Kerek Evan M, Cromwell Christopher R, Hubbard Basil P
Department of Pharmacology, University of Alberta, Edmonton, AB, Canada.
Methods Mol Biol. 2021;2381:227-242. doi: 10.1007/978-1-0716-1740-3_13.
In addition to advancing the development of gene-editing therapeutics, CRISPR/Cas9 is transforming how functional genetic studies are carried out in the lab. By increasing the ease with which genetic information can be inserted, deleted, or edited in cell and organism models, it facilitates genotype-phenotype analysis. Moreover, CRISPR/Cas9 has revolutionized the speed at which new genes underlying a particular phenotype can be identified through its application in genomic screens. Arrayed high-throughput and pooled lentiviral-based CRISPR/Cas9 screens have now been used in a wide variety of contexts, including the identification of essential genes, genes involved in cancer metastasis and tumor growth, and even genes involved in viral response. This technology has also been successfully used to identify drug targets and drug resistance mechanisms. Here, we provide a detailed protocol for performing a genome-wide pooled lentiviral CRISPR/Cas9 knockout screen to identify genetic modulators of a small-molecule drug. While we exemplify how to identify genes involved in resistance to a cytotoxic histone deacetylase inhibitor, Trichostatin A (TSA), the workflow we present can easily be adapted to different types of selections and other types of exogenous ligands or drugs.
除了推动基因编辑疗法的发展，CRISPR/Cas9 还在改变实验室中进行功能基因研究的方式。通过提高在细胞和生物体模型中插入、删除或编辑遗传信息的便捷性，它促进了基因型-表型分析。此外，CRISPR/Cas9 通过在基因组筛选中的应用，彻底改变了鉴定特定表型潜在新基因的速度。基于慢病毒的阵列高通量和混合 CRISPR/Cas9 筛选现已在广泛的背景中使用，包括鉴定必需基因、参与癌症转移和肿瘤生长的基因，甚至参与病毒反应的基因。这项技术也已成功用于鉴定药物靶点和耐药机制。在这里，我们提供了一个详细的方案，用于进行全基因组混合慢病毒 CRISPR/Cas9 敲除筛选，以鉴定小分子药物的遗传调节因子。虽然我们举例说明了如何鉴定参与对细胞毒性组蛋白去乙酰化酶抑制剂曲古抑菌素 A（TSA）耐药的基因，但我们介绍的工作流程可以轻松适用于不同类型的筛选以及其他类型的外源性配体或药物。
