High-throughput screening of a CRISPR/Cas9 library for functional genomics in human cells

Nature. 2014 May 22;509(7501):487-91. doi: 10.1038/nature13166. Epub 2014 Apr 9.

Abstract

Targeted genome editing technologies are powerful tools for studying biology and disease, and have a broad range of research applications. In contrast to the rapid development of toolkits to manipulate individual genes, large-scale screening methods based on the complete loss of gene expression are only now beginning to be developed. Here we report the development of a focused CRISPR/Cas-based (clustered regularly interspaced short palindromic repeats/CRISPR-associated) lentiviral library in human cells and a method of gene identification based on functional screening and high-throughput sequencing analysis. Using knockout library screens, we successfully identified the host genes essential for the intoxication of cells by anthrax and diphtheria toxins, which were confirmed by functional validation. The broad application of this powerful genetic screening strategy will not only facilitate the rapid identification of genes important for bacterial toxicity but will also enable the discovery of genes that participate in other biological processes.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antigens, Bacterial / pharmacology
  • Bacterial Toxins / pharmacology
  • CRISPR-Associated Proteins / genetics*
  • Cell Line
  • Cells / drug effects
  • Cells / metabolism*
  • Clustered Regularly Interspaced Short Palindromic Repeats / genetics*
  • Diphtheria Toxin / pharmacology
  • Gene Library*
  • Genomics / methods*
  • High-Throughput Screening Assays / methods*
  • Humans
  • INDEL Mutation / genetics
  • Lentivirus / genetics
  • Organic Cation Transporter 1 / genetics
  • RNA, Guide / genetics
  • Reproducibility of Results

Substances

  • Antigens, Bacterial
  • Bacterial Toxins
  • CRISPR-Associated Proteins
  • Diphtheria Toxin
  • Organic Cation Transporter 1
  • RNA, Guide
  • anthrax toxin