مقالات پذیرفته شده در هشتمین کنگره بین المللی زیست پزشکی
Engineering Bacteriophages with CRISPR-Cas9 for Targeted Therapy
Engineering Bacteriophages with CRISPR-Cas9 for Targeted Therapy
Mona Arefi,1,*
1. Faculty of Advanced Science and Technology, Tehran Medical Sciences, Islamic Azad University, Tehran, Iran
Introduction: AMR poses a worldwide health problem, requiring options other than antibiotics. Bacterial infections such as Shigella and E. coli lead to significant death rates, particularly among children, due to diarrheal diseases. Treating multidrug-resistant bacteria is challenging due to limited availability of new antimicrobial drugs. Bacteriophages show potential because they can replicate themselves, break down biofilms, and focus on particular hosts. Nevertheless, they exhibit constraints such as limited host ranges and resistance. Genetic modification can improve their effectiveness by surpassing these restrictions. Bacteriophages can serve as carriers for administering antimicrobials, like the CRISPR-Cas9 system, to target and eliminate specific bacterial DNA sequences. In spite of progress in synthetic biology, challenges such as contamination continue to impede their clinical application.
Infection by helper phages can result in the transfer of virulence and antibiotic-resistant genes among bacterial hosts. In order to address this issue, researchers have tried deleting the DNA packaging site from different phages. In this research, a cosmid system based on bacteriophage P4 was created to generate transducing units containing a CRISPR-Cas9 system for targeting particular bacteria. The P4 cosmid system, derived from a prior investigation, removes interference from the helper phage, enabling the creation of uncontaminated transducing units. The P2 lyso mutant strain developed in this research prevents the generation of P2 phage offspring, guaranteeing the production of transducing units that are uncontaminated. Improving the cosmid design and changing the P2 tail fibers enhances the transduction efficiency of cosmid DNA in human gut pathogens, resulting in substantial eradication of targeted bacteria.
Methods: This research included different types of bacteria, growth environments, and techniques for altering the genetic makeup. Bacteria were grown in LB broth. Phage assays were conducted using SM buffer. Specific techniques and primers were utilized to create cosmids, CRISPR spacers, and plasmids. Gene knock-outs were achieved using Lambda-red recombineering. The process of preparing phage lysate included transformation, culture growth, and induction steps under specific conditions.
The research utilizes qPCR for quantifying Cas9 transducing units in bacteria. Chimeric tail fibers in transducing units target and infect certain bacteria, causing instability after transduction. The specific rep gene of cosmid DNA is amplified using qPCR. The measurement of transduction efficiency involves growing bacterial cells with phage lysate and enumerating CFU. Assessment of Cas9-induced cell death involves comparing colony-forming units pre- and post-treatment.
Results: The experiments show that the P4-derived cosmid system produces phage lysates with high titers, containing phagemid transducing units, which help in the Cas9-mediated killing of S. flexneri strains 2457T and 5a M90T. Moreover, adding a chimeric tail, P2-P1(S′), greatly improves both the transduction efficiency and the targeting effectiveness of Cas9 on P4 cosmids in S. flexneri M90T. Furthermore, incorporating a P2-ϕV10 chimeric tail enables the effective introduction of the P4 cosmid into a different host, E. coli O157:H7, resulting in significant Cas9-induced lethality in this strain. These results emphasize the capability of this system for precise manipulation and regulation of bacterial genes.
Conclusion: Utilizing genetic engineering on bacteriophages is an effective method for generating synthetic bacteriophages that possess specific characteristics to enhance therapy. Modified P4 phages are able to transport Cas9 constructs into intestinal bacteria such as S. flexneri and E. coli O157:H7. Modifications to the tail fibers increase transduction efficiency and broaden the range of hosts, which could help overcome resistance to phages. Chimeric phage tail fibers have the ability to transport Cas9 antimicrobial systems to bacteria that are resistant to phages, showcasing the clinical promise of this technology.