CRISPR-Barcoding for Intratumor Genetic Heterogeneity Modeling and Functional Analysis of Oncogenic Driver Mutations

Mol Cell. 2016 Aug 4;63(3):526-38. doi: 10.1016/j.molcel.2016.06.017. Epub 2016 Jul 21.

Abstract

Intratumor genetic heterogeneity underlies the ability of tumors to evolve and adapt to different environmental conditions. Using CRISPR/Cas9 technology and specific DNA barcodes, we devised a strategy to recapitulate and trace the emergence of subpopulations of cancer cells containing a mutation of interest. We used this approach to model different mechanisms of lung cancer cell resistance to EGFR inhibitors and to assess effects of combined drug therapies. By overcoming intrinsic limitations of current approaches, CRISPR-barcoding also enables investigation of most types of genetic modifications, including repair of oncogenic driver mutations. Finally, we used highly complex barcodes inserted at a specific genome location as a means of simultaneously tracing the fates of many thousands of genetically labeled cancer cells. CRISPR-barcoding is a straightforward and highly flexible method that should greatly facilitate the functional investigation of specific mutations, in a context that closely mimics the complexity of cancer.

MeSH terms

  • Animals
  • Antineoplastic Combined Chemotherapy Protocols / pharmacology
  • Biomarkers, Tumor / genetics*
  • CRISPR-Cas Systems*
  • Carcinoma, Non-Small-Cell Lung / drug therapy
  • Carcinoma, Non-Small-Cell Lung / genetics*
  • Carcinoma, Non-Small-Cell Lung / metabolism
  • Carcinoma, Non-Small-Cell Lung / pathology
  • Cell Lineage
  • Clone Cells / drug effects
  • Clone Cells / metabolism
  • Clone Cells / pathology
  • DNA Mutational Analysis
  • DNA, Neoplasm / genetics*
  • Dose-Response Relationship, Drug
  • Drug Resistance, Neoplasm / genetics
  • ErbB Receptors / antagonists & inhibitors
  • ErbB Receptors / genetics
  • ErbB Receptors / metabolism
  • Gene Editing / methods*
  • Genetic Heterogeneity*
  • Genetic Predisposition to Disease
  • HCT116 Cells
  • HEK293 Cells
  • High-Throughput Nucleotide Sequencing
  • Humans
  • Lung Neoplasms / drug therapy
  • Lung Neoplasms / genetics*
  • Lung Neoplasms / metabolism
  • Lung Neoplasms / pathology
  • MCF-7 Cells
  • Male
  • Mice, SCID
  • Multiplex Polymerase Chain Reaction
  • Oncogenes*
  • Phenotype
  • Point Mutation*
  • Protein Kinase Inhibitors / pharmacology
  • Time Factors
  • Tumor Microenvironment
  • Xenograft Model Antitumor Assays

Substances

  • Biomarkers, Tumor
  • DNA, Neoplasm
  • Protein Kinase Inhibitors
  • EGFR protein, human
  • ErbB Receptors