1 Target Sciences Respiratory Therapy Area, Medicines Research Centre, GlaxoSmithKline R&D, Stevenage, United Kingdom.
2 Fibrosis Discovery Performance Unit, Respiratory Therapy Area, Medicines Research Centre, GlaxoSmithKline R&D, Stevenage, United Kingdom.
CRISPR J. 2019 Feb;2(1):31-40. doi: 10.1089/crispr.2018.0047.
Genome editing is a tool that has many applications, including the validation of potential drug targets. However, performing genome editing in low-passage primary human cells with the greatest physiological relevance is notoriously difficult. High editing efficiency is desired because it enables gene knockouts (KO) to be generated in bulk cellular populations and circumvents the problem of having to generate clonal cell isolates. Here, we describe a single-step workflow enabling >90% KO generation in primary human lung fibroblasts via CRISPR ribonucleoprotein delivery in the absence of antibiotic selection or clonal expansion. As proof of concept, we edited two SMAD family members and demonstrated that in response to transforming growth factor beta, SMAD3, but not SMAD2, is critical for deposition of type I collagen in the fibrotic response. The optimization of this workflow can be readily transferred to other primary cell types.
基因组编辑是一种具有多种应用的工具，包括验证潜在的药物靶点。然而，在具有最大生理相关性的低传代原代人细胞中进行基因组编辑是众所周知的困难。高编辑效率是理想的，因为它可以使基因敲除（KO）在大量细胞群体中产生，并避免必须生成克隆细胞分离物的问题。在这里，我们描述了一种单步工作流程，通过 CRISPR 核糖核蛋白传递，在没有抗生素选择或克隆扩增的情况下，使原代人肺成纤维细胞中的 KO 生成率超过 90%。作为概念验证，我们编辑了两个 SMAD 家族成员，并证明在转化生长因子β的作用下，SMAD3 而不是 SMAD2 对于纤维反应中 I 型胶原的沉积至关重要。此工作流程的优化可以很容易地转移到其他原代细胞类型。
