Fig. 9

CRISPR-based lentiviral knockout (KO) libraries. A library of pooled sgRNAs are cloned into a suitable lentiviral vector (here exemplified by a lentiCRISPR-v2 vector), resulting in a plasmid library, which is then used for production of a pooled lentiviral sgRNA library. Transduction of target cells is made at a low MOI ensuring that each cell receives a single unique sgRNA. Transcription of sgRNA and Cas9 results in KO of the target gene in each transduced cell, which results in a large population of cells collectively carrying knockout mutations in all library-targeted genes. Selection (e.g. a chemotherapy drug) is then applied to the pool of cells, which makes it possible to detect genes that affect the drug response. Genes involved in the given drug response is identified by next-generation sequencing (NGS) of sgRNA-containing cassettes and downstream bioinformatic analysis