Nuffield Department of Medicine, Target Discovery Institute, University of Oxford, Oxford, UK.
Wellcome Centre for Human Genetics, University of Oxford, Oxford, UK.
Sci Rep. 2021 Feb 19;11(1):4245. doi: 10.1038/s41598-021-82137-z.
Genome engineering using CRISPR/Cas9 technology enables simple, efficient and precise genomic modifications in human cells. Conventional immortalized cell lines can be easily edited or screened using genome-wide libraries with lentiviral transduction. However, cell types derived from the differentiation of induced Pluripotent Stem Cells (iPSC), which often represent more relevant, patient-derived models for human pathology, are much more difficult to engineer as CRISPR/Cas9 delivery to these differentiated cells can be inefficient and toxic. Here, we present an efficient, lentiviral transduction protocol for delivery of CRISPR/Cas9 to macrophages derived from human iPSC with efficiencies close to 100%. We demonstrate CRISPR/Cas9 knockouts for three nonessential proof-of-concept genes-HPRT1, PPIB and CDK4. We then scale the protocol and validate for a genome-wide pooled CRISPR/Cas9 loss-of-function screen. This methodology enables, for the first time, systematic exploration of macrophage involvement in immune responses, chronic inflammation, neurodegenerative diseases and cancer progression, using efficient genome editing techniques.
利用 CRISPR/Cas9 技术进行基因组工程可在人类细胞中实现简单、高效和精确的基因组修饰。使用慢病毒转导的全基因组文库，可以轻松编辑或筛选常规的永生化细胞系。然而，源自诱导多能干细胞（iPSC）分化的细胞类型则更难以进行工程改造，因为 CRISPR/Cas9 递送至这些分化细胞的效率可能较低且具有细胞毒性。在这里，我们提出了一种高效的慢病毒转导方案，可将 CRISPR/Cas9 递送至源自人类 iPSC 的巨噬细胞，效率接近 100%。我们演示了针对三个非必需的概念验证基因（HPRT1、PPIB 和 CDK4）的 CRISPR/Cas9 敲除。然后，我们按比例放大该方案并对全基因组的 CRISPR/Cas9 功能丧失筛选进行验证。该方法首次使用高效的基因组编辑技术，系统地研究了巨噬细胞在免疫反应、慢性炎症、神经退行性疾病和癌症进展中的作用。
