Project Grant R21EB035772

DNA RECORDER: LIVING BIOSENSOR FOR ARBITRARY DNA - PROJECT SUMMARY RECENT DECADES HAVE REVEALED THE PROFOUND IMPACTS THAT GUT MICROBIOTA HAVE ON HUMAN HEALTH. FAR BEYOND BEING SIMPLY PATHOGENIC VS BENIGN, GUT MICROBES IMPACT A VARIETY OF HEALTH AND DISEASE STATES, INCLUDING METABOLIC, DIGESTIVE, AND MENTAL HEALTH, AS WELL AS PROPENSITY TO CANCER. ADDING TO THE COMPLEXITY, GUT MICROBES CAN INFLUENCE THE METABOLISM, AND THEREBY ACTION, OF DRUGS DEVELOPED TO PROMOTE HEALTH AND TREAT DISEASE. THIS SUGGESTS THAT MANY CONDITIONS WOULD BENEFIT FROM A MORE PERSONALIZED TREATMENT PLAN THAT TAKES INDIVIDUAL GUT MICROBIOTA INTO ACCOUNT. STRATIFYING BY MICROBIAL PROFILE MAY ALSO BENEFIT DRUG DEVELOPMENT, BY REMOVING A CONFOUNDING VARIABLE IN CLINICAL TRIALS. THE CURRENT STANDARD METHOD OF MEASURING THE GUT MICROBIOTA IS VIA FECAL SAMPLES. HOWEVER, WHILE CONVENIENT, INEXPENSIVE, AND NON-INVASIVE, THESE SAMPLES DO NOT ACCURATELY REFLECT THE DETAILS OF GUT COMMUNITIES. UP TO A THIRD OF INTESTINAL MICROBES CAN BE EFFECTIVELY ABSENT FROM FECAL SAMPLES, WHICH ALSO FAIL TO PRESERVE INFORMATION ABOUT SPATIAL STRUCTURE. THEREFORE, NEW MEASUREMENT TECHNIQUES ARE NEEDED TO MORE ACCURATELY PROFILE GUT MICROBIOTA. HERE, WE WILL DEVELOP AN INNOVATIVE APPROACH THAT USES LIVING BIOSENSORS TO RECORD EXTRACELLULAR DNA AS THEY TRAVERSE THE GUT, PRESERVING A RECORD OF THE INTERNAL MICROBIAL COMPOSITION AND SPATIAL STRUCTURE. THIS STRATEGY RELIES ON NATURALLY COMPETENT BACTERIA, WHICH WE RECENTLY USED TO DETECT DNA RELEASED FROM COLORECTAL TUMORS IN VIVO. THE FIRST TWO AIMS WILL DEVELOP COMPLEMENTARY STRATEGIES TO STORE EXTRACELLULAR DNA, AND THE THIRD WILL DEMONSTRATE THEM IN MICE. IN AIM 1, WE WILL STORE SNIPPETS OF ENVIRONMENTAL DNA IN CRISPR ARRAYS FOR LATER READOUT. THIS AIM INCLUDES THE ENDOGENOUS CRISPR-CAS SYSTEM, AS WELL AS ALTERNATIVE SYSTEMS THAT MAY BE BETTER SUITED FOR THE TARGET DNA. IN AIM 2, WE WILL TEST THE HYPOTHESIS THAT ENVIRONMENTAL DNA CAN INSTEAD BE STORED BY CASPOSASES, WHICH COULD ALLOW STORAGE OF LONGER SNIPPETS. THESE TWO AIMS SERVE AS ALTERNATIVE APPROACHES FOR EACH OTHER, AND BOTH APPROACHES WILL ALSO ENCODE BIO-SPATIAL INFORMATION IN THE ORDER OF RECORDED DNA SEQUENCES. IN AIM 3, WE WILL VALIDATE THE GUT DNA RECORDER IN VIVO USING MOUSE MODELS OF DYSBIOSIS. THIS WORK WILL DEVELOP HIGHLY INNOVATIVE APPROACHES, WITH RISK APPROPRIATE FOR THIS MECHANISM AND AMELIORATED BY MULTIPLE ALTERNATIVES. THE RESULT OF THIS WORK WILL BE A NOVEL DIAGNOSTIC TECHNIQUE THAT CAN RECORD AND MEASURE THE MICROBIOTA ACROSS THE ENTIRE INTESTINAL TRACT, WITH NO INVASIVE PRODEDURES REQUIRED.