可扩展的 CRISPR-Cas9 化学遗传筛选在未转化的人类细胞中的应用。

Scalable CRISPR-Cas9 chemical genetic screens in non-transformed human cells.

机构信息

Department of Computer Science and Engineering, University of Minnesota, Minneapolis, MN 55455, USA.

Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN 55455, USA.

出版信息

STAR Protoc. 2022 Dec 16;3(4):101675. doi: 10.1016/j.xpro.2022.101675. Epub 2022 Sep 14.

DOI:10.1016/j.xpro.2022.101675

PMID:36107744

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC9483651/

Abstract

Pooled lentiviral CRISPR-Cas9 screens are utilized for assessing the differential sensitivity or resistance of many single-gene knockouts to a compound. Here, we present a scalable approach for high-throughput compound screening by utilizing a small custom library. We describe steps to perform a proof-of-principle chemical screen in non-transformed hTERT RPE-1 TP53 cells with higher coverage and greater timepoint resolution compared to genome-wide screens. This approach can be adapted for use in various cell lines, compounds, and other focused sgRNA libraries.

摘要

基于慢病毒 CRISPR-Cas9 的细胞文库筛选技术可用于评估多种单基因敲除细胞对化合物的敏感性或耐药性差异。本文介绍了一种利用小型定制文库进行高通量化合物筛选的可扩展方法。我们描述了在未经转化的 hTERT RPE-1 TP53 细胞中进行原理验证性化学筛选的步骤，与全基因组筛选相比，该方法具有更高的覆盖率和更高的时间分辨率。这种方法可以适用于各种细胞系、化合物和其他靶向 sgRNA 文库。