Methodological advances enabled by the construction of a synthetic yeast genome

Abstract

The international Synthetic Yeast Project (Sc2.0) aims to construct the first synthetic designer eukaryote genome. Over the past few years, the Sc2.0 consortium has achieved several significant milestones by synthesizing and characterizing all 16 nuclear chromosomes of the yeast Saccharomyces cerevisiae, as well as a 17^thde novo neochromosome containing all nuclear tRNA genes. In this commentary, we discuss the recent technological advances achieved in this project and provide a perspective on how they will impact the emerging field of synthetic genomics in the future.

Figure 1

Construction of synthetic chromosomes and their systematic “debugging” (A) The stepwise replacement of WT DNA (blue) with its synthetic counterpart in multiple rounds of SwAP-In results in a fully synthetic chromosome (purple). Each round of SwAP-In is a round of transformation and selection, and between each round, the selection marker (orange or green) is swapped. In the final step, the selection marker is replaced with synthetic DNA by a counter selection approach, e.g., 5-FOA selection against URA3. X indicates homologous recombination sites. (B) SwAP-In can be parallelized, and a chromosome can be replaced stepwise from both ends. In this case, the WT DNA is replaced in parallel from both ends of the chromosome in two strains of opposite mating type. The two halves of the synthetic chromosomes are finally fused after mating by induction of double-strand breaks in the WT sequence. The endogenous homologous repair machinery will generate the complete synthetic chromosome by homologous recombination of the synthetic DNA, eliminating the selection markers in the same step. After validation of the synthetic chromosome, the strain must be sporulated and haploid candidates isolated for further validation and characterization. (C) Synthetic chromosome construction can be performed by segmenting the WT chromosome (blue) into n segments. The segments are replaced by synthetic DNA (purple) with alternating selection markers (orange or green) and overlapping sequence regions, resulting in n strains (left panel). In the n strain, the marker is removed to end up with a marker-free synthetic chromosome. The mating type must be altered to allow subsequent chromosome consolidation through successive rounds or mating and sporulation for the MRA (meiotic recombination-mediated assembly) technique, resulting in the final synthetic chromosome (right panel). (D) The wt and synthetic DNA sequences can be distinguished by PCRTags. PCRTags are short, synonymously encoded sequences within coding sequences that allow the generation of specific amplicons to validate the presence or absence of WT or synthetic DNA. PCRTag analysis can be performed by classical PCR followed by gel electrophoresis or by high-throughput endpoint genotyping qPCR. (E) CRISPR D-BUGS allows the identification of sequences responsible for a phenotype. The schematic shows the use of WT PCRTags to identify recessive defects. The method can also be reversed to identify dominant bugs by targeting the synthetic PCRTags. In both cases, the bug can be localized to the sequence region between two PCR tags where the phenotype disappears or appears (called the fitness boundary). The fitness boundary can then be carefully dissected and the phenotype causing sequence repaired. The red bar indicates the phenotype-causing sequence. Not shown is the integration of the URA3 marker downstream of a telomere of the probed chromosome, which allows 5-FOA counter selection for the correct genotype.

Figure 2

Synthetic chromosome consolidation and building a chromosome de novo (A) Multiple synthetic chromosomes can be consolidated into one cell by a process based on mating of semi-synthetic yeast strains and subsequent destabilization of the WT chromosomes. Destabilization of WT chromosomes is achieved by integrating a strong inducible promoter upstream of the centromere. Upon induction, transcription of the centromere region will prevent kinetochore formation and cause loss of the WT chromosome with the inactivated centromere. Endoreduplication may occur, resulting in two copies of the synthetic chromosome in the diploid cells. The diploid strain is then sporulated, and haploid isolates are obtained for further characterization steps and attempts to consolidate increasing numbers of synthetic chromosomes in a cell. (B) Chromoduction was developed to facilitate synthetic chromosome consolidation. The technique relies on the kar1-1 allele, which allows cells of opposite mating types to mate, but their nuclei do not fuse. In some cases, however, individual chromosomes migrate from one nucleus to the other, allowing the selection of cells with a +1 aneuploidy for the intended synthetic chromosome to be transferred with the correct selection scheme. The WT chromosome can then be eliminated using the chromosome destabilization strategy (A). (C) Construction of circular neochromosomes can be achieved by repeated rounds of in vivo homologous recombination in yeast (eSwAP-In). The initial construct is generated by any suitable molecular cloning technique, followed by iterative rounds of DNA sequence integration using in vivo homologous recombination in yeast, facilitated by two homology arms containing the homologous sequences from the previous DNA segment and a universal homologous region (UHR; shown in blue). The sequential increase in size is illustrated by the colored blocks. Selection is achieved by recursive swapping of two markers, shown in orange and green, respectively. In a final step, the construction-based marker is removed, which, in a real case scenario, could be achieved via URA3 and subsequent 5-FOA-mediated counter-selection. The purple bar indicates a plasmid originating region containing at least a centromere, an autonomously replicating sequence (ARS), and one selection marker (e.g., centromeric plasmids of the pRS41X series). (D) Sequence structure and use of the telomerator tool. The telomerator consists of the URA3 marker with a synthetic intron based on the ACT1 intron sequence. In addition, the intron contains two telomeric seed sequence (TSS) regions separated by an I-SceI recognition site. The telomerator can be integrated into almost any position of a circular DNA construct. Upon induction of I-SceI expression, the I-SceI recognition site is cleaved, and the released TSSs can be recognized by telomerase and are extended and maintained as telomeres. Successful telomere formation would be indicated by a URA⁻ and 5-FOA^R phenotype. The telomerator can be used to convert any circular DNA construct into a linear chromosome as long as other sequence requirements, such as a centromere, are present.