The Experts below are selected from a list of 174 Experts worldwide ranked by ideXlab platform
Iain M. Cheeseman - One of the best experts on this subject based on the ideXlab platform.
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CRISPR/Cas9-based gene targeting using synthetic guide RNAs enables robust cell biological analyses.
Molecular Biology of the Cell, 2018Co-Authors: Kuan-chung Su, Mary-jane Tsang, Neil Emans, Iain M. CheesemanAbstract:: A key goal for cell biological analyses is to assess the phenotypes that result from eliminating a target gene. Since the early 1990s, the predominant strategy utilized in human tissue culture cells has been RNA interference (RNAi)-mediated Protein Depletion. However, RNAi suffers well-documented off-target effects as well as incomplete and reversible Protein Depletion. The implementation of CRISPR/Cas9-based DNA cleavage has revolutionized the capacity to conduct functional studies in human cells. However, this approach is still underutilized for conducting visual phenotypic analyses, particularly for essential genes that require conditional strategies to eliminate their gene products. Optimizing this strategy requires effective and streamlined approaches to introduce the Cas9 guide RNA into target cells. Here we assess the efficacy of synthetic guide RNA transfection to eliminate gene products for cell biological studies. On the basis of three representative gene targets (KIF11, CENPN, and RELA), we demonstrate that transfection of synthetic single guide RNA (sgRNA) and CRISPR RNA (crRNA) guides works comparably for Protein Depletion as cell lines stably expressing lentiviral-delivered RNA guides. We additionally demonstrate that synthetic sgRNAs can be introduced by reverse transfection on an array. Together, these strategies provide a robust, flexible, and scalable approach for conducting functional studies in human cells.
Kuan-chung Su - One of the best experts on this subject based on the ideXlab platform.
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CRISPR/Cas9-based gene targeting using synthetic guide RNAs enables robust cell biological analyses.
Molecular Biology of the Cell, 2018Co-Authors: Kuan-chung Su, Mary-jane Tsang, Neil Emans, Iain M. CheesemanAbstract:: A key goal for cell biological analyses is to assess the phenotypes that result from eliminating a target gene. Since the early 1990s, the predominant strategy utilized in human tissue culture cells has been RNA interference (RNAi)-mediated Protein Depletion. However, RNAi suffers well-documented off-target effects as well as incomplete and reversible Protein Depletion. The implementation of CRISPR/Cas9-based DNA cleavage has revolutionized the capacity to conduct functional studies in human cells. However, this approach is still underutilized for conducting visual phenotypic analyses, particularly for essential genes that require conditional strategies to eliminate their gene products. Optimizing this strategy requires effective and streamlined approaches to introduce the Cas9 guide RNA into target cells. Here we assess the efficacy of synthetic guide RNA transfection to eliminate gene products for cell biological studies. On the basis of three representative gene targets (KIF11, CENPN, and RELA), we demonstrate that transfection of synthetic single guide RNA (sgRNA) and CRISPR RNA (crRNA) guides works comparably for Protein Depletion as cell lines stably expressing lentiviral-delivered RNA guides. We additionally demonstrate that synthetic sgRNAs can be introduced by reverse transfection on an array. Together, these strategies provide a robust, flexible, and scalable approach for conducting functional studies in human cells.
Christof Taxis - One of the best experts on this subject based on the ideXlab platform.
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Targeted Protein Depletion in Saccharomyces cerevisiae by activation of a bidirectional degron
BMC Systems Biology, 2010Co-Authors: Marc Jungbluth, Christian Renicke, Christof TaxisAbstract:Background Tools for in vivo manipulation of Protein abundance or activity are highly beneficial for life science research. Protein stability can be efficiently controlled by conditional degrons, which induce target Protein degradation at restrictive conditions. Results We used the yeast Saccharomyces cerevisiae for development of a conditional, bidirectional degron to control Protein stability, which can be fused to the target Protein N-terminally, C-terminally or placed internally. Activation of the degron is achieved by cleavage with the tobacco etch virus (TEV) protease, resulting in quick proteolysis of the target Protein. We found similar degradation rates of soluble substrates using destabilization by the N- or C-degron. C-terminal tagging of essential yeast Proteins with the bidirectional degron resulted in deletion-like phenotypes at non-permissive conditions. Developmental process-specific mutants were created by N- or C-terminal tagging of essential Proteins with the bidirectional degron in combination with sporulation-specific production of the TEV protease. Conclusions We developed a system to influence Protein abundance and activity genetically, which can be used to create conditional mutants, to regulate the fate of single Protein domains or to design artificial regulatory circuits. Thus, this method enhances the toolbox to manipulate Proteins in systems biology approaches considerably.
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Targeted Protein Depletion in Saccharomyces cerevisiae by activation of a bidirectional degron
BMC systems biology, 2010Co-Authors: Marc Jungbluth, Christian Renicke, Christof TaxisAbstract:Background Tools for in vivo manipulation of Protein abundance or activity are highly beneficial for life science research. Protein stability can be efficiently controlled by conditional degrons, which induce target Protein degradation at restrictive conditions.
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Efficient Protein Depletion by genetically controlled deprotection of a dormant N‐degron
Molecular systems biology, 2009Co-Authors: Christof Taxis, Gunter Stier, Roberta Spadaccini, Michael KnopAbstract:Methods that allow for the manipulation of genes or their products have been highly fruitful for biomedical research. Here, we describe a method that allows the control of Protein abundance by a genetically encoded regulatory system. We developed a dormant N-degron that can be attached to the N-terminus of a Protein of interest. Upon expression of a site-specific protease, the dormant N-degron becomes deprotected. The N-degron then targets itself and the attached Protein for rapid proteasomal degradation through the N-end rule pathway. We use an optimized tobacco etch virus (TEV) protease variant combined with selective target binding to achieve complete and rapid deprotection of the N-degron-tagged Proteins. This method, termed TEV protease induced Protein inactivation (TIPI) of TIPI-degron (TDeg) modified target Proteins is fast, reversible, and applicable to a broad range of Proteins. TIPI of yeast Proteins essential for vegetative growth causes phenotypes that are close to deletion mutants. The features of the TIPI system make it a versatile tool to study Protein function in eukaryotes and to create new modules for synthetic or systems biology.
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efficient Protein Depletion by genetically controlled deprotection of a dormant n degron
Molecular Systems Biology, 2009Co-Authors: Christof Taxis, Gunter Stier, Roberta Spadaccini, Michael KnopAbstract:Methods that allow for the manipulation of genes or their products have been highly fruitful for biomedical research. Here, we describe a method that allows the control of Protein abundance by a genetically encoded regulatory system. We developed a dormant N-degron that can be attached to the N-terminus of a Protein of interest. Upon expression of a site-specific protease, the dormant N-degron becomes deprotected. The N-degron then targets itself and the attached Protein for rapid proteasomal degradation through the N-end rule pathway. We use an optimized tobacco etch virus (TEV) protease variant combined with selective target binding to achieve complete and rapid deprotection of the N-degron-tagged Proteins. This method, termed TEV protease induced Protein inactivation (TIPI) of TIPI-degron (TDeg) modified target Proteins is fast, reversible, and applicable to a broad range of Proteins. TIPI of yeast Proteins essential for vegetative growth causes phenotypes that are close to deletion mutants. The features of the TIPI system make it a versatile tool to study Protein function in eukaryotes and to create new modules for synthetic or systems biology.
Mary-jane Tsang - One of the best experts on this subject based on the ideXlab platform.
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CRISPR/Cas9-based gene targeting using synthetic guide RNAs enables robust cell biological analyses.
Molecular Biology of the Cell, 2018Co-Authors: Kuan-chung Su, Mary-jane Tsang, Neil Emans, Iain M. CheesemanAbstract:: A key goal for cell biological analyses is to assess the phenotypes that result from eliminating a target gene. Since the early 1990s, the predominant strategy utilized in human tissue culture cells has been RNA interference (RNAi)-mediated Protein Depletion. However, RNAi suffers well-documented off-target effects as well as incomplete and reversible Protein Depletion. The implementation of CRISPR/Cas9-based DNA cleavage has revolutionized the capacity to conduct functional studies in human cells. However, this approach is still underutilized for conducting visual phenotypic analyses, particularly for essential genes that require conditional strategies to eliminate their gene products. Optimizing this strategy requires effective and streamlined approaches to introduce the Cas9 guide RNA into target cells. Here we assess the efficacy of synthetic guide RNA transfection to eliminate gene products for cell biological studies. On the basis of three representative gene targets (KIF11, CENPN, and RELA), we demonstrate that transfection of synthetic single guide RNA (sgRNA) and CRISPR RNA (crRNA) guides works comparably for Protein Depletion as cell lines stably expressing lentiviral-delivered RNA guides. We additionally demonstrate that synthetic sgRNAs can be introduced by reverse transfection on an array. Together, these strategies provide a robust, flexible, and scalable approach for conducting functional studies in human cells.
Neil Emans - One of the best experts on this subject based on the ideXlab platform.
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CRISPR/Cas9-based gene targeting using synthetic guide RNAs enables robust cell biological analyses.
Molecular Biology of the Cell, 2018Co-Authors: Kuan-chung Su, Mary-jane Tsang, Neil Emans, Iain M. CheesemanAbstract:: A key goal for cell biological analyses is to assess the phenotypes that result from eliminating a target gene. Since the early 1990s, the predominant strategy utilized in human tissue culture cells has been RNA interference (RNAi)-mediated Protein Depletion. However, RNAi suffers well-documented off-target effects as well as incomplete and reversible Protein Depletion. The implementation of CRISPR/Cas9-based DNA cleavage has revolutionized the capacity to conduct functional studies in human cells. However, this approach is still underutilized for conducting visual phenotypic analyses, particularly for essential genes that require conditional strategies to eliminate their gene products. Optimizing this strategy requires effective and streamlined approaches to introduce the Cas9 guide RNA into target cells. Here we assess the efficacy of synthetic guide RNA transfection to eliminate gene products for cell biological studies. On the basis of three representative gene targets (KIF11, CENPN, and RELA), we demonstrate that transfection of synthetic single guide RNA (sgRNA) and CRISPR RNA (crRNA) guides works comparably for Protein Depletion as cell lines stably expressing lentiviral-delivered RNA guides. We additionally demonstrate that synthetic sgRNAs can be introduced by reverse transfection on an array. Together, these strategies provide a robust, flexible, and scalable approach for conducting functional studies in human cells.