Pooled Lentiviral CRISPR-Cas9 Screens for Functional Genomics in Mammalian Cells

Methods Mol Biol. 2019;1869:169-188. doi: 10.1007/978-1-4939-8805-1_15.

Abstract

CRISPR-Cas9 technology provides a simple way to introduce targeted mutations into mammalian cells to induce loss-of-function phenotypes. The CRISPR-Cas9 system has now successfully been applied for genetic screens in many cell types, providing a powerful tool for functional genomics with manifold applications. Genome-wide guide-RNA (gRNA) libraries allow facile generation of a pool of cells, each harboring a gene knockout mutation that can be used for the study of gene function, pathway analysis or the identification of genes required for cellular fitness. Furthermore, CRISPR genetic screens can be applied for the discovery of genes whose knockout sensitizes cells to drug treatments or mediates drug resistance. Here, we provide a detailed protocol discussing the necessary steps for the successful performance of pooled CRISPR-Cas9 screens.

Keywords: CRISPR-Cas9; Fitness; Genetic screen; Knockout; Proliferation; gRNA library.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • CRISPR-Associated Protein 9 / metabolism*
  • CRISPR-Cas Systems / genetics*
  • Cell Line
  • Data Analysis
  • Gene Knockout Techniques
  • Gene Library
  • Genetic Testing*
  • Genomics / methods*
  • High-Throughput Nucleotide Sequencing
  • Lentivirus / genetics*
  • Mammals / genetics*
  • RNA, Guide / genetics

Substances

  • RNA, Guide
  • CRISPR-Associated Protein 9

Grant support