b5
000153073 7001_ $$0P:(DE-He78)3fc9ad1c0cc7a2172b86374faed6edea$$aSchulze-Osthoff, Klaus$$b6$$udkfz
000153073 7001_ $$aKlimiankou, Maksim$$b7
000153073 7001_ $$aWelte, Karl$$b8
000153073 7001_ $$aSkokowa, Julia$$b9
000153073 773__ $$0PERI:(DE-600)2876449-3$$a10.1182/bloodadvances.2017015511$$gVol. 3, no. 1, p. 63 - 71$$n1$$p63 - 71$$tBlood advances$$v3$$x2473-9537$$y2019
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000153073 9141_ $$y2019
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