Project Grant R21AG086869

GENOME ENGINEERING FOR A NOVEL POST-REPRODUCTIVE GENETIC SCREEN FOR INCREASED LONGEVITY - PROJECT SUMMARY THE DISCOVERY OF THE FIRST MUTATIONS KNOWN TO INCREASE LONGEVITY A LITTLE MORE THAN THIRTY YEARS AGO REVOLUTIONIZED THE FIELD OF AGING RESEARCH. DESPITE THEIR FUNDAMENTAL IMPORTANCE, IT HAS BEEN NOTORIOUSLY DIFFICULT TO SCREEN FOR PRO-LONGEVITY MUTATIONS DIRECTLY BECAUSE BY DEFINITION THE EFFECTS OF THE MUTATION CANNOT BE KNOWN UNTIL LATE IN LIFE, AND VERY OLD ANIMALS TEND TO NOT BE ABLE TO REPRODUCE. CROSSES BETWEEN INDIVIDUALS HAVE LONG BEEN FUNDAMENTAL TO GENETICS, BOTH BECAUSE THAT IS WHAT IS NEEDED TO MAP AND IDENTIFY A GIVEN MUTATION AND ALSO BECAUSE REPRODUCTION IS KEY TO PROPAGATING THE MUTATION AFTER IT IS INITIALLY FOUND. THERE ARE STRATEGIES SUCH AS FAMILY SELECTION TO COMPENSATE FOR THIS, BUT THEY TEND TO BE HIGHLY LABORIOUS AND LOW THROUGHPUT. HERE, WE PROPOSE TO BUILD UPON A COMPREHENSIVE GENETIC LIBRARY TRANSGENESIS APPROACH THAT WE HAVE CREATED CALLED "TRANSGENIC ARRAYS RESULTING IN DIVERSITY OF INTEGRATED SEQUENCES" (TARDIS) TO CREATE A CAS9 RNA GUIDE SYSTEM THAT WILL ALLOW COMPREHENSIVE WHOLE GENOME TARGETED MUTAGENESIS IN AN ANIMAL MODEL FOR THE FIRST TIME. TARDIS PROVIDES THE BENEFITS OF LIBRARY-BASED TRANSGENESIS THAT IS FREQUENTLY USED IN MICROBIAL SPECIES, BUT CAPITALIZES UPON THE ABILITY OF THE MODEL NEMATODE CAENORHABDITIS ELEGANS TO PRODUCE AND PROPAGATE EXTRA CHROMOSOMAL ARRAYS. ELEMENTS OF THE ARRAY ARE PULLED AT RANDOM INTO A SINGLE LANDING PAD SITE IN THE GENOME, CONSTRUCTED SO AS GENERATE A SELECTABLE MARKER UPON GUIDE INTEGRATION. THIS SYSTEM HAS THE ADVANTAGE OF ALLOWING THE ENTIRE GENOME TO BE TARGETED USING A CURATED SYNTHETIC LIBRARY IN A DEFINED AND REPRODUCIBLE MANNER. BUT MORE IMPORTANTLY FOR LONGEVITY SCREENS, BECAUSE THE GUIDE IS FOUND AT A WELL-DEFINED LOCATION IN THE GENOME, THE RESULTING MUTATION CAN BE IDENTIFIED VIA SIMPLE SEQUENCING OF A SINGLE SITE IN A SINGLE INDIVIDUAL. REPRODUCTION IS THEREFORE NOT NECESSARY TO MAP OR PROPAGATE THE MUTATION. THIS SYSTEM THEREFORE PROVIDES AN APPROACH FOR THE FIRST FULLY COMPREHENSIVE SCREEN OF PRO-LONGEVITY MUTATIONS IN AN ANIMAL MODEL. SPECIFICALLY, WE AIM TO (1) DEVELOP THIS NEW LIBRARY BASED MUTAGENESIS SYSTEM WITHIN C. ELEGANS, AND (2) USE THIS SYSTEM TO SCREEN FOR NOVEL MUTATIONS THAT INCREASE INDIVIDUAL LONGEVITY. MUTATIONS IDENTIFIED IN THIS FASHION WILL BE CONFIRMED VIA RECAPITULATING THE SAME GENETIC CHANGE IN A STANDARDIZED LINE VIA CRISPR GENOME EDITING, ALLOWING FOR A COMPREHENSIVE ANALYSIS OF THE MUTATIONS EFFECTS ON LONGEVITY. THIS PROJECT WILL CREATE A WHOLLY NEW APPROACH FOR SCREENING FOR MUTATIONS FOR ANY POST-REPRODUCTIVE PHENOTYPE, AS WELL AS ANY MUTATION THAT PRECLUDES REPRODUCTION PER SE. IT WILL ALSO PRODUCE A NEW SET OF GENETIC LINES CONTAINING PRO-LONGEVITY MUTATIONS. BOTH OF THESE OUTCOMES SHOULD BE OF BROAD BENEFIT TO THE GENETICS AND AGING RESEARCH COMMUNITY.