Adeno-associated viruses (AAVs) are gaining traction as delivery vehicles for gene therapy although the molecular understanding of AAV-transgene release is still limited. Typically, the process of viral uncoating is investigated (in vitro) through thermal stress, revealing capsid disintegration at elevated temperatures. To assess the (in)stability of different empty and filled AAV preparations, we used the light-scattering-based interferometric microscopy technique of mass photometry that, on a single-particle basis, determines the molecular weight of AAVs. By introducing a heat-stable DNA plasmid as an internal standard, we quantitatively probed the impact of heat on AAVs. Generally, empty AAVs exhibited greater heat resistance than genome-filled particles. Our data also indicate that upon DNA release, the capsids do not transform into empty AAVs, but seem to aggregate or disintegrate. Strikingly, some AAVs exhibited an intermediate state with disrupted capsids but preserved bound genome, a feature that experimentally only emerged following incubation with a nuclease. Our data demonstrate that the thermal uncoating process is highly AAV specific (i.e., can be influenced by serotype, genome, host system). We argue that nuclease treatment in combination with MP can be used as an additional analytical tool for assessing structural integrity of recombinant and/or clinical AAV vectors.

The WRKY gene family is ubiquitously distributed in plants, serving crucial functions in stress responses. Nevertheless, the structural organization and evolutionary dynamics of WRKY genes in cotton have not been fully elucidated. In this study, a total of 112, 119, 217, and 222 WRKY genes were identified in Gossypium arboreum, Gossypium raimondii, Gossypium hirsutum, and Gossypium barbadense, respectively. These 670 WRKY genes were categorized into seven distinct subgroups and unequally distributed across chromosomes. Examination of conserved motifs, domains, cis-acting elements, and gene architecture collectively highlighted the evolutionary conservation and divergence within the WRKY gene family in cotton. Analysis of synteny and collinearity further confirmed instances of expansion, duplication, and loss events among WRKY genes during cotton evolution. Furthermore, GhWRKY31 transgenic Arabidopsis exhibited heightened germination rates and longer root lengths under drought and salt stress. Silencing GhWRKY31 in cotton led to reduced levels of ABA, proline, POD, and SOD, along with downregulated expression of stress-responsive genes. Yeast one-hybrid and molecular docking assays confirmed the binding capacity of GhWRKY31 to the W box of GhABF1, GhDREB2, and GhRD29. The findings collectively offer a systematic and comprehensive insight into the evolutionary patterns of cotton WRKYs, proposing a suitable regulatory framework for developing cotton cultivars with enhanced resilience to drought and salinity stress.

R2 non-long terminal repeat retrotransposons insert site-specifically into ribosomal RNA genes (rDNA) in a broad range of multicellular eukaryotes. R2-encoded proteins can be leveraged to mediate transgene insertion at 28S rDNA loci in cultured human cells. This strategy, precise RNA-mediated insertion of transgenes (PRINT), relies on the codelivery of an mRNA encoding R2 protein and an RNA template encoding a transgene cassette of choice. Here, we demonstrate that the PRINT RNA template 5\' module, which as a complementary DNA 3\' end will generate the transgene 5\' junction with rDNA, influences the efficiency and mechanism of gene insertion. Iterative design and testing identified optimal 5\' modules consisting of a hepatitis delta virus-like ribozyme fold with high thermodynamic stability, suggesting that RNA template degradation from its 5\' end may limit transgene insertion efficiency. We also demonstrate that transgene 5\' junction formation can be either precise, formed by annealing the 3\' end of first-strand complementary DNA with the upstream target site, or imprecise, by end-joining, but this difference in junction formation mechanism is not a major determinant of insertion efficiency. Sequence characterization of imprecise end-joining events indicates surprisingly minimal reliance on microhomology. Our findings expand the current understanding of the role of R2 retrotransposon transcript sequence and structure, and especially the 5\' ribozyme fold, for retrotransposon mobility and RNA-templated gene synthesis in cells.

The complex morphology of neurons requires precise control of their microtubule cytoskeleton. This is achieved by microtubule-associated proteins (MAPs) that regulate the assembly and stability of microtubules, and transport of molecules and vesicles along them. While many of these MAPs function in all cells, some are specifically or predominantly involved in regulating microtubules in neurons. Here we use the sea anemone Nematostella vectensis as a model organism to provide new insights into the early evolution of neural microtubule regulation. As a cnidarian, Nematostella belongs to an outgroup to all bilaterians and thus occupies an informative phylogenetic position for reconstructing the evolution of nervous system development. We identified an ortholog of the microtubule-binding protein doublecortin-like kinase (NvDclk1) as a gene that is predominantly expressed in neurons and cnidocytes (stinging cells), two classes of cells belonging to the neural lineage in cnidarians. A transgenic NvDclk1 reporter line revealed an elaborate network of neurite-like processes emerging from cnidocytes in the tentacles and the body column. A transgene expressing NvDclk1 under the control of the NvDclk1 promoter suggests that NvDclk1 localizes to microtubules and therefore likely functions as a microtubule-binding protein. Further, we generated a mutant for NvDclk1 using CRISPR/Cas9 and show that the mutants fail to generate mature cnidocytes. Our results support the hypothesis that the elaboration of programs for microtubule regulation occurred early in the evolution of nervous systems.

Legumin A is a seed storage protein that provides nutrients for seed germination. The purpose of this study was to describe the structure and expression pattern of the EuLEGA gene in Eucommia ulmoides Oliver (E. ulmoides) and to infer its functional role. The 1287 bp coding sequence of the EuLEGA CDS of the EuLEGA gene, encoding a protein containing 428 amino acid residues, was cloned. The structure predicted that the protein belonged to the RmlC (deoxythymidine diphosphates, dTDP)-4-dehydrorhamnose 3,5-epimerase)-like cupin conserved domain family, which contains both RmlC, a key enzyme for the synthesis of rhamnose and legumin A. The overexpression (OE) vector of the EuLEGA gene was constructed and genetically transformed into tobacco and E. ulmoides; the RNA interference (RNAi) vector of the EuLEGA gene was constructed and genetically transformed into E. ulmoides; and the contents of legumin A and rhamnose were detected. The results showed that the EuLEGA gene could significantly increase the content of legumin A in transgenic tobacco leaves and transgenic E. ulmoides regenerative buds, and the OE of this gene in E. ulmoides could promote an increase in rhamnose content. RNAi caused a significant decrease in the legumin A content in the regenerated buds of E. ulmoides. These was a significant increase in legumin A in the transgenic tobacco seeds, and these results indicate that the expression of the EuLEGA gene is closely related to the accumulation of legumin A. Subcellular localization studies revealed that EuLEGA is localized to the cytoplasm with the vacuolar membrane. Analysis of the EuLEGA gene expression data revealed that the expression level of the EuLEGA gene in the samaras was significantly greater than that in the leaves and stems. In addition, the study also demonstrated that GA3 can upregulate the expression levels of the EuLEGA gene, while ABA and MeJA can downregulate its expression levels.

Plants respond to biotic and abiotic stress by activating and interacting with multiple defense pathways, allowing for an efficient global defense response. RNA silencing is a conserved mechanism of regulation of gene expression directed by small RNAs important in acquired plant immunity and especially virus and transgene repression. Several RNA silencing pathways in plants are crucial to control developmental processes and provide protection against abiotic and biotic stresses as well as invasive nucleic acids such as viruses and transposable elements. Various notable studies have shed light on the genes, small RNAs, and mechanisms involved in plant RNA silencing. However, published research on the potential interactions between RNA silencing and other plant stress responses is limited. In the present study, we tested the hypothesis that spreading and maintenance of systemic post-transcriptional gene silencing (PTGS) of a GFP transgene are associated with transcriptional changes that pertain to non-RNA silencing-based stress responses. To this end, we analyzed the structure and function of the photosynthetic apparatus and conducted whole transcriptome analysis in a transgenic line of Nicotiana benthamiana that spontaneously initiates transgene silencing, at different stages of systemic GFP-PTGS. In vivo analysis of chlorophyll a fluorescence yield and expression levels of key photosynthetic genes indicates that photosynthetic activity remains unaffected by systemic GFP-PTGS. However, transcriptomic analysis reveals that spreading and maintenance of GFP-PTGS are associated with transcriptional reprogramming of genes that are involved in abiotic stress responses and pattern- or effector-triggered immunity-based stress responses. These findings suggest that systemic PTGS may affect non-RNA-silencing-based defense pathways in N. benthamiana, providing new insights into the complex interplay between different plant stress responses.

Mouse adenoviruses (MAdV) play important roles in studying host-adenovirus interaction. However, easy-to-use reverse genetics systems are still lacking for MAdV. An infectious plasmid pKRMAV1 was constructed by ligating genomic DNA of wild-type MAdV-1 with a PCR product containing a plasmid backbone through Gibson assembly. A fragment was excised from pKRMAV1 by restriction digestion and used to generate intermediate plasmid pKMAV1-ER, which contained E3, fiber, E4, and E1 regions of MAdV-1. CMV promoter-controlled GFP expression cassette was inserted downstream of the pIX gene in pKMAV1-ER and then transferred to pKRMAV1 to generate adenoviral plasmid pKMAV1-IXCG. Replacement of transgene could be conveniently carried out between dual BstZ17I sites in pKMAV1-IXCG by restriction-assembly, and a series of adenoviral plasmids were generated. Recombinant viruses were rescued after transfecting linearized adenoviral plasmids to mouse NIH/3T3 cells. MAdV-1 viruses carrying GFP or firefly luciferase genes were characterized in gene transduction, plaque-forming, and replication in vitro or in vivo by observing the expression of reporter genes. The results indicated that replication-competent vectors presented relevant properties of wild-type MAdV-1 very well. By constructing viruses bearing exogenous fragments with increasing size, it was found that MAdV-1 could tolerate an insertion up to 3.3 kb. Collectively, a replication-competent MAdV-1 vector system was established, which simplified procedures for the change of transgene or modification of E1, fiber, E3, or E4 genes.

Most viruses and transposons serve as effective carriers for the introduction of foreign DNA up to 11 kb into vertebrate genomes. However, their activity markedly diminishes with payloads exceeding 11 kb. Expanding the payload capacity of transposons could facilitate more sophisticated cargo designs, improving the regulation of expression and minimizing mutagenic risks associated with molecular therapeutics, metabolic engineering, and transgenic animal production. In this study, we improved the Tol2 transposon by increasing protein expression levels using a translational enhancer ( QBI SP163, ST) and enhanced the nuclear targeting ability using the nuclear localization protein H2B (SHT). The modified Tol2 and ST transposon efficiently integrated large DNA cargos into human cell cultures (H1299), comparable to the well-established super PiggyBac system. Furthermore, mRNA from ST and SHT showed a significant increase in transgene delivery efficiency of large DNA payloads (8 kb, 14 kb, and 24 kb) into zebrafish ( Danio rerio). This study presents a modified Tol2 transposon as an enhanced nonviral vector for the delivery of large DNA payloads in transgenic applications.
大多数病毒和转座子可将长达11 kb的外源DNA有效引入脊椎动物基因组，但递送超过11 kb的DNA片段时，其活性明显减弱。扩大转座子的有效载荷能力将能够容纳更大的DNA片段、增强调节基因表达的能力，并最大限度地减少分子治疗、代谢工程和转基因动物相关的突变风险。在该研究中，我们通过翻译增强子QBI SP163和核定位蛋白H2B提高蛋白表达水平和细胞核靶向能力，产生了改善的Tol2转座子，ST和SHT。Tol2和ST转座子能有效地将大片段DNA整合到H1299细胞中，其效果可与改善的super PiggyBac转座子系统相媲美。我们发现ST和SHT mRNA能显著提高斑马鱼中大DNA片段（~8 kb， ~14 kb， ~24 kb）的转基因效率。该研究介绍了优化的Tol2转座子可作为一种改进的非病毒载体，应用于递送大DNA片段的转基因实验。.

The development of compact CRISPR systems has facilitated delivery but has concurrently reduced gene editing efficiency, thereby limiting the further utilization of CRISPR systems. Enhancing the efficiency of CRISPR systems poses a challenging task and holds significant implications for the advancement of biotechnology. In our work, we report a synthetic dual-antibody system that can stably exist in the intracellular environment, specifically inhibiting the functions of NF-κB and β-catenin. This not only elevates the transgenic expression of the CRISPR system by suppressing the innate immune response within cells to enhance the gene editing efficiency but also demonstrates a notable tumor inhibitory effect. Based on the specific output expression regulation of CRISPR-CasΦ, we constructed a CRISPR-based gene expression platform, which includes sensor modules for detecting intracellular β-catenin and NF-κB, as well as an SDA module to enhance overall efficiency. In vitro experiments revealed that the CRISPR-based gene expression platform exhibited superior CDK5 expression inhibition efficiency and specific cytotoxicity towards tumor cells. In vitro experiments, we found that CRISPR-based gene expression platforms can selectively kill bladder cancer cells through T cell-mediated cytotoxicity. Our design holds significant assistant potential of transgene therapy and may offer the capability to treat other diseases requiring transgene therapy.

The dosing interval for effective recombinant adeno-associated virus (rAAV)-mediated gene therapy of cystic fibrosis lung disease remains unknown. Here, we assessed the durability of rAAV2.5T-fCFTRΔR-mediated transgene expression and neutralizing antibody (NAb) responses in lungs of adult wild-type ferrets. Within the first 3 months following rAAV2.5T-fCFTRΔR delivery to the lung, CFTRΔR transgene expression declined ∼5.6-fold and then remained stable to 5 months at ∼26% the level of endogenous CFTR. rAAV NAbs in the plasma and bronchoalveolar lavage fluid (BALF) peaked at 21 days, coinciding with peak ELISpot T cell responses to AAV capsid peptides, after which both responses declined and remained stable at 4-5 months post dosing. Administration of reporter vector rAAV2.5T-gLuc (gaussia luciferase) at 5 months following rAAV2.5T-fCFTRΔR dosing gave rise to similar levels of gLuc expression in the BALF as observed in age-matched reporter-only controls, demonstrating that residual BALF NAbs were functionally insignificant. Notably, the second vector administration led to a 2.6-fold greater ELISpot T cell response and ∼2.3-fold decline in fCFTRΔR mRNA and vector genomes derived from the initial rAAV2.5T-fCFTRΔR administration, suggesting selective destruction of transduced cells from the first vector dose. These findings provide insights into humoral and cellular immune response to rAAV that may be useful for optimizing gene therapy to the cystic fibrosis lung.