Rapid Protein Depletion in Human Cells by Auxin-Inducible Degron Tagging with Short Homology Donors
Studying the role of essential proteins is dependent upon a method for rapid inactivation, in order to study the immediate phenotypic consequences. Auxin-inducible degron (AID) technology allows rapid depletion of proteins in animal cells and fungi, but its application to human cells has been limite...
| Main Authors: | , , , |
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| Format: | Article |
| Language: | English |
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Elsevier
2016-04-01
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| Series: | Cell Reports |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S221112471630225X |
| _version_ | 1819044675939467264 |
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| author | Toyoaki Natsume Tomomi Kiyomitsu Yumiko Saga Masato T. Kanemaki |
| author_facet | Toyoaki Natsume Tomomi Kiyomitsu Yumiko Saga Masato T. Kanemaki |
| author_sort | Toyoaki Natsume |
| collection | DOAJ |
| description | Studying the role of essential proteins is dependent upon a method for rapid inactivation, in order to study the immediate phenotypic consequences. Auxin-inducible degron (AID) technology allows rapid depletion of proteins in animal cells and fungi, but its application to human cells has been limited by the difficulties of tagging endogenous proteins. We have developed a simple and scalable CRISPR/Cas-based method to tag endogenous proteins in human HCT116 and mouse embryonic stem (ES) cells by using donor constructs that harbor synthetic short homology arms. Using a combination of AID tagging with CRISPR/Cas, we have generated conditional alleles of essential nuclear and cytoplasmic proteins in HCT116 cells, which can then be depleted very rapidly after the addition of auxin to the culture medium. This approach should greatly facilitate the functional analysis of essential proteins, particularly those of previously unknown function. |
| first_indexed | 2024-12-21T10:16:27Z |
| format | Article |
| id | doaj.art-c586cdd6ddd44290adc0a569742d9a3c |
| institution | Directory Open Access Journal |
| issn | 2211-1247 |
| language | English |
| last_indexed | 2024-12-21T10:16:27Z |
| publishDate | 2016-04-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Cell Reports |
| spelling | doaj.art-c586cdd6ddd44290adc0a569742d9a3c2022-12-21T19:07:35ZengElsevierCell Reports2211-12472016-04-0115121021810.1016/j.celrep.2016.03.001Rapid Protein Depletion in Human Cells by Auxin-Inducible Degron Tagging with Short Homology DonorsToyoaki Natsume0Tomomi Kiyomitsu1Yumiko Saga2Masato T. Kanemaki3Center of Frontier Research, National Institute of Genetics, Research Organization of Information and Systems, Yata 1111, Mishima, Shizuoka 411-8540, JapanDivision of Biological Science, Graduate School of Science, Nagoya University, Chikusa-ku, Nagoya 464-8602, JapanDivision of Mammalian Development, Genetic Strains Research Center, National Institute of Genetics, Yata 1111, Mishima, Shizuoka 411-8540, JapanCenter of Frontier Research, National Institute of Genetics, Research Organization of Information and Systems, Yata 1111, Mishima, Shizuoka 411-8540, JapanStudying the role of essential proteins is dependent upon a method for rapid inactivation, in order to study the immediate phenotypic consequences. Auxin-inducible degron (AID) technology allows rapid depletion of proteins in animal cells and fungi, but its application to human cells has been limited by the difficulties of tagging endogenous proteins. We have developed a simple and scalable CRISPR/Cas-based method to tag endogenous proteins in human HCT116 and mouse embryonic stem (ES) cells by using donor constructs that harbor synthetic short homology arms. Using a combination of AID tagging with CRISPR/Cas, we have generated conditional alleles of essential nuclear and cytoplasmic proteins in HCT116 cells, which can then be depleted very rapidly after the addition of auxin to the culture medium. This approach should greatly facilitate the functional analysis of essential proteins, particularly those of previously unknown function.http://www.sciencedirect.com/science/article/pii/S221112471630225X |
| spellingShingle | Toyoaki Natsume Tomomi Kiyomitsu Yumiko Saga Masato T. Kanemaki Rapid Protein Depletion in Human Cells by Auxin-Inducible Degron Tagging with Short Homology Donors Cell Reports |
| title | Rapid Protein Depletion in Human Cells by Auxin-Inducible Degron Tagging with Short Homology Donors |
| title_full | Rapid Protein Depletion in Human Cells by Auxin-Inducible Degron Tagging with Short Homology Donors |
| title_fullStr | Rapid Protein Depletion in Human Cells by Auxin-Inducible Degron Tagging with Short Homology Donors |
| title_full_unstemmed | Rapid Protein Depletion in Human Cells by Auxin-Inducible Degron Tagging with Short Homology Donors |
| title_short | Rapid Protein Depletion in Human Cells by Auxin-Inducible Degron Tagging with Short Homology Donors |
| title_sort | rapid protein depletion in human cells by auxin inducible degron tagging with short homology donors |
| url | http://www.sciencedirect.com/science/article/pii/S221112471630225X |
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