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A Journal of the Gesellschaft Deutscher Chemiker
International Edition
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
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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*
Taihe Hospital, Hubei University of Medicine, Shiyan, Hubei 442000, China
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.
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Zhongnan Hospital, Wuhan University, Wuhan, Hubei 430071, China
Angewandte Chemie International Edition
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;[1]
since then, the pursuit of longer synthetic DNA molecules has continued. In the 1980s,
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small viral genomes, advances in DNA assembly and rewriting have enabled the
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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.[2] 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. [3] 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
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support matrix, such as controlled pore glass (CPG) or polystyrene (PS), followed by
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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.[4] 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.[5] 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,[6] BioBricksTM,[7] Golden Gate,[8] and
methylation-assisted tailorable ends rational (MASTER) ligation[9] are assembly
techniques that rely on restriction enzymes and are popular for standardized
biological component assembly. Gateway,[10] InFusionTM,[11] uracil-specific excision
reagent (USER) cloning,[12] sequence and ligation independent cloning (SLIC),[13]
circular polymerase extension cloning (CPEC),[14] and Gibson assembly[15] are
sequence-independent overlap methods that are better options for larger
polynucleotide assemblies. In contrast to the in vitro assembly methods described
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synthesis technology, tens of thousands of distinct oligonucleotides can be
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above, transformation-associated recombination (TAR) in S. cerevisiae, the “domino
method” in Bacillus subtilis,[16] and Cas9-facilitated homologous recombination
assembly (CasHRA)[17] 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.[18]
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.[19] Poliovirus, the
causative agent of poliomyelitis, is a small icosahedral Picornaviridae enterovirus,
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The choice of DNA assembly method is largely a matter of preference, and multiple
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and its 7,740 nt genome consists of positive-sense, single-stranded RNA.[20] 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.[19] 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,
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plasmid vector for sequencing. Three larger DNA fragments measuring 1.9 kb, 2.7 kb,
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was chosen because of its compact genomic organization.[21] 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).[18] M.
genitalium is a bacterium with the smallest genome of organisms that can be grown in
pure culture,[22] but its genome is still 100 times larger than that of ΦX174. Using
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(55°C) to prevent incorrect pairing, and (3) assembly of the ligation products to yield
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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.[24]
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.[25] Recent technical advancements
have accelerated our ability to manipulate the information encoded in genomes,
including the conjugative assembly genome engineering (CAGE)[26], replicon excision
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exceeds 300 kb.[23] The last two phases of the half-genome and whole-genome
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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).[28] 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.[29] 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.[27] 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.
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2013, Lajoie et al. synonymously replaced all known 321 TAG stop codons in the E.
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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 ([31] 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
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essential gene functionality examined was retained.[30] The recoding and repurposing
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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).[31]
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.[32] As a pilot study, the right arm of chromosome
IX, the smallest chromosome arm in the genome, was designed and chemically
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oligonucleotides using PCA. The BBs are assembled into overlapping DNA
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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).[33] 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.[35] 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
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synonymous codons were recoded to produce “PCR-Tags,” enabling the distinction
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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
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to replace native chromosomes.
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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
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chromosomes maintained the same average trajectories as native counterparts within
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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.[37] 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.[37] 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.
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minimal cell machine or chassis. The small genome of Mycoplasma represents the
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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,[19] 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).[38] When the synonymous
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expanded genetic codons and desirable functions.
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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,[39]
influenza virus,[40] Chikungunya virus (CHIKV),[41] human immunodeficiency virus type
1 (HIV-1),[42] 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,[43] intellectual disabilities,[43b] leukemia,[44] microcephaly[45]
and others.[46] Considering the complexity of inheritance and the pleiotropy associated
with human ring chromosomes, the ability to create a stable and modifiable yeast
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immune response. Through exploiting codon and codon pair biases, synonymous
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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,[47] 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
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
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potentially responsible for the disorder into the synthesized genome to mimic the
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animals. Additionally, considering their regulatory role in cell processes, epigenetic
modifications[48] 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.
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,
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chromosomes, from rare codon replacements to genome-wide recoding, the
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which has the potential to create new designer genomes, minimal cells, and even new
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artificial life forms.
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Table 1. A glossary of techniques mentioned in this review
Techniques in
Golden Gate
USER cloning
Gibson assembly
Domino method
Full name
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
Conjugative assembly genome engineering
Replicon excision enhanced recombination
A hierarchical assembly method for
merging modified chromosomal
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
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DNA assembly
modification by loxP-mediated evolution
Switching auxotrophies progressively for integration
Pooled PCRTag mapping
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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.
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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.
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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.
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Figure 4. Designed genomes enable the creation of genomes through various
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We thank Professor Peter C. Dedon for his suggestions and Ms. Yizhou Zhang for
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