Abstract
Long non-coding RNAs (lncRNAs) are emerging as key regulators in the initiation, growth, and progression of cancer. High-throughput CRISPR-based techniques systematically assess the function of genes or regulatory elements present in the human genome. Here, we present a protocol for identifying essential lncRNAs in cancer using CRISPRi-based dropout screens. We describe steps to select target sites, design guide RNAs, and generate CRISPRi cell lines. We then detail the execution and analysis of CRISPRi-based dropout screens.