Rapid generation of drug-resistance alleles at endogenous loci using CRISPR-Cas9 indel mutagenesis

PLoS One. 2017 Feb 23;12(2):e0172177. doi: 10.1371/journal.pone.0172177. eCollection 2017.

Abstract

Genetic alterations conferring resistance to the effects of chemical inhibitors are valuable tools for validating on-target effects in cells. Unfortunately, for many therapeutic targets such alleles are not available. To address this issue, we evaluated whether CRISPR-Cas9-mediated insertion/deletion (indel) mutagenesis can produce drug-resistance alleles at endogenous loci. This method takes advantage of the heterogeneous in-frame alleles produced following Cas9-mediated DNA cleavage, which we show can generate rare alleles that confer resistance to the growth-arrest caused by chemical inhibitors. We used this approach to identify novel resistance alleles of two lysine methyltransferases, DOT1L and EZH2, which are each essential for the growth of MLL-fusion leukemia cells. We biochemically characterized the DOT1L mutation, showing that it is significantly more active than the wild-type enzyme. These findings validate the on-target anti-leukemia activities of existing DOT1L and EZH2 inhibitors and reveal a simple method for deriving drug-resistance alleles for novel targets, which may have utility during early stages of drug development.

MeSH terms

  • Alleles*
  • Animals
  • Antineoplastic Agents / pharmacology
  • Benzimidazoles / pharmacology
  • CRISPR-Cas Systems*
  • Cell Line
  • Cell Line, Tumor
  • Cell Proliferation / drug effects
  • Clustered Regularly Interspaced Short Palindromic Repeats
  • Drug Resistance, Neoplasm*
  • Enhancer of Zeste Homolog 2 Protein / genetics*
  • Enhancer of Zeste Homolog 2 Protein / metabolism
  • HEK293 Cells
  • Histone-Lysine N-Methyltransferase
  • Humans
  • INDEL Mutation*
  • Methyltransferases / genetics*
  • Methyltransferases / metabolism
  • Mice
  • Models, Molecular
  • Mutagenesis*
  • Neoplasms / drug therapy
  • Neoplasms / genetics
  • Neoplasms / metabolism

Substances

  • Antineoplastic Agents
  • Benzimidazoles
  • EPZ-5676
  • DOT1L protein, human
  • Methyltransferases
  • EZH2 protein, human
  • Enhancer of Zeste Homolog 2 Protein
  • Histone-Lysine N-Methyltransferase