Abstract
Discovery of clustered, regularly interspaced, short palindromic repeats and the Cas9 RNA-guided nuclease (CRISPR/Cas9) system provides a new opportunity to create programmable gene-specific antimicrobials that are far less likely to drive resistance than conventional antibiotics. However, the practical therapeutic use of CRISPR/Cas9 is still questionable due to current shortcomings in phage-based delivery systems such as inefficient delivery, narrow host range, and potential transfer of virulence genes by generalized transduction. In this study, we demonstrate genetic engineering strategies to overcome these shortcomings by integrating CRISPR/Cas9 system into a temperate phage genome, removing major virulence genes from the host chromosome, and expanding host specificity of the phage by complementing tail fiber protein. This significantly improved the efficacy and safety of CRISPR/Cas9 antimicrobials to therapeutic levels in both in vitro and in vivo assays. The genetic engineering tools and resources established in this study are expected to provide an efficacious and safe CRISPR/Cas9 antimicrobial, broadly applicable to Staphylococcus aureus.
Highlights
The CRISPR (Clustered, regularly interspaced, short palindromic repeats) and CRISPR associated (Cas) genes serve as a bacterial immune system to resist foreign DNA7,8
Phage-based delivery systems may deliver a plasmid or phagemid harboring CRISPR/Cas system, and host chromosomal segments by generalized and specialized transduction to target cells[17]. This is important for phage-based delivery systems using S. aureus since many important staphylococcal virulence factors such as superantigens and cytolysins are commonly located in mobile genetic elements (MGEs) and are transferred to other S. aureus and Listeria monocytogenes by temperate phage-mediated generalized transduction[18,19], thereby raising the safety issues[20,21,22]
To generate a programmable CRISPR/Cas system, synthetic oligonucleotides containing a CRISPR array encoding a promoter, leader sequence, and direct repeats interspaced with two BbsI restriction sites (Supplemental Figure S1) was cloned into pMK4, resulting pKS1 (Fig. 1A)
Summary
The CRISPR (Clustered, regularly interspaced, short palindromic repeats) and CRISPR associated (Cas) genes serve as a bacterial immune system to resist foreign DNA7,8. Recent studies demonstrated that a plasmid or phagemid harboring a CRISPR/Cas[9] system programmed to target an antibiotic resistance gene or a specific pathogen could be delivered by a temperate phage and could successfully control antibiotic resistant Escherichia coli or MRSA with minimal effects on non-targeted bacteria[12,13,14,15,16]. Phage-based delivery systems may deliver a plasmid or phagemid harboring CRISPR/Cas system, and host chromosomal segments by generalized and specialized transduction to target cells[17]. The modifications allow for improved efficiency of delivery to target cells, expanded host specificity by complementing the tail fiber protein of the phage, and removal of virulence factor genes from the host strain to prevent contamination of harmful bacterial products in the phage lysates and subsequent spread of virulence genes by generalized transduction
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