Transgene Silencing Research Articles

CRISPR associated enzymes are mislocalized to the cytoplasm in iPSC-derived neurons resulting in KRAB-specific degradation.

The use of CRISPR-associated enzymes in iPSC-derived neurons for precise gene targeting and high-throughput gene perturbation screens offers great potential but presents unique challenges compared to dividing cell lines. CRISPRi screens in iPSC-derived neurons and glia have already been successful in relating gene function to neurological phenotypes; however, loss of dCas9-KRAB expression after differentiation has been observed by many labs and has been largely ascribed to transgene silencing after differentiation. Here, we investigated the expression levels of different CRISPR enzymes in iPSC and Ngn2-derived neurons using piggybac delivery. We found that the commonly used dCas9-KRAB (using the KOX1 domain) displayed dramatic reduction in protein expression levels following neuronal differentiation, yet surprisingly, nCas9 constructs retained comparable protein expression between iPSCs and neurons. We further found that CRISPR constructs, primarily relying on the SV40 Nuclear Localization Signal (NLS), fail to efficiently localize to the nuclei of neurons, despite having robust nuclear levels in iPSCs, leading to KRAB-specific cytoplasmic degradation. By adding a neuronal-specific NLS, we were able to correct neuronal nuclear localization and protein expression, confirming the contribution of mislocalization to the instability of dCas9-KRAB in neurons. As the lack of nuclear localization can have a profound impact on editing and gene perturbation efficiency, we suggest further investigation across both cultured and in-vivo postmitotic cell models.

Development of an RNA virus-based episomal vector with artificial aptazyme for gene silencing

RNA virus-based episomal vector (REVec), engineered from Borna disease virus, is an innovative gene delivery tool that enables sustained gene expression in transduced cells. However, the difficulty in controlling gene expression and eliminating vectors has limited the practical use of REVec. In this study, we overcome these shortcomings by inserting artificial aptazymes into the untranslated regions of foreign genes carried in vectors or downstream of the viral phosphoprotein gene, which is essential for vector replication. Non-transmissive REVec carrying GuaM8HDV or the P1-F5 aptazyme showed immediate suppression of gene expression in a guanine or theophylline concentration-dependent manner. Continuous compound administration also markedly reduced the percentage of vector-transduced cells and eventually led to the complete elimination of the vectors from the transduced cells. This new REVec is a safe gene delivery technology that allows fine-tuning of gene expression and could be a useful platform for gene therapy and gene-cell therapy, potentially contributing to the cure of many genetic disorders.Key points• We developed a bornavirus vector capable of silencing transgene expression by insertion of aptazyme• Transgene expression was markedly suppressed in a compound concentration-dependent manner• Artificial aptazyme systems allowed complete elimination of the vector from transduced cells

STRAIGHT-IN Dual: a platform for dual, single-copy integrations of DNA payloads and gene circuits into human induced pluripotent stem cells.

Targeting DNA payloads into human (h)iPSCs involves multiple time-consuming, inefficient steps that must be repeated for each construct. Here, we present STRAIGHT-IN Dual, which enables simultaneous, allele-specific, single-copy integration of two DNA payloads with 100% efficiency within one week. Notably, STRAIGHT-IN Dual leverages the STRAIGHT-IN platform to allow near-scarless cargo integration, facilitating the recycling of components for subsequent cellular modifications. Using STRAIGHT-IN Dual, we investigated how promoter choice and gene syntax influences transgene silencing, and demonstrate the impact of these design features on forward programming of hiPSCs into neurons. Furthermore, we designed a grazoprevir-inducible synZiFTR system to complement the widely-used tetracycline-inducible system, providing independent, tunable, and temporally controlled expression of both transcription factors and functional reporters. The unprecedented efficiency and speed with which STRAIGHT-IN Dual generates homogenous genetically engineered hiPSC populations represents a major advancement for synthetic biology in stem cell applications and opens opportunities for precision cell engineering.

Resistance gene Ty-1 restricts TYLCV infection in tomato by increasing RNA silencing

A major antiviral mechanism in plants is mediated by RNA silencing through the action of DICER-like (DCL) proteins, which cleave dsRNA into discrete small RNA fragments, and ARGONAUTE (AGO) proteins, which use the small RNAs to target single-stranded RNA. RNA silencing can also be amplified through the action of RNA-dependent RNA polymerases (RDRs), which use single stranded RNA to generate dsRNA that in turn is targeted by DCL proteins. As a counter-defense, plant viruses encode viral suppressors of RNA silencing (VSRs) that target different components in the RNA silencing pathway. The tomato Ty-1 gene confers resistance to the DNA virus tomato yellow leaf curl virus (TYLCV) and has been reported to encode an RDRγ protein. However, the molecular mechanisms by which Ty-1 controls TYLCV infection, including whether Ty-1 is involved in RNA silencing, are unknown. Here, by using a transient expression assay, we have confirmed that Ty-1 shows antiviral activity against TYLCV in Nicotiana benthamiana. Also, in transient expression-based silencing assays, Ty-1 augmented systemic transgene silencing in GFP transgenic N. benthamiana plants. Furthermore, co-expression of Ty-1 or other RDRγ proteins from N. benthamiana or Arabidopsis with various proteins resulted in lower protein expression. These results are consistent with a model wherein Ty-1-mediated resistance to TYLCV is due, at least in part, to an increase in RNA silencing activity.

Use of tRNA gene barriers improves stability of transgene expression in CHO cells.

Instability of transgene expression is a major challenge for the biopharmaceutical industry, which can impact yields and regulatory approval. Some tRNA genes (tDNAs) can resist epigenetic silencing, the principal mechanism of expression instability, and protect adjacent genes against the spread of repressive heterochromatin. We have taken two naturally occurring clusters of human tDNAs and tested their ability to reduce epigenetic silencing of transgenes integrated into the genome of Chinese hamster ovary (CHO) cells. We find sustained improvements in productivity both in adherent CHO-K1 cells and in an industrially relevant CHO-DG44 expression system (Apollo X, FUJIFILM Diosynth Biotechnologies). We conclude that specific tDNA clusters offer potential to mitigate the widespread problem of production instability.

Stable long-term germline transmission of GFP transgenic rat via PiggyBac transposon mediated gene transfer

Transgene silencing provides a significant challenge in animal model production via gene engineering using viral vectors or transposons. Selecting an appropriate strategy, contingent upon the species is crucial to circumvent transgene silencing, necessitating long-term observation of in vivo gene expression. This study employed the PiggyBac transposon to create a GFP rat model to address transgene silencing in rats. Surprisingly, transgene silencing occurred while using the CAG promoter, contrary to conventional understanding, whereas the Ef1α promoter prevented silencing. GFP expression remained stable through over five generations, confirming efficacy of the Ef1α promoter for long-term protein expression in rats. Additionally, GFP expression was consistently maintained at the cellular level in various cellular sources produced from the GFP rats, thereby validating the in vitro GFP expression of GFP rats. Whole-genome sequencing identified a stable integration site in Akap1 between exons 1 and 2, mitigating sequence-independent mechanism-mediated transgene silencing. This study established an efficient method for producing transgenic rat models using PiggyBac transposon. Our GFP rats represent the first model to exhibit prolonged expression of foreign genes over five generations, with implications for future research in gene-engineered rat models.

Stress-related transcriptomic changes associated with GFP transgene expression and active transgene silencing in plants

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.

Study on chromatin regulation patterns of expression vectors in the PhiC31 integration site

ABSTRACT The PhiC31 integration system allows for targeted and efficient transgene integration and expression by recognizing pseudo attP sites in mammalian cells and integrating the exogenous genes into the open chromatin regions of active chromatin. In order to investigate the regulatory patterns of efficient gene expression in the open chromatin region of PhiC31 integration, this study utilized Ubiquitous Chromatin Opening Element (UCOE) and activating RNA (saRNA) to modulate the chromatin structure in the promoter region of the PhiC31 integration vector. The study analysed the effects of DNA methylation and nucleosome occupancy changes in the integrated promoter on gene expression levels. The results showed that for the OCT4 promoter with moderate CG density, DNA methylation had a smaller impact on expression compared to changes in nucleosome positioning near the transcription start site, which was crucial for enhancing downstream gene expression. On the other hand, for the SOX2 promoter with high CG density, increased methylation in the CpG island upstream of the transcription start site played a key role in affecting high expression, but the positioning and clustering of nucleosomes also had an important influence. In conclusion, analysing the DNA methylation patterns, nucleosome positioning, and quantity distribution of different promoters can determine whether the PhiC31 integration site possesses the potential to further enhance expression or overcome transgene silencing effects by utilizing chromatin regulatory elements.

Tissue-specific silencing of integrated transgenes achieved through endogenous RNA interference in Caenorhabditis elegans

ABSTRACT Transgene silencing is a common phenomenon observed in Caenorhabditis elegans, particularly in the germline, but the precise mechanisms underlying this process remain elusive. Through an analysis of the transcription factors profile of C. elegans, we discovered that the expression of several transgenic reporter lines exhibited tissue-specific silencing, specifically in the intestine of C. elegans. Notably, this silencing could be reversed in mutants defective in endogenous RNA interference (RNAi). Further investigation using knock-in strains revealed that these intestine-silent genes were indeed expressed in vivo, indicating that the organism itself regulates the intestine-specific silencing. This tissue-specific silencing appears to be mediated through the endo-RNAi pathway, with the main factors of this pathway, mut-2 and mut-16, are significantly enriched in the intestine. Additionally, histone modification factors, such as met-2, are involved in this silencing mechanism. Given the crucial role of the intestine in reproduction alongside the germline, the transgene silencing observed in the intestine reflects the self-protective mechanisms employed by the organisms. In summary, our study proposed that compared to other tissues, the transgenic silencing of intestine is specifically regulated by the endo-RNAi pathway.

An RRM domain protein SOE suppresses transgene silencing in rice.

Gene expression is regulated at multiple levels, including RNA processing and DNA methylation/demethylation. How these regulations are controlled remains unclear. Here, through analysis of a suppressor for the OsEIN2 over-expressor, we identified an RNA recognition motif protein SUPPRESSOR OF EIN2 (SOE). SOE is localized in nuclear speckles and interacts with several components of the spliceosome. We find SOE associates with hundreds of targets and directly binds to a DNA glycosylase gene DNG701 pre-mRNA for efficient splicing and stabilization, allowing for subsequent DNG701-mediated DNA demethylation of the transgene promoter for proper gene expression. The V81M substitution in the suppressor mutant protein mSOE impaired its protein stability and binding activity to DNG701 pre-mRNA, leading to transgene silencing. SOE mutation enhances grain size and yield. Haplotype analysis in c. 3000 rice accessions reveals that the haplotype 1 (Hap 1) promoter is associated with high 1000-grain weight, and most of the japonica accessions, but not indica ones, have the Hap 1 elite allele. Our study discovers a novel mechanism for the regulation of gene expression and provides an elite allele for the promotion of yield potentials in rice.

Calcineurin contributes to RNAi-mediated transgene silencing and small interfering RNA production in the human fungal pathogen Cryptococcus neoformans.

Adaptation to external environmental challenges at the cellular level requires rapid responses and involves relay of information to the nucleus to drive key gene expression changes through downstream transcription factors. Here, we describe an alternative route of adaptation through a direct role for cellular signaling components in governing gene expression via RNA interference-mediated small RNA production. Calcium-calcineurin signaling is a highly conserved signaling cascade that plays central roles in stress adaptation and virulence of eukaryotic pathogens, including the human fungal pathogen Cryptococcus neoformans. Upon activation in C. neoformans, calcineurin localizes to P-bodies, membraneless organelles that are also the site for RNA processing. Here, we studied the role of calcineurin and its substrates in RNAi-mediated transgene silencing. Our results reveal that calcineurin regulates both the onset and the reversion of transgene silencing. We found that some calcineurin substrates that localize to P-bodies also regulate transgene silencing but in opposing directions. Small RNA sequencing in mutants lacking calcineurin or its targets revealed a role for calcineurin in small RNA production. Interestingly, the impact of calcineurin and its substrates was found to be different in genome-wide analysis, suggesting that calcineurin may regulate small RNA production in C. neoformans through additional pathways. Overall, these findings define a mechanism by which signaling machinery induced by external stimuli can directly alter gene expression to accelerate adaptative responses and contribute to genome defense.

Silencing of a NRDE-3 transgene in C. elegans germ cells and early embryos is mediated by the RNAi pathway.

Transgenes are highly susceptible to gene silencing in the C. elegans germline. Here, we examine the expression of the nuclear Argonaute protein NRDE-3 , comparing two GFP::NRDE-3 strains, one constructed by bombardment and one by CRISPR. We found that the GFP::NRDE-3 strain constructed by bombardment displays transgene silencing in germline and early embryos and that NRDE-3 expression can be restored in a rde-3 mutant, which disrupts the RNAi pathway. This finding reveals that NRDE-3 is not a soma-specific Argonaute protein and is, in fact, expressed in the proximal germline and early embryos.

The C. elegans SET1 histone methyltransferase SET-2 is not required for transgenerational memory of silencing.

The SET-2 /SET1 histone H3K4 methyltransferase and RNAi pathway components are required to maintain fertility across generations in C. elegans . SET-2 preserves the germline transcriptional program transgenerationally, and RNAi pathways rely on small RNAs to establish and maintain transgenerational gene silencing. We investigated whether the functionality of RNAi-induced transgenerational silencing and the composition of pools of endogenous small RNA are affected by the absence of SET-2 . Our results suggest that defects in RNAi pathways are not responsible for the transcriptional misregulation observed in the absence of SET-2 .

10000-SPE-1 STEM CELL-BASED GENE THERAPY FOR MALIGNANT GLIOMA USING GENOME-EDITED HUMAN INDUCED PLURIPOTENT STEM CELLS

Abstract Glioblastoma is the most aggressive primary brain tumor, and is characterized by diffuse infiltration into the normal brain parenchyma. New therapeutic approaches targeting invasive biological behaviour are warranted. In the present study, we show that neural stem cells (NSCs) derived from CRISRP/Cas9-edited induced pluripotent stem cells (iPSCs) have high tumor-trophic migratory capacity and stable constitutive therapeutic transgene expression, which leads to strong anti-tumor effects against glioma stem cell (GSC) models. The present study provides answers to some important research questions associated with stem cell-based gene therapy. First, the tumor-trophic migratory capacities of human iPSC-derived NSCs (iPSC-NSCs), fetal NSCs, and mesenchymal stem cells (MSCs) were quantitatively evaluated by spatiotemporal methodologies. We demonstrated that iPSC-NSCs have a higher tumor-trophic migratory capacity than MSCs in the brain. Self-repulsive action and pathotropism were important for the migration of iPSC-NSCs: ephrin ligand/receptor mediated repulsion of iPSC-NSCs and CXCL12-CXCR4 interactions between GSCs and iPSC-NSCs. Second, a prodrug converting enzyme fusion gene was selected as a therapeutic gene in human iPSCs. In general, stable constitutive transgene expression by viral vectors was difficult in human iPSCs. Furthermore, viral vectors integrate randomly into the host genome, which raises concerns about transgene silencing, insertional mutagenesis, and oncogene activation. In the present study, several common insertion sites including GAPDH, ACTB, and AAVS1, were compared. The most appropriate gene locus that achieved stable constitutive transgene expression was determined via CRISPR/Cas9-mediated genome editing. Third, we revealed the novel mechanism of action using iPSC-NSCs expressing CD-UPRT, in which ferroptosis was associated with enhanced anti-tumor immune responses. We demonstrated that the established iPSC-NSCs had strong therapeutic efficacy in GSC animal models. Finally, predictive biomarkers for the efficacy of the present treatment strategy were established. We will conduct a clinical trial of this treatment strategy. This research concept can disseminate biological, medical and engineering advances.

Successful reversal of transgene silencing in Chlamydomonas reinhardtii.

Chlamydomonas reinhardtii has been successfully engineered to produce compounds of interest following transgene integration and heterologous protein expression. The advantages of this model include the availability of validated tools for bioengineering, its photosynthetic ability, and its potential use as biofuel. Despite this, breakthroughs have been hindered by its ability to silence transgene expression through epigenetic changes. Histone deacetylases (HDAC) are main players in gene expression. We hypothesized that transgene silencing can be reverted with chemical treatments using HDAC inhibitors. To analyze this, we transformed C. reinhardtii, integrating into its genome the mVenus reporter gene under the HSP70-rbcs2 promoter. From 384 transformed clones, 88 (22.9%) displayed mVenus positive (mVenus+ ) cells upon flow-cytometry analysis. Five clones with different fluorescence intensities were selected. The number of integrated copies was measured by qPCR. Transgene expression levels were followed over the growth cycle and upon SAHA treatment, using a microplate reader, flow cytometry, RT-qPCR, and western blot analysis. First, we observed that expression varies with the cell cycle, reaching a maximum level just before the stationary phase in all clones. Second, we uncovered that supplementation with HDAC inhibitors of the hydroxamate family, such as vorinostat (suberoylanilide-hydroxamic-acid, SAHA) at the initiation of culture increases the frequency (% of mVenus+ cells) and the level of transgene expression per cell over the whole growth cycle, through histone deacetylase inhibition. Thus, we propose a new tool to successfully trigger the expression of heterologous proteins in the green algae C. reinhardtii, overcoming its main obstacle as an expression platform.

Rice requires a chromatin remodeler for Polymerase IV-small interfering RNA production and genomic immunity.

Transgenes are often spontaneously silenced, which hinders the application of genetic modifications to crop breeding. While gene silencing has been extensively studied in Arabidopsis (Arabidopsis thaliana), the molecular mechanism of transgene silencing remains elusive in crop plants. We used rice (Oryza sativa) plants silenced for a 35S::OsGA2ox1 (Gibberellin 2-oxidase 1) transgene to isolate five elements mountain (fem) mutants showing restoration of transgene expression. In this study, we isolated multiple fem2 mutants defective in a homolog of Required to Maintain Repression 1 (RMR1) of maize (Zea mays) and CLASSY (CLSY) of Arabidopsis. In addition to failing to maintain transgene silencing, as occurs in fem3, in which mutation occurs in NUCLEAR RNA POLYMERASE E1 (OsNRPE1), the fem2 mutant failed to establish transgene silencing of 35S::OsGA2ox1. Mutation in FEM2 eliminated all RNA POLYMERASE IV (Pol-IV)-FEM1/OsRDR2 (RNA-DEPENDENT RNA POLYMERASE 2)-dependent small interfering RNAs (siRNAs), reduced DNA methylation on genome-wide scale in rice seedlings, caused pleiotropic developmental defects, and increased disease resistance. Simultaneous mutation in 2 FEM2 homologous genes, FEM2-Like 1 (FEL1) and FEL2, however, did not affect DNA methylation and rice development and disease resistance. The predominant expression of FEM2 over FEL1 and FEL2 in various tissues was likely caused by epigenetic states. Overexpression of FEL1 but not FEL2 partially rescued hypomethylation of fem2, indicating that FEL1 maintains the cryptic function. In summary, FEM2 is essential for establishing and maintaining gene silencing; moreover, FEM2 is solely required for Pol IV-FEM1 siRNA biosynthesis and de novo DNA methylation.

Evidence of a novel silencing effect on transgenes in the Arabidopsis thaliana sperm cell.

We encountered unexpected transgene silencing in Arabidopsis thaliana sperm cells; transgenes encoding proteins with no specific intracellular localization (cytoplasmic proteins) were silenced transcriptionally or posttranscriptionally. The mRNA of cytoplasmic protein transgenes tagged with a fluorescent protein gene was significantly reduced, resulting in undetectable fluorescent protein signals in the sperm cell. Silencing of the cytoplasmic protein transgenes in the sperm cell did not affect the expression of either its endogenous homologous genes or cotransformed transgenes encoding a protein with targeted intracellular localization. This transgene silencing in the sperm cell persisted in mutants of the major gene silencing machinery including DNA methylation. The incomprehensible, yet real, transgene silencing phenotypes occurring in the sperm cell could mislead the interpretation of experimental results in plant reproduction, and this Commentary calls attention to that risk and highlights details of this novel cytoplasmic protein transgene silencing.

Transgene silencing in the moss, Physcomitrella (Physcomitrium patens)

Gene targeting is a powerful tool for functional genetic analysis. It has proved especially effective in the model plant, Physcomitrella ( Physcomitrium patens (Hedw.) Mitt.), in which integration of exogenous DNA into the genome occurs with very high efficiency at targeted positions by homologous recombination. Here, we present a preliminary characterisation of a poorly documented property of Physcomitrella targeted gene knockout lines, namely, silencing of reporter and selective marker genes of integrated targeting constructs. We discovered a high incidence of transgene silencing among Physcomitrella knockout lines and, as is the case in seed plants, cytosine (DNA) methylation of the transgenes is correlated with their silencing. Silencing in Physcomitrella is characterised by metastable epigenetic inheritance through repetitive mitosis and variable stability through meiosis.

Condensate cooperativity underlies transgenerational gene silencing

Biomolecular condensates have been shown to interact invivo, yet it is unclear whether these interactions are functionally meaningful. Here, we demonstrate that cooperativity between two distinct condensates-germ granules and P bodies-is required for transgenerational gene silencing in C.elegans. We find that P bodies form a coating around perinuclear germ granules and that P body components CGH-1/DDX6 and CAR-1/LSM14 are required for germ granules to organize into sub-compartments and concentrate small RNA silencing factors. Functionally, while the P body mutant cgh-1 is competent to initially trigger gene silencing, it is unable to propagate the silencing to subsequent generations. Mechanistically, we trace this loss of transgenerational silencing to defects in amplifying secondary small RNAs and the stability of WAGO-4 Argonaute, both known carriers of gene silencing memories. Together, these data reveal that cooperation between condensates results in an emergent capability of germ cells to establish heritable memory.

RTEL1 is required for silencing and epigenome stability.

Transcriptional silencing is an essential mechanism for controlling the expression of genes, transgenes and heterochromatic repeats through specific epigenetic marks on chromatin that are maintained during DNA replication. In Arabidopsis, silenced transgenes and heterochromatic sequences are typically associated with high levels of DNA methylation, while silenced genes are enriched in H3K27me3. Reactivation of these loci is often correlated with decreased levels of these repressive epigenetic marks. Here, we report that the DNA helicase REGULATOR OF TELOMERE ELONGATION 1 (RTEL1) is required for transcriptional silencing. RTEL1 deficiency causes upregulation of many genes enriched in H3K27me3 accompanied by a moderate decrease in this mark, but no loss of DNA methylation at reactivated heterochromatic loci. Instead, heterochromatin exhibits DNA hypermethylation and increased H3K27me3 in rtel1. We further find that loss of RTEL1 suppresses the release of heterochromatin silencing caused by the absence of the MOM1 silencing factor. RTEL1 is conserved among eukaryotes and plays a key role in resolving DNA secondary structures during DNA replication. Inducing such aberrant DNA structures using DNA cross-linking agents also results in a loss of transcriptional silencing. These findings uncover unappreciated roles for RTEL1 in transcriptional silencing and in stabilizing DNA methylation and H3K27me3 patterns.