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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...
Autores principales: | , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
John Wiley and Sons Inc.
2021
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8574044/ https://www.ncbi.nlm.nih.gov/pubmed/34747560 http://dx.doi.org/10.15252/msb.202110512 |
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author | Anderson, Daniel A Voigt, Christopher A |
author_facet | Anderson, Daniel A Voigt, Christopher A |
author_sort | Anderson, Daniel A |
collection | PubMed |
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. |
format | Online Article Text |
id | pubmed-8574044 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | John Wiley and Sons Inc. |
record_format | MEDLINE/PubMed |
spelling | pubmed-85740442021-11-12 Competitive dCas9 binding as a mechanism for transcriptional control Anderson, Daniel A Voigt, Christopher A Mol Syst Biol Articles 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. John Wiley and Sons Inc. 2021-11-08 /pmc/articles/PMC8574044/ /pubmed/34747560 http://dx.doi.org/10.15252/msb.202110512 Text en © 2021 The Authors. Published under the terms of the CC BY 4.0 license https://creativecommons.org/licenses/by/4.0/This is an open access article under the terms of the http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Articles 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 |
topic | Articles |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8574044/ https://www.ncbi.nlm.nih.gov/pubmed/34747560 http://dx.doi.org/10.15252/msb.202110512 |
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