Details

Autor(en) / Beteiligte

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

Titel

An Inducible Lentiviral Guide RNA Platform Enables the Identification of Tumor-Essential Genes and Tumor-Promoting Mutations In Vivo

Ist Teil von

• Cell reports (Cambridge), 2015-03, Vol.10 (8), p.1422-1432

Ort / Verlag

United States: Elsevier Inc

Erscheinungsjahr

2015

Link zum Volltext

Quelle

MEDLINE

Beschreibungen/Notizen

• 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. [Display omitted] •A lentiviral CRISPR/Cas9 platform for the conditional targeting of essential genes•Efficient mutation of genes in mouse and human cell lines and primary mouse cells•Identification of cancer-promoting mutations in the tumor suppressor p53 The CRISPR/Cas9 system is an exciting methodology for genetic modification. Aubrey et al. have advanced this technology by developing an inducible lentiviral system. This platform facilitates efficient gene targeting that permits phenotypic assessment following deletion of essential genes and identification of tumor-promoting mutations in vivo.