CRISPR transduction of iPS cells

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 non-essential 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.EGA study EGAS00001005102

基于CRISPR/Cas9技术（CRISPR/Cas9 technology）的基因组工程操作，可实现人类细胞内简便、高效且精准的基因组修饰。常规永生化细胞系可通过全基因组文库（genome-wide libraries）结合慢病毒转导（lentiviral transduction），轻松完成基因编辑与筛选。然而，由诱导多能干细胞（induced Pluripotent Stem Cells, iPSC）分化而来的细胞类型——这类细胞通常是更贴合临床的患者源性人类病理模型——其基因工程化操作难度显著更高，原因在于CRISPR/Cas9向这类分化细胞的递送效率低下且具有细胞毒性。本研究开发了一套高效的慢病毒转导方案，可将CRISPR/Cas9递送至人诱导多能干细胞分化得到的巨噬细胞中，编辑效率接近100%。我们针对三个非必需的概念验证基因——HPRT1、PPIB及CDK4——成功实现了CRISPR/Cas9介导的基因敲除。随后我们对该方案进行规模化优化，并通过全基因组混合池化CRISPR/Cas9功能缺失筛选（genome-wide pooled CRISPR/Cas9 loss-of-function screen）验证了其有效性。该方法首次借助高效基因组编辑技术，实现了对巨噬细胞在免疫应答、慢性炎症、神经退行性疾病及癌症进展中作用的系统性探索。欧洲基因组表型档案馆（European Genome-phenome Archive, EGA）研究编号：EGAS00001005102