Translational Oncology Division, Internal Medicine II, University Hospital Tuebingen, Tuebingen, Germany.
Department of General Paediatrics, Oncology/Haematology, The University Children's Hospital Tuebingen, Tuebingen, Germany.
Methods Mol Biol. 2020;2115:455-469. doi: 10.1007/978-1-0716-0290-4_26.
In this chapter, we present an optimized CRISPR/Cas9 RNP nucleofection approach for gene knockout (KO) in hematopoietic stem and progenitor cells (HSPCs). With experimentally proved active locus-specific sgRNAs, we routinely reach over 80% gene KO in HSPCs, thus avoiding the need for cell sorting or enrichment of targeted cell population. Additionally, we provide a protocol for in vitro granulocytic differentiation of HSPCs after gene KO and detailed description of granulocyte function tests which can be applied to study the effects of a particular gene KO.
在本章中，我们提出了一种优化的 CRISPR/Cas9 RNP 核转染方法，用于造血干细胞和祖细胞（HSPC）中的基因敲除（KO）。使用经过实验验证的活性基因座特异性 sgRNA，我们通常可以在 HSPC 中实现超过 80%的基因 KO，从而避免了对靶细胞群进行细胞分选或富集的需要。此外，我们还提供了一种在基因 KO 后进行 HSPC 体外粒细胞分化的方案，以及详细描述的粒细胞功能测试，可以应用于研究特定基因 KO 的影响。
