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CRISPR/Cas9-based gene targeting using synthetic guide RNAs enables robust cell biological analyses

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 of...

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Autores principales: Su, Kuan-Chung, Tsang, Mary-Jane, Emans, Neil, Cheeseman, Iain M.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: The American Society for Cell Biology 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6233062/
https://www.ncbi.nlm.nih.gov/pubmed/30091644
http://dx.doi.org/10.1091/mbc.E18-04-0214
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author Su, Kuan-Chung
Tsang, Mary-Jane
Emans, Neil
Cheeseman, Iain M.
author_facet Su, Kuan-Chung
Tsang, Mary-Jane
Emans, Neil
Cheeseman, Iain M.
author_sort Su, Kuan-Chung
collection PubMed
description 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.
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spelling pubmed-62330622018-12-16 CRISPR/Cas9-based gene targeting using synthetic guide RNAs enables robust cell biological analyses Su, Kuan-Chung Tsang, Mary-Jane Emans, Neil Cheeseman, Iain M. Mol Biol Cell Brief Reports 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. The American Society for Cell Biology 2018-10-01 /pmc/articles/PMC6233062/ /pubmed/30091644 http://dx.doi.org/10.1091/mbc.E18-04-0214 Text en © 2018 Su et al. “ASCB®,” “The American Society for Cell Biology®,” and “Molecular Biology of the Cell®” are registered trademarks of The American Society for Cell Biology. http://creativecommons.org/licenses/by-nc-sa/3.0 This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License.
spellingShingle Brief Reports
Su, Kuan-Chung
Tsang, Mary-Jane
Emans, Neil
Cheeseman, Iain M.
CRISPR/Cas9-based gene targeting using synthetic guide RNAs enables robust cell biological analyses
title CRISPR/Cas9-based gene targeting using synthetic guide RNAs enables robust cell biological analyses
title_full CRISPR/Cas9-based gene targeting using synthetic guide RNAs enables robust cell biological analyses
title_fullStr CRISPR/Cas9-based gene targeting using synthetic guide RNAs enables robust cell biological analyses
title_full_unstemmed CRISPR/Cas9-based gene targeting using synthetic guide RNAs enables robust cell biological analyses
title_short CRISPR/Cas9-based gene targeting using synthetic guide RNAs enables robust cell biological analyses
title_sort crispr/cas9-based gene targeting using synthetic guide rnas enables robust cell biological analyses
topic Brief Reports
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6233062/
https://www.ncbi.nlm.nih.gov/pubmed/30091644
http://dx.doi.org/10.1091/mbc.E18-04-0214
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