Synthetic Genome Research Articles

Environmental plasticity of wheat lines derived from synthetic genomes ABD

Introduction. In order to improve wheat by breeding, bread spring wheat was crossed with the synthetics Triticum durum Desf. – Aegilops tauschii Coss. created in Mexico, CIMMYT, and introgressive lines were obtained.Purpose of the study was to evaluate the environmental plasticity and stability of the lines derived from by hybridization of bread wheat with synthetics by yield and its important components – grain weight per spike and 1000-grain weight.Material and methods. Twenty seven wheat-like lines derived from hybridization of bread spring wheat Kharkivska 26 with the synthetics were taken as the test material. The lines were grown in 2015, 2016 and 2017, of which 2015 and 2017 were drier and 2016 was wetter. The environmental plasticity and stability were evaluated using the Eberhart S.A. and Russel W.A. method (1966).Results and discussion. The highest genotypic effect and environmental plasticity of yield were intrinsic to the DK 4 and DK 6 lines; high yield stability combined with increased yields was observed in the DK 2, DK 27 and DK 30 lines. DK 34 and DK 39 showed high genotypic effects, environmental plasticity and stability by high grain weight per spike and 1000-grain weight. The lines with high plasticity are recommended for breeding of intensive cultivars with positive response to improved growing conditions, whereas stable lines – as sources for more rigorous conditions.Conclusions. Crossing of synthetics with genome ABD with spring bread wheat allows improving wheat by its genotypic effect, plasticity and stability of the yield and its main components - grain weight per spike and 1000-grain weight.

Synthetic Genomes.

DNA synthesis technology has progressed to the point that it is now practical to synthesize entire genomes. Quite a variety of methods have been developed, first to synthesize single genes but ultimately to massively edit or write from scratch entire genomes. Synthetic genomes can essentially be clones of native sequences, but this approach does not teach us much new biology. The ability to endow genomes with novel properties offers special promise for addressing questions not easily approachable with conventional gene-at-a-time methods. These include questions about evolution and about how genomes are fundamentally wired informationally, metabolically, and genetically. The techniques and technologies relating to how to design, build, and deliver big DNA at the genome scale are reviewed here. A fuller understanding of these principles may someday lead to the ability to truly design genomes from scratch.

WUHAN COVID-19 SYNTHETIC ORIGINS AND EVOLUTION

The main result of this updated release is the formal proof that 2019-nCoV coronavirus is partially a SYNTHETIC genome. We proof the CONCENTRATION in a small région of wuhan New genome (300bp) of 3 different régions from HIV1 ENVELOPPE gene and 3 others from HIV2 and SIV (ENV and POL RT). All this is remarkable and bears the mark of a desire for organization of a human nature: LOGIC, SYMETRIES.
 In this article, we demonstrate also that there is a kind of global human hosts adaptation strategy of SARS viruses as well as a strategy of global evolution of the genomes of the different strains of SARS which have emerged, mainly in China, between years 2003 first SARS genomes and the last 2019 COVID-19 Wuhan seafood market pneumonia virus isolate Wuhan-Hu-1, complete genome.
 This global strategy, this temporal link, is materialized in our demonstration by highlighting stationary numerical waves controlling the entire sequence of their genomes.
 Curiously, these digital waves characterizing the 9 SARS genomes studied here are characteristic whole numbers: the "Fibonacci numbers", omnipresent in the forms of Nature, and which our research for several decades has shown strong links with the proportions of nucleotides in DNA.
 Here we demonstrate that the complexity and fractal multiplicity of these Fibonacci numerical waves increases over the years of the emergence of new SARS strains.
 We suggest that this increase in the overall organization of the SARS genomes over the years reflects a better adaptation of SARS genomes to the human host.
 The question of a link with pathogenicity remains open.
 However, we believe that this overall strategy for the evolution of the SARS genomes ensures greater unity, consistency and integrity of the genome.
 Finally, we ask ourselves the question of a possible artificial origin of this genome, in particular because of the presence of fragments of HIV1, HIV2 and SIV retroviruses.

Development and Molecular Cytogenetic Characterization of Cold-Hardy Perennial Wheatgrass Adapted to Northeastern China.

Cold-hardy perennial wheatgrass plays an important role in the use of barren land for farming, soil and water conservation, variety improvement, and also for increasing grass yield. By crossing octoploid tritelytrigia (2n = 8x = 56, AABBDDEE) with Thinopyrum intermedium (2n = 6x = 42, StStJJJSJS), we developed 34 lines of perennial wheatgrass from F1 to F6 generations, which had vigorous regrowth and cold hardiness. The cold-hardy, perennial wheatgrass lines were well-adapted to the cold environment and developed root and rhizomes, with a longevity between 5 and 11 years and a better seed set. Some of them maintained wheat chromosomes beneficial for breeding perennial wheat. Molecular cytogenetic analysis demonstrated that the Th. intermedium chromosomes contributed the most to the synthetic genome of the wheatgrass hybrids and were associated with the perennial growth habit and winter hardiness. They were also preferentially maintained and transmitted to the progenies. Some wheat chromosomes were also transmitted from the F1 to F6 generations, although they were eliminated in each life cycle of the wheatgrass hybrids. The numbers of wheat and Th. intermedium chromosomes affected seed set and perennial growth habit. Seed set increased with the establishment of a more balanced genomic constitution in later generations. The cold-hardy and perennial wheatgrass lines were produced, which can be the starting point of domestication effort aimed at producing well-adapted ground cover plants under extreme environments.

Rapid reconstruction of SARS-CoV-2 using a synthetic genomics platform.

Reverse genetics has been an indispensable tool to gain insights into viral pathogenesis and vaccine development. The genomes of large RNA viruses, such as those from coronaviruses, are cumbersome to clone and manipulate in Escherichia coli owing to the size and occasional instability of the genome1-3. Therefore, an alternative rapid and robust reverse-genetics platform for RNA viruses would benefit the research community. Here we show the full functionality of a yeast-based synthetic genomics platform to genetically reconstruct diverse RNA viruses, including members of the Coronaviridae, Flaviviridae and Pneumoviridae families. Viral subgenomic fragments were generated using viral isolates, cloned viral DNA, clinical samples or synthetic DNA, and these fragments were then reassembled in one step in Saccharomyces cerevisiae using transformation-associated recombination cloning to maintain the genome as a yeast artificial chromosome. T7 RNA polymerase was then used to generate infectious RNA to rescue viable virus. Using this platform, we were able to engineer and generate chemically synthesized clones of the virus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)4, which has caused the recent pandemic of coronavirus disease (COVID-19), in only a week after receipt of the synthetic DNA fragments. The technical advance that we describe here facilitates rapid responses to emerging viruses as it enables the real-time generation and functional characterization of evolving RNA virus variants during an outbreak.

Prudently conduct the engineering and synthesis of the SARS-CoV-2 virus

In response to the outbreak of COVID-19 that has been sweeping the world, scientists reconstructed the SARS-CoV-2 rapidly using a synthetic genomics platform, in order to accelerate therapeutics and vaccine development. However, given the dual-use nature of this technology, there exists a high biosecurity risk. This paper points out the potential risks of the engineering SARS-CoV-2 virus and puts forward 6 questions to this work. The authors emphasize that the two basic values of safety/security and intellectual freedom of research must be considered evenly. From the perspective of responsible development of biotechnology, this paper calls for a careful assessment to the risks of the technology, replacing risky technologies with safe ones. The risks of publication also need to be strictly assessed. The authors believe, in addition to enhancing the “self-government” and self-discipline of scientists and scientific communities, government supervision must be reinforced, laws and regulations should be improved, and global regulation framework ought to be constructed.

Construction of the mycobacterium phage Giles genome through transformation‐associated recombination (TAR) cloning in Saccharomyces cerevisiae

The rise of antibiotic resistance in pathogenic bacteria has lead to a renewed interest in bacteriophages. These naturally occurring antimicrobial agents can potentially be exploited as novel therapeutic agents. Existing limitations such as bacterial resistance and narrow host range; however, are problematic. The advent of new techniques in genome science and synthetic biology have provided mechanisms to build unique phage genomes that confer new functions that may overcome these difficulties. Building upon methods developed in the Build‐a‐Genome course at Johns Hopkins University and the Synthetic Yeast Project ( www.syntheticyeast.org), we present the complete synthetic genome of the mycobacterium phage Giles. We adapted the novel assembly line approach introduced by Oldfield et al in 2017. We set out to construct the complete genome from 12 overlapping DNA fragments of the phage using the yeast based transformation‐associated recombination (TAR) system. Individual mini‐chunks of DNA were incorporated via polymerase chain reaction (PCR) with a TAR cloning vector using specifically designed primers. The DNA fragments were then assembled into the full length phage genome inside yeast. Genetic modifications were attempted by inserting novel DNA sequences into one or more fragments. The final objective of this project is to transform the synthetic phage into bacterial cells and measure its infectivity. The results of this project are expected to show successfully engineered and infectious clone of phage Giles. The significance of which will be to demonstrate the optimization of novel methods to construct a synthetic phage genome.

Discovering and genotyping genomic structural variations by yeast genome synthesis and inducible evolution.

Genomic structural variations (SVs) promote the evolution of Saccharomyces cerevisiae, and play an important role in phenotypic diversities. Yeast genomic structures can be remodeled by design and bottom-up synthesis. The synthesis of yeast genome creates novel copy number variations (CNVs) and SVs and develops new strategies to discover gene functions. Further, an inducible evolution system SCRaMbLE, consisted of 3,932 loxPsym sites, was incorporated on synthetic yeast genome. SCRaMbLE enables genomic rearrangements at will and rapidly generates chromosomal number variations, and massive SVs under customized conditions. The impacts of genetic variations on phenotypes can be revealed by genome analysis and chromosome restructuring. Yeast genome synthesis and SCRaMbLE provide a new research paradigm to explore the genotypic mechanisms of phenotype diversities, and can be used to improve biological traits and optimize industrial chassis.

A Tandem Evolutionary Algorithm for Identifying Causal Rules from Complex Data.

We propose a new evolutionary approach for discovering causal rules in complex classification problems from batch data. Key aspects include (a) the use of a hypergeometric probability mass function as a principled statistic for assessing fitness that quantifies the probability that the observed association between a given clause and target class is due to chance, taking into account the size of the dataset, the amount of missing data, and the distribution of outcome categories, (b) tandem age-layered evolutionary algorithms for evolving parsimonious archives of conjunctive clauses, and disjunctions of these conjunctions, each of which have probabilistically significant associations with outcome classes, and (c) separate archive bins for clauses of different orders, with dynamically adjusted order-specific thresholds. The method is validated on majority-on and multiplexer benchmark problems exhibiting various combinations of heterogeneity, epistasis, overlap, noise in class associations, missing data, extraneous features, and imbalanced classes. We also validate on a more realistic synthetic genome dataset with heterogeneity, epistasis, extraneous features, and noise. In all synthetic epistatic benchmarks, we consistently recover the true causal rule sets used to generate the data. Finally, we discuss an application to a complex real-world survey dataset designed to inform possible ecohealth interventions for Chagas disease.

Transforming Ocean Conservation: Applying the Genetic Rescue Toolkit.

Although oceans provide critical ecosystem services and support the most abundant populations on earth, the extent of damage impacting oceans and the diversity of strategies to protect them is disconcertingly, and disproportionately, understudied. While conventional modes of conservation have made strides in mitigating impacts of human activities on ocean ecosystems, those strategies alone cannot completely stem the tide of mounting threats. Biotechnology and genomic research should be harnessed and developed within conservation frameworks to foster the persistence of viable ocean ecosystems. This document distills the results of a targeted survey, the Ocean Genomics Horizon Scan, which assessed opportunities to bring novel genetic rescue tools to marine conservation. From this Horizon Scan, we have identified how novel approaches from synthetic biology and genomics can alleviate major marine threats. While ethical frameworks for biotechnological interventions are necessary for effective and responsible practice, here we primarily assessed technological and social factors directly affecting technical development and deployment of biotechnology interventions for marine conservation. Genetic insight can greatly enhance established conservation methods, but the severity of many threats may demand genomic intervention. While intervention is controversial, for many marine areas the cost of inaction is too high to allow controversy to be a barrier to conserving viable ecosystems. Here, we offer a set of recommendations for engagement and program development to deploy genetic rescue safely and responsibly.

In vitro self-replication and multicistronic expression of large synthetic genomes

The generation of a chemical system capable of replication and evolution is a key objective of synthetic biology. This could be achieved by in vitro reconstitution of a minimal self-sustaining central dogma consisting of DNA replication, transcription and translation. Here, we present an in vitro translation system, which enables self-encoded replication and expression of large DNA genomes under well-defined, cell-free conditions. In particular, we demonstrate self-replication of a multipartite genome of more than 116 kb encompassing the full set of Escherichia coli translation factors, all three ribosomal RNAs, an energy regeneration system, as well as RNA and DNA polymerases. Parallel to DNA replication, our system enables synthesis of at least 30 encoded translation factors, half of which are expressed in amounts equal to or greater than their respective input levels. Our optimized cell-free expression platform could provide a chassis for the generation of a partially self-replicating in vitro translation system.

SCRaMbLE-in: A Fast and Efficient Method to Diversify and Improve the Yields of Heterologous Pathways in Synthetic Yeast.

The synthetic chromosome rearrangement and modification by LoxP-mediated evolution (SCRaMbLE) system is a key component of the synthetic yeast genome (Sc2.0) project, an international effort to construct an entire synthetic genome in yeast. SCRaMbLE involves the introduction of thousands of symmetrical LoxP (LoxPsym) recombination sites downstream of every nonessential gene in all 16 chromosomes, enabling numerous genome rearrangements in the form of deletions, inversions, duplications, and translocations by the Cre-LoxPsym recombination system. We highlight a two-step protocol for SCRaMbLE-in (Liu, Nat Commun 9(1):1936, 2018), a recombinase-based combinatorial method to expedite genetic engineering and exogenous pathway optimization, using a synthetic β-carotene pathway as an example. First, an in vitro phase uses a recombinase toolkit to diversify gene expression by integrating various regulatory elements into the target pathway. This combinatorial pathway library can be transformed directly into yeast for traditional screening. Once an optimized pathway which is flanked by LoxPsym sites is identified, it is transformed into Sc2.0 yeast for the in vivo SCRaMbLE phase, where LoxPsym sites in the synthetic yeast genome and Cre recombinase catalyze massive genome rearrangements. We describe all the conditions necessary to perform SCRaMbLE and post-SCRaMbLE experiments including screening, spot test analysis, and PCRTag analysis to elucidate genotype-phenotype relationships.

Discrete Circular Distributions with Applications to Shared Orthologs of Paired Circular Genomes

For structural comparisons of paired prokaryotic genomes, an important topic in synthetic and evolutionary biology, the locations of shared orthologous genes (henceforth orthologs) are observed as binned data. This and other data, e.g., wind directions recorded at monitoring sites and intensive care unit arrival times on the 24-hour clock, are counted in binned circular arcs, thus modeling them by discrete circular distributions (DCDs) is required. We propose a novel method to construct a DCD from a base continuous circular distribution (CCD). The probability mass function is defined to take the normalized values of the probability density function at some pre-fixed equidistant points on the circle. Five families of constructed DCDs which have normalizing constants in closed form are presented. Simulation studies show that DCDs outperform the corresponding CCDs in modeling grouped (discrete) circular data, and minimum chi-square estimation outperforms maximum likelihood estimation for parameters. We apply the constructed DCDs, invariant wrapped Poisson and wrapped discrete skew Laplace to compare the structures of paired bacterial genomes. Specifically, discrete four-parameter wrapped Cauchy (nonnegative trigonometric sums) distribution models multi-modal shared orthologs in <i>Clostridium</i> (<i>Sulfolobus</i>) better than the others considered, in terms of AIC and Freedman’s goodness-of-fit test. The result that different DCDs fit the shared orthologs is consistent with the fact they belong to two kingdoms. Nevertheless, these prokaryotes have a common favored site around 70° on the unit circle; this finding is important for building synthetic prokaryotic genomes in synthetic biology. These DCDs can also be applied to other binned circular data.

In-Yeast Assembly of Coronavirus Infectious cDNA Clones Using a Synthetic Genomics Pipeline.

The Escherichia coli and vaccinia virus-based reverse genetics systems have been widely applied for the manipulation and engineering of coronavirus genomes. These systems, however, present several limitations and are sometimes difficult to establish in a timely manner for (re-)emerging viruses. In this chapter, we present a new universal reverse genetics platform for the assembly and engineering of infectious full-length cDNAs using yeast-based transformation-associated recombination cloning. This novel assembly method not only results in stable coronavirus infectious full-length cDNAs cloned in the yeast Saccharomyces cerevisiae but also fosters and accelerates the manipulation of their genomes. Such a platform is widely applicable for the scientific community, as it requires no specific equipment and can be performed in a standard laboratory setting. The protocol described can be easily adapted to virtually all known or emerging coronaviruses, such as Middle East respiratory syndrome coronavirus (MERS-CoV).

Meaning of Ambiguity: A Japanese Survey on Synthetic Biology and Genome Editing.

Synthetic biology and genome editing have become increasingly controversial issues, necessitating careful attention and engagement with the public. Our study examined ambiguity in public perception about emerging biotechnologies through the use of several intermediate response options in a survey. To understand the relationship between respondents' thoughts and attitudes, we also examined how respondents' indecision is related to their cognitive concept of “self” as well as their interpretation of “future generations.” An online survey of 994 respondents living in Japan revealed that around 80% hold intermediate attitudes (two-sided, non-judgmental, or reserved attitudes) toward synthetic biology and genome editing. These results revealed that respondents who have a narrow self-concept tend to postpone decisions about the application of emerging technologies. In contrast, those with a broad self-concept tend to adopt an ambivalent attitude and are more short-sighted, but make judgments based on the impact of their decisions on current and future generations. This study thus demonstrates that public views are more diverse and nuanced than those obtained from conventional public surveys for policy making.

Definitive demonstration by synthesis of genome annotation completeness

We develop a method for completing the genetics of natural living systems by which the absence of expected future discoveries can be established. We demonstrate the method using bacteriophage øX174, the first DNA genome to be sequenced. Like many well-studied natural organisms, closely related genome sequences are available-23 Bullavirinae genomes related to øX174. Using bioinformatic tools, we first identified 315 potential open reading frames (ORFs) within the genome, including the 11 established essential genes and 82 highly conserved ORFs that have no known gene products or assigned functions. Using genome-scale design and synthesis, we made a mutant genome in which all 11 essential genes are simultaneously disrupted, leaving intact only the 82 conserved but cryptic ORFs. The resulting genome is not viable. Cell-free gene expression followed by mass spectrometry revealed only a single peptide expressed from both the cryptic ORF and wild-type genomes, suggesting a potential new gene. A second synthetic genome in which 71 conserved cryptic ORFs were simultaneously disrupted is viable but with ∼50% reduced fitness relative to the wild type. However, rather than finding any new genes, repeated evolutionary adaptation revealed a single point mutation that modulates expression of gene H, a known essential gene, and fully suppresses the fitness defect. Taken together, we conclude that the annotation of currently functional ORFs for the øX174 genome is formally complete. More broadly, we show that sequencing and bioinformatics followed by synthesis-enabled reverse genomics, proteomics, and evolutionary adaptation can definitely establish the sufficiency and completeness of natural genome annotations.

Development of a multiplex real-time PCR surveillance assay for monitoring the health status of Ecuadorian amphibians at risk of extinction

Chytrid fungi and viruses within the genus Ranavirus have been associated with mass mortality events and declines in amphibian populations worldwide. The fungus Batrachochytrium dendrobatidis (Bd) was reported in Ecuador; however, other chytrid fungi like Batrachochytrium salamandrivorans (Bsal) or ranaviruses have not been described in the country so far. To prevent the introduction of pathogens into amphibian populations under conservation programs and to implement a successful disease surveillance program, the development of a sensitive and specific diagnostic assay was required. We describe here the optimization of one TaqMan probe-based multiplex quantitative polymerase chain reaction (qPCR) assay that enables the simultaneous detection of Bsal and ranavirus, and one monoplex TaqMan qPCR assay for the detection of Bd. Standard curves, with a high linear correlation (r2 > 0.995), were generated using a synthetic genome template (gBlocks®) containing the target sequences from all three pathogens. Different samples from skin, liver, kidney, spleen, and lung from six different amphibian species were tested, and both qPCR assays showed highly reproducible and reliable results. To our knowledge, this method is the first multiplex qPCR system developed in Ecuador for identifying amphibian pathogens and represents a valuable tool for the early detection of these pathogens and for infection and co-infection monitoring in future epidemiological surveillance of amphibian species at risk of extinction.

Technological challenges and milestones for writing genomes.

Synthetic genomics requires improved technologies

Whole genome transplantation: bringing natural or synthetic bacterial genomes back to life

The development of synthetic genomics (SG) allowed the emergence of several groundbreaking techniques including the synthesis, assembly and engineering of whole bacterial genomes. The successful implantation of those methods, which culminated in the creation of JCVI-syn3.0 the first nearly minimal bacterium with a synthetic genome, mainly results from the use of the yeast Saccharomyces cerevisiae as a transient host for bacterial genome replication and modification. Another method played a key role in the resounding success of this project: bacterial genome transplantation (GT). GT consists in the transfer of bacterial genomes cloned in yeast, back into a cellular environment suitable for the expression of their genetic content. While successful using many mycoplasma species, a complete understanding of the factors governing GT will most certainly help unleash the power of the entire SG pipeline to other genetically intractable bacteria.

Synthetic chromosomes: rewriting the code of life

The past decade has seen vast improvements in DNA synthesis and assembly methods. The creation of synthetic DNA molecules is becoming easier and more affordable, such that entire chromosomes can now be synthesized. These advances mark the beginning of synthetic genomics, a new discipline interested in the construction of complete genomes tailored for the study and application of biological systems. From viral genome synthesis to the reconstruction of the yeast 16 chromosomes, we discuss the main discoveries, the regulations and ethical considerations along with the potential of this emerging discipline for the future.