Theragnostic application of nanoparticle and CRISPR against food-borne multi-drug resistant pathogens

Abstract

Foodborne infection is one of the leading sources of infections spreading across the world. Foodborne pathogens are recognized as multidrug-resistant (MDR) pathogens posing a significant problem in the food industry and healthy consumers resulting in enhanced economic burden, and nosocomial infections. The continued search for enhanced microbial detection tools has piqued the interest of the CRISPR-Cas system and Nanoparticles. CRISPR-Cas system is present in the bacterial genome of some prokaryotes and is repurposed as a theragnostic tool against MDR pathogens. Nanoparticles and composites have also emerged as an efficient tool in theragnostic applications against MDR pathogens. The diagnostic limitations of the CRISPR-Cas system are believed to be overcome by a synergistic combination of the nanoparticles system and CRISPR-Cas using nanoparticles as vehicles. In this review, we have discussed the diagnostic application of CRISPR-Cas technologies along with their potential usage in applications like phage resistance, phage vaccination, strain typing, genome editing, and antimicrobial. we have also elucidated the antimicrobial and detection role of nanoparticles against foodborne MDR pathogens. Moreover, the novel combinatorial approach of CRISPR-Cas and nanoparticles for their synergistic effects in pathogen clearance and drug delivery vehicles has also been discussed.

Keywords: Antimicrobial activity; CRISPR-Cas system; Diagnosis; Nanoparticle and MDR food-Borne- pathogens.

Graphical abstract

Fig. 1

Illustrative representation of CRISPR-Cas general mechanism. 1. Invasion of DNA where a foreign DNA from a plasmid invades the cell, 2. Incorporation of the DNA fragments from the invading DNA into the CRISPR locus as spacers, 3. pre-cRNA transcription occurs where the cell constitutively transcribes a spacer group, 4. Formation of guide RNA takes place where transactivating (tracrRNA) base pairs with the CRISPR repeat sequences on the pre-crRNA, 5. Activation of Cas9 protein takes place where inactivated Cas9 protein complex binds to the guide RNA and thus gets activated, 6. The activated guide RNA/Cas9 protein complex binds with the target DNA, 7. Inactivation of the target DNA which is cleaved by the Cas9 protein.

Fig. 2

Detection mechanisms of using CRISPR-Cas against different food-borne pathogens. Schematic illustrating ASFV detection in suspected swine serum samples using the CASLFA technique along with, the CRISPR/Cas9-mediated test strip based on the DNA HRP-AuNP probes. DNA-HRP-AuNP probes are pre-embedded in conjugate pads. Cas9/sgRNA recognizes the biotinylated DNA product, is added dropwise to the sample pad and then flows through the conjugate pad and hybridizes with DNA-HRP-AuNP probes. The resulting complexes flow through the test line and are captured. Excess DNA-HRP-AuNP probes flow through the control line and are captured by precoated single-stranded DNA probes. When the target DNA is detected, the test strip will show two colored bands that form through a reaction catalyzed by HRP with the peroxidase substrate DAB, and only one control line band will appear when no target DNA is detected (Figures adapted and edited from Refs. [136,159]).

Fig. 3

Graphical model demonstrating CRISPR-based delivery through three different mechanisms targeting antimicrobial activity 1. Conjugate-based delivery, 2. Plasmid-based delivery and 3. Polymer-based nanoparticle delivery.

Fig. 4

Genome editing for inactivation of MDR pathogens via CRISPR-Cas system. (1) Endogenous mechanism - pathogens containing a native CRISPR-Cas-type II without any spacer focuses on genome editing at desired location. crRNA-tracrRNA present inside bacterial cells forms a complex when come in contact with Cas9 protein. This complex further goes and bind to the bacterial gene at the site where DNA repairing is must and interrupt this repair mechanism, resulting into mutation at cleavage site. (2) Exogenous mechanism - Pathogens which do not have native CRISPR are incorporated with CRISPR (with the help of nano-carriers), where it acts as a scissors to cut the MDR gene, resulting into antibacterial sensitivity.

Fig. 5

(A) Different types of nanoparticles as nano carriers used for detection and antimicrobial activity of food-borne pathogens. (B) DNA-functionalized gold nanoparticles combined with the trans-cleavage activity of the CRISPRCas12a would release the short-distance gold nanoparticles, greatly increasing the fluorescence intensity. (C) Schematic illstrating the CRISPR/Cas13a detection system based on the AuNP-RNA-FAM probes (Figures adapted and edited from Refs. [159,160]).

Fig. 6

Mechanism of Antibacterial action of different nanoparticles. The naoparticles act agai.

Fig. 7

A. Nanotechnologies in Food Science: Applications, Recent Trends, and Future Perspectives. B. Specific capture probes (antibodies) were immobilized onto the porous surface to provide the active component of the biosensor. The biosensor was then exposed to the target bacteria to directly capture the bacteria cells onto the antibody-modified PSiO₂ surface. A drop in the intensity of the thin-film optical interference spectrum of the biosensor results from bacteria capture. Microscopy tools (light microscope and HRSEM) and real-time PCR methods were used to confirm the presence of bacteria on the biosensor surface. C. Representative biosensing experiments and the corresponding HRSEM images of the biosensors immediately after the experiments. Spiked process water (104 ​cell/mL E. coli). Control - original process water (no E. coli). The corresponding HRSEM images (in two different magnifications) of the biosensor after a biosensing experiment with spiked process water, demonstrating bacteria capture. The inset presents enlargement of a captured bacterium on the biosensor surface. A corresponding HRSEM image of the biosensor after the control experiment (original process water, no E. coli) showing a negligible amount of cells. The inset presents enlargement of the biosensor surface (Figures adapted and edited from Refs. [161,162]).

Fig. 8

Different strategies of CRISPR-Cas complex delivery by nanomaterials as a therapeutic solution against food-borne pathogens. (A) The perspective of nanotechnology assisted CRISPR/Cas system for efficient diagnostics and treatment of a viral infection/diseases, overall, towards personalized health management. (B) Antimicrobial Photodynamic Therapy (pharmaceutics-13-01995-v3). (C) A pictorial representation illustrating the mechanism of photochemical internalization technology. The photosensitizer bind to the surface of the membrane. The gene/drug delivery system along with photosensitizer molecules internalize into the cells through endocytic pathway. The photosensitizers are integrated into the membranes of the endosomes and remain inactive. The photosensitizers becomess activated after light exposure and generates highly reactive singlet oxygen (1O2), causing rupture of the endosomal membranes. (Figures adapted and edited from Refs. [136,163]).

Fig. 9

Mechanism of Nano-CRISPR synergy for pathogen clearance.

Fig. 10

Modified Organic nanomaterials for their use as CRISPR delivery and their mechanistic application with detailed molecular understanding of genome editing upon delivery.