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Programming CRISPRi to control the lifecycle of bacteriophage T7
1Department of Biosciences, Faculty of Health and Life Sciences, University of Exeter, Exeter, United Kingdom.
Frontiers in Microbiology
|February 27, 2025
Summary
Clustered Regularly Interspaced Short Palindromic Repeats interference (CRISPRi) effectively controls bacteriophage T7 lifecycle and infectivity by targeting key promoters and T7 RNA polymerase. Multiplex CRISPRi significantly increased lysis time and reduced plaque size, demonstrating its potential for phage manipulation.
Area of Science:
- Molecular Biology
- Microbiology
- Bacteriophage Research
Background:
- Clustered Regularly Interspaced Short Palindromic Repeats interference (CRISPRi) utilizes catalytically dead Cas9 nuclease to block gene expression.
- CRISPRi is a versatile tool for investigating gene function and essentiality in bacteria.
- Bacteriophage T7 is a highly virulent, obligate lytic phage of Escherichia coli.
Purpose of the Study:
- To program CRISPRi for controlling the life cycle and infectivity of Escherichia coli bacteriophage T7.
- To investigate the impact of targeting specific T7 promoters and genes on phage progression.
- To expand the application of dCas9-dependent CRISPRi for manipulating phage biology.
Main Methods:
- CRISPRi was programmed to target promoters within the T7 internalisation signal region and the T7 RNA polymerase (T7 RNAP) promoter C.
- Fluorescent reporter assays were used to assess the efficiency of promoter targeting.
- Multiplex single-guide RNAs (sgRNAs) were employed to target multiple phage regions simultaneously.
Main Results:
- CRISPRi targeting of T7 RNAP expression was highly efficient, while targeting internalisation signal promoters showed moderate effectiveness.
- Targeting the left-most promoter or T7 RNAP most significantly affected the time to lysis.
- Multiplex CRISPRi increased lysis time by 25% and reduced plaque size by up to 8-fold.
Conclusions:
- CRISPRi is an effective tool for non-invasively manipulating the lifecycle and infectivity of bacteriophage T7.
- Targeting T7 RNAP expression is a highly efficient strategy for controlling T7 phage.
- Multiplex CRISPRi enhances the stringency and effectiveness of phage manipulation.
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