LISTERIA MONOCYTOGENES IS A BACTERIAL FOODBORNE PATHOGEN WHICH REMAINS A MAJOR CAUSE OF DEATHS DUE TO FOODBORNE DISEASE IN THE UNITED STATES. INFECTION CAN RESULT IN INVASIVE, SYSTEMIC ILLNESS (LISTERIOSIS) WITH SEVERE SYMPTOMS AND HIGH CASE FATALITY RATE OF APPROXIMATELY 16%, EVEN UNDER APPROPRIATE ANTIMICROBIAL TREATMENT. IT AFFLICTS PRIMARILY INDIVIDUALS SUFFERING FROM SEVERE IMMUNOCOMPROMISING CONDITIONS, THE ELDERLY, AND PREGNANT WOMEN IN WHOM ILLNESS CAN RESULT IN ABORTIONS AND STILLBIRTHS. LISTERIA HAS BECOME NOTORIOUS FOR ITS CAPACITY TO PERSIST IN FOOD PROCESSING ENVIRONMENTS OVER LONG PERIODS OF TIME, SOMETIMES OVER MANY YEARS. IN SPITE OF IMPROVEMENTS IN LISTERIA SURVEILLANCE AND CONTROL, OUTBREAKS OF FOODBORNE LISTERIOSIS AND MULTIPLE FOOD RECALLS CONTINUE TO TAKE PLACE IN THE UNITED STATES AND OTHER INDUSTRIALIZED NATIONS, SUGGESTING THE NEED FOR IMPROVED INTERVENTIONS. IN THIS PROJECT WE WILL TAKE ADVANTAGE OF RECENT ADVANCES IN THE USE OF CRISPR TECHNOLOGY TOWARDS THE GENETIC ENGINEERING OF VIRUSES (ALSO REFERRED TO AS BACTERIOPHAGE OR PHAGE) SPECIFIC FOR LISTERIA. LISTERIA-SPECIFIC PHAGE HAVE THE CAPACITY TO SERVE AS POWERFUL BIOLOGICAL CONTROLS AGAINST LISTERIA, AND HAVE ALREADY GAINED REGULATORY APPROVAL IN THE UNITED STATES FOR THEIR USE IN FOODS AND FOOD PROCESSING ENVIRONMENTS. HOWEVER, THEIR EFFICACY CAN BE COMPROMISED BY THE FACT THAT CERTAIN STRAINS OF LISTERIA, INCLUDING ALL MEMBERS OF A MAJOR CLONAL GROUP IMPLICATED IN NUMEROUS OUTBREAKS, ARE RESISTANT TO PHAGE. WE WILL EMPLOY AN ARRAY OF MOLECULAR BIOLOGIC PROCEDURES TO ENGINEER THE GENOMES OF LISTERIA-SPECIFIC PHAGE SO THAT THEY ENCODE CRISPR SYSTEMS PROGRAMMED TO DEGRADE HIGHLY-CONSERVED GENES OF LISTERIA MONOCYTOGENES, ESSENTIALLY CAUSING LISTERIA TO SELF-DESTRUCT UPON INFECTION BY THE PHAGE. THE CRISPR SYSTEM WILL BE INTRODUCED IN PHAGE GENOMES WHICH WILL BE ADDITIONALLY MODIFIED TO ALLOW MAXIMUM HOST RANGE, SO THAT LISTERIA STRAINS OF DIVERSE SEROTYPES AND GENOTYPES CAN BE EFFECTIVELY INFECTED AND KILLED. SPECIFIC PROCEDURES WILL BE EMPLOYED TO ALLOW THE PHAGE TO EVADE THE RESISTANCE EXHIBITED BY CERTAIN PROBLEMATIC STRAINS OF LISTERIA MONOCYTOGENES. THE ENGINEERED CRISPR-EQUIPPED PHAGES WILL BE VALIDATED AGAINST A LARGE LISTERIA PANEL INCLUDING STRAINS FROM FOOD PROCESSING ENVIRONMENTS, READY-TO-EAT FOODS AND RECENT OUTBREAKS OF HUMAN LISTERIOSIS. WE EXPECT THAT THE ENGINEERED PHAGE WILL SERVE WELL AS A HIGHLY-TARGETED, EFFECTIVE BIOCONTROL TOOL FOR ELIMINATING PERSISTENT LISTERIA MONOCYTOGENES FROM FOOD-PROCESSING ENVIRONMENTS, THUS SIGNIFICANTLY ENHANCING THE SAFETY OF THE FOOD SUPPLY.