CRISPR RNA-Guided Transposases Facilitate Dispensable Gene Study in Phage

Author:

Liu Yanmei1,Liang Zizhen1,Yu Shuting1,Ye Yanrui1ORCID,Lin Zhanglin12

Affiliation:

1. School of Biology and Biological Engineering, South China University of Technology, Guangzhou 510006, China

2. Shenzhen Institute of Synthetic Biology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, China

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.

Funder

National Key R&D Program of China

Publisher

MDPI AG

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