Abstract
Efforts have been made to reduce the genomes of living cells, but phage genome reduction remains challenging. It is of great interest to investigate whether genome reduction can make phages obtain new infectious properties. We developed a CRISPR/Cas9-based iterative phage genome reduction (CiPGr) approach and applied this to four distinct phages, thereby obtaining heterogeneous genome-reduced mutants. We isolated and sequenced 200 mutants with loss of up to 8-23% (3.3-35 kbp) of the original sequences. This allowed the identification of non-essential genes for phage propagation, although loss of these genes is mostly detrimental to phage fitness to various degrees. Notwithstanding this, mutants with higher infectious efficiency than their parental strains were characterized, indicating a trade-off between genome reduction and infectious fitness for phages. In conclusion, this study provides a foundation for future work to leverage the information generated by CiPGr in phage synthetic biology research.
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