Large-Scale Analysis of CRISPR/Cas9 Cell-Cycle Knockouts Reveals the Diversity of p53-Dependent Responses to Cell-Cycle Defects

Dev Cell. 2017 Feb 27;40(4):405-420.e2. doi: 10.1016/j.devcel.2017.01.012. Epub 2017 Feb 16.

Abstract

Defining the genes that are essential for cellular proliferation is critical for understanding organismal development and identifying high-value targets for disease therapies. However, the requirements for cell-cycle progression in human cells remain incompletely understood. To elucidate the consequences of acute and chronic elimination of cell-cycle proteins, we generated and characterized inducible CRISPR/Cas9 knockout human cell lines targeting 209 genes involved in diverse cell-cycle processes. We performed single-cell microscopic analyses to systematically establish the effects of the knockouts on subcellular architecture. To define variations in cell-cycle requirements between cultured cell lines, we generated knockouts across cell lines of diverse origins. We demonstrate that p53 modulates the phenotype of specific cell-cycle defects through distinct mechanisms, depending on the defect. This work provides a resource to broadly facilitate robust and long-term depletion of cell-cycle proteins and reveals insights into the requirements for cell-cycle progression.

Keywords: CRISPR/Cas9; DNA replication; centromere; kinetochore; microtubule; mitosis; multipolarity; p53; spindle.

Publication types

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

MeSH terms

  • CRISPR-Cas Systems / genetics*
  • Cell Cycle / genetics*
  • Cell Polarity
  • Cell Proliferation
  • Cell Survival
  • Cellular Senescence
  • Centrioles / metabolism
  • Cyclin-Dependent Kinase Inhibitor p21 / metabolism
  • Gene Knockout Techniques*
  • HeLa Cells
  • Humans
  • Phenotype
  • Signal Transduction
  • Spindle Apparatus / metabolism
  • Tumor Suppressor Protein p53 / metabolism*

Substances

  • Cyclin-Dependent Kinase Inhibitor p21
  • Tumor Suppressor Protein p53