P1 Bacteriophage-Enabled Delivery of CRISPR-Cas9 Antimicrobial Activity Against Shigella flexneri

Abstract

The discovery of clustered, regularly interspaced, short palindromic repeats (CRISPR) and the Cas9 RNA-guided nuclease provides unprecedented opportunities to selectively kill specific populations or species of bacteria. However, the use of CRISPR-Cas9 to clear bacterial infections in vivo is hampered by the inefficient delivery of cas9 genetic constructs into bacterial cells. Here, we use a broad-host-range P1-derived phagemid to deliver the CRISPR-Cas9 chromosomal-targeting system into Escherichia coli and the dysentery-causing Shigella flexneri to achieve DNA sequence-specific killing of targeted bacterial cells. We show that genetic modification of the helper P1 phage DNA packaging site (pac) significantly enhances the purity of packaged phagemid and improves the Cas9-mediated killing of S. flexneri cells. We further demonstrate that P1 phage particles can deliver chromosomaltargeting cas9 phagemids into S. flexneri in vivo using a zebrafish larvae infection model, where they significantly reduce the bacterial load and promote host survival. Our study highlights the potential of combining P1 bacteriophage-based delivery with the CRISPR chromosomal-targeting system to achieve DNA sequence-specific cell lethality and efficient clearance of bacterial infection.

Transcriptional profiling of zebrafish identifies host factors controlling susceptibility to Shigella flexneri

“…The zebrafish larva is a powerful non-mammalian vertebrate model that has been instrumental in the study of the innate immune response to bacterial infection ( Gomes and Mostowy, 2020 ; Stream and Madigan, 2022 ; Torraca and Mostowy, 2018 ). We have previously used S. flexneri infection of zebrafish to study bacterial autophagy ( Mostowy et al, 2013 ), predator-prey interactions ( Willis et al, 2016 ), P1-bacteriophage-mediated immunity ( Huan et al, 2023 ), septin-mediated immunity ( Mazon-Moya et al, 2017 ; Torraca et al, 2023a ; Van Ngo et al, 2023 ), innate immune training ( Gomes et al, 2023 ; Willis et al, 2018 ), Shigella speciation ( Torraca et al, 2019 ) and Shigella persistent infection ( Torraca et al, 2023b ). Here, using RNA-seq, we transcriptionally capture the host response to S. flexneri infection over time.…”

Engineering probioticEscherichia coliNissle 1917 to block transfer of multiple antibiotic resistance genes by exploiting a type I CRISPR-Cas system

“…Our work also showed growth advantage of EcN expressing Cas3-Cascade over that expressing Cas9. Although the type II CRISPR-Cas9 system is toxic to many host bacteria, previous work applied this system in killing pathogens or eliminating ARGs 15, 17, 20, 63 . Whereas, an excellent probiotic should not only exert a beneficial effect, but also out-compete other bacteria in the complex ecosystem of the gut microbiome.…”

An automated microscopy workflow to studyShigella–neutrophil interactions and antibiotic efficacyin vivo

“…Our results indicate that antibiotics are working with the immune system in an additive manner to control S. flexneri infection. These results after the use of antibiotics can be compared to those after the use of the P1 bacteriophage ( Huan et al, 2022 ) or predatory bacteria ( Bdellovibrio bacteriovorus ) to treat against S. flexneri infection in zebrafish ( Willis et al, 2016 ). In the latter case, injection of Bdellovibrio triggers neutrophil recruitment and promotes direct interaction of Bdellovibrio with S. flexneri , whereas antibiotics diluted into the bath water require time to diffuse through the larvae to reach the bacteria.…”