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...
| Main Authors: | , , , |
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| Format: | Article |
| Language: | English |
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American Society for Cell Biology (ASCB)
2020
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| Online Access: | https://hdl.handle.net/1721.1/126142 |
| _version_ | 1811092658109546496 |
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| author | Su, Kuan-Chung Tsang, Mary-Jane Emans, Neil Cheeseman, Iain M |
| author2 | Massachusetts Institute of Technology. Department of Biology |
| author_facet | Massachusetts Institute of Technology. Department of Biology Su, Kuan-Chung Tsang, Mary-Jane Emans, Neil Cheeseman, Iain M |
| author_sort | Su, Kuan-Chung |
| collection | MIT |
| 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. |
| first_indexed | 2024-09-23T15:21:35Z |
| format | Article |
| id | mit-1721.1/126142 |
| institution | Massachusetts Institute of Technology |
| language | English |
| last_indexed | 2024-09-23T15:21:35Z |
| publishDate | 2020 |
| publisher | American Society for Cell Biology (ASCB) |
| record_format | dspace |
| spelling | mit-1721.1/1261422022-09-29T14:27:36Z 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 Massachusetts Institute of Technology. Department of Biology 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. National Institute of General Medical Sciences (Grant GM088313) National Institute of General Medical Sciences (Grant GM108718) 2020-07-10T20:38:30Z 2020-07-10T20:38:30Z 2018-10 2018-07 2019-12-02T15:04:58Z Article http://purl.org/eprint/type/JournalArticle 1059-1524 1939-4586 https://hdl.handle.net/1721.1/126142 Su, Kuan-Chung et al. "CRISPR/Cas9-based gene targeting using synthetic guide RNAs enables robust cell biological analyses." Molecular Biology of the Cell 29, 20 (October 2018): 2359-2507 © 2018 The American Society for Cell Biology en http://dx.doi.org/10.1091/mbc.e18-04-0214 Molecular Biology of the Cell Creative Commons Attribution Noncommercial 3.0 unported license https://creativecommons.org/licenses/by-nc/3.0/ application/pdf American Society for Cell Biology (ASCB) Molecular Biology of the Cell |
| spellingShingle | 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 |
| url | https://hdl.handle.net/1721.1/126142 |
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