Taming genome complexity via bacteriophage X174 synthetic genome design

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Paul Jaschke, Erica K. Lieberman, Diane Liu, Jon Rodriguez, Adrian Sierra, Drew Endy

Stanford University, United States

Our capacity to read and write DNA has outpaced our ability to design useful and predictable DNA sequences. Synthesizing genomes could enable powerful new approaches to biological discovery, however, due to the overwhelming complexity of biological systems, most designs have largely recapitulated natural sequences. To advance synthetic genome design methods we focused on the 5.4 kb X174 bacteriophage genome because it is: (1) small enough to enable rapid prototyping of new designs, and (2) complex, displaying an intricate architecture encoding 11 overlapped genes spanning all reading frames. Two approaches were used to reduce the complexity of the X174 genome while addressing important biological questions. Gene overlaps are common in nearly every genome and their functional significance is controversial. We investigated the origin of gene overlaps by designing a fully decompressed synthetic genome ‘X174.1f’, that contained no overlapping genetic elements. We describe our genome design, construction, and successful redeployment. Phenotypic comparisons between wild type and X174.1f showed there were minimal observable differences between the two viruses, and no essential information in the gene overlaps. Additionally, the results suggest that gene decompression is a viable approach to reducing genome complexity. Secondly, we developed a method of genome simplification called ‘negative genomics’ that seeks to address a fundamental question of genome annotation: at what point can we confidently say we have found all the genes? To establish all potential open reading frames (ORFs), other than the 11 known genes, we searched the X174 genome using computational gene-finding tools. Finding 85 candidate cryptic ORFs we designed a new genome, called ‘X_clean’, with 88% of these cryptic ORFs erased via synonymous changes. Testing the prototype X_clean genome revealed that it is capable of forming plaques but is phenotypically different from wild type, suggesting previously undiscovered functions associated with the disrupted ORFs.