dCas9 regulator to neutralize competition in CRISPRi circuits
CRISPRi-mediated gene regulation allows simultaneous control of many genes. However, highly specific sgRNA-promoter binding is, alone, insufficient to achieve independent transcriptional regulation of multiple targets. Indeed, due to competition for dCas9, the repression ability of one sgRNA changes...
| Main Authors: | , , , , , , |
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| Other Authors: | |
| Format: | Article |
| Language: | English |
| Published: |
Springer Science and Business Media LLC
2021
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| Online Access: | https://hdl.handle.net/1721.1/138546 |
| _version_ | 1826213168987242496 |
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| author | Huang, Hsin-Ho Bellato, Massimo Qian, Yili Cárdenas, Pablo Pasotti, Lorenzo Magni, Paolo Del Vecchio, Domitilla |
| author2 | Massachusetts Institute of Technology. Department of Mechanical Engineering |
| author_facet | Massachusetts Institute of Technology. Department of Mechanical Engineering Huang, Hsin-Ho Bellato, Massimo Qian, Yili Cárdenas, Pablo Pasotti, Lorenzo Magni, Paolo Del Vecchio, Domitilla |
| author_sort | Huang, Hsin-Ho |
| collection | MIT |
| description | CRISPRi-mediated gene regulation allows simultaneous control of many genes. However, highly specific sgRNA-promoter binding is, alone, insufficient to achieve independent transcriptional regulation of multiple targets. Indeed, due to competition for dCas9, the repression ability of one sgRNA changes significantly when another sgRNA becomes expressed. To solve this problem and decouple sgRNA-mediated regulatory paths, we create a dCas9 concentration regulator that implements negative feedback on dCas9 level. This allows any sgRNA to maintain an approximately constant dose-response curve, independent of other sgRNAs. We demonstrate the regulator performance on both single-stage and layered CRISPRi-based genetic circuits, zeroing competition effects of up to 15-fold changes in circuit I/O response encountered without the dCas9 regulator. The dCas9 regulator decouples sgRNA-mediated regulatory paths, enabling concurrent and independent regulation of multiple genes. This allows predictable composition of CRISPRi-based genetic modules, which is essential in the design of larger scale synthetic genetic circuits. |
| first_indexed | 2024-09-23T15:44:42Z |
| format | Article |
| id | mit-1721.1/138546 |
| institution | Massachusetts Institute of Technology |
| language | English |
| last_indexed | 2024-09-23T15:44:42Z |
| publishDate | 2021 |
| publisher | Springer Science and Business Media LLC |
| record_format | dspace |
| spelling | mit-1721.1/1385462023-01-11T21:32:51Z dCas9 regulator to neutralize competition in CRISPRi circuits Huang, Hsin-Ho Bellato, Massimo Qian, Yili Cárdenas, Pablo Pasotti, Lorenzo Magni, Paolo Del Vecchio, Domitilla Massachusetts Institute of Technology. Department of Mechanical Engineering Massachusetts Institute of Technology. Department of Biological Engineering Massachusetts Institute of Technology. Synthetic Biology Center CRISPRi-mediated gene regulation allows simultaneous control of many genes. However, highly specific sgRNA-promoter binding is, alone, insufficient to achieve independent transcriptional regulation of multiple targets. Indeed, due to competition for dCas9, the repression ability of one sgRNA changes significantly when another sgRNA becomes expressed. To solve this problem and decouple sgRNA-mediated regulatory paths, we create a dCas9 concentration regulator that implements negative feedback on dCas9 level. This allows any sgRNA to maintain an approximately constant dose-response curve, independent of other sgRNAs. We demonstrate the regulator performance on both single-stage and layered CRISPRi-based genetic circuits, zeroing competition effects of up to 15-fold changes in circuit I/O response encountered without the dCas9 regulator. The dCas9 regulator decouples sgRNA-mediated regulatory paths, enabling concurrent and independent regulation of multiple genes. This allows predictable composition of CRISPRi-based genetic modules, which is essential in the design of larger scale synthetic genetic circuits. 2021-12-17T17:40:26Z 2021-12-17T17:40:26Z 2021 2021-12-17T17:38:33Z Article http://purl.org/eprint/type/JournalArticle https://hdl.handle.net/1721.1/138546 Huang, Hsin-Ho, Bellato, Massimo, Qian, Yili, Cárdenas, Pablo, Pasotti, Lorenzo et al. 2021. "dCas9 regulator to neutralize competition in CRISPRi circuits." Nature Communications, 12 (1). en 10.1038/S41467-021-21772-6 Nature Communications Creative Commons Attribution 4.0 International license https://creativecommons.org/licenses/by/4.0/ application/pdf Springer Science and Business Media LLC Nature |
| spellingShingle | Huang, Hsin-Ho Bellato, Massimo Qian, Yili Cárdenas, Pablo Pasotti, Lorenzo Magni, Paolo Del Vecchio, Domitilla dCas9 regulator to neutralize competition in CRISPRi circuits |
| title | dCas9 regulator to neutralize competition in CRISPRi circuits |
| title_full | dCas9 regulator to neutralize competition in CRISPRi circuits |
| title_fullStr | dCas9 regulator to neutralize competition in CRISPRi circuits |
| title_full_unstemmed | dCas9 regulator to neutralize competition in CRISPRi circuits |
| title_short | dCas9 regulator to neutralize competition in CRISPRi circuits |
| title_sort | dcas9 regulator to neutralize competition in crispri circuits |
| url | https://hdl.handle.net/1721.1/138546 |
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