This project focuses on the inactivation of bacteria with specific genes using CRISPR-Cas9 technology in complex matrices. The growing problem of antibiotic resistance and the need to manipulate microbial communities in various environments (such as food, the gut microbiome, or environmental samples) are driving the exploration of alternative and specific methods. CRISPR-Cas9 technology, a recently discovered bacterial adaptive immunity system, offers the potential to edit or eliminate target genes in bacteria with high specificity. This project will focus on designing, delivering, and validating CRISPR-Cas9 systems targeting selected bacteria in environments with high bacterial richness, with the aim of increasing knowledge about their application in uncontrolled conditions. The results of this research may serve as a basis for the development of a versatile tool for research and possible biotechnological applications.
 

• Leartiker

• Design and optimisation of specific gRNAs.
• Bioinformatic analysis and whole genome sequencing (WGS) of relevant strains.
• Evaluation of CRISPR-Cas9 delivery methods in bacteria, including heat shock and other approaches.
• Construction of functional CRISPR plasmids.
• In vitro assays and preliminary tests on milk matrices (pasteurised and raw milk).
• Analysis of the impact on enterotoxin production and assessment of the specificity of the system.