Synthetic Genome Research Articles

Introduction to Applied Biosafety’s Second Special Issue on Synthetic Genomics

Introduction to <i>Applied Biosafety</i>’s Second Special Issue on Synthetic Genomics

Prioritizing genomic variants through neuro-symbolic, knowledge-enhanced learning.

Whole-exome and genome sequencing have become common tools in diagnosing patients with rare diseases. Despite their success, this approach leaves many patients undiagnosed. A common argument is that more disease variants still await discovery, or the novelty of disease phenotypes results from a combination of variants in multiple disease-related genes. Interpreting the phenotypic consequences of genomic variants relies on information about gene functions, gene expression, physiology, and other genomic features. Phenotype-based methods to identify variants involved in genetic diseases combine molecular features with prior knowledge about the phenotypic consequences of altering gene functions. While phenotype-based methods have been successfully applied to prioritizing variants, such methods are based on known gene-disease or gene-phenotype associations as training data and are applicable to genes that have phenotypes associated, thereby limiting their scope. In addition, phenotypes are not assigned uniformly by different clinicians, and phenotype-based methods need to account for this variability. We developed an Embedding-based Phenotype Variant Predictor (EmbedPVP), a computational method to prioritize variants involved in genetic diseases by combining genomic information and clinical phenotypes. EmbedPVP leverages a large amount of background knowledge from human and model organisms about molecular mechanisms through which abnormal phenotypes may arise. Specifically, EmbedPVP incorporates phenotypes linked to genes, functions of gene products, and the anatomical site of gene expression, and systematically relates them to their phenotypic effects through neuro-symbolic, knowledge-enhanced machine learning. We demonstrate EmbedPVP's efficacy on a large set of synthetic genomes and genomes matched with clinical information. EmbedPVP and all evaluation experiments are freely available at https://github.com/bio-ontology-research-group/EmbedPVP.

Design and Application of Biosafe Coronavirus Engineering Systems without Virulence.

In the last twenty years, three deadly zoonotic coronaviruses (CoVs)-namely, severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and SARS-CoV-2-have emerged. They are considered highly pathogenic for humans, particularly SARS-CoV-2, which caused the 2019 CoV disease pandemic (COVID-19), endangering the lives and health of people globally and causing unpredictable economic losses. Experiments on wild-type viruses require biosafety level 3 or 4 laboratories (BSL-3 or BSL-4), which significantly hinders basic virological research. Therefore, the development of various biosafe CoV systems without virulence is urgently needed to meet the requirements of different research fields, such as antiviral and vaccine evaluation. This review aimed to comprehensively summarize the biosafety of CoV engineering systems. These systems combine virological foundations with synthetic genomics techniques, enabling the development of efficient tools for attenuated or non-virulent vaccines, the screening of antiviral drugs, and the investigation of the pathogenic mechanisms of novel microorganisms.

Synthesis of the Genome of Bacteriophage N4

At present, bacteriophages are considered as an alternative to antibiotics in prevention and treatment of bacterial infections, in particular cholera.The aim of the work was to demonstrate a method to obtain synthetic bacteriophage against Vibrio cholerae. Vibriophage N4 was selected as a subject for the study.Materials and methods. The genome sequence of vibriophage N4 (38.5 kb) was taken from the NCBI GenBank database. The sequence was divided into gene blocks of 1500–2000 bp. The gene blocks, in turn, were split into oligonucleotides. Sequence partitioning was carried out using the BAC-browser software that we have developed. Oligonucleotides were chemically synthesized; gene blocks were assembled from them. After that, the complete genome of vibriophage N4 was synthesized from the obtained gene blocks. The assembly of the synthetic genome took place in two stages. At the first stage, gene block cassettes of 5–7 pieces with sizes ranging from 7 to 10.5 thousand bp were generated via homologous recombination in yeast. The resulting cassettes were then amplified and used for in vitro assembly using 5’-3’ exonuclease and thermostable DNA polymerase. The resulting preparation was used for electroporation of V. cholerae cells.Results and discussion. The synthetic genome of vibriophage N4 was delivered into the V. cholerae strain M818 O1 biovar El Tor using electroporation. As a result, the formation of lytic plaques on the lawn of V. cholerae was observed. The range of technologies we have developed: software for assembly design, enzymes and buffers for the synthesis of gene blocks and their crosslinking by homologous recombination in vitro, the method for producing large-sized assemblies in yeast can be used to obtain artificial bacteriophages with a rational genome design.

Introduction to Applied Biosafety's Special Issue on Synthetic Genomics: Part 1

Introduction to <i>Applied Biosafety's</i> Special Issue on Synthetic Genomics: Part 1

Methodological advances enabled by the construction of a synthetic yeast genome

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

Abstract 7415: Assigning mutational signatures to individual samples and individual somatic mutations with SigProfilerAssignment

Abstract Analysis of mutational signatures is a powerful approach for understanding the mutagenic processes that have shaped the evolution of a cancer genome. To evaluate the mutational signatures operative in a cancer genome, one first needs to quantify their activities by estimating the number of mutations imprinted by each signature, a process known as refitting of mutational signatures. Although multiple tools for refitting known signatures were developed in the past decade, most provide support almost exclusively for single base substitution signatures and lack an online interface. Further, these tools have never been compared, and no existing tool supports probabilistically assigning signatures to a single somatic mutation. Here, we present SigProfilerAssignment, a desktop and online computational framework for assigning all types of mutational signatures to individual samples. SigProfilerAssignment is the first tool that allows both analysis of copy-number signatures and probabilistic assignment of signatures to individual somatic mutations. As its computational engine, the tool uses a custom implementation of the forward stagewise algorithm for sparse regression and nonnegative least squares for numerical optimization. The analysis of 2,700 synthetic cancer genomes with and without noise demonstrates that SigProfilerAssignment outperforms four commonly used approaches for assigning mutational signatures, with consistent top performance across cancer types and most signatures. SigProfilerAssignment stands out as the most precise and sensitive tool while maintaining high computational performance and bringing novel capabilities. SigProfilerAssignment is distributed as an open-source Python/R package available at https://github.com/AlexandrovLab/SigProfilerAssignment, with an online interface available as part of the COSMIC Mutational Signatures website at https://cancer.sanger.ac.uk/signatures/assignment/. Citation Format: Raviteja Vangara, Marcos Diaz-Gay, Mark D. Barnes, Xi Wang, S M Ashiqul Islam, Ian Vermes, Stephen Duke, Nithish Bharadhwaj Narasimman, Ting Yang, Zichen Jiang, Sarah Moody, Sergey Senkin, Paul Brennan, Michael R. Stratton, Ludmil B. Alexandrov. Assigning mutational signatures to individual samples and individual somatic mutations with SigProfilerAssignment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7415.

Minimal Bacterial Cell JCVI-syn3B as a Chassis to Investigate Interactions between Bacteria and Mammalian Cells.

Mycoplasmas are atypical bacteria with small genomes that necessitate colonization of their respective animal or plant hosts as obligate parasites, whether as pathogens, or commensals. Some can grow axenically in specialized complex media yet show only host-cell-dependent growth in cell culture, where they can survive chronically and often through interactions involving surface colonization or internalization. To develop a mycoplasma-based system to identify genes mediating such interactions, we exploited genetically tractable strains of the goat pathogen Mycoplasma mycoides (Mmc) with synthetic designer genomes representing the complete natural organism (minus virulence factors; JCVI-syn1.0) or its reduced counterpart (JCVI-syn3B) containing only those genes supporting axenic growth. By measuring growth of surviving organisms, physical association with cultured human cells (HEK-293T, HeLa), and induction of phagocytosis by human myeloid cells (dHL-60), we determined that JCVI-syn1.0 contained a set of eight genes (MMSYN1-0179 to MMSYN1-0186, dispensable for axenic growth) conferring survival, attachment, and phagocytosis phenotypes. JCVI-syn3B lacked these phenotypes, but insertion of these genes restored cell attachment and phagocytosis, although not survival. These results indicate that JCVI-syn3B may be a powerful living platform to analyze the role of specific gene sets, from any organism, on the interaction with diverse mammalian cells in culture.

Using synthetic genome readers/regulators to interrogate chromatin processes: A brief review

Aberrant gene expression underlies numerous human ailments. Hence, developing small molecules to target and remedy dysfunctional gene regulation has been a long-standing goal at the interface of chemistry and medicine. A major challenge for designing small molecule therapeutics aimed at targeting desired genomic loci is the minimization of widescale disruption of genomic functions. To address this challenge, we rationally design polyamide-based multi-functional molecules, i.e., Synthetic Genome Readers/Regulators (SynGRs), which, by design, target distinct sequences in the genome. Herein, we briefly review how SynGRs access chromatin-bound and chromatin-free genomic sites, then highlight the methods for the study of chromatin processes using SynGRs on positioned nucleosomes in vitro or disease-causing repressive genomic loci in vivo.

A designer synthetic chromosome fragment functions in moss.

Rapid advances in DNA synthesis techniques have enabled the assembly and engineering of viral and microbial genomes, presenting new opportunities for synthetic genomics in multicellular eukaryotic organisms. These organisms, characterized by larger genomes, abundant transposons and extensive epigenetic regulation, pose unique challenges. Here we report the in vivo assembly of chromosomal fragments in the moss Physcomitrium patens, producing phenotypically virtually wild-type lines in which one-third of the coding region of a chromosomal arm is replaced by redesigned, chemically synthesized fragments. By eliminating 55.8% of a 155 kb endogenous chromosomal region, we substantially simplified the genome without discernible phenotypic effects, implying that many transposable elements may minimally impact growth. We also introduced other sequence modifications, such as PCRTag incorporation, gene locus swapping and stop codon substitution. Despite these substantial changes, the complex epigenetic landscape was normally established, albeit with some three-dimensional conformation alterations. The synthesis of a partial multicellular eukaryotic chromosome arm lays the foundation for the synthetic moss genome project (SynMoss) and paves the way for genome synthesis in multicellular organisms.

GMOCU: Digital Documentation, Management, and Biological Risk Assessment of Genetic Parts.

The continuous evolution of molecular biology and gene synthesis methods paired with an ever-increasing potential of synthetic biology approaches and genome engineering toolkits enables the rapid design of genetic bioparts and genetically modified organisms. Although various software solutions assist with specific design tasks and challenges, lab internal documentation and ensuring compliance with governmental regulations on biosafety assessment of the generated organisms remain the responsibility of individual academic researchers. This results in inconsistent and redundant documentation regimes and a significant time and labor burden. GMOCU (GMO documentation) is a standardized semi-automatic user-oriented software approach -written in Python and freely available- that unifies lab internal data documentation on genetic parts and genetically modified organisms (GMOs). It automatizes biological risk evaluations and maintains a shared up-to-date inventory of bioparts for team-wide data navigation and sharing. GMOCU further enables data export into customizable formats suitable for scientific publications, official biosafety documents, and the research community.

Plant kinetochore complex: composition, function, and regulation.

The kinetochore complex, an important protein assembly situated on the centromere, plays a pivotal role in chromosome segregation during cell division. Like in animals and fungi, the plant kinetochore complex is important for maintaining chromosome stability, regulating microtubule attachment, executing error correction mechanisms, and participating in signaling pathways to ensure accurate chromosome segregation. This review summarizes the composition, function, and regulation of the plant kinetochore complex, emphasizing the interactions of kinetochore proteins with centromeric DNAs (cenDNAs) and RNAs (cenRNAs). Additionally, the applications of the centromeric histone H3 variant (the core kinetochore protein CENH3, first identified as CENP-A in mammals) in the generation of ploidy-variable plants and synthesis of plant artificial chromosomes (PACs) are discussed. The review serves as a comprehensive roadmap for researchers delving into plant kinetochore exploration, highlighting the potential of kinetochore proteins in driving technological innovations in synthetic genomics and plant biotechnology.

Rapid in vitro method to assemble and transfer DNA fragments into the JCVI-syn3B minimal synthetic bacterial genome through Cre/&lt;i&gt;loxP&lt;/i&gt; system

Rapid in vitro method to assemble and transfer DNA fragments into the JCVI-syn3B minimal synthetic bacterial genome through Cre/&lt;i&gt;loxP&lt;/i&gt; system

Advances, opportunities, and challenges in methods for interrogating the structure activity relationships of natural products.

Time span in literature: 1985-early 2024Natural products play a key role in drug discovery, both as a direct source of drugs and as a starting point for the development of synthetic compounds. Most natural products are not suitable to be used as drugs without further modification due to insufficient activity or poor pharmacokinetic properties. Choosing what modifications to make requires an understanding of the compound's structure-activity relationships. Use of structure-activity relationships is commonplace and essential in medicinal chemistry campaigns applied to human-designed synthetic compounds. Structure-activity relationships have also been used to improve the properties of natural products, but several challenges still limit these efforts. Here, we review methods for studying the structure-activity relationships of natural products and their limitations. Specifically, we will discuss how synthesis, including total synthesis, late-stage derivatization, chemoenzymatic synthetic pathways, and engineering and genome mining of biosynthetic pathways can be used to produce natural product analogs and discuss the challenges of each of these approaches. Finally, we will discuss computational methods including machine learning methods for analyzing the relationship between biosynthetic genes and product activity, computer aided drug design techniques, and interpretable artificial intelligence approaches towards elucidating structure-activity relationships from models trained to predict bioactivity from chemical structure. Our focus will be on these latter topics as their applications for natural products have not been extensively reviewed. We suggest that these methods are all complementary to each other, and that only collaborative efforts using a combination of these techniques will result in a full understanding of the structure-activity relationships of natural products.

Synthetic yeast genome project and beyond

Synthetic yeast genome project and beyond

Elementary and Advanced Mechanisms for Genetic Engineering in Crops

Presently, the field of plant breeding is in the genomics era, where innovative techniques are being integrated to accelerate and enhance the efficiency of breeding. Traditional plant breeding methods rely on maintaining plant germplasm with desirable agronomic traits from distinct plants produced through crosses or mutagenesis. However, advancements in genetic engineering encompass all forms of genetic modification through recombinant DNA technology (RDT) and cell fusion mechanisms. These approaches shed light on areas involving mutant organisms, DNA replication, genetic linkage resolution, genetically modified organisms (GMOs), protein sequencing, functional genomics, and computational genomics alterations in genetic engineering. The integration of structural genomics into breeding and eugenics analysis has resulted in a vast knowledge base on crop genetics, species divergence, and molecular origin of traits, as well as the evolutionary history of crop lineage from ancient ancestral species. The genomic data and advancements have proven essential in identifying rare genes, alleles, or local lesions crucial to significant agronomic traits, thereby expediting breeding cycles. This article aims to explore the potential of emerging genetic engineering technologies, including synthetic biology and genome editing, to further advance crop genetics and plant breeding.

Transformation-associated recombination (TAR) cloning and its applications for gene function; genome architecture and evolution; biotechnology and biomedicine.

Transformation-associated recombination (TAR) cloning represents a unique tool to selectively and efficiently recover a given chromosomal segment up to several hundred kb in length from complex genomes (such as animals and plants) and simple genomes (such as bacteria and viruses). The technique exploits a high level of homologous recombination in the yeast Sacharomyces cerevisiae. In this review, we summarize multiple applications of the pioneering TAR cloning technique, developed previously for complex genomes, for functional, evolutionary, and structural studies, and extended the modified TAR versions to isolate biosynthetic gene clusters (BGCs) from microbes, which are the major source of pharmacological agents and industrial compounds, and to engineer synthetic viruses with novel properties to design a new generation of vaccines. TAR cloning was adapted as a reliable method for the assembly of synthetic microbe genomes for fundamental research. In this review, we also discuss how the TAR cloning in combination with HAC (human artificial chromosome)- and CRISPR-based technologies may contribute to the future.

An Expanded Genetic Toolbox to Accelerate the Creation of Acholeplasma laidlawii Driven by Synthetic Genomes.

We have developed genetic tools for the atypical bacterium Acholeplasma laidlawii. A. laidlawii is a member of the class Mollicutes, which lacks cell walls, has small genomes, and has limited metabolic capabilities, requiring many metabolites from their hosts. Several of these traits have facilitated the development of genome transplantation for some Mollicutes, consequently enabling the generation of synthetic cells. Here, we propose the development of genome transplantation for A. laidlawii. We first investigated a donor-recipient relationship between two strains, PG-8A and PG-8195, through whole-genome sequencing. We then created multihost shuttle plasmids and used them to optimize an electroporation protocol. We also evolved a superior strain for DNA uptake via electroporation. We created a PG-8A donor strain with a Tn5 transposon carrying a tetracycline resistance gene. These tools will enhance Acholeplasma research and accelerate the effort toward creating A. laidlawii strains with synthetic genomes.

Orchestration and theranostic applications of synthetic genome with Hachimoji bases/building blocks.

Synthetic genomics is a novel field of chemical biology where the chemically modified genetic alphabets have been considered in central dogma of life. Tweaking of chemical compositions of natural nucleotide bases could be developed as novel building blocks of DNA/RNA. The modified bases (dP, dZ, dS, and dB etc.) have been demonstrated to be adaptable for replication, transcription and follow Darwinism law of evolution. With advancement of chemical biology especially nucleotide chemistry, synthetic genetic codes have been discovered and Hachimoji nucleotides are the most important and significant one among them. These additional nucleotide bases can form orthogonal base-pairing, and also follow Darwinian evolution and other structural features. In the Hachimoji base pairing, synthetic building blocks are formed using eight modified nucleotide (DNA/RNA) letters (hence the name "Hachimoji"). Their structural conformations, like polyelectrolyte backbones and stereo-regular building blocks favor thermodynamic stability and confirm Schrodinger aperiodic crystal. From the structural genomics aspect, these synthetic bases could be incorporated into the central dogma of life. Researchers have shown Hachimoji building blocks were transcribed to its RNA counterpart as a functional fluorescent Hachimoji aptamer. Apart from several unnatural nucleotide base pairs maneuvered into its invitro and invivo applications, this review describes future perspective towards the development and therapeutic utilization of the genetic codes, a primary objective of synthetic and chemical biology.

Application and Technical Challenges in Design, Cloning, and Transfer of Large DNA.

In the field of synthetic biology, rapid advancements in DNA assembly and editing have made it possible to manipulate large DNA, even entire genomes. These advancements have facilitated the introduction of long metabolic pathways, the creation of large-scale disease models, and the design and assembly of synthetic mega-chromosomes. Generally, the introduction of large DNA in host cells encompasses three critical steps: design-cloning-transfer. This review provides a comprehensive overview of the three key steps involved in large DNA transfer to advance the field of synthetic genomics and large DNA engineering.