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Crispr chromosome translocation

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https://www.readbyqxmd.com/read/28572162/crispr-cas9-induced-t-11-19-mll-enl-translocations-initiate-leukemia-in-human-hematopoietic-progenitor-cells-in-vivo
#1
Jana Reimer, Sabine Knoess, Maurice Labuhn, Emmanuelle M Charpentier, Gudrun Göhring, Brigitte Schlegelberger, Jan-Henning Klusmann, Dirk Heckl
Chromosomal translocations generating oncogenic fusion proteins are causative for most pediatric leukemias and frequently affect the MLL/KMT2A gene. In vivo modeling of bona fide chromosomal translocations in human hematopoietic stem and progenitor cells (HSPCs) is challenging but essential to determine their actual leukemogenic potential. We therefore developed an advanced lentiviral CRISPR-Cas9 vector that efficiently transduced human CD34+-HSPCs and induced the t(11;19)/MLL-ENL translocation. Leveraging this system we could demonstrate that HSPCs harboring the translocation showed only a transient clonal growth advantage in vitro...
June 1, 2017: Haematologica
https://www.readbyqxmd.com/read/28522325/generation-of-chromosomal-translocations-that-lead-to-conditional-fusion-protein-expression-using-crispr-cas9-and-homology-directed-repair
#2
Fabio Vanoli, Maria Jasin
Recurrent chromosomal translocations often lead to expression of fusion proteins associated with oncogenic transformation. To study translocations and downstream events, genome editing techniques have been developed to generate chromosomal translocations through non-homologous end joining of DNA double-strand breaks introduced at the two participating endogenous loci. However, the frequencies at which these events occur is usually too low to efficiently clone cells carrying the translocation. This article provides a detailed method using CRISPR-Cas9 technology and homology-directed repair to efficiently isolate cells harboring a chromosomal translocation...
May 15, 2017: Methods: a Companion to Methods in Enzymology
https://www.readbyqxmd.com/read/28494941/efficient-recreation-of-t-11-22-ewsr1-fli1-in-human-stem-cells-using%C3%A2-crispr-cas9
#3
Raul Torres-Ruiz, Marta Martinez-Lage, Maria C Martin, Aida Garcia, Clara Bueno, Julio Castaño, Juan C Ramirez, Pablo Menendez, Juan C Cigudosa, Sandra Rodriguez-Perales
Efficient methodologies for recreating cancer-associated chromosome translocations are in high demand as tools for investigating how such events initiate cancer. The CRISPR/Cas9 system has been used to reconstruct the genetics of these complex rearrangements at native loci while maintaining the architecture and regulatory elements. However, the CRISPR system remains inefficient in human stem cells. Here, we compared three strategies aimed at enhancing the efficiency of the CRISPR-mediated t(11;22) translocation in human stem cells, including mesenchymal and induced pluripotent stem cells: (1) using end-joining DNA processing factors involved in repair mechanisms, or (2) ssODNs to guide the ligation of the double-strand break ends generated by CRISPR/Cas9; and (3) all-in-one plasmid or ribonucleoprotein complex-based approaches...
May 9, 2017: Stem Cell Reports
https://www.readbyqxmd.com/read/28480303/a-one-step-pcr-based-assay-to-evaluate-the-efficiency-and-precision-of-genomic-dna-editing-tools
#4
Diego Germini, Yara Bou Saada, Tatiana Tsfasman, Kristina Osina, Chloé Robin, Nikolay Lomov, Mikhail Rubtsov, Nikolajs Sjakste, Mar Lipinski, Yegor Vassetzky
Despite rapid progress, many problems and limitations persist and limit the applicability of gene-editing techniques. Making use of meganucleases, TALENs, or CRISPR/Cas9-based tools requires an initial step of pre-screening to determine the efficiency and specificity of the designed tools. This step remains time consuming and material consuming. Here we propose a simple, cheap, reliable, time-saving, and highly sensitive method to evaluate a given gene-editing tool based on its capacity to induce chromosomal translocations when combined with a reference engineered nuclease...
June 16, 2017: Molecular Therapy. Methods & Clinical Development
https://www.readbyqxmd.com/read/28325870/crispr-cas9-guided-oncogenic-chromosomal-translocations-with-conditional-fusion-protein-expression-in-human-mesenchymal-cells
#5
Fabio Vanoli, Mark Tomishima, Weiran Feng, Khadija Lamribet, Loelia Babin, Erika Brunet, Maria Jasin
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...
April 4, 2017: Proceedings of the National Academy of Sciences of the United States of America
https://www.readbyqxmd.com/read/28325290/rna-guided-crispr-cas9-system-mediated-engineering-of-acute-myeloid-leukemia-mutations
#6
Oliver Brabetz, Vijay Alla, Linus Angenendt, Christoph Schliemann, Wolfgang E Berdel, Maria-Francisca Arteaga, Jan-Henrik Mikesch
Current acute myeloid leukemia (AML) disease models face severe limitations because most of them induce un-physiological gene expressions that do not represent conditions in AML patients and/or depend on external promoters for regulation of gene expression/repression. Furthermore, many AML models are based on reciprocal chromosomal translocations that only reflect the minority of AML patients, whereas more than 50% of patients have a normal karyotype. The majority of AML, however, is driven by somatic mutations...
March 17, 2017: Molecular Therapy. Nucleic Acids
https://www.readbyqxmd.com/read/28258182/annealing-of-complementary-dna-sequences-during-double-strand-break-repair-in-drosophila-is-mediated-by-the-ortholog-of-smarcal1
#7
Julie Korda Holsclaw, Jeff Sekelsky
DNA double-strand breaks (DSBs) pose a serious threat to genomic integrity. If unrepaired, they can lead to chromosome fragmentation and cell death. If repaired incorrectly, they can cause mutations and chromosome rearrangements. DSBs are repaired using end-joining or homology-directed repair strategies, with the predominant form of homology-directed repair being synthesis-dependent strand annealing (SDSA). SDSA is the first defense against genomic rearrangements and information loss during DSB repair, making it a vital component of cell health and an attractive target for chemotherapeutic development...
May 2017: Genetics
https://www.readbyqxmd.com/read/28241139/transcription-control-by-the-enl-yeats-domain-in-acute-leukaemia
#8
Michael A Erb, Thomas G Scott, Bin E Li, Huafeng Xie, Joshiawa Paulk, Hyuk-Soo Seo, Amanda Souza, Justin M Roberts, Shiva Dastjerdi, Dennis L Buckley, Neville E Sanjana, Ophir Shalem, Behnam Nabet, Rhamy Zeid, Nana K Offei-Addo, Sirano Dhe-Paganon, Feng Zhang, Stuart H Orkin, Georg E Winter, James E Bradner
Recurrent chromosomal translocations producing a chimaeric MLL oncogene give rise to a highly aggressive acute leukaemia associated with poor clinical outcome. The preferential involvement of chromatin-associated factors as MLL fusion partners belies a dependency on transcription control. Despite recent progress made in targeting chromatin regulators in cancer, available therapies for this well-characterized disease remain inadequate, prompting the need to identify new targets for therapeutic intervention. Here, using unbiased CRISPR-Cas9 technology to perform a genome-scale loss-of-function screen in an MLL-AF4-positive acute leukaemia cell line, we identify ENL as an unrecognized gene that is specifically required for proliferation in vitro and in vivo...
March 9, 2017: Nature
https://www.readbyqxmd.com/read/28188619/generation-of-conditional-oncogenic-chromosomal-translocations-using-crispr-cas9-genomic-editing-and-homology-directed-repair
#9
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...
May 2017: Journal of Pathology
https://www.readbyqxmd.com/read/28138962/pax3-foxo1-is-essential-for-tumour-initiation-and-maintenance-but-not-recurrence-in-a-human-myoblast-model-of-rhabdomyosarcoma
#10
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 immortalized human myoblasts. Although myoblasts expressing PAX3-FOXO1 or MYCN alone were not transformed in focus formation assays, combined PAX3-FOXO1 and MYCN expression resulted in transformation...
April 2017: Journal of Pathology
https://www.readbyqxmd.com/read/28124028/optimized-crispr-cas9-genome-editing-for-leishmania-and-its-use-to-target-a-multigene-family-induce-chromosomal-translocation-and-study-dna-break-repair-mechanisms
#11
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
https://www.readbyqxmd.com/read/27694798/interference-driven-spacer-acquisition-is-dominant-over-naive-and-primed-adaptation-in-a-native-crispr-cas-system
#12
Raymond H J Staals, Simon A Jackson, Ambarish Biswas, Stan J J Brouns, Chris M Brown, Peter C Fineran
CRISPR-Cas systems provide bacteria with adaptive immunity against foreign nucleic acids by acquiring short, invader-derived sequences called spacers. Here, we use high-throughput sequencing to analyse millions of spacer acquisition events in wild-type populations of Pectobacterium atrosepticum. Plasmids not previously encountered, or plasmids that had escaped CRISPR-Cas targeting via point mutation, are used to provoke naive or primed spacer acquisition, respectively. The origin, location and order of spacer acquisition show that spacer selection through priming initiates near the site of CRISPR-Cas recognition (the protospacer), but on the displaced strand, and is consistent with 3'-5' translocation of the Cas1:Cas2-3 acquisition machinery...
October 3, 2016: Nature Communications
https://www.readbyqxmd.com/read/27640184/efficient-generation-and-reversion-of-chromosomal-translocations-using-crispr-cas-technology
#13
Sergey Lekomtsev, Sofia Aligianni, Ana Lapao, Tilmann Bürckstümmer
BACKGROUND: Chromosomal translocations are a hallmark of cancer cells and give rise to fusion oncogenes. To gain insight into the mechanisms governing tumorigenesis, adequate model cell lines are required. RESULTS: We employ the versatile CRISPR/Cas system to engineer cell lines in which chromosomal translocations are either generated de novo (CD74-ROS1) or existing translocations are reverted back to the original configuration (BCR-ABL1). To this end, we co-apply two guide RNAs to artificially generate two breakpoints and screen for spontaneous fusion events by PCR...
September 17, 2016: BMC Genomics
https://www.readbyqxmd.com/read/27622539/genome-wide-assessment-of-efficiency-and-specificity-in-crispr-cas9-mediated-multiple-site-targeting-in-arabidopsis
#14
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
https://www.readbyqxmd.com/read/27497066/in-vivo-and-in-vitro-disease-modeling-with-crispr-cas9
#15
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
https://www.readbyqxmd.com/read/27440725/jak1-stat3-signals-are-essential-effectors-of-the-usp6-tre17-oncogene-in-tumorigenesis
#16
Laura Quick, Robert Young, Ian C Henrich, Xiaoke Wang, Yan W Asmann, Andre M Oliveira, Margaret M Chou
Bone and soft tissue tumors (BSTT) are relatively poorly understood, hampering the development of effective therapies. Here we report a role for the ubiquitin-specific protease 6 (USP6)/TRE17 oncogene, which is overexpressed upon chromosome translocation in various human tumors, including aneurysmal bone cyst (ABC), and the related benign lesion nodular fasciitis. Ectopic expression of USP6 is known to drive formation of tumors, which recapitulate key features of ABC and nodular fasciitis; however, the identity of USP6's relevant substrates has been obscure...
September 15, 2016: Cancer Research
https://www.readbyqxmd.com/read/27264557/dna-polymerase-%C3%AE-polq-double-strand-break-repair-and-cancer
#17
REVIEW
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
https://www.readbyqxmd.com/read/27016031/genome-editing-of-structural-variations-modeling-and-gene-correction
#18
REVIEW
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
https://www.readbyqxmd.com/read/26916719/multiplexed-pancreatic-genome-engineering-and-cancer-induction-by-transfection-based-crispr-cas9-delivery-in-mice
#19
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...
February 26, 2016: Nature Communications
https://www.readbyqxmd.com/read/26898344/induction-of-site-specific-chromosomal-translocations-in-embryonic-stem-cells-by-crispr-cas9
#20
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
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