Phagemid-based capsid system for CRISPR-Cas13a antimicrobials targeting methicillin-resistant Staphylococcus aureus

Abstract

In response to the escalating antibiotic resistance in multidrug-resistant pathogens, we propose an innovative phagemid-based capsid system to generate CRISPR-Cas13a-loaded antibacterial capsids (AB-capsids) for targeted therapy against multidrug-resistant Staphylococcus aureus. Our optimized phagemid system maximizes AB-capsid yield and purity, showing a positive correlation with phagemid copy number. Notably, an 8.65-fold increase in copy number results in a 2.54-fold rise in AB-capsid generation. Phagemids carrying terL-terS-rinA-rinB (prophage-encoded packaging site genes) consistently exhibit high packaging efficiency, and the generation of AB-capsids using lysogenized hosts with terL-terS deletion resulted in comparatively lower level of wild-type phage contamination, with minimal compromise on AB-capsid yield. These generated AB-capsids selectively eliminate S. aureus strains carrying the target gene while sparing non-target strains. In conclusion, our phagemid-based capsid system stands as a promising avenue for developing sequence-specific bactericidal agents, offering a streamlined approach to combat antibiotic-resistant pathogens within the constraints of efficient production and targeted efficacy.

Fig. 1. Generation of phagemid-based bactericidal capsids carrying mecA-targeting CRISPR-Cas13a.

a Schematic representation of the generation of the phagemid-based SA-capsid. S. aureus cells with an integrated prophage in their chromosome are transformed with the phagemid carrying the phage packaging site. Post-transformation, mitomycin C induction facilitates the excision of the prophage genome and initiates the translation of phage structural proteins, leading to phage assembly. This process results in the phagemid being loaded into capsids, as the phagemid carries packaging signals that can be recognized by phage, thereby packaging them and yielding phagemid-based SA-capsid. b Gene structure of the Tan2 phage packaging site, consisting of genes encoding the proteins RinB, RinA, TerS, and TerL. c Schematic representation of the generation of bactericidal phagemid-based SA-Capsid Cas13a:: CpR_mecA. The phagemid carrying mecA-targeting spacer, CRISPR-Cas13a, phage packaging site genes, and chloramphenicol-resistant (CpR) gene is packaged into capsids, resulting in phagemid-based SA-Capsid Cas13a::CpR_mecA. d Schematic depiction of targeted killing assay by soft agar overlay method. The packaged capsid filtrate and the host S. aureus bacterial culture were mixed along with TSB soft top agar, poured on the top of chloramphenicol TSA plate, and incubated for 12 h at 37 °C to examine the transduction efficacy and any sequence-specific bactericidal activity. e MRSA strain USA300 or MSSA strain USA300ΔmecA were independently treated with mecA-targeting and non-targeting capsids. The representative plate images (left panel) and the colony counts of bacterial grown on these plates (right panel) are shown. Note that the mecA-targeting SA-CapsidCas13a killed the target cells MRSA that carries target gene mecA. Mean log CFU/ml of surviving target strain USA300 transformed with mecA-targeting/non-targeting capsids were compared with that of non-target strain USA300ΔmecA via Student’s t test (n = 3). ***p  <  0.001; ****p  <  0.0001. Error bars represent standard deviation of the mean.

Fig. 2. Relationship between phagemid copy number and transduction efficiency (log TFU/ml).

The x-axis indicates copy number comparison per chromosome for phagemids with different staphylococcal replication origins (ori) (n = at least 4): repC 187 C-A (pLK7-623), repB (pLK12_1), repM (pLK11-KAT), and repC Δ183-362 (pLK7-608). NC represents the negative control without phagemid. The y-axis indicates the transduction efficiency of phagemids (in log TFU/ml) with different ori. The correlation coefficient (r²) calculated using Pearson’s correlation was determined to be 0.86 (p < 0.05). Error bars represent standard deviation of the mean.

Fig. 3. Impact of TerS packaging site on transduction efficiency and natural phage contamination.

a Schematic representation illustrating the reduction in natural phage contamination during the packaging of phagemids when the terS gene is knocked out in the integrated prophage within host cells. b Quantification of transduced colony-forming unit (TFU) and plaque-forming unit (PFU) of capsids packaged in host cells, RN4220^{ΦTan2 WT}, and RN4220^ΦTan2ΔTerS, transformed with either of the two phagemids, pLK12::TerS and pLK12::TerL-TerS-RinA-RinB, or without phagemid transformation (n = 3). White bars and black bars represent log PFU/ml and log TFU/ml, respectively. Mean differences in log TFU/ml or log PFU/ml were determined using Student’s t test. *p  <  0.05; **p  <  0.01. Error bars represent standard deviation of the mean.

Fig. 4. Elimination of contaminating natural phage during phagemid-capsid packaging.

a Schematic maps depicting wild-type or various knockout mutants (packaging site genes deletion from the prophages of host cells) constructed in this study. b Schematic maps illustrating the packaging site genes retained on each constructed phagemid. c Comparison of PFU or TFU of SA-Capsids packaged by five phagemids (pLK12::TerS, pLK12::TerL-TerS, pLK12::TerL-TerS-RinA, pLK12::TerL-TerS-RinA-RinB, and pLK12::Empty as a negative control), transformed into five different host cells (RN4220^{ΦTan2 WT}, RN4220^ΦTan2ΔTerS, RN4220^{ΦTan2ΔTerL-TerS}, RN4220^{ΦTan2ΔTerL-TerS-RinA}, RN4220^{ΦTan2ΔTerL-TerS-RinA-RinB}). This resulted in a total of twenty-five combinations. White and black bars represent log PFU/ml and log TFU/ml, respectively (n = 3). n.d. not detected. Error bars represent standard deviation of the mean.

Fig. 5. Sequence-specific bacterial killing by phagemid-based SA-CapsidCas13a.

a Schematic representation of the experimental method to assess capsid-mediated bactericidal activity (spot test). Various clinical isolates of S. aureus were added to TSB top agar and poured onto the TSA-Cp plate. After the top agar solidified, tenfold serial dilutions of phagemid-based SA-CapsidCas13a::CpR;TetR_X (where X represents target genes) were spotted on different S. aureus clinical isolate bacterial lawns and incubated for 12 hours at 37 °C to visualize bactericidal activity. b–e Infection experiments were conducted against clinical isolates of S. aureus JMUB1278, JMUB4958, JMUB3007, and JMUB4975 using SA-CapsidCas13a::CpR;TetR_X (X represents the targeted antibiotic resistance genes). The test results were assessed by observing bacterial growth on TSB top agar plates supplemented with Cp. Anibiotic resistance gene presence in clinical isolates is highlighted in red font. Each gene listed on the left of plate scans signifies the presence of target spacers in the phagemid-based SA-CapsidCas13a::CpR;TetR_X corresponding to the genes. Non-T represents non-targeting capsids that lack spacers and serve as control. f Spot assay was performed against S. aureus laboratory strain RN4220 as a control that does not carry any of the resistance genes that is targeted in this work. No growth arrested by antimicrobial capsid was observed in this strain.