Project Grant R43HG012745

DEVELOPMENT OF GENOMEBUILD AS A UNIVERSAL METHOD TO SYNTHESIZE GENOMES - PROJECT SUMMARY DEVELOPMENT OF GENOMEBUILD AS A UNIVERSAL METHOD TO SYNTHESIZE GENOMES OPPORTUNITY NUMBER: RFA-HG-20-016 PIS: CHRISTOPHER L. WARREN AND MARY S. OZERS SYNTHETIC BIOLOGY ENCOMPASSES THE ABILITY TO DE NOVO SYNTHESIZE AND ASSEMBLE LARGE OLIGONUCLEOTIDES INTO GENOMES OF WHOLE ORGANISMS, WITH A BROAD RANGE OF APPLICATIONS IN MEDICINE, THERAPEUTICS, ENVIRONMENTAL SCIENCES, AND HUMAN GENOMICS. HOWEVER, THE ABILITY TO REALIZE THE FULL POTENTIAL OF SYNTHETIC BIOLOGY HAS BEEN HAMPERED BY THE TIME AND COST LIMITATIONS OF BUILDING SYNTHETIC GENOMES AND LARGE MAMMALIAN CHROMOSOMES USING CURRENT TECHNIQUES. ADDITIONALLY, THERE IS NO COST EFFECTIVE METHOD TO INTRODUCE EXTENSIVE GENETIC EDITS INTO GENOMES. THE "DESIGN-BUILD-TEST-LEARN-REPEAT" APPROACH IS FUNDAMENTAL TO SYNTHETIC BIOLOGY BUT THE INABILITY TO "BUILD" QUICKLY OR COST-EFFECTIVELY INHIBITS OUR ABILITY TO "LEARN," MUCH LESS "REPEAT." FURTHERMORE, WITH AFFORDABILITY, A DIVERSE ARRAY OF RESEARCHERS CAN BE INCLUDED IN THIS PROCESS, NOT ONLY THE MOST HIGHLY FUNDED LABORATORIES AND COMPANIES, TO PROPEL THE FIELD FORWARD AND MAXIMIZE ITS GAINS. THE ABILITY TO CUSTOM- DESIGN AN ENTIRE GENOME CANNOT BE DONE WITH CURRENT GENOMIC EDITING TECHNIQUES SUCH AS CRISPR, AND SOLID- STATE SYNTHESIS METHODS ON THE GENOME SCALE ARE TOO EXPENSIVE. IN THIS PROPOSAL, WE DETAIL A NOVEL METHOD, GENOMEBUILD, TO INEXPENSIVELY AND RAPIDLY SYNTHESIZE A HIGH FIDELITY AND COMPLETELY CUSTOMIZABLE VIRAL GENOME FROM A STANDARD DNA MICROARRAY. AS PROOF-OF-PRINCIPLE, WE WILL SYNTHESIZE A MODIFIED FORM OF THE 150 KB HSV-1 GENOME THAT REPRODUCES THE GENETIC ALTERATIONS DESIGNED IN TALIMOGENE LAHERPAREPVEC (TVEC). TVEC, MARKETED UNDER THE BRAND NAME IMLYGIC, IS THE FIRST FDA APPROVED ONCOLYTIC VIRUS USED FOR THE TREATMENT OF ADVANCED MELANOMA. ALTHOUGH DNA MICROARRAYS CAN SERVE AS A DIRECT AND INEXPENSIVE SOURCE FOR A COMPLEX LIBRARY OF OLIGONUCLEOTIDES, THEIR HIGH ERROR-RATE RELATIVE TO SOLID PHASE SYNTHESIS HAVE PRECLUDED THEIR EFFECTIVE USE IN SYNTHETIC BIOLOGY. OUR TECHNOLOGY WILL CIRCUMVENT THIS PROBLEM BY HARNESSING AN UNPRECEDENTED HIGH-DENSITY PEPTIDE NUCLEIC ACID (PNA) ARRAY TO REMOVE IMPERFECT DNA SEQUENCES OBTAINED FROM A CORRESPONDING DNA MICROARRAY. OUR AIMS WILL SYNTHESIZE HIGH-FIDELITY MICROARRAY-EXTRACTED GENOMIC OLIGONUCLEOTIDES THAT CAN BE ASSEMBLED INTO A MODIFIED VIRAL GENOME WITH LESS THAN ONE ERROR PER 75 KB OF SEQUENCE ON AVERAGE. FINALLY, THIS CUSTOM GENOME WILL BE INTRODUCED INTO A CELL LINE USING STANDARD PROTOCOLS AND ASSESSED FOR INFECTIVITY AS COMPARED TO A GENETICALLY EQUIVALENT NON-SYNTHESIZED VIRUS. THE INEXPENSIVE COST AND FAST TURNAROUND TIME OF OUR GENOMEBUILD PLATFORM WILL ALLOW PRODUCTION OF >100 KB SYNTHETIC OLIGONUCLEOTIDES AT A SIGNIFICANT TIME AND COST SAVINGS COMPARED TO COMMERCIALLY AVAILABLE PRODUCTS. UPON SUCCESSFUL DEVELOPMENT, THE GENOMEBUILD TECHNOLOGY CAN BE EXTRAPOLATED FROM THESE EFFORTS TO ADDITIONAL ORGANISMS SUCH AS OTHER VIRUSES AND BACTERIA WITH LARGER GENOMIC SIZES, MAKING SYNTHETIC BIOLOGY OF WHOLE GENOMES, AND HUMAN CHROMOSOMES, MORE ATTAINABLE FOR ANY LABORATORY.