Identification of Genes Regulating Hepatocyte Injury by a Genome-Wide CRISPR-Cas9 Screen.
Gene editing introduces stable mutations into the genome and has powerful applications extending from research to clinical gene therapy. CRISPR-Cas9 gene editing can be employed to study directly the functional impact of stable gene knockout, activation, and knockdown. Here, we describe the end-to-end methodology by which we employ genome-wide CRISPR-Cas9 knockout to study drug toxicity using acetaminophen (APAP) in a hepatocellular carcinoma liver model as an example. This methodology can be extended to other proliferative cell types and chemical metabolic and toxicity models. By employing a massively parallelized genome-wide knockout model, the genes responsible for cellular toxicity and proliferation may be assayed concurrently. Resultant data are interrogated in the context of existing gene expression data, pathway analysis, drug-gene interactions, and orthogonal confirmatory assays to better understand the metabolic mechanisms.
Methods in molecular biology (Clifton, N.J.)
Acetaminophen; CRISPR-Cas Systems; Gene Editing; Genome; Hepatocytes
CRISPR-Cas9; Drug screen; Gene-editing; Genome-wide; Hepatocytes; Next-generation sequencing; Toxicity
Shortt K, Heruth DP. Identification of Genes Regulating Hepatocyte Injury by a Genome-Wide CRISPR-Cas9 Screen. Methods Mol Biol. 2022;2544:227-251. doi:10.1007/978-1-0716-2557-6_17