Designing a synthetic moss genome using GenoDesigner.

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.

Apple Glycosyltransferase MdUGT73AR4 Glycosylates ABA to Regulate Stomatal Movement Involved in Drought Stress.

Abscisic acid (ABA) is a drought-stress-responsive hormone that plays an important role in the stomatal activity of plant leaves. Currently, ABA glycosides have been identified in apples, but their glycosyltransferases for glycosylation modification of ABA are still unidentified. In this study, the mRNA expression of glycosyltransferase gene MdUGT73AR4 was significantly up-regulated in mature apple leaves which were treated in drought stress by Real-Time PCR. It was hypothesised that MdUGT73AR4 might play an important role in drought stress. In order to further characterise the glycosylation modification substrate of glycosyltransferase MdUGT73AR4, we demonstrated through in vitro and in vivo functional validation that MdUGT73AR4 can glycosylate ABA. Moreover, the overexpression lines of MdUGT73AR4 significantly enhance its drought stress resistance function. We also found that the adversity stress transcription factor AREB1B might be an upstream transcription factor of MdUGT73AR4 by bioinformatics, EMSA, and ChIP experiments. In conclusion, this study found that the adversity stress transcription factor AREB1B was significantly up-regulated at the onset of drought stress, which in turn positively regulated the downstream glycosyltransferase MdUGT73AR4, causing it to modify ABA by mass glycosylation and promoting the ABA synthesis pathway, resulting in the accumulation of ABA content, and displaying a stress-resistant phenotype.

Ultra-Sensitive Detection of the SARS-CoV-2 Nucleocapsid Protein via a Clustered Regularly Interspaced Short Palindromic Repeat/Cas12a-Mediated Immunoassay.

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.

First Clarification of the Involvement of Glycosyltransferase MdUGT73CG22 in the Detoxification Metabolism of Nicosulfuron in Apple.

Nicosulfuron, an acetolactate synthase (ALS) inhibitor herbicide, is a broad-spectrum and highly effective post-emergence herbicide. Glycosyltransferases (GTs) are widely found in organisms and transfer sugar molecules from donors to acceptors to form glycosides or sugar esters, thereby altering the physicochemical properties of the acceptor molecule, such as participating in detoxification. In this study, nine glycosyltransferases in group D of the apple glycosyltransferase family I were predicted to possibly be involved in the detoxification metabolism of ALS-inhibiting herbicides based on gene chip data published online. In order to confirm this, we analysed whether the expression of the nine glycosyltransferase genes in group D was induced by the previously reported ALS-inhibiting herbicides by real-time PCR (polymerase chain reaction). It was found that the ALS-inhibiting herbicide nicosulfuron significantly increased the expression of the MdUGT73CG22 gene in group D. Further investigation of the mechanism of action revealed that the apple glycosyltransferase MdUGT73CG22 glycosylated and modified nicosulfuron both in vivo and ex vivo to form nicosulfuron glycosides, which were involved in detoxification metabolism. In conclusion, a new glycosyltransferase, MdUGT73CG22, was identified for the first time in this study, which can glycosylate modifications of the ALS-inhibiting herbicide nicosulfuron and may be involved in the detoxification process in plants, which can help to further improve the knowledge of the non-targeted mechanism of herbicides.

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.

High plasticity of ribosomal DNA organization in budding yeast.

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.

Large-scale genomic rearrangements boost SCRaMbLE in Saccharomyces cerevisiae.

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.

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.

Near telomere-to-telomere genome of the model plant Physcomitrium patens.

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.

Programmable Aptasensor for Regulating CRISPR/Cas12a Activity.

CRISPR-mediated aptasensors have gained prevalence for detecting non-nucleic acid targets. However, there is an urgent need to develop an easily customizable design to improve the signal-to-noise ratio, enhance universality, and expand the detection range. In this article, we report a CRISPR-mediated programmable aptasensor (CPAS) platform. The platform includes single-stranded DNA comprising the aptamer sequence, locker DNA, and a crRNA recognition region, forming a hairpin structure through complementary hybridization. With T4 DNA polymerase, the crRNA recognition region was transformed into a complete double-stranded DNA through stem-loop extension, thereby activating the trans-cleavage activity of Cas 12a and generating fluorescence signals. The specific binding between the target molecule and aptamer disrupted the formation of the hairpin structure, altering the fluorescence signals. Notably, the CPAS platform allows for easy customization by simply changing the aptamer sequence and locker DNA, without entailing adjustments to the crRNA. The optimal number of bases in the locker DNA was determined to be seven nucleotides for the SARS-CoV-2 spike (S) protein and four nucleotides for ATP. The CPAS platform exhibited high sensitivity for S protein and ATP detection. Integration with a lateral flow assay enabled sensitive detection within 1 h, revealing its excellent potential for portable analysis.

Precise Synthesis of Organic Cocrystal Alloys with Full-Spectrum Emission Characteristics for the Stepless Color Changing Display.

Organic luminescent materials are indispensable in optoelectronic displays and solid-state luminescence applications. Compared with single-component, multi-component crystalline materials can improve optoelectronic characteristics. This work forms a series of full-spectrum tunable luminescent charge-transfer (CT) cocrystals ranging from 400 to 800 nm through intermolecular collaborative self-assembly. What is even more interesting is that o-TCP-Cor(x)-Pe(1-x), p-TCP-Cor(x)-Pe(1-x), and o-TCP-AN(x)-TP(1-x) alloys are prepared based on cocrystals by doping strategies, which correspondingly achieve the stepless color change from blue (CIE [0.22, 0.44]) to green (CIE [0.16, 0.14]), from green (CIE [0.27, 0.56]) to orange (CIE [0.58, 0.42]), from yellow (CIE [0.40, 0.57]) to red (CIE [0.65, 0.35]). The work provides an efficient method for precisely synthesizing new luminescent organic semiconductor materials and lays a solid foundation for developing advanced organic solid-state displays.

Building a eukaryotic chromosome arm by de novo design and synthesis.

The genome of an organism is inherited from its ancestor and continues to evolve over time, however, the extent to which the current version could be altered remains unknown. To probe the genome plasticity of Saccharomyces cerevisiae, here we replace the native left arm of chromosome XII (chrXIIL) with a linear artificial chromosome harboring small sets of reconstructed genes. We find that as few as 12 genes are sufficient for cell viability, whereas 25 genes are required to recover the partial fitness defects observed in the 12-gene strain. Next, we demonstrate that these genes can be reconstructed individually using synthetic regulatory sequences and recoded open-reading frames with a "one-amino-acid-one-codon" strategy to remain functional. Finally, a synthetic neochromsome with the reconstructed genes is assembled which could substitute chrXIIL for viability. Together, our work not only highlights the high plasticity of yeast genome, but also illustrates the possibility of making functional eukaryotic chromosomes from entirely artificial sequences.

Development of dual-mode ELISA based on ALP-catalyzed APP hydrolysis for IL-6 detection.

Sensitive and accurate detection of interleukin 6 (IL-6) is crucial for the early diagnosis of cerebral infarction to improve patient survival rates. However, the low-abundance of IL-6 in cerebral infarction presents a significant challenge in developing effective diagnosis method. Herein, we studied and analyzed the strong fluorescence property of 4-aminophenol phosphate (APP) and developed an enzyme-linked immunosorbent assay (ELISA) for IL-6 detection. The detection was based on the integration of optical signal change induced by alkaline phosphatase (ALP)-catalyzed APP hydrolysis and ALP-mediated ELISA. The generated colorimetric signal of 4-aminophenol, APP hydrolysis product, was used for ELISA of IL-6 with a detection limit of 0.1 ng/mL, and the visual detection of IL-6 was achieved. The changes in APP fluorescence have a good linear relationship with the logarithm of IL-6 concentration in the range of 0.005 ng/mL to 5.0 ng/mL, with a detection limit of 0.001 ng/mL, which was 100 times lower than that of conventional pNPP-based ELISA. Furthermore, the constructed ELISA effectively distinguished between samples from patients with cerebral infarction and volunteers with non-cerebral infarction, and the severity of symptoms was well distinguished based on IL-6 measurement. The dual-mode ELISA demonstrated high feasibility of low-abundance biomarker detection and displayed good potential for accurate in vitro diagnosis.

Combining hybrid nanoflowers with hybridization chain reaction for highly sensitive detection of SARS-CoV-2 nucleocapsid protein.

BACKGROUND: COVID-19 (coronavirus disease 2019) pandemic has had enormous social and economic impacts so far. The nucleocapsid protein (N protein) is highly conserved and is a key antigenic marker for the diagnosis of early SARS-CoV-2 infection. RESULTS: In this study, the N protein was first captured by an aptamer (Aptamer 58) coupled to magnetic beads (MBs), which in turn were bound to another DNA sequence containing the aptamer (Aptamer 48-Initiator). After adding 5'-biotinylated hairpin DNA Amplifier 1 and Amplifier 2 with cohesive ends for complementary hybridization, the Initiator in the Aptamer 48-Initiator began to trigger the hybridization chain reaction (HCR), generating multiple biotin-labeled DNA concatamers. When incubated with synthetic streptavidin-invertase-Ca3(PO4)2 hybrid nanoflower (SICa), DNA concatamers could specifically bind to SICa through biotin-streptavidin interaction with high affinity. After adding sucrose, invertase in SICa hydrolyzed sucrose to glucose, whose concentration could be directly read with a portable glucometer, and its concentration was positively correlated with the amount of captured N protein. The method is highly sensitive with a detection limit as low as 1 pg/mL. SIGNIFICANCE: We believe this study provided a practical solution for the early detection of SARS-CoV-2 infection, and offered a new method for detecting other viruses through different target proteins.

Symmetry or asymmetry: which one is the platform of nitrogen vacancies for alkaline hydrogen evolution.

Conventional nitrogen vacancies with a symmetric coordination of metal cations (i.e., M1-Nv-M1) play a crucial role in tuning the local environment of the metal sites in metal nitrides and improving their electrochemical activity in the hydrogen evolution reaction (HER). However, the symmetric Nv sites, which feature a uniform charge distribution on adjacent metal sites, suffer from sluggish water dissociation kinetics and a poor capability for hydrogen desorption. Here, we fabricated Cr-doped and Nv-rich Co4N nanorods grown on a Ni foam (Cr-Co4N-Nv/NF) with asymmetric Cr-Nv-Co sites to effectively catalyze hydrogen evolution under alkaline conditions, with a low overpotential of 33 mV at a current density of 10 mA cm-2 and a small Tafel slope of 37 mV dec-1. The experimental characterizations and theoretical simulations collectively reveal that the construction of asymmetric Cr-Nv-Co sites gives rise to the upshift of the d-band center, thus promoting water adsorption and activation. Moreover, asymmetric Nv sites allow a balance between hydrogen adsorption and desorption, which avoids the limited desorption process over the symmetric Co-Nv-Co sites.

Synergistic effect-triggered fluorescence quenching enables rapid and sensitive detection of alkaline phosphatase.

The development of biosensors mediated by synergistic quenching effect is of great significance for rapid and accurate clinical diagnosis. Hence, we prepared a cyan-emitting fluorescent Si dots for alkaline phosphatase (ALP) detection through the synergistic quenching effect of inner filter effect (IFE) and photo-induced electron transfer (PET). Si dots were prepared by microwave-assisted method, which displayed high quantum yield (28.7%), as well as good physiochemical properties, such as photo-stability, pH stability, and chemical stability. As the hydrolysate of 4-nitrophenyl phosphate disodium salt hexahydrate catalyzed by ALP, both IFE and PET of 4-nitrophenyl to Si dots were used for the turn-off mode detection of ALP. The linear relationships were established between the change of fluorescence intensity and ALP concentration in the range of 0.05 U L-1 to 5.0 U L-1, and 5.0 U L-1 to 80.0 U L-1, respectively. The detection limit was 0.01 U L-1. The synergistic quenching effect caused the turn-off mode detection to be more sensitive, and it can also be used for the accurate detection of ALP in human serum, thereby showing great anti-interference ability in complex environments.

Fluorescence and Colorimetric Analysis of African Swine Fever Virus Based on the RPA-Assisted CRISPR/Cas12a Strategy.

It is well-established that different detection modes are necessary for corresponding applications, which can effectively reduce matrix interference and improve the detection accuracy. Here, we reported a magnetic separation method based on recombinase polymerase amplification (RPA)-assisted clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a for dual-mode analysis of African swine fever virus (ASFV) genes, including colorimetry and fluorescence. The ASFV gene was selected as the initial RPA template to generate the amplicon. The RPA amplicon was then recognized by CRISPR-associated RNA (crRNA), activating the trans-cleavage activity of Cas12a and leading to the nonspecific cleavage of ssDNA as well as a significant release of alkaline phosphatase (ALP) in the ALP-ssDNA modified magnetic bead. The released ALP can catalyze para-nitrophenyl phosphate to generate para-nitrophenol, resulting in substantial changes in absorbance and fluorescence, both of which can be used for detection with the naked eye. This strategy allows the sensitive detection of ASFV DNA, with a 20 copies/mL detection limit; no cross-reactivity with other viruses was observed. A good linear relationship was obtained in serum. In addition, this sensor displayed 100% specificity and sensitivity for clinical sample analysis. This method integrates the high sensitivity of fluorescence with easy readout of colorimetry and enables a simple, low-cost, and highly sensitive dual-mode detection of viral nucleic acid, thereby providing a broad prospect for the practical application in the diagnosis of virus infection.

Electrochemical Production of Glycolate Fuelled By Polyethylene Terephthalate Plastics with Improved Techno-Economics.

Electrochemical valorization of polyethylene terephthalate (PET) waste streams into commodity chemicals offers a potentially sustainable route for creating a circular plastic economy. However, PET wastes upcycling into valuable C2 product remains a huge challenge by the lack of an electrocatalyst that can steer the oxidation economically and selectively. Here, it is reported a catalyst comprising Pt nanoparticles hybridized with γ-NiOOH nanosheets supported on Ni foam (Pt/γ-NiOOH/NF) that favors electrochemical transformation of real-word PET hydrolysate into glycolate with high Faradaic efficiency (> 90%) and selectivity (> 90%) across wide reactant (ethylene glycol, EG) concentration ranges under a marginal applied voltage of 0.55 V, which can be paired with cathodic hydrogen production. Computational studies combined with experimental characterizations elucidate that the Pt/γ-NiOOH interface with substantial charge accumulation gives rise to an optimized adsorption energy of EG and a decreased energy barrier of potential determining step. A techno-economic analysis demonstrates that, with the nearly same amount of resource investment, the electroreforming strategy towards glycolate production can raise revenue by up to 2.2 times relative to conventional chemical process. This work may thus serve as a framework for PET wastes valorization process with net-zero carbon footprint and high economic viability.

Oriented Self-Assembly of Hierarchical Branch Organic Crystals for Asymmetric Photonics.

Organic hierarchical branch micro/nanostructures constituted by single crystals with inherent multichannel characteristics exhibit superior potential in regulating photon transmission for photonic circuits. However, organic branch micro/nanostructures with precise branch positions are extremely difficult to achieve due to the randomness of the nucleation process. Herein, by taking advantage of the dislocation stress field-impurity interaction that solute molecules deposit preferentially along the dislocation line, twinning deformation was introduced into microcrystals to induce oriented nucleation sites, and ultimately organic branch microstructures with controllable branch sites were fabricated. The growth mechanism of these controllable single crystals with an angle of 140° between trunk and branch is attributed to the low lattice mismatching ratio (η) of 4.8%. These as-prepared hierarchical branch single crystals with asymmetrical optical waveguide characteristics have been demonstrated as an optical logic gate with multiple input/out channels, which provides a route to command the nucleation sites and offers potential applications in the organic optoelectronics at the micro/nanoscale.