Mantwill Klaus, Nawroth Roman
Department of Urology, Klinikum rechts der Isar, Technical University of Munich, Munich, Germany.
Department of Urology, University Hospital Rechts der Isar, Technical University Munich, Munich, Germany.
Methods Mol Biol. 2023;2684:155-165. doi: 10.1007/978-1-0716-3291-8_9.
The application of CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 technology with pooled guide RNA libraries enables genome-wide screening, which has some advantages over other screening methods using chemical DNA mutagens for inducing genetic changes, RNA interference, or arrayed screens. Here we describe the use of genome-wide knockout and transcriptional activation screening enabling the CRISPR-Cas9 system to discover resistance mechanisms to CDK4/6 inhibition in bladder cancer along with next-generation sequencing (NGS) analysis. We will describe the approach for transcriptional activation in the bladder cancer cell line T24 and provide guidance on critical points during the experimental workflow.
将CRISPR（成簇规律间隔短回文重复序列）-Cas9技术与混合引导RNA文库相结合的应用能够实现全基因组筛选，相较于其他使用化学DNA诱变剂诱导基因变化、RNA干扰或阵列筛选的筛选方法具有一些优势。在此，我们描述了利用全基因组敲除和转录激活筛选，使CRISPR-Cas9系统能够发现膀胱癌中对CDK4/6抑制的耐药机制，并结合下一代测序（NGS）分析。我们将描述在膀胱癌细胞系T24中进行转录激活的方法，并为实验流程中的关键点提供指导。
