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CRISPR RNA-Guided Transposases Facilitate Dispensable Gene Study in Phage
- Source :
- Viruses, Vol 16, Iss 3, p 422 (2024)
- Publication Year :
- 2024
- Publisher :
- MDPI AG, 2024.
-
Abstract
- Phages provide a potential therapy for multi-drug-resistant (MDR) bacteria. However, a significant portion of viral genes often remains unknown, posing potential dangers. The identification of non-essential genes helps dissect and simplify phage genomes, but current methods have various limitations. In this study, we present an in vivo two-plasmid transposon insertion system to assess the importance of phage genes, which is based on the V. cholerae transposon Tn6677, encoding a nuclease-deficient type I-F CRISPR–Cas system. We first validated the system in Pseudomonas aeruginosa PAO1 and its phage S1. We then used the selection marker AcrVA1 to protect transposon-inserted phages from CRISPR-Cas12a and enriched the transposon-inserted phages. For a pool of selected 10 open-reading frames (2 known functional protein genes and 8 hypothetical protein genes) of phage S1, we identified 5 (2 known functional protein genes and 3 hypothetical protein genes) as indispensable genes and the remaining 5 (all hypothetical protein genes) as dispensable genes. This approach offers a convenient, site-specific method that does not depend on homologous arms and double-strand breaks (DSBs), holding promise for future applications across a broader range of phages and facilitating the identification of the importance of phage genes and the insertion of genetic cargos.
Details
- Language :
- English
- ISSN :
- 19994915
- Volume :
- 16
- Issue :
- 3
- Database :
- Directory of Open Access Journals
- Journal :
- Viruses
- Publication Type :
- Academic Journal
- Accession number :
- edsdoj.01714a187f054434968ee6468a75e776
- Document Type :
- article
- Full Text :
- https://doi.org/10.3390/v16030422