Programmed chromosome fission and fusion enable precise large-scale genome rearrangement and assembly

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Abstract

The design and creation of synthetic genomes provide a powerful approach to understanding and engineering biology. However, it is often limited by the paucity of methods for precise genome manipulation. Here, we demonstrate the programmed fission of the Escherichia coli genome into diverse pairs of synthetic chromosomes and the programmed fusion of synthetic chromosomes to generate genomes with user-defined inversions and translocations. We further combine genome fission, chromosome transplant, and chromosome fusion to assemble genomic regions from different strains into a single genome. Thus, we program the scarless assembly of new genomes with nucleotide precision, a key step in the convergent synthesis of genomes from diverse progenitors. This work provides a set of precise, rapid, large-scale (megabase) genome-engineering operations for creating diverse synthetic genomes.

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Precise manipulation of chromosomes in vivo enables genome-wide codon replacement.

Farren J. Isaacs, Peter A Carr, Harris Wang … (2011)

We present genome engineering technologies that are capable of fundamentally reengineering genomes from the nucleotide to the megabase scale. We used multiplex automated genome engineering (MAGE) to site-specifically replace all 314 TAG stop codons with synonymous TAA codons in parallel across 32 Escherichia coli strains. This approach allowed us to measure individual recombination frequencies, confirm viability for each modification, and identify associated phenotypes. We developed hierarchical conjugative assembly genome engineering (CAGE) to merge these sets of codon modifications into genomes with 80 precise changes, which demonstrate that these synonymous codon substitutions can be combined into higher-order strains without synthetic lethal effects. Our methods treat the chromosome as both an editable and an evolvable template, permitting the exploration of vast genetic landscapes.

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Synthetic chromosome arms function in yeast and generate phenotypic diversity by design.

Junbiao Dai, Bryan Richardson, Robert Blake … (2011)

Recent advances in DNA synthesis technology have enabled the construction of novel genetic pathways and genomic elements, furthering our understanding of system-level phenomena. The ability to synthesize large segments of DNA allows the engineering of pathways and genomes according to arbitrary sets of design principles. Here we describe a synthetic yeast genome project, Sc2.0, and the first partially synthetic eukaryotic chromosomes, Saccharomyces cerevisiae chromosome synIXR, and semi-synVIL. We defined three design principles for a synthetic genome as follows: first, it should result in a (near) wild-type phenotype and fitness; second, it should lack destabilizing elements such as tRNA genes or transposons; and third, it should have genetic flexibility to facilitate future studies. The synthetic genome features several systemic modifications complying with the design principles, including an inducible evolution system, SCRaMbLE (synthetic chromosome rearrangement and modification by loxP-mediated evolution). We show the utility of SCRaMbLE as a novel method of combinatorial mutagenesis, capable of generating complex genotypes and a broad variety of phenotypes. When complete, the fully synthetic genome will allow massive restructuring of the yeast genome, and may open the door to a new type of combinatorial genetics based entirely on variations in gene content and copy number. © 2011 Macmillan Publishers Limited. All rights reserved