CRISPRactivation-SMS, a message for PAM sequence independent gene up-regulation in Escherichia coli.
Journal
Nucleic acids research
ISSN: 1362-4962
Titre abrégé: Nucleic Acids Res
Pays: England
ID NLM: 0411011
Informations de publication
Date de publication:
14 10 2022
14 10 2022
Historique:
accepted:
08
09
2022
revised:
10
08
2022
received:
04
07
2022
pubmed:
23
9
2022
medline:
18
10
2022
entrez:
22
9
2022
Statut:
ppublish
Résumé
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.
Identifiants
pubmed: 36134715
pii: 6711140
doi: 10.1093/nar/gkac804
pmc: PMC9561276
doi:
Substances chimiques
RNA, Guide
0
Recombinant Proteins
0
CRISPR-Associated Protein 9
EC 3.1.-
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
10772-10784Informations de copyright
© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.
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