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

• 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

SummaryThe 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.

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