P1 phagemid-based delivery of Cas9 antimicrobial into Shigella flexneri
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Date
22/12/2022Author
Huan, Yang Wei
Metadata
Abstract
A bacteriophage P1 phagemid system allowed efficient packaging of a DNA-sequence
specific, Cas9 antimicrobial into transducing particles, which could be used to infect and kill
pathogenic bacteria, such as Shigella flexneri. Although the transduction of a cas9 phagemid
yielded significant antimicrobial effect on S. flexneri, the phagemid lysates produced a
significant non-Cas9-mediated cytotoxic effect, which might be attributed to the presence of
wildtype P1 phage and/or other cytotoxic factors in phagemid lysates. Treatment of
phagemid lysates with a low concentration of polythene glycol (PEG) reduced the titre of
wildtype P1 phage and led to a lower cytotoxicity of phagemid lysates on S. flexneri cells.
Mutations introduced into the P1 lytic origin of replication (oriL) inhibited its lytic stage DNA
replication, yet the mutant was incapable of producing phagemid transducing particles.
Deletion of the DNA packaging site, pac, from the P1 genome significantly reduced the titre
of wildtype P1 phage. A Δpac mutation, however, did not reduce the cytotoxicity of phagemid
lysates significantly, when used for treatment of S. flexneri in vitro and in a zebrafish larvae
infection model. An alternative P4 cosmid system allowed efficient packaging of cas9
construct into transducing units without the contamination of host and/or phage DNA.
Contrastingly, treatment of S. flexneri with P4 cosmid lysates gave negligible cytotoxic killing
of the bacterial cells, suggesting that the non-Cas9-mediated killing effect might be attributed
to impurities associated with the P1 phagemid transducing units and/or due to inherent
properties of P1 transduction. Nevertheless, the versatility of the P1 phagemid system
allowed an inducible Cpf1-mediated, vector curing function to be incorporated into the
phagemid while retaining its Cas9-mediated antimicrobial effect on S. flexneri cells.
Furthermore, the P1 cas9 phagemid was compatible with the ΔnadC autotrophy
complementation system, providing antibiotic-free maintenance and selection of the modified
vector in ΔnadC E. coli. Taken together, the phagemid and/or cosmid system that produces
pure transducing particles might be the key to achieving a high Cas9 antimicrobial effect on
S. flexneri cells with negligible lysate cytotoxicity.