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cas9 chromosome translocation

Alexanda K Ling, Clare C So, Michael X Le, Audrey Y Chen, Lisa Hung, Alberto Martin
Activation-induced cytidine deaminase (AID) inflicts DNA damage at Ig genes to initiate class switch recombination (CSR) and chromosomal translocations. However, the DNA lesions formed during these processes retain an element of randomness, and thus knowledge of the relationship between specific DNA lesions and AID-mediated processes remains incomplete. To identify necessary and sufficient DNA lesions in CSR, the Cas9 endonuclease and nickase variants were used to program DNA lesions at a greater degree of predictability than is achievable with conventional induction of CSR...
February 22, 2018: Proceedings of the National Academy of Sciences of the United States of America
Zubair Ahmed Ratan, Young-Jin Son, Mohammad Faisal Haidere, Bhuiyan Mohammad Mahtab Uddin, Md Abdullah Yusuf, Sojib Bin Zaman, Jong-Hoon Kim, Laila Anjuman Banu, Jae Youl Cho
Bacteria and archaea possess adaptive immunity against foreign genetic materials through clustered regularly interspaced short palindromic repeat (CRISPR) systems. The discovery of this intriguing bacterial system heralded a revolutionary change in the field of medical science. The CRISPR and CRISPR-associated protein 9 (Cas9) based molecular mechanism has been applied to genome editing. This CRISPR-Cas9 technique is now able to mediate precise genetic corrections or disruptions in in vitro and in vivo environments...
2018: Therapeutic Advances in Medical Oncology
Pei-Chi Wei, Cheng-Sheng Lee, Zhou Du, Bjoern Schwer, Yuxiang Zhang, Jennifer Kao, Jeffrey Zurita, Frederick W Alt
We recently discovered 27 recurrent DNA double-strand break (DSB) clusters (RDCs) in mouse neural stem/progenitor cells (NSPCs). Most RDCs occurred across long, late-replicating RDC genes and were found only after mild inhibition of DNA replication. RDC genes share intriguing characteristics, including encoding surface proteins that organize brain architecture and neuronal junctions, and are genetically implicated in neuropsychiatric disorders and/or cancers. RDC identification relies on high-throughput genome-wide translocation sequencing (HTGTS), which maps recurrent DSBs based on their translocation to "bait" DSBs in specific chromosomal locations...
February 5, 2018: Proceedings of the National Academy of Sciences of the United States of America
Katsufumi Dejima, Sayaka Hori, Satoru Iwata, Yuji Suehiro, Sawako Yoshina, Tomoko Motohashi, Shohei Mitani
Balancer chromosomes are critical tools for genetic research. In C. elegans, reciprocal translocations that lead to aneuploidy have been widely used to maintain lethal and sterile mutations in stable stocks. Here, we generated a set of aneuploidy-free and structurally defined crossover suppressors that contain two overlapping inversions using the CRISPR/Cas9 system. The toolkit includes 13 crossover suppressors and covers approximately 63% of all C. elegans coding genes. Together with the classical intrachromosomal crossover suppressors, the system now covers 89% of the coding genes...
January 2, 2018: Cell Reports
Azza Al Ismail, Afzal Husain, Maki Kobayashi, Tasuku Honjo, Nasim A Begum
Activation-induced cytidine deaminase (AID) is essential for class-switch recombination (CSR) and somatic hypermutation (SHM) of immunoglobulin (Ig) genes. Studies on in vitro mutagenized AID as well as its mutations in human patients with Hyper-IgM (HIGM)-syndrome type II revealed that C-terminal AID mutations were defective in CSR whereas their DNA cleavage and SHM activities remained intact. The C-terminal mutants of AID were speculated to exert the dominant negative effect on wild type WT AID whereas its mechanism remains unknown...
November 10, 2017: International Immunology
Pedro A Mateos-Gomez, Tatiana Kent, Sarah K Deng, Shane McDevitt, Ekaterina Kashkina, Trung M Hoang, Richard T Pomerantz, Agnel Sfeir
Mammalian polymerase theta (Polθ) is a multifunctional enzyme that promotes error-prone DNA repair by alternative nonhomologous end joining (alt-NHEJ). Here we present structure-function analyses that reveal that, in addition to the polymerase domain, Polθ-helicase activity plays a central role during double-strand break (DSB) repair. Our results show that the helicase domain promotes chromosomal translocations by alt-NHEJ in mouse embryonic stem cells and also suppresses CRISPR-Cas9- mediated gene targeting by homologous recombination (HR)...
December 2017: Nature Structural & Molecular Biology
Xiaoyu Chen, Josephine M Janssen, Jin Liu, Ignazio Maggio, Anke E J 't Jong, Harald M M Mikkers, Manuel A F V Gonçalves
Precise genome editing involves homologous recombination between donor DNA and chromosomal sequences subjected to double-stranded DNA breaks made by programmable nucleases. Ideally, genome editing should be efficient, specific, and accurate. However, besides constituting potential translocation-initiating lesions, double-stranded DNA breaks (targeted or otherwise) are mostly repaired through unpredictable and mutagenic non-homologous recombination processes. Here, we report that the coordinated formation of paired single-stranded DNA breaks, or nicks, at donor plasmids and chromosomal target sites by RNA-guided nucleases based on CRISPR-Cas9 components, triggers seamless homology-directed gene targeting of large genetic payloads in human cells, including pluripotent stem cells...
September 22, 2017: Nature Communications
Jana Reimer, Sabine Knöß, Maurice Labuhn, Emmanuelle M Charpentier, Gudrun Göhring, Brigitte Schlegelberger, Jan-Henning Klusmann, Dirk Heckl
Chromosomal translocations that generate 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 is challenging but essential to determine their actual leukemogenic potential. We therefore developed an advanced lentiviral CRISPR-Cas9 vector that efficiently transduced human CD34+ hematopoietic stem and progenitor cells and induced the t(11;19)/MLL-ENL translocation...
September 2017: Haematologica
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
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
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
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
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
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
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
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
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
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
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