利用全基因组 CRISPR-Cas9 筛选鉴定耐药机制。

Identification of Drug Resistance Mechanisms Using Genome-Wide CRISPR-Cas9 Screens.

机构信息

Leslie Dan Faculty of Pharmacy, University of Toronto, Toronto, ON, Canada.

Department of Biochemistry, University of Toronto, Toronto, ON, Canada.

出版信息

Methods Mol Biol. 2022;2535:141-156. doi: 10.1007/978-1-0716-2513-2_12.

DOI:10.1007/978-1-0716-2513-2_12

PMID:35867229

Abstract

CRISPR-Cas9 genome editing provides a means for simple and scalable production of gene knockouts in mammalian cell lines. The development of guide RNA (gRNA) libraries targeting tens of thousands of genes has allowed researchers to produce pools of cells, each containing a single gene knockout for use in genetic screens. In addition to assessing the effect of gene knockout on cell proliferation, CRISPR-Cas9 genetic screens can be used to assess gene-drug interactions. Here, we outline a protocol for performing positive and negative selection genome-wide CRISPR-Cas9 screens for identifying gene knockouts that cause drug resistance and hypersensitivity. This protocol is designed for the use of the TKOv3 library in human cell lines, but can be readily adapted for different libraries.

摘要

CRISPR-Cas9 基因组编辑为在哺乳动物细胞系中简单且可扩展地生产基因敲除提供了一种手段。针对数万个基因的向导 RNA (gRNA) 文库的开发，使研究人员能够产生含有单个基因敲除的细胞池，用于遗传筛选。除了评估基因敲除对细胞增殖的影响外，CRISPR-Cas9 遗传筛选还可用于评估基因-药物相互作用。在这里，我们概述了一种用于进行正向和负向选择全基因组 CRISPR-Cas9 筛选的方案，以鉴定导致药物抗性和敏感性的基因敲除。该方案专为人类细胞系中的 TKOv3 文库设计，但可以很容易地适应不同的文库。