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

Brandon J Aubrey; Gemma L Kelly; Andrew J Kueh; Margs S Brennan; Liam O'Connor; Liz Milla; Stephen Wilcox; Lin Tai; Andreas Strasser; Marco J Herold

doi:10.1016/j.celrep.2015.02.002

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.

Authors

Affiliations

¹ Walter and Eliza Hall Institute of Medical Research, Parkville, VIC 3052, Australia; Department of Medical Biology, University of Melbourne, Parkville, VIC 3050, Australia; Department of Clinical Haematology and Bone Marrow Transplant Service, the Royal Melbourne Hospital, Parkville, VIC 3050, Australia.
² Walter and Eliza Hall Institute of Medical Research, Parkville, VIC 3052, Australia; Department of Medical Biology, University of Melbourne, Parkville, VIC 3050, Australia.
³ Walter and Eliza Hall Institute of Medical Research, Parkville, VIC 3052, Australia.
⁴ Walter and Eliza Hall Institute of Medical Research, Parkville, VIC 3052, Australia; Department of Medical Biology, University of Melbourne, Parkville, VIC 3050, Australia. Electronic address: herold@wehi.edu.au.

PMID: 25732831
DOI: 10.1016/j.celrep.2015.02.002

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, CRISPR-Cas Systems / 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, CRISPR-Cas Systems
Tumor Suppressor Protein p53
Doxycycline