Fluorescent labeling of CRISPR/Cas9 RNP for gene knockout in HSPCs and iPSCs reveals an essential role for GADD45b in stress response
Fluorescent labeling of CRISPR/Cas9 RNP for gene knockout in HSPCs and iPSCs reveals an essential role for GADD45b in stress response
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DOI：
10.1182/bloodadvances.2017015511
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发表时间：
2019-01-08
期刊：
影响因子：
7.5
通讯作者：
Skokowa, Julia
中科院分区：
文献类型：
--
作者：
Nasri, Masoud;Mir, Perihan;Skokowa, Julia
CRISPR/Cas9介导的干细胞和原代细胞类型的基因编辑对于临床应用有几个限制。由Cas9核酸酶和指导RNA（gRNA）组成的核糖核蛋白（RNP）复合物的直接递送改善了无DNA和无病毒的基因修饰，但它不能实现基因编辑细胞的基本富集。在这里，我们建立了一种用于在原代人类造血干细胞和祖细胞（HSPC）和诱导多能干细胞（iPSC）中荧光标记和递送CRISPR/Cas9-gRNA RNP的方案。作为具有低丰度转录物和上下文依赖性诱导表达的基因的原理证明，我们成功地删除了生长停滞和DNA损伤诱导β（GADD 45 B）。我们发现GADD 45 B对于DNA损伤保护和干细胞存活是不可或缺的。因此，我们描述了一种简单有效的方案，即对难以靶向的转录物进行无DNA基因编辑，并富集通常难以转染的基因修饰细胞。
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 beta (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.