Competitive dCas9 binding as a mechanism for transcriptional control
Catalytically dead Cas9 (dCas9) is a programmable transcription factor that can be targeted to promoters through the design of small guide RNAs (sgRNAs), where it can function as an activator or repressor. Natural promoters use overlapping binding sites as a mechanism for signal integration, where t...
| Main Authors: | , |
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| Other Authors: | |
| Format: | Article |
| Language: | English |
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EMBO
2022
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| Online Access: | https://hdl.handle.net/1721.1/141264 |
| _version_ | 1811083328270368768 |
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| author | Anderson, Daniel A Voigt, Christopher A |
| author2 | Massachusetts Institute of Technology. Synthetic Biology Center |
| author_facet | Massachusetts Institute of Technology. Synthetic Biology Center Anderson, Daniel A Voigt, Christopher A |
| author_sort | Anderson, Daniel A |
| collection | MIT |
| description | Catalytically dead Cas9 (dCas9) is a programmable transcription factor that can be targeted to promoters through the design of small guide RNAs (sgRNAs), where it can function as an activator or repressor. Natural promoters use overlapping binding sites as a mechanism for signal integration, where the binding of one can block, displace, or augment the activity of the other. Here, we implemented this strategy in Escherichia coli using pairs of sgRNAs designed to repress and then derepress transcription through competitive binding. When designed to target a promoter, this led to 27-fold repression and complete derepression. This system was also capable of ratiometric input comparison over two orders of magnitude. Additionally, we used this mechanism for promoter sequence-independent control by adopting it for elongation control, achieving 8-fold repression and 4-fold derepression. This work demonstrates a new genetic control mechanism that could be used to build analog circuit or implement cis-regulatory logic on CRISPRi-targeted native genes. |
| first_indexed | 2024-09-23T12:31:19Z |
| format | Article |
| id | mit-1721.1/141264 |
| institution | Massachusetts Institute of Technology |
| language | English |
| last_indexed | 2024-09-23T12:31:19Z |
| publishDate | 2022 |
| publisher | EMBO |
| record_format | dspace |
| spelling | mit-1721.1/1412642023-04-07T20:49:03Z Competitive dCas9 binding as a mechanism for transcriptional control Anderson, Daniel A Voigt, Christopher A Massachusetts Institute of Technology. Synthetic Biology Center Catalytically dead Cas9 (dCas9) is a programmable transcription factor that can be targeted to promoters through the design of small guide RNAs (sgRNAs), where it can function as an activator or repressor. Natural promoters use overlapping binding sites as a mechanism for signal integration, where the binding of one can block, displace, or augment the activity of the other. Here, we implemented this strategy in Escherichia coli using pairs of sgRNAs designed to repress and then derepress transcription through competitive binding. When designed to target a promoter, this led to 27-fold repression and complete derepression. This system was also capable of ratiometric input comparison over two orders of magnitude. Additionally, we used this mechanism for promoter sequence-independent control by adopting it for elongation control, achieving 8-fold repression and 4-fold derepression. This work demonstrates a new genetic control mechanism that could be used to build analog circuit or implement cis-regulatory logic on CRISPRi-targeted native genes. 2022-03-17T18:42:07Z 2022-03-17T18:42:07Z 2021-11 2022-03-17T18:35:48Z Article http://purl.org/eprint/type/JournalArticle https://hdl.handle.net/1721.1/141264 Anderson, Daniel A and Voigt, Christopher A. 2021. "Competitive dCas9 binding as a mechanism for transcriptional control." Molecular Systems Biology, 17 (11). en 10.15252/msb.202110512 Molecular Systems Biology Creative Commons Attribution 4.0 International license https://creativecommons.org/licenses/by/4.0/ application/pdf EMBO Wiley |
| spellingShingle | Anderson, Daniel A Voigt, Christopher A Competitive dCas9 binding as a mechanism for transcriptional control |
| title | Competitive dCas9 binding as a mechanism for transcriptional control |
| title_full | Competitive dCas9 binding as a mechanism for transcriptional control |
| title_fullStr | Competitive dCas9 binding as a mechanism for transcriptional control |
| title_full_unstemmed | Competitive dCas9 binding as a mechanism for transcriptional control |
| title_short | Competitive dCas9 binding as a mechanism for transcriptional control |
| title_sort | competitive dcas9 binding as a mechanism for transcriptional control |
| url | https://hdl.handle.net/1721.1/141264 |
| work_keys_str_mv | AT andersondaniela competitivedcas9bindingasamechanismfortranscriptionalcontrol AT voigtchristophera competitivedcas9bindingasamechanismfortranscriptionalcontrol |