MICROBES ARE INCREASINGLY BEING ENGINEERED TO BENEFIT SOCIETY IN AREAS SUCH AS AGRICULTURE AND HEALTH. FOR EXAMPLE, NEW MICROBES ARE BEING DESIGNED TO RELEASE NUTRIENTS TO CROPS, SUPPRESS PLANT DISEASES, OR CLEAN UP POLLUTANTS. HOWEVER, ONCE RELEASED INTO SOIL, THESE ENGINEERED MICROBES CAN SHARE THEIR SYNTHETIC DNA WITH THE MANY WILD MICROBES ALREADY LIVING THERE. THIS UNINTENDED TRANSFER OF DNA COULD DISRUPT NATURAL SOIL COMMUNITIES, POTENTIALLY AFFECTING SOIL HEALTH, CROP PRODUCTIVITY, AND THE BROADER ECOSYSTEM. WHILE SCIENTISTS HAVE DEVELOPED STRATEGIES TO LIMIT THE ESCAPE OF SYNTHETIC DNA, THESE SAFEGUARDS ARE NOT PERFECT, AND WE CURRENTLY LACK SIMPLE WAYS TO MONITOR WHEN AND HOW ENGINEERED DNA SPREADS IN REAL SOILS.THIS PROJECT WILL DEVELOP NEW RNA-BASED TOOLS TO TRACK THE MOVEMENT OF ENGINEERED DNA IN COMPLEX SOIL COMMUNITIES. USING AN APPROACH CALLED RNA-ADDRESSABLE MODIFICATION (RAM), WE WILL BE ABLE TO RECORD WHEN ENGINEERED DNA IS TRANSFERRED INTO NEW MICROBES, AND THEN READ OUT THESE EVENTS USING STANDARD DNA SEQUENCING. WE WILL APPLY THIS TOOL TO TEST HOW DIFFERENT FACTORS--SUCH AS SOIL NUTRIENTS, THE PRESENCE OF FARM CHEMICALS, OR SOIL MOISTURE--AFFECT GENE TRANSFER. WE WILL ALSO TEST WHETHERSAFETY SYSTEMS BUILT INTO MICROBESCAN EFFECTIVELY PREVENT DNA FROM SPREADING. FINALLY, WE WILL USE THE DATA COLLECTED TO IMPROVE MODELS THAT PREDICT THE RISKS OF GENE TRANSFER IN REAL SOILS.BY THE END OF THIS PROJECT, WE WILL DELIVER EASY-TO-USE MONITORING TOOLS, NEW SCIENTIFIC KNOWLEDGE, AND PREDICTIVE MODELS THAT CAN HELP RESEARCHERS, FARMERS, AND REGULATORS ASSESS THE SAFETY OF ENGINEERED MICROBES IN SOILS. THESE OUTCOMES WILL STRENGTHEN THE RESPONSIBLE DEVELOPMENT OF AGRICULTURAL BIOTECHNOLOGY, PROTECT SOILHEALTH, AND PROVIDE DATA TO GUIDE EVIDENCE-BASED POLICY DECISIONS.