An inducible lentiviral guide RNA platform enables the identification of tumor-essential genes and tumor-promoting mutations in vivo

Cell Rep. 2015 Mar 3;10(8):1422-32. doi: 10.1016/j.celrep.2015.02.002. Epub 2015 Feb 26.

Abstract

The CRISPR/Cas9 technology enables the introduction of genomic alterations into almost any organism; however, systems for efficient and inducible gene modification have been lacking, especially for deletion of essential genes. Here, we describe a drug-inducible small guide RNA (sgRNA) vector system allowing for ubiquitous and efficient gene deletion in murine and human cells. This system mediates the efficient, temporally controlled deletion of MCL-1, both in vitro and in vivo, in human Burkitt lymphoma cell lines that require this anti-apoptotic BCL-2 protein for sustained survival and growth. Unexpectedly, repeated induction of the same sgRNA generated similar inactivating mutations in the human Mcl-1 gene due to low mutation variability exerted by the accompanying non-homologous end-joining (NHEJ) process. Finally, we were able to generate hematopoietic cell compartment-restricted Trp53-knockout mice, leading to the identification of cancer-promoting mutants of this critical tumor suppressor.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Apoptosis Regulatory Proteins / genetics
  • Apoptosis Regulatory Proteins / metabolism
  • Base Sequence
  • Bcl-2-Like Protein 11
  • Cell Line
  • Cell Survival / drug effects
  • Clustered Regularly Interspaced Short Palindromic Repeats / genetics
  • DNA End-Joining Repair
  • Doxycycline / pharmacology
  • Genes, Essential / genetics*
  • Genes, Neoplasm / genetics*
  • Humans
  • Lentivirus / genetics*
  • Membrane Proteins / genetics
  • Membrane Proteins / metabolism
  • Mice
  • Mice, Inbred C57BL
  • Mice, Knockout
  • Mice, Transgenic
  • Mutation
  • Myeloid Cell Leukemia Sequence 1 Protein / genetics
  • Myeloid Cell Leukemia Sequence 1 Protein / metabolism
  • Neoplasms / genetics*
  • Neoplasms / metabolism
  • Neoplasms / pathology
  • Proto-Oncogene Proteins / genetics
  • Proto-Oncogene Proteins / metabolism
  • Proto-Oncogene Proteins c-bcl-2 / metabolism
  • RNA, Guide / metabolism*
  • Transplantation, Heterologous
  • Tumor Suppressor Protein p53 / deficiency
  • Tumor Suppressor Protein p53 / genetics
  • Tumor Suppressor Protein p53 / metabolism

Substances

  • Apoptosis Regulatory Proteins
  • BCL2L11 protein, human
  • Bcl-2-Like Protein 11
  • Bcl2l11 protein, mouse
  • Membrane Proteins
  • Myeloid Cell Leukemia Sequence 1 Protein
  • Proto-Oncogene Proteins
  • Proto-Oncogene Proteins c-bcl-2
  • RNA, Guide
  • Tumor Suppressor Protein p53
  • Doxycycline