Bug mapping and fitness testing of chemically synthesized chromosome X

Wu, Yi; Li, Bing Zhi; Zhao, Meng; Mitchell, Leslie A.; Xie, Ze‐Xiong; Lin, Qiu Hui; Wang, Xia; Xiao, Wen; Wang, Ying; Zhou, Xiao Dong; Liu, Hong; Li, Xia; Ding, Ming; Liu, Duo; Zhang, Lu; Liu, Bao Li; Wu, Xiao Le; Li, Fei Fei; Dong, Xiu Tao; Jia, Bin; Zhang, Wen Zheng; Jiang, Guo Zhen; Liu, Yue; Bai, Xue; Song, Tian Qing; Yan, Cheng; Zhou, Si; Zhu, Rui; Gao, Feng; Kuang, Zheng; Wang, Xuya; Shen, Michael; Yang, Kun; Stracquadanio, Giovanni; Richardson, Sarah M.; Lin, Yicong; Wang, Lihui; Walker, Roy; Luo, Yisha; Sheng, Ping; Yang, Huanming; Cai, Yizhi; Dai, Junbiao; Bader, Joel S.; Boeke, Jef D.; Yuan, Ying

doi:10.1126/science.aaf4706

Cited by 177 publications

(129 citation statements)

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“…Third, synonymous recoding within an open reading frame is generally well tolerated. We found at least one case during the process of replacing megachunk E, in which the function of MMM1 was impaired as a result of recoding to generate a PCRTag (YLL006W.1F) (9,10). Fourth, deletion of a hypothetical intron within the 5′ UTR of COQ9 led to a transcriptional block ( fig.…”

Section: Synxii Debuggingmentioning

confidence: 92%

Engineering the ribosomal DNA in a megabase synthetic chromosome

Zhang

Zhao

Luo

et al. 2017

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INTRODUCTION It has long been an interesting question whether a living cell can be constructed from scratch in the lab, a goal that may not be realized anytime soon. Nonetheless, with advances in DNA synthesis technology, the complete genetic material of an organism can now be synthesized chemically. Hitherto, genomes of several organisms including viruses, phages, and bacteria have been designed and constructed. These synthetic genomes are able to direct all normal biological functions, capable of self-replication and production of offspring. Several years ago, a group of scientists worldwide formed an international consortium to reconstruct the genome of budding yeast, Saccharomyces cerevisiae . RATIONALE The synthetic yeast genome, designated Sc2.0, was designed according to a set of arbitrary rules, including the elimination of transposable elements and incorporation of specific DNA elements to facilitate further genome manipulation. Among the 16 S. cerevisiae chromosomes, chromosome XII is unique as one of the longest yeast chromosomes (~1 million base pairs) and additionally encodes the highly repetitive ribosomal DNA locus, which forms the well-organized nucleolus. We report on the design, construction, and characterization of chromosome XII, the physically largest chromosome in S. cerevisiae. RESULTS A 976,067–base pair linear chromosome, synXII, was designed based on the native chromosome XII sequence of S. cerevisiae , and chemically synthesized. SynXII was assembled using a two-step method involving, successive megachunk integration to produce six semisynthetic strains, followed by meiotic recombination–mediated assembly, yielding a full-length functional chromosome in S. cerevisiae. Minor growth defect “bugs” detected in synXII were caused by deletion of tRNA genes and were corrected by introducing an ectopic copy of a single tRNA gene. The ribosomal gene cluster (rDNA) on synXII was left intact during the assembly process and subsequently replaced by a modified rDNA unit. The same synthetic rDNA unit was also used to regenerate rDNA at three distinct chromosomal locations. The rDNA signature sequences of the internal transcribed spacer (ITS), often used to determine species identity by standard DNA barcoding procedures, were swapped to generate a Saccharomyces synXII strain that would be identified as S. bayanus. Remarkably, these substantial DNA changes had no detectable phenotypic consequences under various laboratory conditions. CONCLUSION The rDNA locus of synXII is highly plastic; not only can it be moved to other chromosomal loci, it can also be altered in its ITS region to masquerade as a distinct species as defined by DNA barcoding, used widely in taxonomy. The ability to perform “species morphing” reported here presumably reflects the degree of evolutionary flexibility by which these ITS regions change. However, this barcoding region is clearly not infinitely flexible, as only relatively modest intragenus base changes were tolerated. More severe intergenus differences in ITS sequence did not result in functional rDNAs, probably because of defects in rRNA processing. The ability to design, build, and debug a megabase-sized chromosome, together with the flexibility in rDNA locus position, speaks to the remarkable overall flexibility of the yeast genome. Hierarchical assembly and subsequent restructuring of synXII. SynXII was assembled in two steps: First, six semisynthetic synXII strains were built in which segments of native XII DNA were replaced with the corresponding designer sequences. Next, the semisynthetic strains were combined withmultiple rounds ofmating/sporulation, eventually generating a single strain encoding fulllength synXII.The rDNA repeats were removed, modified, and subsequently regenerated at distinct chromosomal locations for species morphing and genome restructuring.

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Section: Synxii Debuggingmentioning

confidence: 92%

Engineering the ribosomal DNA in a megabase synthetic chromosome

Zhang

Zhao

Luo

et al. 2017

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“…The Saccharomyces cerevisiae 2.0 project (Sc2.0), taking a similar de novo approach, aims to modify the yeast genome with a series of densely spaced designer changes. Several synthetic chromosomes have been shown to function in yeast, including the synthetic yeast chromosome arm (synIXR), the entirely synthetic chromosome (synIII) (6,7), and four additional synthetic chromosomes described in this issue (8)(9)(10)(11). For designer genome synthesis projects, precisely matching the physical sequence to the specified design is important for the systematic evaluation of underlying design principles.…”

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“Perfect” designer chromosome V and behavior of a ring derivative

Xie

Mitchell

et al. 2017

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INTRODUCTION The Saccharomyces cerevisiae 2.0 project (Sc2.0) aims to modify the yeast genome with a series of densely spaced designer changes. Both a synthetic yeast chromosome arm (synIXR) and the entirely synthetic chromosome (synIII) function with high fitness in yeast. For designer genome synthesis projects, precise engineering of the physical sequence to match the specified design is important for the systematic evaluation of underlying design principles. Yeast can maintain nuclear chromosomes as rings, occurring by chance at repeated sequences, although the cyclized format is unfavorable in meiosis given the possibility of dicentric chromosome formation from meiotic recombination. Here, we describe the de novo synthesis of synthetic yeast chromosome V (synV) in the “Build-A-Genome China” course, perfectly matching the designer sequence and bearing loxPsym sites, distinguishable watermarks, and all the other features of the synthetic genome. We generated a ring synV derivative with user-specified cyclization coordinates and characterized its performance in mitosis and meiosis. RATIONALE Systematic evaluation of underlying Sc2.0 design principles requires that the final assembled synthetic genome perfectly match the designed sequence. Given the size of yeast chromosomes, synthetic chromosome construction is performed iteratively, and new mutations and unpredictable events may occur during synthesis; even a very small number of unintentional nucleotide changes across the genome could have substantial effects on phenotype. Therefore, precisely matching the physical sequence to the designed sequence is crucial for verification of the design principles in genome synthesis. Ring chromosomes can extend those design principles to provide a model for genomic rearrangement, ring chromosome evolution, and human ring chromosome disorders. RESULTS We chemically synthesized, assembled, and incorporated designer chromosome synV (536,024 base pairs) of S. cerevisiae according to Sc2.0 principles, based on the complete nucleotide sequence of native yeast chromosome V (576,874 base pairs). This work was performed as part of the “Build-A-Genome China” course in Tianjin University. We corrected all mutations found—including duplications, substitutions, and indels—in the initial synV strain by using integrative cotransformation of the precise desired changes and by means of a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)–based method. Altogether, 3331 corrected base pairs were required to match to the designed sequence. We generated a strain that exactly matches all designer sequence changes that displays high fitness under a variety of culture conditions. All corrections were verified with whole-genome sequencing; RNA sequencing revealed only minor changes in gene expression—most notably, decreases in expression of genes relocated near synthetic telomeres as a result of design. We constructed a functional circular synV (ring_synV) derivative in yeast by precisely joining both chromosome ends (telomeres) at specified coordinates. The ring chromosome showed restoration of subtelomeric gene expression levels. The ring_synV strain exhibited fitness comparable with that of the linear synV strain, revealed no change in sporulation frequency, but notably reduced spore viability. In meiosis, heterozygous or homozygous diploid ring_wtV and ring_synV chromosomes behaved similarly, exhibiting substantially higher frequency of the formation of zero-spore tetrads, a type that was not seen in the rod chromosome diploids. Rod synV chromosomes went through meiosis with high spore viability, despite no effort having been made to preserve meiotic competency in the design of synV. CONCLUSION The perfect designer-matched synthetic chromosome V provides strategies to edit sequence variants and correct unpredictable events, such as off-target integration of extra copies of synthetic DNA elsewhere in the genome. We also constructed a ring synthetic chromosome derivative and evaluated its fitness and stability in yeast. Both synV and synVI can be circularized and can power yeast cell growth without affecting fitness when gene content is maintained. These fitness and stability phenotypes of the ring synthetic chromosome in yeast provide a model system with which to probe the mechanism of human ring chromosome disorders. Synthesis, cyclization, and characterization of synV . ( A ) Synthetic chromosome V (synV, 536,024 base pairs) was designed in silico from native chromosome V (wtV, 576,874 base pairs), with extensive genotype modification designed to be phenotypically neutral. ( B ) CRISPR/Cas9 strategy for multiplex repair. ( C ) Colonies of wtV, synV, and ring_synV strains.

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“…Recently, two papers, one on the design and synthesis of a chromosome arm and the other about the first completely synthetic chromosome were published [32,34]. Excitingly, seven papers which covering five synthetic chromosomes were published in recent issue of Science, setting another milestone in the journey of eukaryotic genome synthesis [35][36][37][38][39][40][41]. Work on the synthesis of remaining yeast chromosomes is ongoing around the world.…”

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confidence: 99%

“…The BAG course served as a great teaching vehicle to educate the young students, giving them chance to experience real scientific research and synthetic biology. Up to now, through collaboration, the consortium has ChrII, ChrV, ChrVI, ChrX and ChrXII completely synthesized [36,[38][39][40][41] while several other chromosomes are in progress or near completion. In addition, several tools/technologies have been developed through the execution of Sc2.0 [26,43,44].…”

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confidence: 99%