The Experts below are selected from a list of 4083 Experts worldwide ranked by ideXlab platform
Mitsuo Oshimura - One of the best experts on this subject based on the ideXlab platform.
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crispr cas9 induced transgene insertion and telomere associated truncation of a single human Chromosome for Chromosome Engineering in cho and a9 cells
Scientific Reports, 2017Co-Authors: Narumi Uno, Kei Hiramatsu, Shinya Komoto, Yasuhiro Kazuki, Katsuhiro Uno, Mitsuo OshimuraAbstract:Chromosome Engineering techniques including gene insertion, telomere-associated truncation and microcell-mediated Chromosome transfer (MMCT) are powerful tools for generation of humanised model animal, containing megabase-sized genomic fragments. However, these techniques require two cell lines: homologous recombination (HR)-proficient DT40 cells for Chromosome modification, and CHO cells for transfer to recipient cells. Here we show an improved technique using a combination of CRISPR/Cas9-induced HR in CHO and mouse A9 cells without DT40 cells following MMCT to recipient cells. Transgene insertion was performed in CHO cells with the insertion of enhanced green fluorescence protein (EGFP) using CRISPR/Cas9 and a circular targeting vector containing two 3 kb HR arms. Telomere-associated truncation was performed in CHO cells using CRISPR/Cas9 and a linearised truncation vector containing a single 7 kb HR arm at the 5′ end, a 1 kb artificial telomere at the 3′ end. At least 11% and 6% of the targeting efficiency were achieved for transgene insertion and telomere-associated truncation, respectively. The transgene insertion was also confirmed in A9 cells (29%). The modified Chromosomes were transferrable to other cells. Thus, this CHO and A9 cell-mediated Chromosome Engineering using the CRISPR/Cas9 for direct transfer of the modified Chromosome is a rapid technique that will facilitate Chromosome manipulation.
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CRISPR/Cas9-induced transgene insertion and telomere-associated truncation of a single human Chromosome for Chromosome Engineering in CHO and A9 cells
Scientific Reports, 2017Co-Authors: Kei Hiramatsu, Shinya Komoto, Yasuhiro Kazuki, Mitsuo OshimuraAbstract:Chromosome Engineering techniques including gene insertion, telomere-associated truncation and microcell-mediated Chromosome transfer (MMCT) are powerful tools for generation of humanised model animal, containing megabase-sized genomic fragments. However, these techniques require two cell lines: homologous recombination (HR)-proficient DT40 cells for Chromosome modification, and CHO cells for transfer to recipient cells. Here we show an improved technique using a combination of CRISPR/Cas9-induced HR in CHO and mouse A9 cells without DT40 cells following MMCT to recipient cells. Transgene insertion was performed in CHO cells with the insertion of enhanced green fluorescence protein (EGFP) using CRISPR/Cas9 and a circular targeting vector containing two 3 kb HR arms. Telomere-associated truncation was performed in CHO cells using CRISPR/Cas9 and a linearised truncation vector containing a single 7 kb HR arm at the 5′ end, a 1 kb artificial telomere at the 3′ end. At least 11% and 6% of the targeting efficiency were achieved for transgene insertion and telomere-associated truncation, respectively. The transgene insertion was also confirmed in A9 cells (29%). The modified Chromosomes were transferrable to other cells. Thus, this CHO and A9 cell-mediated Chromosome Engineering using the CRISPR/Cas9 for direct transfer of the modified Chromosome is a rapid technique that will facilitate Chromosome manipulation.
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Studies of Tumor Suppressor Genes via Chromosome Engineering.
Cancers, 2015Co-Authors: Hiroyuki Kugoh, Takahito Ohira, Mitsuo OshimuraAbstract:The development and progression of malignant tumors likely result from consecutive accumulation of genetic alterations, including dysfunctional tumor suppressor genes. However, the signaling mechanisms that underlie the development of tumors have not yet been completely elucidated. Discovery of novel tumor-related genes plays a crucial role in our understanding of the development and progression of malignant tumors. Chromosome Engineering technology based on microcell-mediated Chromosome transfer (MMCT) is an effective approach for identification of tumor suppressor genes. The studies have revealed at least five tumor suppression effects. The discovery of novel tumor suppressor genes provide greater understanding of the complex signaling pathways that underlie the development and progression of malignant tumors. These advances are being exploited to develop targeted drugs and new biological therapies for cancer.
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Localization of an hTERTrepressor region on human Chromosome 3p21.3 using Chromosome Engineering
Genome Integrity, 2010Co-Authors: Satoshi Abe, Mitsuo Oshimura, Hiromi Tanaka, Tomomi Notsu, Shin-ichi Horike, Chikako Fujisaki, Takahito Ohhira, David Gilley, Hiroyuki KugohAbstract:Telomerase is a ribonucleoprotein enzyme that synthesizes telomeric DNA. The reactivation of telomerase activity by aberrant upregulation/expression of its catalytic subunit hTERT is a major pathway in human tumorigenesis. However, regulatory mechanisms that control hTERT expression are largely unknown. Previously, we and others have demonstrated that the introduction of human Chromosome 3, via microcell-mediated Chromosome transfer (MMCT), repressed transcription of the hTERT gene. These results suggested that human Chromosome 3 contains a regulatory factor(s) involved in the repression of hTERT . To further localize this putative hTERT repressor(s), we have developed a unique experimental approach by introducing various truncated Chromosome 3 regions produced by a novel chromosomal Engineering technology into the renal cell carcinoma cell line (RCC23 cells). These cells autonomously express ectopic hTERT (exohTERT) promoted by a retroviral LTR promoter in order to permit cellular division after repression of endogenous hTERT . We found a telomerase repressor region located within a 7-Mb interval on Chromosome 3p21.3. These results provide important information regarding hTERT regulation and a unique method to identify hTERT repressor elements.
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Localization of an hTERT repressor region on human Chromosome 3p21.3 using Chromosome Engineering.
Genome integrity, 2010Co-Authors: Satoshi Abe, Mitsuo Oshimura, Hiromi Tanaka, Tomomi Notsu, Shin-ichi Horike, Chikako Fujisaki, Takahito Ohhira, David Gilley, Hiroyuki KugohAbstract:Telomerase is a ribonucleoprotein enzyme that synthesizes telomeric DNA. The reactivation of telomerase activity by aberrant upregulation/expression of its catalytic subunit hTERT is a major pathway in human tumorigenesis. However, regulatory mechanisms that control hTERT expression are largely unknown. Previously, we and others have demonstrated that the introduction of human Chromosome 3, via microcell-mediated Chromosome transfer (MMCT), repressed transcription of the hTERT gene. These results suggested that human Chromosome 3 contains a regulatory factor(s) involved in the repression of hTERT. To further localize this putative hTERT repressor(s), we have developed a unique experimental approach by introducing various truncated Chromosome 3 regions produced by a novel chromosomal Engineering technology into the renal cell carcinoma cell line (RCC23 cells). These cells autonomously express ectopic hTERT (exohTERT) promoted by a retroviral LTR promoter in order to permit cellular division after repression of endogenous hTERT. We found a telomerase repressor region located within a 7-Mb interval on Chromosome 3p21.3. These results provide important information regarding hTERT regulation and a unique method to identify hTERT repressor elements.
Allan Bradley - One of the best experts on this subject based on the ideXlab platform.
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Chromosome Engineering in zygotes with crispr cas9
Genesis, 2016Co-Authors: Katharina Boroviak, Ruby Banerjee, Fengtang Yang, Brendan Doe, Allan BradleyAbstract:Deletions, duplications, and inversions of large genomic regions covering several genes are an important class of disease causing variants in humans. Modeling these structural variants in mice requires multistep processes in ES cells, which has limited their availability. Mutant mice containing small insertions, deletions, and single nucleotide polymorphisms can be reliably generated using CRISPR/Cas9 directly in mouse zygotes. Large structural variants can be generated using CRISPR/Cas9 in ES cells, but it has not been possible to generate these directly in zygotes. We now demonstrate the direct generation of deletions, duplications and inversions of up to one million base pairs by zygote injection.
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Chromosome Engineering in zygotes with CRISPR/Cas9
Genesis, 2016Co-Authors: Katharina Boroviak, Ruby Banerjee, Fengtang Yang, Allan BradleyAbstract:SUMMARY Deletions, duplications, and inversions of large genomic regions covering several genes are an important class of disease causing variants in humans. Modeling these structural variants in mice requires multistep processes in ES cells, which has limited their availability. Mutant mice containing small insertions, deletions, and single nucleotide polymorphisms can be reliably generated using CRISPR/Cas9 directly in mouse zygotes. Large structural variants can be generated using CRISPR/Cas9 in ES cells, but it has not been possible to generate these directly in zygotes. We now demonstrate the direct generation of deletions, duplications and inversions of up to one million base pairs by zygote injection. genesis 54:78–85, 2016. © 2016 The Authors. genesis Published by Wiley Periodicals, Inc.
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Chromosome Engineering in zygotes with CRISPR/Cas9
Genesis, 2016Co-Authors: Katharina Boroviak, Ruby Banerjee, Fengtang Yang, Allan BradleyAbstract:SUMMARY Deletions, duplications, and inversions of large genomic regions covering several genes are an important class of disease causing variants in humans. Modeling these structural variants in mice requires multistep processes in ES cells, which has limited their availability. Mutant mice containing small insertions, deletions, and single nucleotide polymorphisms can be reliably generated using CRISPR/Cas9 directly in mouse zygotes. Large structural variants can be generated using CRISPR/Cas9 in ES cells, but it has not been possible to generate these directly in zygotes. We now demonstrate the direct generation of deletions, duplications and inversions of up to one million base pairs by zygote injection. genesis 54:78–85, 2016. © 2016 The Authors. genesis Published by Wiley Periodicals, Inc.
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Chromosome Engineering in zygotes with CRISPR/Cas9
Genesis (New York N.Y. : 2000), 2016Co-Authors: Katharina Boroviak, Ruby Banerjee, Fengtang Yang, Brendan Doe, Allan BradleyAbstract:Deletions, duplications, and inversions of large genomic regions covering several genes are an important class of disease causing variants in humans. Modeling these structural variants in mice requires multistep processes in ES cells, which has limited their availability. Mutant mice containing small insertions, deletions, and single nucleotide polymorphisms can be reliably generated using CRISPR/Cas9 directly in mouse zygotes. Large structural variants can be generated using CRISPR/Cas9 in ES cells, but it has not been possible to generate these directly in zygotes. We now demonstrate the direct generation of deletions, duplications and inversions of up to one million base pairs by zygote injection.
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Chromosome Engineering in ES cells.
Methods in molecular biology (Clifton N.J.), 2009Co-Authors: Louise Van Der Weyden, Charles Shaw-smith, Allan BradleyAbstract:Chromosomal rearrangements, such as deletions, duplications, inversions and translocations, occur frequently in humans and can be disease-associated or phenotypically neutral. To understand the genetic consequences of such genomic changes, these mutations need to be modelled in experimentally tractable systems. The mouse is an excellent organism for this analysis because of its biological and genetic similarity to humans, the ease with which its genome can be manipulated and the similarity of observed affects. Through Chromosome Engineering, defined rearrangements can be introduced into the mouse genome. The resulting mouse models are leading to a better understanding of the molecular and cellular basis of dosage alterations in human disease phenotypes, in turn opening new diagnostic and therapeutic opportunities.
Holger Puchta - One of the best experts on this subject based on the ideXlab platform.
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CRISPR–Cas-mediated Chromosome Engineering for crop improvement and synthetic biology
Nature Plants, 2021Co-Authors: Michelle Rönspies, Patrick Schindele, Annika Dorn, Holger PuchtaAbstract:This Review summarizes current strategies for manipulating genetic linkages in plants, with a focus on the use of CRISPR–Cas technology to generate heritable and targeted chromosomal rearrangements for crop improvement and synthetic biology. Plant breeding relies on the presence of genetic variation, as well as on the ability to break or stabilize genetic linkages between traits. The development of the genome-editing tool clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated protein (Cas) has allowed breeders to induce genetic variability in a controlled and site-specific manner, and to improve traits with high efficiency. However, the presence of genetic linkages is a major obstacle to the transfer of desirable traits from wild species to their cultivated relatives. One way to address this issue is to create mutants with deficiencies in the meiotic recombination machinery, thereby enhancing global crossover frequencies between homologous parental Chromosomes. Although this seemed to be a promising approach at first, thus far, no crossover frequencies could be enhanced in recombination-cold regions of the genome. Additionally, this approach can lead to unintended genomic instabilities due to DNA repair defects. Therefore, efforts have been undertaken to obtain predefined crossovers between homologues by inducing site-specific double-strand breaks (DSBs) in meiotic, as well as in somatic plant cells using CRISPR–Cas tools. However, this strategy has not been able to produce a substantial number of heritable homologous recombination-based crossovers. Most recently, heritable chromosomal rearrangements, such as inversions and translocations, have been obtained in a controlled way using CRISPR–Cas in plants. This approach unlocks a completely new way of manipulating genetic linkages, one in which the DSBs are induced in somatic cells, enabling the formation of chromosomal rearrangements in the megabase range, by DSB repair via non-homologous end-joining. This technology might also enable the restructuring of genomes more globally, resulting in not only the obtainment of synthetic plant Chromosome, but also of novel plant species.
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CRISPR-Cas-mediated Chromosome Engineering for crop improvement and synthetic biology.
Nature plants, 2021Co-Authors: Michelle Rönspies, Patrick Schindele, Annika Dorn, Holger PuchtaAbstract:Plant breeding relies on the presence of genetic variation, as well as on the ability to break or stabilize genetic linkages between traits. The development of the genome-editing tool clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein (Cas) has allowed breeders to induce genetic variability in a controlled and site-specific manner, and to improve traits with high efficiency. However, the presence of genetic linkages is a major obstacle to the transfer of desirable traits from wild species to their cultivated relatives. One way to address this issue is to create mutants with deficiencies in the meiotic recombination machinery, thereby enhancing global crossover frequencies between homologous parental Chromosomes. Although this seemed to be a promising approach at first, thus far, no crossover frequencies could be enhanced in recombination-cold regions of the genome. Additionally, this approach can lead to unintended genomic instabilities due to DNA repair defects. Therefore, efforts have been undertaken to obtain predefined crossovers between homologues by inducing site-specific double-strand breaks (DSBs) in meiotic, as well as in somatic plant cells using CRISPR-Cas tools. However, this strategy has not been able to produce a substantial number of heritable homologous recombination-based crossovers. Most recently, heritable chromosomal rearrangements, such as inversions and translocations, have been obtained in a controlled way using CRISPR-Cas in plants. This approach unlocks a completely new way of manipulating genetic linkages, one in which the DSBs are induced in somatic cells, enabling the formation of chromosomal rearrangements in the megabase range, by DSB repair via non-homologous end-joining. This technology might also enable the restructuring of genomes more globally, resulting in not only the obtainment of synthetic plant Chromosome, but also of novel plant species.
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Novel CRISPR/Cas applications in plants: from prime editing to Chromosome Engineering
Transgenic Research, 2021Co-Authors: Teng-kuei Huang, Holger PuchtaAbstract:In the last years, tremendous progress has been made in the development of CRISPR/Cas-mediated genome editing tools. A number of natural CRISPR/Cas nuclease variants have been characterized. Engineered Cas proteins have been developed to minimize PAM restrictions, off-side effects and temperature sensitivity. Both kinds of enzymes have, by now, been applied widely and efficiently in many plant species to generate either single or multiple mutations at the desired loci by multiplexing. In addition to DSB-induced mutagenesis, specifically designed CRISPR/Cas systems allow more precise gene editing, resulting not only in random mutations but also in predefined changes. Applications in plants include gene targeting by homologous recombination, base editing and, more recently, prime editing. We will evaluate these different technologies for their prospects and practical applicability in plants. In addition, we will discuss a novel application of the Cas9 nuclease in plants, enabling the induction of heritable chromosomal rearrangements, such as inversions and translocations. This technique will make it possible to change genetic linkages in a programmed way and add another level of genome Engineering to the toolbox of plant breeding. Also, strategies for tissue culture free genome editing were developed, which might be helpful to overcome the transformation bottlenecks in many crops. All in all, the recent advances of CRISPR/Cas technology will help agriculture to address the challenges of the twenty-first century related to global warming, pollution and the resulting food shortage.
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novel crispr cas applications in plants from prime editing to Chromosome Engineering
Transgenic Research, 2021Co-Authors: Teng-kuei Huang, Holger PuchtaAbstract:In the last years, tremendous progress has been made in the development of CRISPR/Cas-mediated genome editing tools. A number of natural CRISPR/Cas nuclease variants have been characterized. Engineered Cas proteins have been developed to minimize PAM restrictions, off-side effects and temperature sensitivity. Both kinds of enzymes have, by now, been applied widely and efficiently in many plant species to generate either single or multiple mutations at the desired loci by multiplexing. In addition to DSB-induced mutagenesis, specifically designed CRISPR/Cas systems allow more precise gene editing, resulting not only in random mutations but also in predefined changes. Applications in plants include gene targeting by homologous recombination, base editing and, more recently, prime editing. We will evaluate these different technologies for their prospects and practical applicability in plants. In addition, we will discuss a novel application of the Cas9 nuclease in plants, enabling the induction of heritable chromosomal rearrangements, such as inversions and translocations. This technique will make it possible to change genetic linkages in a programmed way and add another level of genome Engineering to the toolbox of plant breeding. Also, strategies for tissue culture free genome editing were developed, which might be helpful to overcome the transformation bottlenecks in many crops. All in all, the recent advances of CRISPR/Cas technology will help agriculture to address the challenges of the twenty-first century related to global warming, pollution and the resulting food shortage.
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crispr cas mediated Chromosome Engineering opening up a new avenue for plant breeding
Journal of Experimental Botany, 2021Co-Authors: Michelle Rönspies, Patrick Schindele, Holger PuchtaAbstract:The advent of powerful site-specific nucleases, particularly the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) system, which enables precise genome manipulation, has revolutionized plant breeding. Until recently, the main focus of researchers has been to simply knock-in or knock-out single genes, or to induce single base changes, but constant improvements of this technology have enabled more ambitious applications that aim to improve plant productivity or other desirable traits. One long-standing aim has been the induction of targeted chromosomal rearrangements (crossovers, inversions, or translocations). The feasibility of this technique has the potential to transform plant breeding, because natural rearrangements, like inversions, for example, typically present obstacles to the breeding process. In this way, genetic linkages between traits could be altered to combine or separate favorable and deleterious genes, respectively. In this review, we discuss recent breakthroughs in the field of Chromosome Engineering in plants and their potential applications in the field of plant breeding. In the future, these approaches might be applicable in shaping plant Chromosomes in a directed manner, based on plant breeding needs.
Daryl L Klindworth - One of the best experts on this subject based on the ideXlab platform.
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development and characterization of wheat lines carrying stem rust resistance gene sr43 derived from thinopyrum ponticum
Theoretical and Applied Genetics, 2014Co-Authors: Zhixia Niu, Daryl L Klindworth, Shiaoman Chao, Timothy L Friesen, Yue Jin, Xiwen Cai, Jaebom Ohm, Jack B RasmussenAbstract:Key message Wheat lines carrying Ug99-effective stem rust resistance geneSr43on shortened alien Chromosome segments were produced using Chromosome Engineering, and molecular markers linked toSr43were identified for marker-assisted selection.
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targeted introgression of a wheat stem rust resistance gene by dna marker assisted Chromosome Engineering
Genetics, 2011Co-Authors: Daryl L Klindworth, Shiaoman Chao, Timothy L Friesen, Steven S. XuAbstract:Chromosome Engineering is a useful strategy for transfer of alien genes from wild relatives into modern crops. However, this strategy has not been extensively used for alien gene introgression in most crops due to low efficiency of conventional cytogenetic techniques. Here, we report an improved scheme of Chromosome Engineering for efficient elimination of a large amount of goatgrass (Aegilops speltoides) chromatin surrounding Sr39, a gene that provides resistance to multiple stem rust races, including Ug99 (TTKSK) in wheat. The wheat ph1b mutation, which promotes meiotic pairing between homoeologous Chromosomes, was employed to induce recombination between wheat Chromosome 2B and goatgrass 2S chromatin using a backcross scheme favorable for inducing and detecting the homoeologous recombinants with small goatgrass Chromosome segments. Forty recombinants with Sr39 with reduced surrounding goatgrass chromatin were quickly identified from 1048 backcross progenies through disease screening and molecular marker analysis. Four of the recombinants carrying Sr39 with a minimal amount of goatgrass chromatin (2.87–9.15% of the translocated Chromosomes) were verified using genomic in situ hybridization. Approximately 97% of the goatgrass chromatin was eliminated in one of the recombinants, in which a tiny goatgrass Chromosome segment containing Sr39 was retained in the wheat genome. Localization of the goatgrass chromatin in the recombinants led to rapid development of three molecular markers tightly linked to Sr39. The new wheat lines and markers provide useful resources for the ongoing global effort to combat Ug99. This study has demonstrated great potential of Chromosome Engineering in genome manipulation for plant improvement.
Jack B Rasmussen - One of the best experts on this subject based on the ideXlab platform.
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development and characterization of wheat lines carrying stem rust resistance gene sr43 derived from thinopyrum ponticum
Theoretical and Applied Genetics, 2014Co-Authors: Zhixia Niu, Daryl L Klindworth, Shiaoman Chao, Timothy L Friesen, Yue Jin, Xiwen Cai, Jaebom Ohm, Jack B RasmussenAbstract:Key message Wheat lines carrying Ug99-effective stem rust resistance geneSr43on shortened alien Chromosome segments were produced using Chromosome Engineering, and molecular markers linked toSr43were identified for marker-assisted selection.