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| Status |
Public on Nov 04, 2024 |
| Title |
Virulence regulates and boosts CRISPR-Cas9 immunity in Group B Streptococcus [CrispRseq] |
| Organisms |
Escherichia coli; Streptococcus agalactiae |
| Experiment type |
Other
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| Summary |
Bacterial CRISPR-Cas9 immune systems protect against foreign DNA. However, immune efficiency is constrained by Cas9 off-target effects and toxicity. Here, we demonstrate that CRISPR-Cas9 immunity is regulated by CovR, the major regulator of virulence in Group B Streptococcus, a pathobiont responsible for neonatal invasive infections. We show that CovR binds to and represses a distal promoter of the cas operon, embedding immunity in the virulence regulatory network. Releasing CovR repression enhances immune efficiency against suboptimal spacers, originating from old immune memory or mutations, thereby transiently expanding the sequence space recognized and cleaved by Cas9. Furthermore, CovR inactivation promotes the acquisition of new spacers, enhancing immune memory. CovR-mediated immune regulation is conserved at the species level, with lineage-specific variability in the constitutive cas promoter and Cas9 variants, suggesting different evolutionary trajectories. Overall, we describe a coordinated regulatory mechanism between immunity and virulence that enhances the immune repertoire during acute infection phases.
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| Overall design |
The experiment investigates how single mismatch in the protospacer affect immunity in the BM110 wild-type strain and in the isogenic ∆covR mutant. We first constructed in vitro a vector collection (Input pool) containing the protospacer 4 or 8 with single mutation at each base position with all four nucleotide options (A, T, G, C). We then transformed the pool of vector into GBS and selected for kanamycin resistant clones. Transformants were pooled and plasmid purified (Output pools).
Purified pools of vectors were used as matrix to amplify by PCR the protospacers. The primers included a common annealing sequence and an unique identifiers (index) for multiplex sequencing. Amplicon (197 bp) were pooled and Illumina adapters added (PCR-free). After amplicon sequencing, Illumina adapters were removed and amplicon demultiplexed.
PCR amplification and sequencing were done starting from 3 independent plasmid purification of the input pool (before and after long term -80°C storage). This should be considered as technical replicate.
As a control, the input pool was transformed into a double mutant ∆covR ∆P2cas in which the immune system is inactive. A single experiment was done.
Input pool were transformed indepently two times in the WT and in the ∆covR mutant to give two biological duplicate of the corresponding output pools.
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| Contributor(s) |
MAZZUOLI M, JACQUEMET E, LEGENDRE R, SISMEIRO O, FIRON A |
| Citation missing |
Has this study been published? Please login to update or notify GEO. |
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| Submission date |
Jun 10, 2024 |
| Last update date |
Nov 04, 2024 |
| Contact name |
Rachel Legendre |
| E-mail(s) |
[email protected]
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| Organization name |
Institut Pasteur
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| Department |
Research and Resource Center for Scientific Informatics
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| Lab |
Hub of Bioinformatics and Biostatistics
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| Street address |
28, rue du docteur Roux
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| City |
Paris |
| ZIP/Postal code |
75724 |
| Country |
France |
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| Platforms (2) |
| GPL25368 |
Illumina NovaSeq 6000 (Escherichia coli) |
| GPL28679 |
Illumina NovaSeq 6000 (Streptococcus agalactiae) |
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| Samples (8)
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| GSM8316159 |
Output pool, BM110 WT, Biological Replicate 1 |
| GSM8316160 |
Output pool, BM110 WT, Biological Replicate 2 |
| GSM8316161 |
Output pool, ∆covR, Biological Replicate 1 |
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| This SubSeries is part of SuperSeries: |
| GSE269473 |
Virulence regulates and boosts CRISPR-Cas9 immunity in Group B Streptococcus. |
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| Relations |
| BioProject |
PRJNA1122103 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE269471_Data_files.xlsx |
29.1 Kb |
(ftp)(http) |
XLSX |
SRA Run Selector |
| Raw data are available in SRA |
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