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cas9 chromosome translocation

Lee Spraggon, Luciano G Martelotto, Julija Hmeljak, Tyler D Hitchman, Jiang Wang, Lu Wang, Emily K Slotkin, Pang-Dian Fan, Jorge S Reis-Filho, Marc Ladanyi
Chromosomal rearrangements encoding oncogenic fusion proteins are found in a wide variety of malignancies. The use of programmable nucleases to generate specific double-strand breaks in endogenous loci, followed by non-homologous end joining DNA repair, has allowed several of these translocations to be generated as constitutively expressed fusion genes within a cell population. Here, we describe a novel approach that combines CRISPR-Cas9 technology with homology-directed repair to engineer, capture and modulate the expression of chromosomal translocation products in a human cell line...
February 11, 2017: Journal of Pathology
Puspa R Pandey, Bishwanath Chatterjee, Mary E Olanich, Javed Khan, Markku M Miettinen, Stephen M Hewitt, Frederic G Barr
The PAX3-FOXO1 fusion gene is generated by a 2;13 chromosomal translocation and is a characteristic feature of an aggressive subset of rhabdomyosarcoma (RMS). To dissect the mechanism of oncogene action during RMS tumourigenesis and progression, doxycycline-inducible PAX3-FOXO1 and constitutive MYCN expression constructs were introduced into immortalised human myoblasts. Though myoblasts expressing PAX3-FOXO1 or MYCN alone were not transformed in focus formation assays, combined PAX3-FOXO1 and MYCN expression resulted in transformation...
January 31, 2017: Journal of Pathology
Wen-Wei Zhang, Patrick Lypaczewski, Greg Matlashewski
CRISPR-Cas9-mediated genome editing has recently been adapted for Leishmania spp. parasites, the causative agents of human leishmaniasis. We have optimized this genome-editing tool by selecting for cells with CRISPR-Cas9 activity through cotargeting the miltefosine transporter gene; mutation of this gene leads to miltefosine resistance. This cotargeting strategy integrated into a triple guide RNA (gRNA) expression vector was used to delete all 11 copies of the A2 multigene family; this was not previously possible with the traditional gene-targeting method...
January 2017: MSphere
Brenda A Peterson, David C Haak, Marc T Nishimura, Paulo J P L Teixeira, Sean R James, Jeffery L Dangl, Zachary L Nimchuk
Simultaneous multiplex mutation of large gene families using Cas9 has the potential to revolutionize agriculture and plant sciences. The targeting of multiple genomic sites at once raises concerns about the efficiency and specificity in targeting. The model Arabidopsis thaliana is widely used in basic plant research. Previous work has suggested that the Cas9 off-target rate in Arabidopsis is undetectable. Here we use deep sequencing on pooled plants simultaneously targeting 14 distinct genomic loci to demonstrate that multiplex targeting in Arabidopsis is highly specific to on-target sites with no detectable off-target events...
2016: PloS One
Tomoko Kato, Shuji Takada
In the past few years, extensive progress has been made in the development of genome-editing technology. Among several genome-editing tools, the clustered regularly interspaced short palindrome repeat-associated Cas9 nuclease (CRISPR/Cas9) system is particularly widely used owing to the ease of sequence-specific nuclease construction and the highly efficient introduction of mutations. The CRISPR/Cas9 system was originally constructed to induce small insertion and deletion mutations, but various methods have been developed to introduce point mutations, deletions, insertions, chromosomal translocations and so on...
January 2017: Briefings in Functional Genomics
Richard D Wood, Sylvie Doublié
DNA polymerase theta (pol θ) is encoded in the genomes of many eukaryotes, though not in fungi. Pol θ is encoded by the POLQ gene in mammalian cells. The C-terminal third of the protein is a family A DNA polymerase with additional insertion elements relative to prokaryotic homologs. The N-terminal third is a helicase-like domain with DNA-dependent ATPase activity. Pol θ is important in the repair of genomic double-strand breaks (DSBs) from many sources. These include breaks formed by ionizing radiation and topoisomerase inhibitors, breaks arising at stalled DNA replication forks, breaks introduced during diversification steps of the mammalian immune system, and DSB induced by CRISPR-Cas9...
August 2016: DNA Repair
Chul-Yong Park, Jin Jea Sung, Dong-Wook Kim
The analysis of chromosomal structural variations (SVs), such as inversions and translocations, was made possible by the completion of the human genome project and the development of genome-wide sequencing technologies. SVs contribute to genetic diversity and evolution, although some SVs can cause diseases such as hemophilia A in humans. Genome engineering technology using programmable nucleases (e.g., ZFNs, TALENs, and CRISPR/Cas9) has been rapidly developed, enabling precise and efficient genome editing for SV research...
July 2016: Trends in Biotechnology
Roman Maresch, Sebastian Mueller, Christian Veltkamp, Rupert Öllinger, Mathias Friedrich, Irina Heid, Katja Steiger, Julia Weber, Thomas Engleitner, Maxim Barenboim, Sabine Klein, Sandra Louzada, Ruby Banerjee, Alexander Strong, Teresa Stauber, Nina Gross, Ulf Geumann, Sebastian Lange, Marc Ringelhan, Ignacio Varela, Kristian Unger, Fengtang Yang, Roland M Schmid, George S Vassiliou, Rickmer Braren, Günter Schneider, Mathias Heikenwalder, Allan Bradley, Dieter Saur, Roland Rad
Mouse transgenesis has provided fundamental insights into pancreatic cancer, but is limited by the long duration of allele/model generation. Here we show transfection-based multiplexed delivery of CRISPR/Cas9 to the pancreas of adult mice, allowing simultaneous editing of multiple gene sets in individual cells. We use the method to induce pancreatic cancer and exploit CRISPR/Cas9 mutational signatures for phylogenetic tracking of metastatic disease. Our results demonstrate that CRISPR/Cas9-multiplexing enables key applications, such as combinatorial gene-network analysis, in vivo synthetic lethality screening and chromosome engineering...
2016: Nature Communications
Junfeng Jiang, Li Zhang, Xingliang Zhou, Xi Chen, Guanyi Huang, Fengsheng Li, Ruizhe Wang, Nancy Wu, Youzhen Yan, Chang Tong, Sankalp Srivastava, Yue Wang, Houqi Liu, Qi-Long Ying
Chromosomal translocation is the most common form of chromosomal abnormality and is often associated with congenital genetic disorders, infertility, and cancers. The lack of cellular and animal models for chromosomal translocations, however, has hampered our ability to understand the underlying disease mechanisms and to develop new therapies. Here, we show that site-specific chromosomal translocations can be generated in mouse embryonic stem cells (mESCs) via CRISPR/Cas9. Mouse ESCs carrying translocated chromosomes can be isolated and expanded to establish stable cell lines...
February 22, 2016: Scientific Reports
Guangqing Lu, Jinzhi Duan, Sheng Shu, Xuxiang Wang, Linlin Gao, Jing Guo, Yu Zhang
In eukaryotes, DNA double-strand breaks (DSBs), one of the most harmful types of DNA damage, are repaired by homologous repair (HR) and nonhomologous end-joining (NHEJ). Surprisingly, in cells deficient for core classic NHEJ factors such as DNA ligase IV (Lig4), substantial end-joining activities have been observed in various situations, suggesting the existence of alternative end-joining (A-EJ) activities. Several putative A-EJ factors have been proposed, although results are mostly controversial. By using a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system, we generated mouse CH12F3 cell lines in which, in addition to Lig4, either Lig1 or nuclear Lig3, representing the cells containing a single DNA ligase (Lig3 or Lig1, respectively) in their nucleus, was completely ablated...
February 2, 2016: Proceedings of the National Academy of Sciences of the United States of America
P-Y Hsu, H-K Hsu, T-H Hsiao, Z Ye, E Wang, A L Profit, I Jatoi, Y Chen, N B Kirma, V X Jin, Z D Sharp, T H-M Huang
Recruitment of transcription machinery to target promoters for aberrant gene expression has been well studied, but underlying control directed by distant-acting enhancers remains unclear in cancer development. Our previous study demonstrated that distant estrogen response elements (DEREs) located on chromosome 20q13 are frequently amplified and translocated to other chromosomes in ERα-positive breast cancer cells. In this study, we used three-dimensional interphase fluorescence in situ hybridization to decipher spatiotemporal gathering of multiple DEREs in the nucleus...
May 5, 2016: Oncogene
Haiwei Mou, Zachary Kennedy, Daniel G Anderson, Hao Yin, Wen Xue
The cancer genome is highly complex, with hundreds of point mutations, translocations, and chromosome gains and losses per tumor. To understand the effects of these alterations, precise models are needed. Traditional approaches to the construction of mouse models are time-consuming and laborious, requiring manipulation of embryonic stem cells and multiple steps. The recent development of the clustered regularly interspersed short palindromic repeats (CRISPR)-Cas9 system, a powerful genome-editing tool for efficient and precise genome engineering in cultured mammalian cells and animals, is transforming mouse-model generation...
2015: Genome Medicine
Kalpana Kannan, Cristian Coarfa, Pei-Wen Chao, Liming Luo, Yan Wang, Amy E Brinegar, Shannon M Hawkins, Aleksandar Milosavljevic, Martin M Matzuk, Laising Yen
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...
March 17, 2015: Proceedings of the National Academy of Sciences of the United States of America
Irina V Lagutina, Virginia Valentine, Fabrizio Picchione, Frank Harwood, Marcus B Valentine, Barbara Villarejo-Balcells, Jaime J Carvajal, Gerard C Grosveld
Many recurrent chromosome translocations in cancer result in the generation of fusion genes that are directly implicated in the tumorigenic process. Precise modeling of the effects of cancer fusion genes in mice has been inaccurate, as constructs of fusion genes often completely or partially lack the correct regulatory sequences. The reciprocal t(2;13)(q36.1;q14.1) in human alveolar rhabdomyosarcoma (A-RMS) creates a pathognomonic PAX3-FOXO1 fusion gene. In vivo mimicking of this translocation in mice is complicated by the fact that Pax3 and Foxo1 are in opposite orientation on their respective chromosomes, precluding formation of a functional Pax3-Foxo1 fusion via a simple translocation...
2015: PLoS Genetics
Richard L Frock, Jiazhi Hu, Robin M Meyers, Yu-Jui Ho, Erina Kii, Frederick W Alt
Although great progress has been made in the characterization of the off-target effects of engineered nucleases, sensitive and unbiased genome-wide methods for the detection of off-target cleavage events and potential collateral damage are still lacking. Here we describe a linear amplification-mediated modification of a previously published high-throughput, genome-wide, translocation sequencing (HTGTS) method that robustly detects DNA double-stranded breaks (DSBs) generated by engineered nucleases across the human genome based on their translocation to other endogenous or ectopic DSBs...
February 2015: Nature Biotechnology
Rafael B Blasco, Elif Karaca, Chiara Ambrogio, Taek-Chin Cheong, Emre Karayol, Valerio G Minero, Claudia Voena, Roberto Chiarle
Generation of genetically engineered mouse models (GEMMs) for chromosomal translocations in the endogenous loci by a knockin strategy is lengthy and costly. The CRISPR/Cas9 system provides an innovative and flexible approach for genome engineering of genomic loci in vitro and in vivo. Here, we report the use of the CRISPR/Cas9 system for engineering a specific chromosomal translocation in adult mice in vivo. We designed CRISPR/Cas9 lentiviral vectors to induce cleavage of the murine endogenous Eml4 and Alk loci in order to generate the Eml4-Alk gene rearrangement recurrently found in non-small-cell lung cancers (NSCLCs)...
November 20, 2014: Cell Reports
Benjamin Renouf, Marion Piganeau, Hind Ghezraoui, Maria Jasin, Erika Brunet
Recurrent chromosomal translocations are found in numerous tumor types, often leading to the formation and expression of fusion genes with oncogenic potential. Creating chromosomal translocations at the relevant endogenous loci, rather than ectopically expressing the fusion genes, opens new possibilities for better characterizing molecular mechanisms driving tumor formation. In this chapter, we describe methods to create cancer translocations in human cells. DSBs or paired nicks generated by either wild-type Cas9 or the Cas9 nickase, respectively, are used to induce translocations at the relevant loci...
2014: Methods in Enzymology
Danilo Maddalo, Eusebio Manchado, Carla P Concepcion, Ciro Bonetti, Joana A Vidigal, Yoon-Chi Han, Paul Ogrodowski, Alessandra Crippa, Natasha Rekhtman, Elisa de Stanchina, Scott W Lowe, Andrea Ventura
Chromosomal rearrangements have a central role in the pathogenesis of human cancers and often result in the expression of therapeutically actionable gene fusions. A recently discovered example is a fusion between the genes echinoderm microtubule-associated protein like 4 (EML4) and anaplastic lymphoma kinase (ALK), generated by an inversion on the short arm of chromosome 2: inv(2)(p21p23). The EML4-ALK oncogene is detected in a subset of human non-small cell lung cancers (NSCLC) and is clinically relevant because it confers sensitivity to ALK inhibitors...
December 18, 2014: Nature
Hind Ghezraoui, Marion Piganeau, Benjamin Renouf, Jean-Baptiste Renaud, Annahita Sallmyr, Brian Ruis, Sehyun Oh, Alan E Tomkinson, Eric A Hendrickson, Carine Giovannangeli, Maria Jasin, Erika Brunet
Breakpoint junctions of the chromosomal translocations that occur in human cancers display hallmarks of nonhomologous end-joining (NHEJ). In mouse cells, translocations are suppressed by canonical NHEJ (c-NHEJ) components, which include DNA ligase IV (LIG4), and instead arise from alternative NHEJ (alt-NHEJ). Here we used designer nucleases (ZFNs, TALENs, and CRISPR/Cas9) to introduce DSBs on two chromosomes to study translocation joining mechanisms in human cells. Remarkably, translocations were altered in cells deficient for LIG4 or its interacting protein XRCC4...
September 18, 2014: Molecular Cell
R Torres, M C Martin, A Garcia, Juan C Cigudosa, J C Ramirez, S Rodriguez-Perales
Cancer-related human chromosomal translocations are generated through the illegitimate joining of two non-homologous chromosomes affected by double-strand breaks (DSB). Effective methodologies to reproduce precise reciprocal tumour-associated chromosomal translocations are required to gain insight into the initiation of leukaemia and sarcomas. Here we present a strategy for generating cancer-related human chromosomal translocations in vitro based on the ability of the RNA-guided CRISPR-Cas9 system to induce DSBs at defined positions...
2014: Nature Communications
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