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

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.

Hepatocyte growth factor renders BRAF mutant human melanoma cell lines resistant to PLX4032 by downregulating the pro-apoptotic BH3-only proteins PUMA and BIM

“…Efficient targeting of BIM and/or PUMA with the sgRNAs was already validated previously. 20,21 Knockout of BIM and PUMA in both, single and double knockout cell lines, was verified, with these proteins being either undetectable or at least markedly reduced in all cell lines after treatment with PLX4032 ( Figure 2b and Supplementary Figure 1b). As expected, PLX4032-induced upregulation of BIM and PUMA occurred normally in the control sgRNA transduced cell lines.…”

“…38 The generation of the Cas9 expression vector and sgRNA constructs was described recently. 20 The sgRNA sequences are as follows:…”

“…Successful target gene disruption was verified by DNA sequence analysis as described previously. 20 Inducible BIM S overexpression variants in patient melanoma cell lines. Patient-derived cell lines LM-MEL-28, -33, -34 (ref.…”

Combination of IAP antagonist and IFNγ activates novel caspase-10- and RIPK1-dependent cell death pathways

“…We therefore generated singlecell HT29 clones deficient for apoptotic (CASP8 − / − ) or necroptotic (RIPK1 − / − , RIPK3 − / − or MLKL − / − ) cell death pathways using CRISPR/Cas9 45,46 and validated them using next-generation sequencing (Supplementary Figure S5). Similar to their wild-type counterparts, CASP8 − / − HT29 cells were sensitive to IFNγ/SM treatment (Figure 4b).…”

“…46 Infected cells were selected with 5 μg/ml of puromycin (for IκBα SR , SOCS1, PKR selection) and/or 10-50 μg/ml of hygromycin (for GEV16 selection) or single cells were sorted for GFP and mCherry (selection of CRISPR/Cas9 cell lines) into 96-well plates. Lentiviral constructs were induced with 10 nM of 4-hydroxy tamoxifen or 20 ng/ml (for pFTRE 3G human PKR vector) and 1 μg/ml (for pF GH1t UTG vector) doxycycline.…”