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CRISPRactivation-SMS, a message for PAM sequence independent gene up-regulation in Escherichia coli
Governance of the endogenous gene regulatory network enables the navigation of cells towards beneficial traits for recombinant protein production. CRISPRactivation and interference provides the basis for gene expression modulation but is primarily applied in eukaryotes. Particularly the lack of wide...
Autores principales: | , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Oxford University Press
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9561276/ https://www.ncbi.nlm.nih.gov/pubmed/36134715 http://dx.doi.org/10.1093/nar/gkac804 |
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author | Klanschnig, Marco Cserjan-Puschmann, Monika Striedner, Gerald Grabherr, Reingard |
author_facet | Klanschnig, Marco Cserjan-Puschmann, Monika Striedner, Gerald Grabherr, Reingard |
author_sort | Klanschnig, Marco |
collection | PubMed |
description | Governance of the endogenous gene regulatory network enables the navigation of cells towards beneficial traits for recombinant protein production. CRISPRactivation and interference provides the basis for gene expression modulation but is primarily applied in eukaryotes. Particularly the lack of wide-ranging prokaryotic CRISPRa studies might be attributed to intrinsic limitations of bacterial activators and Cas9 proteins. While bacterial activators need accurate spatial orientation and distancing towards the target promoter to be functional, Cas9-based CRISPR tools only bind sites adjacent to NGG PAM sequences. These circumstances hampered Cas9-guided activators from mediating the up-regulation of endogenous genes at precise positions in bacteria. We could overcome this limitation by combining the PAM independent Cas9 variant SpRY and a CRISPRa construct using phage protein MCP fused to transcriptional activator SoxS. This CRISPRa construct, referred to as SMS, was compared with previously reported CRISPRa constructs and showed up-regulation of a reporter gene library independent of its PAM sequence in Escherichia coli. We also demonstrated down-regulation and multi-gene expression control with SMS at non-NGG PAM sites. Furthermore, we successfully applied SMS to up-regulate endogenous genes, and transgenes at non-NGG PAM sites, which was impossible with the previous CRISPRa construct. |
format | Online Article Text |
id | pubmed-9561276 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | Oxford University Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-95612762022-10-18 CRISPRactivation-SMS, a message for PAM sequence independent gene up-regulation in Escherichia coli Klanschnig, Marco Cserjan-Puschmann, Monika Striedner, Gerald Grabherr, Reingard Nucleic Acids Res Synthetic Biology and Bioengineering Governance of the endogenous gene regulatory network enables the navigation of cells towards beneficial traits for recombinant protein production. CRISPRactivation and interference provides the basis for gene expression modulation but is primarily applied in eukaryotes. Particularly the lack of wide-ranging prokaryotic CRISPRa studies might be attributed to intrinsic limitations of bacterial activators and Cas9 proteins. While bacterial activators need accurate spatial orientation and distancing towards the target promoter to be functional, Cas9-based CRISPR tools only bind sites adjacent to NGG PAM sequences. These circumstances hampered Cas9-guided activators from mediating the up-regulation of endogenous genes at precise positions in bacteria. We could overcome this limitation by combining the PAM independent Cas9 variant SpRY and a CRISPRa construct using phage protein MCP fused to transcriptional activator SoxS. This CRISPRa construct, referred to as SMS, was compared with previously reported CRISPRa constructs and showed up-regulation of a reporter gene library independent of its PAM sequence in Escherichia coli. We also demonstrated down-regulation and multi-gene expression control with SMS at non-NGG PAM sites. Furthermore, we successfully applied SMS to up-regulate endogenous genes, and transgenes at non-NGG PAM sites, which was impossible with the previous CRISPRa construct. Oxford University Press 2022-09-22 /pmc/articles/PMC9561276/ /pubmed/36134715 http://dx.doi.org/10.1093/nar/gkac804 Text en © The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research. https://creativecommons.org/licenses/by/4.0/This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Synthetic Biology and Bioengineering Klanschnig, Marco Cserjan-Puschmann, Monika Striedner, Gerald Grabherr, Reingard CRISPRactivation-SMS, a message for PAM sequence independent gene up-regulation in Escherichia coli |
title | CRISPRactivation-SMS, a message for PAM sequence independent gene up-regulation in Escherichia coli |
title_full | CRISPRactivation-SMS, a message for PAM sequence independent gene up-regulation in Escherichia coli |
title_fullStr | CRISPRactivation-SMS, a message for PAM sequence independent gene up-regulation in Escherichia coli |
title_full_unstemmed | CRISPRactivation-SMS, a message for PAM sequence independent gene up-regulation in Escherichia coli |
title_short | CRISPRactivation-SMS, a message for PAM sequence independent gene up-regulation in Escherichia coli |
title_sort | crispractivation-sms, a message for pam sequence independent gene up-regulation in escherichia coli |
topic | Synthetic Biology and Bioengineering |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9561276/ https://www.ncbi.nlm.nih.gov/pubmed/36134715 http://dx.doi.org/10.1093/nar/gkac804 |
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