Engineering human tumour-associated chromosomal translocations with the RNA-guided CRISPR–Cas9 system

Torres-Ruíz, Raúl; Martín, Maria Carmen; García, Andrés Pun; Cigudosa, Juan C.; Ramı́rez, Juan Carlos; Rodríguez-Perales, Sandra

doi:10.1038/ncomms4964

Cited by 218 publications

(172 citation statements)

References 28 publications

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“…5D). Thus, 60-80% of the isolated foci carry the BCAM-AKT2 translocation, and this is far greater than the expected low rate of long-range chromosome translocation that can be induced by the CRISPR/Cas9 system (1-4%) in cells (27). The observed focus formation, therefore, is not a random event but largely associated with BCAM-AKT2 expression.…”

Section: Bcam-akt2 Is Translated Into An In-frame Fusion Protein In Pmentioning

confidence: 88%

Recurrent BCAM-AKT2 fusion gene leads to a constitutively activated AKT2 fusion kinase in high-grade serous ovarian carcinoma

Kannan

Coarfa

Chao

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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High-grade serous ovarian cancer (HGSC) is among the most lethal forms of cancer in women. Excessive genomic rearrangements, which are expected to create fusion oncogenes, are the hallmark of this cancer. Here we report a cancer-specific gene fusion between BCAM, a membrane adhesion molecule, and AKT2, a key kinase in the PI3K signaling pathway. This fusion is present in 7% of the 60 patient cancers tested, a significant frequency considering the highly heterogeneous nature of this malignancy. Further, we provide direct evidence that BCAM-AKT2 is translated into an in-frame fusion protein in the patient's tumor. The resulting AKT2 fusion kinase is membrane-associated, constitutively phosphorylated, and activated as a functional kinase in cells. Unlike endogenous AKT2, whose activity is tightly regulated by external stimuli, BCAM-AKT2 escapes the regulation from external stimuli. Moreover, a BCAM-AKT2 fusion gene generated via chromosomal translocation using the CRISPR/Cas9 system leads to focus formation in both OVCAR8 and HEK-293T cell lines, suggesting that BCAM-AKT2 is oncogenic. Together, the results indicate that BCAM-AKT2 expression is a new mechanism of AKT2 kinase activation in HGSC. BCAM-AKT2 is the only fusion gene in HGSC that is proven to translate an aberrant yet functional kinase fusion protein with oncogenic properties. This recurrent genomic alteration is a potential therapeutic target and marker of a clinically relevant subtype for tailored therapy of HGSC.cancer-specific fusion gene | high-grade serous ovarian cancer | AKT2 | BCAM | fusion kinase O varian cancer is responsible for the death of ∼140,200 women yearly, and 70% of these deaths are due to the high-grade serous ovarian cancer (HGSC) subtype (1), which is typically detected only after it has metastasized. Genomic characterization of HGSC tumors shows that TP53 mutations are present in 96% and BRCA1/2 in 22% of HGSC (2). These mutations are thought to occur early in pathogenesis (3) and promote genomic instability, thus giving rise to the high degree of heterogeneity and genomic rearrangements that are characteristic of these cancers. The characteristic genome rearrangements in HGSC imply that recombination events such as gene fusions should be common. Moreover, if the activity of a fusion gene represents a common oncogenic mechanism, the same fusion gene is likely to occur in many patients.Recurrent fusion genes are important for understanding cancer mechanisms and developing useful clinical biomarkers and anticancer therapies. For instance, the BCR-ABL fusion gene in chronic myeloid leukemia is known to initiate oncogenesis through formation of a misregulated BCR-ABL fusion kinase. The BCR-ABL fusion gene is also a clinical biomarker of high diagnostic and prognostic utility in chronic myeloid leukemia. In addition, this fusion protein serves as a therapeutic target for the successful drug imatinib (4). In solid human tumors, fusion genes such as the TMPRSS2-ERG fusion in prostate cancer (5), FGFR-TACC in glioblastoma (6), and DNAJB1...

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Section: Bcam-akt2 Is Translated Into An In-frame Fusion Protein In Pmentioning

confidence: 88%

Recurrent BCAM-AKT2 fusion gene leads to a constitutively activated AKT2 fusion kinase in high-grade serous ovarian carcinoma

Kannan

Coarfa

Chao

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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“…More recently, the discovery of RNAguided nucleases in bacterial adaptive immunity (CRISPR-Cas9) has facilitated DSB introduction at desired genomic loci (18,19). Using these various programmable nucleases, a number of cancer translocations have been generated in a variety of human cell lines, including EWSR1-FLI1 (ZFNs, CRISPR-Cas9) (11,20,21), NPM1-ALK (TALENs, CRISPR-Cas9) (11,12), and CD74-ROS1 (CRISPR-Cas9) (22). Oncogenic chromosomal inversions have also been engineered in mouse tissues, leading to tumorigenesis (EML4-ALK) (23).…”

mentioning

confidence: 99%

CRISPR-Cas9–guided oncogenic chromosomal translocations with conditional fusion protein expression in human mesenchymal cells

Vanoli

Tomishima

Feng

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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Gene editing techniques have been extensively used to attempt to model recurrent genomic rearrangements found in tumor cells. These methods involve the induction of double-strand breaks at endogenous loci followed by the identification of breakpoint junctions within a population, which typically arise by nonhomologous end joining. The low frequency of these events, however, has hindered the cloning of cells with the desired rearrangement before oncogenic transformation. Here we present a strategy combining CRISPR-Cas9 technology and homology-directed repair to allow for the selection of human mesenchymal stem cells harboring the oncogenic translocation EWSR1-WT1 found in the aggressive desmoplastic small round cell tumor. The expression of the fusion transcript is under the control of the endogenous EWSR1 promoter and, importantly, can be conditionally expressed using Cre recombinase. This method is easily adapted to generate any cancer-relevant rearrangement.CRISPR-Cas9 | chromosomal translocation | homology-directed repair | EWSR1-WT1 | DSRCT

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“…Examples of some of the outcomes that have become routine because of the emergence of targeted nucleases include gene knockout (Santiago et al 2008;Mali et al 2013b), gene deletion (Lee et al 2010), gene inversion (Xiao et al 2013), gene correction (Urnov et al 2005;Ran et al 2013), gene addition (Moehle et al 2007;Hockemeyer et al 2011;Hou et al 2013), and even chromosomal translocation (Fig. 2) (Torres et al 2014). In addition to cell line engineering, targeted nucleases have also expedited the generation of genetically modified organisms, facilitating the rapid creation of transgenic zebrafish (Doyon et al 2008;Sander et al 2011a;Hwang et al 2013), mice (Cui et al 2011;Wang et al 2013;Wu et al 2013), rats (Geurts et al 2009;Tesson et al 2011;Li et al 2013), monkeys (Liu et al 2014c), and livestock (Hauschild et al 2011;Carlson et al 2012), which together have the capacity to accelerate human disease modeling and the discovery of new therapeutics.…”

Section: Genome-editing Applications Engineering Cell Lines and Organmentioning

confidence: 99%

Genome-Editing Technologies: Principles and Applications

Gaj

Sirk

Shui

et al. 2016

Cold Spring Harb Perspect Biol

295

142

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Targeted nucleases have provided researchers with the ability to manipulate virtually any genomic sequence, enabling the facile creation of isogenic cell lines and animal models for the study of human disease, and promoting exciting new possibilities for human gene therapy. Here we review three foundational technologies-clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9), transcription activator-like effector nucleases (TALENs), and zinc-finger nucleases (ZFNs). We discuss the engineering advances that facilitated their development and highlight several achievements in genome engineering that were made possible by these tools. We also consider artificial transcription factors, illustrating how this technology can complement targeted nucleases for synthetic biology and gene therapy.

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Engineering human tumour-associated chromosomal translocations with the RNA-guided CRISPR–Cas9 system

Cited by 218 publications

References 28 publications

Recurrent BCAM-AKT2 fusion gene leads to a constitutively activated AKT2 fusion kinase in high-grade serous ovarian carcinoma

Recurrent BCAM-AKT2 fusion gene leads to a constitutively activated AKT2 fusion kinase in high-grade serous ovarian carcinoma

CRISPR-Cas9–guided oncogenic chromosomal translocations with conditional fusion protein expression in human mesenchymal cells

Genome-Editing Technologies: Principles and Applications

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