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Synthetic genomics involves the design, assembly, and transfer of artificially synthesized DNA fragments into target hosts to replace the native genome and construct viable forms of life. With advances in DNA synthesis and assembly techniques, the application of synthetic genomics in viruses, bacteria, and yeast has improved our knowledge of genome organization and function. Multicellular eukaryotic organisms are characterized by larger genomes, more complex epigenetic regulation, and widespread transposable elements, making genome synthesis challenging. Recently, the first synthetic multicellular eukaryotic organism was generated in the model plant Physcomitrium patens with a partially synthetic chromosome arm. Here, we introduce the design and assembly principles of moss genome synthesis. We also discuss the remaining technical barriers in the application of synthetic genomics in seed plants.
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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.
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SARS-CoV-2 , Humanos , Animais , Virulência , SARS-CoV-2/patogenicidade , SARS-CoV-2/genética , Contenção de Riscos Biológicos , COVID-19/virologia , Antivirais/farmacologiaRESUMO
Deoxyribonucleic acid (DNA) has been suggested as a very promising medium for data storage in recent years. Although numerous studies have advocated for DNA data storage, its practical application remains obscure and there is a lack of a user-oriented platform. Here, we developed a DNA data storage platform, named Storage-D, which allows users to convert their data into DNA sequences of any length and vice versa by selecting algorithms, error-correction, random-access, and codec pin strategies in terms of their own choice. It incorporates a newly designed "Wukong" algorithm, which provides over 20 trillion codec pins for data privacy use. This algorithm can also control GC content to the selected standard, as well as adjust the homopolymer run length to a defined level, while maintaining a high coding potential of ~1.98 bis/nt, allowing it to outperform previous algorithms. By connecting to a commercial DNA synthesis and sequencing platform with "Storage-D," we successfully stored "Diagnosis and treatment protocol for COVID-19 patients" into 200 nt oligo pools in vitro, and 500 bp genes in vivo which replicated in both normal and extreme bacteria. Together, this platform allows for practical and personalized DNA data storage, potentially with a wide range of applications.
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Tracking trace protein analytes in precision diagnostics is an ongoing challenge. Here, we developed an ultrasensitive detection method for the detection of SARS-CoV-2 nucleocapsid (N) protein by combining enzyme-linked immunosorbent assay (ELISA) with the clustered regularly interspaced short palindromic repeat/CRISPR-associated protein (CRISPR/Cas) system. First, the SARS-CoV-2 N protein bound by the capture antibody adsorbed on the well plate was sequentially coupled with the primary antibody, biotinylated secondary antibody, and streptavidin (SA), followed by biotin primer binding to SA. Subsequently, rolling circle amplification was initiated to generate ssDNA strands, which were targeted by CRISPR/Cas12a to cleave the FAM-ssDNA-BHQ1 probe in trans to generate fluorescence signals. We observed a linear relationship between fluorescence intensity and the logarithm of N protein concentration ranging from 3 fg/mL to 3 × 107 fg/mL. The limit of detection (LOD) was 1 fg/mL, with approximately nine molecules in 1 µL of the sample. This detection sensitivity was 4 orders magnitude higher than that of commercially available ELISA kits (LOD: 5.7 × 104 fg/mL). This method was highly specific and sensitive and could accurately detect SARS-CoV-2 pseudovirus and clinical samples, providing a new approach for ultrasensitive immunoassay of protein biomarkers.
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Proteínas do Nucleocapsídeo de Coronavírus , Limite de Detecção , SARS-CoV-2 , SARS-CoV-2/imunologia , SARS-CoV-2/genética , Humanos , Proteínas do Nucleocapsídeo de Coronavírus/imunologia , Proteínas do Nucleocapsídeo de Coronavírus/análise , Ensaio de Imunoadsorção Enzimática/métodos , Imunoensaio/métodos , COVID-19/diagnóstico , COVID-19/virologia , Sistemas CRISPR-Cas/genética , Fosfoproteínas/imunologia , Fosfoproteínas/química , Proteínas Associadas a CRISPR/química , Endodesoxirribonucleases/química , Proteínas do Nucleocapsídeo/imunologia , Proteínas de BactériasRESUMO
DNA-based data storage is a new technology in computational and synthetic biology, that offers a solution for long-term, high-density data archiving. Given the critical importance of medical data in advancing human health, there is a growing interest in developing an effective medical data storage system based on DNA. Data integrity, accuracy, reliability, and efficient retrieval are all significant concerns. Therefore, this study proposes an Effective DNA Storage (EDS) approach for archiving medical MRI data. The EDS approach incorporates three key components (i) a novel fraction strategy to address the critical issue of rotating encoding, which often leads to data loss due to single base error propagation; (ii) a novel rule-based quaternary transcoding method that satisfies bio-constraints and ensure reliable mapping; and (iii) an indexing technique designed to simplify random search and access. The effectiveness of this approach is validated through computer simulations and biological experiments, confirming its practicality. The EDS approach outperforms existing methods, providing superior control over bio-constraints and reducing computational time. The results and code provided in this study open new avenues for practical DNA storage of medical MRI data, offering promising prospects for the future of medical data archiving and retrieval.
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Functionalizing materials with biomacromolecules such as enzymes has broad applications in biotechnology and biomedicine. Here, we introduce a grafting method mediated by living cells to functionalize materials. We use polymeric scaffolds to trap engineered bacteria and micron-sized particles with chemical groups serving as active sites for grafting. The bacteria synthesize the desired protein for grafting and autonomously lyse to release it. The released functional moieties are locally grafted onto the active sites, generating the materials engineered by living grafting (MELGs). MELGs are resilient to perturbations because of both the bonding and the regeneration of functional domains synthesized by living cells. The programmability of the bacteria enables us to fabricate MELGs that can respond to external input, decompose a pollutant, reconstitute synthetic pathways for natural product synthesis, and purify mismatched DNA. Our work establishes a bacteria-assisted grafting strategy to functionalize materials with a broad range of biological activities in an integrated, flexible, and modular manner. A record of this paper's transparent peer review process is included in the supplemental information.
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Biotecnologia , Engenharia Genética , Proteínas , Biologia Sintética , Bactérias/genéticaRESUMO
In eukaryotic genomes, rDNA generally resides as a highly repetitive and dynamic structure, making it difficult to study. Here, a synthetic rDNA array on chromosome III in budding yeast was constructed to serve as the sole source of rRNA. Utilizing the loxPsym site within each rDNA repeat and the Cre recombinase, we were able to reduce the copy number to as few as eight copies. Additionally, we constructed strains with two or three rDNA arrays and found that the presence of multiple arrays did not affect the formation of a single nucleolus. Although alteration of the position and number of rDNA arrays did impact the three-dimensional genome structure, the additional rDNA arrays had no deleterious influence on cell growth or transcriptomes. Overall, this study sheds light on the high plasticity of rDNA organization and opens up opportunities for future rDNA engineering.
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Saccharomycetales , Saccharomycetales/genética , Ciclo Celular , Nucléolo Celular , Proliferação de Células , DNA Ribossômico/genéticaRESUMO
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.
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Bryopsida , Epigênese Genética , Cromossomos , Genômica/métodos , Bryopsida/genética , Elementos de DNA TransponíveisRESUMO
The model plant Physcomitrium patens has played a pivotal role in enhancing our comprehension of plant evolution and development. However, the current genome harbours numerous regions that remain unfinished and erroneous. To address these issues, we generated an assembly using Oxford Nanopore reads and Hi-C mapping. The assembly incorporates telomeric and centromeric regions, thereby establishing it as a near telomere-to-telomere genome except a region in chromosome 1 that is not fully assembled due to its highly repetitive nature. This near telomere-to-telomere genome resolves the chromosome number at 26 and provides a gap-free genome assembly as well as updated gene models to aid future studies using this model organism.
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Centrômero , Telômero , Centrômero/genética , Telômero/genética , Genoma de PlantaRESUMO
DNA has emerged as an appealing material for information storage due to its great storage density and durability. Random reading and rewriting are essential tasks for practical large-scale data storage. However, they are currently difficult to implement simultaneously in a single DNA-based storage system, strongly limiting their practicability. Here, a "Cell Disk" storage system is presented, achieving high-density in vivo DNA data storage that enables both random reading and rewriting. In this system, each yeast cell is used as a chamber to store information, similar to a "disk block" but with the ability to self-replicate. Specifically, each genome of yeast cell has a customized CRISPR/Cas9-based "lock-and-key" module inserted, which allows selective retrieval, erasure, or rewriting of the targeted cell "block" from a pool of cells ("disk"). Additionally, a codec algorithm with lossless compression ability is developed to improve the information density of each cell "block". As a proof of concept, target-specific reading and rewriting of the compressed data from a mimic cell "disk" comprising up to 105 "blocks" are demonstrated and achieve high specificity and reliability. The "Cell Disk" system described here concurrently supports random reading and rewriting, and it should have great scalability for practical data storage use.
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Leitura , Saccharomyces cerevisiae , Reprodutibilidade dos Testes , Saccharomyces cerevisiae/genética , DNA/genética , Armazenamento e Recuperação da InformaçãoRESUMO
Synthetic Chromosome Rearrangement and Modification by LoxP-mediated Evolution (SCRaMbLE) is a promising tool to study genomic rearrangements. However, the potential of SCRaMbLE to study genomic rearrangements is currently hindered, because a strain containing all 16 synthetic chromosomes is not yet available. Here, we construct SparLox83R, a yeast strain containing 83 loxPsym sites distributed across all 16 chromosomes. SCRaMbLE of SparLox83R produces versatile genome-wide genomic rearrangements, including inter-chromosomal events. Moreover, when combined with synthetic chromosomes, SCRaMbLE of hetero-diploids with SparLox83R leads to increased diversity of genomic rearrangements and relatively faster evolution of traits compared to hetero-diploids only with wild-type chromosomes. Analysis of the SCRaMbLEd strain with increased tolerance to nocodazole demonstrates that genomic rearrangements can perturb the transcriptome and 3D genome structure and consequently impact phenotypes. In summary, a genome with sparsely distributed loxPsym sites can serve as a powerful tool for studying the consequence of genomic rearrangements and accelerating strain engineering in Saccharomyces cerevisiae.
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Genoma Fúngico , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Rearranjo Gênico/genética , Cromossomos , GenômicaRESUMO
The de novo synthesis of genomes has made unprecedented progress and achieved milestones, particularly in bacteria and yeast. However, the process of synthesizing a multicellular plant genome has not progressed at the same pace, due to the complexity of multicellular plant genomes, technical difficulties associated with large genome size and structure, and the intricacies of gene regulation and expression in plants. Here we outline the bottom-up design principles for the de novo synthesis of the Physcomitrium patens (that is, earthmoss) genome. To facilitate international collaboration and accessibility, we have developed and launched a public online design platform called GenoDesigner. This platform offers an intuitive graphical interface enabling users to efficiently manipulate extensive genome sequences, even up to the gigabase level. This tool is poised to greatly expedite the synthesis of the P. patens genome, offering an essential reference and roadmap for the synthesis of plant genomes.
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Bryopsida , Genoma de Planta , Bryopsida/genética , Biologia Sintética/métodos , SoftwareRESUMO
The Microbiome Protocols eBook (MPB) serves as a crucial bridge, filling gaps in microbiome protocols for both wet experiments and data analysis. The first edition, launched in 2020, featured 152 meticulously curated protocols, garnering widespread acclaim. We now extend a sincere invitation to researchers to participate in the upcoming 2nd version of MPB, contributing their valuable protocols to advance microbiome research.
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Polycyclic aromatic hydrocarbons (PAHs) are a class of persistent pollutants with adverse biological effects and pose a serious threat to ecological environments and human health. The previously isolated phenanthrene-degrading bacterial consortium (PDMC) consists of the genera Sphingobium and Pseudomonas and can degrade a wide range of PAHs. To identify the degradation mechanism of PAHs in the consortium PDMC, metagenomic binning was conducted and a Sphingomonadales assembly genome with 100% completeness was obtained. Additionally, Sphingobium sp. SHPJ-2, an efficient degrader of PAHs, was successfully isolated from the consortium PDMC. Strain SHPJ-2 has powerful degrading abilities and various degradation pathways of high-molecular-weight PAHs, including fluoranthene, pyrene, benzo[a]anthracene, and chrysene. Two ring-hydroxylating dioxygenases, five cytochrome P450s, and a pair of electron transfer chains associated with PAH degradation in strain SHPJ-2, which share 83.0%-99.0% similarity with their corresponding homologous proteins, were identified by a combination of Sphingomonadales assembly genome annotation, reverse-transcription quantitative polymerase chain reaction and heterologous expression. Furthermore, when coexpressed in Escherichia coli BL21(DE3) with the appropriate electron transfer chain, PhnA1B1 could effectively degrade chrysene and benzo[a]anthracene, while PhnA2B2 degrade fluoranthene. Altogether, these results provide a comprehensive assessment of strain SHPJ-2 and contribute to a better understanding of the molecular mechanism responsible for the PAH degradation.