Genome-Wide CRISPR/Cas9 Screening for Identification of Cancer Genes in Cell Lines

Methods Mol Biol. 2019:1907:125-136. doi: 10.1007/978-1-4939-8967-6_10.

Abstract

In this protocol, pooled sgRNA libraries targeting thousands of genes are computationally designed, generated using microarray-based synthesis techniques, and packaged into lentiviral particles. Target cells of interest are transduced with the lentiviral sgRNA pools to generate a collection of knockout mutants-via Cas9-mediated genomic cleavage-and screened for a phenotype of interest. The relative abundance of each mutant in the population can be monitored over time through high-throughput sequencing of the integrated sgRNA expression cassettes. Using this technique, we outline strategies for the identification of cancer driver genes and genes mediating drug response.

Keywords: CRISPR/Cas9 mutagenesis screens; Drug sensitivity; Loss-of-function gene discovery; sgRNA libraries.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • CRISPR-Cas Systems*
  • Gene Knockout Techniques
  • Genome, Human*
  • Genome-Wide Association Study / methods*
  • Genomics / methods*
  • High-Throughput Nucleotide Sequencing / methods*
  • Humans
  • Neoplasm Proteins / antagonists & inhibitors
  • Neoplasm Proteins / genetics*
  • Neoplasms / diagnosis
  • Neoplasms / genetics*
  • Phenotype
  • Tumor Cells, Cultured

Substances

  • Neoplasm Proteins