Angewandte A Journal of the Gesellschaft Deutscher Chemiker International Edition Chemie www.angewandte.org Accepted Article Title: Synthetic genomes Authors: Shi Chen, Lianrong Wang, Susu Jiang, Chao Chen, Wei He, Xiaolin Wu, Fei Wang, Tong Tong, Xuan Zou, Zhiqiang Li, Jie Luo, and Zixin Deng This manuscript has been accepted after peer review and appears as an Accepted Article online prior to editing, proofing, and formal publication of the final Version of Record (VoR). This work is currently citable by using the Digital Object Identifier (DOI) given below. The VoR will be published online in Early View as soon as possible and may be different to this Accepted Article as a result of editing. Readers should obtain the VoR from the journal website shown below when it is published to ensure accuracy of information. The authors are responsible for the content of this Accepted Article. To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201708741 Angew. Chem. 10.1002/ange.201708741 Link to VoR: http://dx.doi.org/10.1002/anie.201708741 http://dx.doi.org/10.1002/ange.201708741 10.1002/anie.201708741 Angewandte Chemie International Edition Synthetic genomes from DNA synthesis to genome design Lianrong Wang1,2,3, Susu Jiang1,2,3, Chao Chen1,2,3, Wei He3, Xiaolin Wu2,3, Fei Wang3, Tong Tong1,2,3, Xuan Zou1,2,3, Zhiqiang Li1, Jie Luo2, Zixin Deng3, Shi Chen1,2,3* 1 2 Taihe Hospital, Hubei University of Medicine, Shiyan, Hubei 442000, China 3 Key Laboratory of Combinatorial Biosynthesis and Drug Discovery, School of Pharmaceutical Sciences, Ministry of Education, Wuhan University, Wuhan, Hubei 430071, China Correspondence to: Shi Chen, Wuhan University, Wuhan, Hubei 430071, China. E-mail: email@example.com 1 This article is protected by copyright. All rights reserved. Accepted Manuscript Zhongnan Hospital, Wuhan University, Wuhan, Hubei 430071, China 10.1002/anie.201708741 Angewandte Chemie International Edition Abstract Rapid technological advances enabling the construction of designer gene networks, biosynthetic pathways, and even entire genomes are moving the fields of genetics and genomics from descriptive to synthetic applications. Following the synthesis of hierarchical synthesis of bacterial genomes, such as Mycoplasma genitalium, as well as the recoding of the Escherichia coli genome by reducing the number of codons from 64 to 57. The field has advanced to the point of synthesizing an entire eukaryotic genome. The Synthetic Yeast Genome (Sc2.0) Project is underway and aims to rewrite all 16 Saccharomyces cerevisiae chromosomes by 2018; to date, 6.5 chromosomes have been designed and synthesized. Using bottom-up assembly and applying genome-wide alterations will improve our understanding of genome structure and function. This approach will not only provide a platform for systematic studies of eukaryotic chromosomes but will also generate diverse “streamlined” strains that are potentially suitable for medical and industrial applications. Here, we review the current state of synthetic genome research and discuss potential applications of this emerging technology. 1. Nucleic acid synthesis and DNA assembly The field of synthetic genomics began in 1970, when Khorana and co-workers successfully synthesized a 77 bp double-stranded DNA encoding yeast tRNAAla; since then, the pursuit of longer synthetic DNA molecules has continued. In the 1980s, 2 This article is protected by copyright. All rights reserved. Accepted Manuscript small viral genomes, advances in DNA assembly and rewriting have enabled the 10.1002/anie.201708741 Angewandte Chemie International Edition phosphoramidite chemistry was developed for nucleic acid synthesis. The synthetic method was later enhanced with solid-phase supports and automation, and it remains the method of choice for oligonucleotide manufacturing. In general, the 3’-most dimethoxytrityl (DMT)-protected nucleoside phosphoramidite is attached to a solid a series of four-step chain elongation cycles until the 5’-most nucleotide is attached (Figure 1). In the first deprotection (or detritylation) step, the DMT-protected 3’-most nucleotide phosphoramidite anchored to the solid support is deprotected using trichloroacetic acid (TCA). In the second coupling step, the next base, in the form of a DMT-protected nucleoside phosphoramidite monomer, is coupled to the 5’-hydroxyl group to form a phosphite triester linkage. In the third capping step, unreacted 5’-hydroxyl groups are capped by acylation to render them inert to subsequent reactions. The fourth stabilization step is an oxidation reaction to convert the phosphite triester to a cyanoethyl-protected phosphate triester with iodine solution. The synthesis cycle then repeats for the next base in the designed sequence.  After the desired sequence has been synthesized, the oligonucleotide is chemically cleaved from the solid support, and the protecting groups on the bases and backbones are removed, which is followed by oligonucleotide purification steps, e.g., oligonucleotide purification cartridge filtration, polyacrylamide gel electrophoresis, or high-performance liquid chromatography. Even though phosphoramidite chemistry has >99% efficiency, errors may occur during successive chemical synthesis, arising from side reactions during synthesis, e.g., incomplete 3 This article is protected by copyright. All rights reserved. couplings and Accepted Manuscript support matrix, such as controlled pore glass (CPG) or polystyrene (PS), followed by 10.1002/anie.201708741 Angewandte Chemie International Edition misincorporations. Using conventional column-based approaches, each oligonucleotide is synthesized individually on a separate column or in a multi-well plate, which gives high yields but is costly and time consuming. Recent advancements in microarray synthesized simultaneously on a single chip. Additionally, microfluidic technology has been adopted to potentially address limitations of microarray oligonucleotide synthesis for error rates and yields. Lee et al. reported the synthesis of 16 oligonucleotides in parallel on a single microfluidic device; approximately 100 pmol of each oligonucleotide were synthesized, sufficient enough yields to directly assemble a 200 bp long DNA construct. These strategies and methods will assuredly lead to the development of high-throughput gene synthesis techniques. Upon the collection of sequence-verified de novo synthesized or amplified gene fragments, larger DNA constructs and even whole chromosomes can be obtained using diverse assembly techniques. BglBricks, BioBricksTM, Golden Gate, and methylation-assisted tailorable ends rational (MASTER) ligation are assembly techniques that rely on restriction enzymes and are popular for standardized biological component assembly. Gateway, InFusionTM, uracil-specific excision reagent (USER) cloning, sequence and ligation independent cloning (SLIC), circular polymerase extension cloning (CPEC), and Gibson assembly are sequence-independent overlap methods that are better options for larger polynucleotide assemblies. In contrast to the in vitro assembly methods described 4 This article is protected by copyright. All rights reserved. Accepted Manuscript synthesis technology, tens of thousands of distinct oligonucleotides can be 10.1002/anie.201708741 Angewandte Chemie International Edition above, transformation-associated recombination (TAR) in S. cerevisiae, the “domino method” in Bacillus subtilis, and Cas9-facilitated homologous recombination assembly (CasHRA) are in vivo cloning protocols that take advantage of powerful DNA recombination systems and enable the assembly of megabase-sized genomes. approaches are often applied sequentially to achieve the final goal. For instance, synthesis of the M. genitalium JCVI-1.0 genome exploited in vitro Gibson assembly, standard cloning in E. coli, and TAR assembly in yeast. 2. Synthesis of viral and bacterial genomes Advancements in high-throughput DNA writing (synthesis) and large-scale editing are enabling more complex manipulation of genes, pathways, and even entire genomes. Since the early 21st century, we have witnessed a series of synthetic genomics milestones (Figure 2), which open a new avenue for understanding life using bottom-up assembly and will boost research and development across diverse areas, such as vaccines, minimal cells, therapeutics, and bio-industrial products. 2.1 Synthesis of poliovirus cDNA (2002) The chemical synthesis of poliovirus cDNA in the absence of a natural template, which generated an infectious virus, received global attention. Poliovirus, the causative agent of poliomyelitis, is a small icosahedral Picornaviridae enterovirus, 5 This article is protected by copyright. All rights reserved. Accepted Manuscript The choice of DNA assembly method is largely a matter of preference, and multiple 10.1002/anie.201708741 Angewandte Chemie International Edition and its 7,740 nt genome consists of positive-sense, single-stranded RNA. The de novo synthesis of poliovirus cDNA began with the assembly of oligonucleotides with an average length of 69 nt using terminal overlapping complementary sequences to yield 400-600 bp segments; these segments were then individually ligated into a and 3.0 kb were then generated by ligating the overlapping 400-600 bp segments. Finally, the full-length poliovirus cDNA carrying a T7 RNA polymerase promoter was assembled from the three overlapping DNA fragments via insertion into a plasmid vector. After chemical synthesis, the cDNA was transcribed into viral RNA, generating infectious poliovirus in a HeLa cell extract. This groundbreaking work not only showed the feasibility of generating infectious virus using chemically synthesized oligonucleotides as starting material but also demonstrated the realistic possibility of creating and modifying more complex genomes in the laboratory. 2.2 Synthesis of a bacteriophage genome (2003) Despite the minuscule size of viral genomes, it took many months to synthesize the 7,740 bp poliovirus cDNA. Microbial genomes comprising millions of base pairs represent a greater challenge. Venter’s team improved the available methodology and dramatically decreased the time required to assemble 5-6 kb segments from a single pool of chemically synthesized oligonucleotides (Figure 3). To test the feasibility of this approach, bacteriophage ΦX174, with a genome size of 5,386 bp, 6 This article is protected by copyright. All rights reserved. Accepted Manuscript plasmid vector for sequencing. Three larger DNA fragments measuring 1.9 kb, 2.7 kb, 10.1002/anie.201708741 Angewandte Chemie International Edition was chosen because of its compact genomic organization. The strategy for synthesizing the ΦX174 genome involved three key steps: (1) gel purification of pooled oligonucleotides to avoid contamination by incorrect chain-length molecules, (2) ligation of the purified oligonucleotides using a stringent annealing temperature full-length genomes by polymerase chain assembly (PCA) (Figure 3). Electroporation of the chemically synthesized ΦX174 genome into E. coli resulted in the formation of plaques and yielded new phage particles, as observed for native ΦX174 infections. By transforming E. coli, the authors introduced a round of functional selection in which incomplete or incorrect assemblies were efficiently removed from the total population. In contrast to the poliovirus synthesis, the artificial ΦX174 genome was created in only 14 days.[19, 21] This elegant work enabled the rapid and accurate synthesis of viral genomes and paved the way for synthesizing larger DNA assemblies, such as bacterial genomes. 2.3 Synthesis of bacterial genomes (2008, 2010) The synthesis of poliovirus and ΦX174 provided the initial validation of whole-genome synthesis and encouraged scientists to build more complex life forms from scratch. Using the experience gained from building the ΦX174 genome, Gibson et al. synthesized the 582,970 bp Mycobacterium genitalium genome (Figure 3). M. genitalium is a bacterium with the smallest genome of organisms that can be grown in pure culture, but its genome is still 100 times larger than that of ΦX174. Using 7 This article is protected by copyright. All rights reserved. Accepted Manuscript (55°C) to prevent incorrect pairing, and (3) assembly of the ligation products to yield 10.1002/anie.201708741 Angewandte Chemie International Edition assembled 5-7-kb DNA segments from commercial providers, scientists generated intermediate assemblies of ~24 kb, 72 kb (“1/8 genome”), and 144 kb (“1/4 genome”) using in vitro recombination and cloned these assemblies into E. coli as bacterial artificial chromosomes (BACs). The maximum insert size that BACs can hold barely assembly were therefore performed in S. cerevisiae using TAR cloning to produce the synthetic M. genitalium JCVI-1.0 genome. This monumental work established that chromosome-sized DNA synthesis is achievable from chemically synthesized pieces, and JCVI-1.0 was regarded as a landmark in the history of synthetic genomics. However, the synthesis of entire genomes became more realistic when the 1.1 Mbp synthesized Mycoplasma mycoides genome JCVI-syn1.0 was successfully transplanted and shown to be functional in a Mycoplasma capricolum recipient, generating new M. cycoides cells. 3. Genome recoding (2013, 2016) Synthetic genomes not only imitate template DNA but also allow for genetic code reprogramming. Lajoie et al. swapped all instances of 13 rare codons synonymously in 42 highly expressed essential genes across 80 E. coli strains, showing the feasibility of recoding at a whole-genome scale in living cells. Recent technical advancements have accelerated our ability to manipulate the information encoded in genomes, including the conjugative assembly genome engineering (CAGE), replicon excision 8 This article is protected by copyright. All rights reserved. Accepted Manuscript exceeds 300 kb. The last two phases of the half-genome and whole-genome 10.1002/anie.201708741 Angewandte Chemie International Edition for enhanced genome engineering through programmed recombination (REXER)  , and de novo design and genome building techniques. The hierarchical CAGE method allows for the step-wise incorporation of individually modified genomic modules into a single genome via conjugal transfer. In coli MG1655 genome, as well as release factor 1 (RF1, which terminates translation at TAG), generating a genomically recoded organism (GRO). Substitution of TAA for TAG permits the reassignment of TAG stop codons as sense codons capable of incorporating nonstandard amino acids (nsAAs), such as p-acetylphenylalanine, p-azidophenylalanine or 2-naphthalalanine into proteins via orthogonal aminoacyl-tRNA synthases and tRNAs. The metabolic dependence of GROs on nsAAs provides an alternative biocontainment design strategy, though practical evaluations are still required. Different from CAGE, REXER couples the CRISPR/Cas9 system to lambda Red recombineering, which enables programmable and scarless chromosomal replacement with long (>100 kb) synthetic DNA in E. coli. Using REXER, Wang et al. investigated the consequence of 1,468 codon changes (serine, leucine and alanine) and observed clear differences in the extent to which synonymously replaced codons are tolerated. For instance, the 407 TCG to AGT replacements in the ftsA gene were found to be found deleterious, but TCG could be synonymously recoded to TCT. These strategies and methods not only prove the plasticity of genomes but also pave the way for the rational design of recoded genomes for de novo synthesis. 9 This article is protected by copyright. All rights reserved. Accepted Manuscript 2013, Lajoie et al. synonymously replaced all known 321 TAG stop codons in the E. 10.1002/anie.201708741 Angewandte Chemie International Edition Ostrov et al. designed, de novo synthesized and assembled a 57 codon E. coli genome in which up to 62,214 instances of seven codons (TAG (stop), AGG & AGA (Arg), AGC (Ser), AGT (Ser), TTG (Leu) and TTA (Leu)) were synonymously replaced. In this synthetic genome, 63% (2.5 Mb) was experimentally validated, and 91% of the of genetic codons provides a powerful approach for creating GROs capable of utilizing nsAAs to generate products not commonly found in nature, as well as for impairing infection by multiple viruses (see Section 6) and horizontal gene transfer. 4. Synthesis of yeast chromosomes (2011, 2014, 2017) In parallel with the efforts to synthesize viral and bacterial genomes, the Sc2.0 project was initiated under the leadership of Jef Boeke and Srinivasan Chandrasegaran and has grown into an international collaborative project among global research institutes. Sc2.0 aims to design and completely chemically synthesize 16 chromosomes containing 12.5 million bases from S. cerevisiae and an additional “neochromosome” with all of the tRNA genes (http://syntheticyeast.org/). This project will not only provide a platform for the systematic investigation of eukaryotic chromosomes but will also extend the limits of our biological knowledge through its “build-to-understand” process. 4.1 Synthesis and assembly 10 This article is protected by copyright. All rights reserved. Accepted Manuscript essential gene functionality examined was retained. The recoding and repurposing 10.1002/anie.201708741 Angewandte Chemie International Edition Prior to de novo synthesis, native chromosomes were first edited in silico using the BioStudio platform, which coordinates segmenting, deletion, insertion, and base substitution to generate “designer” sequences. Hierarchical construction typically starts with the assembly of ~750 bp “building blocks” (BBs) from overlapping “minichunks” measuring 2-4 kb, which are subsequently combined into chunks (≤10 kb) using Golden Gate assembly, Gibson assembly, or regular cloning. To cope with the challenge of replacing an entire native chromosome with a synthetic one in a single step, 30-60 kb “megachunks” are assembled from the “chunks” and swapped with their counterparts in the native chromosome, yielding recombinant semisynthetic strains. Multiple rounds of sequential, endogenous homologous recombination steps complete the refactored chromosome (a process that was named switching auxotrophies progressively for integration or SwAP-In) (Figure 3). Many characterized nonessential genes and unstable elements occur in the yeast genome. To balance the desire to maintain a wild-type phenotype while introducing genetic flexibility and deleting destabilizing elements, the Sc2.0 project follows three design principles: (1) a synthetic chromosome should lead to a phenotype and fitness similar to those of the wild-type yeast, (2) a synthetic chromosome should not encode elements such as tRNA genes, introns, and transposons to improve stability and (3) a synthetic chromosome should have genetic flexibility to facilitate future research. As a pilot study, the right arm of chromosome IX, the smallest chromosome arm in the genome, was designed and chemically 11 This article is protected by copyright. All rights reserved. Accepted Manuscript oligonucleotides using PCA. The BBs are assembled into overlapping DNA 10.1002/anie.201708741 Angewandte Chemie International Edition synthesized to replace the native 89,299 bp sequence in yeast. In accordance with the design principles, the following modifications were introduced into the synthetic chromosome arm synIXR: (1) all TAG stop codons were changed to TAA, allowing the expansion of TAG-coded translational functions in the future; (2) short pairs of between designer and wild-type chromosomes; and (3) loxPsym sites were included 3 bp after the stop codons of nonessential genes and fragments to enable inducible genome reduction and combinatorial diversity (a process termed synthetic chromosome rearrangement and modification by loxP-mediated evolution or SCRaMbLE). The good fitness of the final synIXR swap strains encourages the refactoring of entire yeast chromosomes. In March 2014, the Sc2.0 international consortium reported the first synthesis of a complete designer yeast chromosome, synIII, which is 272,871 bp and includes 182 open reading frames (ORFs). Despite more than 500 alterations in synIII, the swap strain resembles native cells in terms of colony size, growth rate, morphology, and transcript profiling under various growth conditions. Three years later, the consortium published seven papers as a package describing the successful synthesis of five additional yeast chromosomes: synII, synV, synVI, synX, and synXII.[31, 34] Chromosome XII, 60% of which encodes ribosomal RNA sequences, is the largest of the 16 S. cerevisiae chromosomes. Dai’s group recently decreased the size of this chromosome from 2.5 Mb to nearly 1 Mb by removing all of the ribosomal gene clusters (rDNA) and all of the tRNA genes except for TRR4(tR(CCG)L) as well as 28 12 This article is protected by copyright. All rights reserved. Accepted Manuscript synonymous codons were recoded to produce “PCR-Tags,” enabling the distinction 10.1002/anie.201708741 Angewandte Chemie International Edition introns and 15 repeat clusters, and they also performed 123 TAG stop codon conversions and 299 loxPsym site insertions.[34a] To date, 6.5 Sc2.0 designer chromosomes accounting for 40% of all yeast chromosomes have been constructed 4.2 Debugging and consolidation Of the six synthetic chromosomes, synV perfectly matches the designer sequence and upholds the Sc2.0 design principles. Other designer chromosomes, however, have been found to encode different fitness-reducing “bugs” after undergoing the “design-build-test” process. One efficient strategy for identifying these bugs is the correlation of step-by-step SwAP-In with fitness assessment, enabling the rapid identification and assignment of bugs to a specific megachunk. For example, the progressive swapping of chromosome XII led to the identification of an impaired MMM1 gene that resulted from synonymous recoding in megachunk E.[34a] This strategy is most suitable for identifying bugs that cause slow growth. As previously mentioned, “PCR-Tags” were introduced genome-wide by synonymous recoding, enabling the generation of amplicons using only synthetic genomes as PCR templates.[34b] Based on the “PCR Tags” embedded in the designer genomes, Wu et al. developed a high-throughput bug-mapping strategy, pooled PCRTag mapping (PoPM), which utilizes a pooling method and the PCRTags to compare strains with patchworks of synthetic and native sequences to identify bugs that lead to growth defects under 13 This article is protected by copyright. All rights reserved. Accepted Manuscript to replace native chromosomes. 10.1002/anie.201708741 Angewandte Chemie International Edition selective stress conditions.[34c] Although it requires extensive multi-round testing, the bugs can be corrected by reverting the sequence to that of the wild-type, completing the “design-build-test-debug” process. Although thousands of designer changes were made in Sc2.0, the synthetic the nucleus and have exhibited no significant effects on overall genome organization  . To date, Mitchell et al. have consolidated the synthetic chromosomes synIII, synVI, and synIXR into a single strain, yielding a triple-synthetic strain.[34e] Although nearly 70 kb have been deleted and 12 kb have been recoded, the triple designer chromosomes are exceptionally well tolerated by yeast cells and exhibit good fitness.[34e] No major global changes have been observed in the poly-synthetic strain by phenotypic, transcriptomic, or proteomic analysis, bolstering the resolve to complete the synthesis and consolidation of a designer eukaryotic genome. Upon induction, a variety of SCRaMbLE-mediated genome arrangements occurred, resulting in a highly reorganized structure. At this juncture, it is still too early to speculate on the biotechnological applications of Sc2.0. However, one plan is to use SCRaMbLE to arrive at the minimal genome and to generate “streamlined” yeast strains with reduced metabolic burdens as “chassis” organisms for the production of industrial or pharmaceutical compounds. 5. Minimal genomes 14 This article is protected by copyright. All rights reserved. Accepted Manuscript chromosomes maintained the same average trajectories as native counterparts within 10.1002/anie.201708741 Angewandte Chemie International Edition Successful de novo genome synthesis enables a bottom-up approach to design-build-test a variety of reduced genomes in search of the minimal genome in recipient cells. The identification of essential genes expands our understanding of the core functions needed to sustain life and provides direction for the development of the smallest known cultivable bacterial genome and thus is close to a minimal autonomous genome, making it a model for minimal genome exploration. Following the successful creation of M. mycoides JCVI-syn1.0, Venter’s team continued their efforts to design and develop a minimal synthetic cell. The hypothetical minimal genome (HMG) was designed using Tn5 transposon mutagenesis data, which provides better identification of essential and nonessential genes. It should be noted that a class of quasi-essential genes, which are otherwise nonessential but are required for robust growth, should be assessed during genome minimization, as they will create the need to compromise between genome size and growth rate. Using the hierarchical strategy described above, the HMG was designed and divided into eight segments that were built from synthesized oligonucleotides and assessed in a seven-eighths JCVI-syn1.0 genome background for viability. After four rounds of design-build-test cycles, 428 genes were stripped from the 1079 kb JCVI-1.0 template, yielding JCVI-syn3.0 with a 531 kb genome encoding 473 essential and quasi-essential genes. The development from JCVI-syn1.0 to JCVI-syn3.0 demonstrates the feasibility of assembling a complete functional genome and forming a viable cell through the deletion of nonessential genes under laboratory conditions. 15 This article is protected by copyright. All rights reserved. Accepted Manuscript minimal cell machine or chassis. The small genome of Mycoplasma represents the 10.1002/anie.201708741 Angewandte Chemie International Edition Compared with conventional top-down mutagenesis, the approach provides another strategy for developing a microbial chassis equipped with a minimal genome of 6. Application of synthesized genomes for disease research Since the first complete synthesis of poliovirus cDNA in 2002, efforts have continuously been underway to apply gene/genome synthesis technology to clinical therapeutics. The most immediate application is the synthesis of genetically modified viruses to generate viral vaccines. In October 2010, Novartis, JCVI and SGI/Synthetic Genomics Vaccines Inc. (SGVI) announced a collaboration to apply synthetic genomics technologies to accelerate influenza vaccine production. The ultimate goal of this collaboration is to develop a "bank" of synthetically constructed vaccine viruses ready for production when the WHO identifies specific pandemic or influenza strains. The first successful outcome occurred in 2013, when robust synthetic vaccine viruses for influenza were accurately and rapidly constructed in just 4.5-5.5 days using the gene sequences of two antigens, hemagglutinin and neuraminidase, as blueprints and oligonucleotides as starting materials. Compared with the unreliability of conventional vaccine virus isolation using chicken eggs, the synthetic approach will enable more rapid and reliable responses to pandemics. After the de novo synthesis of poliovirus, Wimmer et al. continued using this model virus to conduct synthetic genome recoding, introducing up to 631 synonymous mutations in the 2643 nt virus capsid coding region (P1). When the synonymous 16 This article is protected by copyright. All rights reserved. Accepted Manuscript expanded genetic codons and desirable functions. 10.1002/anie.201708741 Angewandte Chemie International Edition mutations were replaced by under-represented codon pairs, the virus was not viable (631 substitutions) or showed reduced replication capacity (407 and 224 substitutions), even though the encoded protein sequence was identical to that of the wild-type virus. However, the codon pair-deoptimized viruses were still able to provoke a protective virus genome recoding has been reported to attenuate vesicular stomatitis virus, influenza virus, Chikungunya virus (CHIKV), human immunodeficiency virus type 1 (HIV-1), etc., and therefore has been regarded as an exciting new strategy to produce live-attenuated vaccine candidates. By applying synthetic genomes, we can synthesize the “correct” DNA sequence or even the “correct” chromosome and replace the defective one. Furthermore, synthetic genomics enables the structural alteration of chromosomes. Xie et al. eliminated both telomeres and circularized yeast synV by homologous recombination, generating the synV ring derivative ring_synV. Ring chromosomes have been found for nearly all human chromosomes generated through different mechanisms, such as breaks in the chromosome arms followed by fusion of the proximal broken ends, the fusion of two subtelomeric regions, or telomere-telomere fusion, among others. In addition, ring instability triggers secondary aberrations, including the loss or gain of genetic material, as well as other structural conformations, resulting in highly variable syndromes, e.g., epilepsy, intellectual disabilities,[43b] leukemia, microcephaly and others. Considering the complexity of inheritance and the pleiotropy associated with human ring chromosomes, the ability to create a stable and modifiable yeast 17 This article is protected by copyright. All rights reserved. Accepted Manuscript immune response. Through exploiting codon and codon pair biases, synonymous 10.1002/anie.201708741 Angewandte Chemie International Edition ring_synV chromosome in which changes can be tracked might provide an alternative model for exploring the mechanisms of ring chromosome disorders. We could build disease models for disorders such as Lesch-Nyhan syndrome, which involves neurological and kidney problems, by integrating all of the genes disease networks. Furthermore, we could build a super immunological cell by programming its DNA sequence and use it for cancer therapy. Once we can redesign and synthesize cells from more complex species, we can install different disease models and revolutionize diagnosis from “top-down” to “bottom-up”. The synthesis of large regions of mammalian genomes, mammalian artificial chromosomes, and even mammalian cell lines is possible and might be a precious resource for medical research. 7. Outlook We should be aware that the DNA synthesis capabilities available today have lagged far behind the advances in DNA sequencing. Sc2.0 has also spurred other synthetic genome projects, such as Genome Project-Write, which aims to understand the genetic blueprint of plant, animal and human genomes. The implementation of Sc2.0 and Genome Project-Write will push current technical limits to help narrow the DNA reading-writing gap. Following breakthroughs in next-generation high-throughput DNA sequencing, a future next-generation DNA synthesis/manipulation technology is essential to efficiently design, edit and build the genomes of microbes, plants and 18 This article is protected by copyright. All rights reserved. Accepted Manuscript potentially responsible for the disorder into the synthesized genome to mimic the 10.1002/anie.201708741 Angewandte Chemie International Edition animals. Additionally, considering their regulatory role in cell processes, epigenetic modifications should be considered during synthetic genome design, especially for the complex organisms. From the synthesis of small viral genomes to the de novo Sc2.0 yeast feasibility and capability of synthetic genomics have been demonstrated time and again. Sc2.0 highlights the development of a yeast “chassis” for the production of non-native pharmaceutical and industrial compounds such as artemisinin.  Based on synthetic chromosomes, it is possible to directly integrate multiple synthesized heterologous pathways in the yeast genome. Synthetic genomics also makes it feasible to shuffle genomes to rapidly generate new genomes and screen them for desired properties. Can we reduce or increase the number of chromosomes in certain cells? Can we integrate chromosomes from two or more species? Synthetic chromosomes from different organisms could be used as “modules” that could be added, deleted, and exchanged to obtain a hybrid cell. Finally, one might ask, “Why bother to synthesize a genome that naturally exists?” In short, during the bottom-up assembly process, synthetic genomics improves our understanding of how the genetic blueprint works and will accelerate research and development over a broad range of areas, including pharmaceuticals, vaccines, and disease therapies. Recent advances in biotechnology have accelerated the transition from genome reading to genome editing and, most importantly, to genome writing and design. These advances mark the beginning of a new era of synthetic genomics, 19 This article is protected by copyright. All rights reserved. Accepted Manuscript chromosomes, from rare codon replacements to genome-wide recoding, the 10.1002/anie.201708741 Angewandte Chemie International Edition which has the potential to create new designer genomes, minimal cells, and even new Accepted Manuscript artificial life forms. 20 This article is protected by copyright. All rights reserved. 10.1002/anie.201708741 Angewandte Chemie International Edition Table 1. A glossary of techniques mentioned in this review Genome recording Techniques in Sc2.0 Abbreviation TM BioBricks BglBricks Golden Gate MASTER Gateway TM InFusion USER cloning SLIC CPEC Gibson assembly PCA TAR Domino method CasHRA Full name Description In vitro DNA assembly methods involving restriction enzymes Polymerase chain assembly Transformation-associated recombination A BAC-based domino method Cas9-facilitated homologous recombination assembly PCR-related DNA assembly technology Homologous recombination assembly methods in vivo  CAGE Conjugative assembly genome engineering  REXER Replicon excision enhanced recombination A hierarchical assembly method for merging modified chromosomal segments Lambda red-associated recombination system coupled with CRISPR/Cas9 A watermark system to distinguish synthetic and native chromosomes Genome rearrangement system involving loxPsym A programmed method to assemble synthetic chromosomes in vivo PCR-related bug mapping on the genomic scale Methylation-assisted tailorable ends rational Sequence-independent overlap DNA assembly methods in vitro Uracil-specific excision reagent cloning Sequence and ligase independent cloning Circular polymerase extension cloning PCR-Tags SCRaMbLE Accepted Manuscript Techniques DNA assembly SwAP-In Synthetic chromosome rearrangement and modification by loxP-mediated evolution Switching auxotrophies progressively for integration PoPM Pooled PCRTag mapping 21 This article is protected by copyright. All rights reserved. references                  [34c] 10.1002/anie.201708741 Angewandte Chemie International Edition Figure 1. Cycle of phosphoramidite-based synthesis of oligonucleotides. Phosphoramidite synthesis begins with the 3’-most nucleotide and proceeds through a series of cycles, each of which involves four steps—deprotection, coupling, capping and oxidation—until the 5’-most nucleotide is attached. 22 This article is protected by copyright. All rights reserved. Accepted Manuscript Figures and Figure legends Figure 2. Timeline of synthetic genomics milestones from the 1900s to 2017. Green indicates milestones in genome synthesis and rewriting. Purple represents progress in sequencing technology and related projects. Orange indicates theoretical knowledge supporting synthetic biology. 23 This article is protected by copyright. All rights reserved. Accepted Manuscript 10.1002/anie.201708741 Angewandte Chemie International Edition Figure 3. Schematic flowchart of synthetic genome assembly. The de novo synthesis and assembly of bacteriophage ΦX174, M. genitalium and S. cerevisiae using oligonucleotides as starting materials. 24 This article is protected by copyright. All rights reserved. Accepted Manuscript 10.1002/anie.201708741 Angewandte Chemie International Edition Figure 4. Designed genomes enable the creation of genomes through various methods. 25 This article is protected by copyright. All rights reserved. Accepted Manuscript 10.1002/anie.201708741 Angewandte Chemie International Edition 10.1002/anie.201708741 Angewandte Chemie International Edition ACKNOWLEDGMENTS We thank Professor Peter C. Dedon for his suggestions and Ms. Yizhou Zhang for preparation of Figure 2. This work was supported by grants from the 973 Program of the Ministry of Science and Technology (2013CB734003), the National Natural 31670072), and the Young One Thousand Talent program of China. REFERENCES  K. L. Agarwal, H. Buchi, M. H. 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