Engineering Forward Genetics into Cultured Cancer Cells for Chemical Target Identification

Cell Chem Biol. 2019 Sep 19;26(9):1315-1321.e3. doi: 10.1016/j.chembiol.2019.06.006. Epub 2019 Jul 11.

Abstract

Target identification for biologically active small molecules remains a major barrier for drug discovery. Cancer cells exhibiting defective DNA mismatch repair (dMMR) have been used as a forward genetics system to uncover compound targets. However, this approach has been limited by the dearth of cancer cell lines that harbor naturally arising dMMR. Here, we establish a platform for forward genetic screening using CRISPR/Cas9 to engineer dMMR into mammalian cells. We demonstrate the utility of this approach to identify mechanisms of drug action in mouse and human cancer cell lines using in vitro selections against three cellular toxins. In each screen, compound-resistant alleles emerged in drug-resistant clones, supporting the notion that engineered dMMR enables forward genetic screening in mammalian cells.

Keywords: Ewing sarcoma; forward genetics; genetically-engineered mouse model (GEMM); hypermutation; mismatch repair; mismatch repair deficiency; phenotypic screening; target identification.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Animals
  • CRISPR-Cas Systems / genetics
  • Cell Line, Tumor
  • Clustered Regularly Interspaced Short Palindromic Repeats / genetics
  • DNA Mismatch Repair / genetics
  • Drug Discovery / methods*
  • Genetic Engineering / methods*
  • Genetic Testing / methods*
  • Humans
  • Mice
  • Neoplasms / genetics