Silencing System Research Articles

Expression and functional analysis of adipokinetic hormone reveal its different roles in larval development and female fecundity in Panonychus citri (McGregor) (Acari: Tetranychidae).

Adipokinetic hormone (AKH), a crucial neuropeptide, participates in the important physiological processes by specially binding to its receptor to activate the AKH signalling pathway. AKH regulates energy metabolism. However, it remains unknown whether AKH affects larval development and adult reproduction by influencing energy metabolism. In the present study, the AKH was identified from Panonychus citri and contained the conserved functional domain 'Q-[LIV]-[NT]-F-[ST]-X (2)-W' that characterises the AKH family. The relative expression levels of PcAKH revealed different patterns of AKH expression at different developmental stages of P. citri. Feeding of double-standard RNA against PcAKH induced decreased fecundity and reduced survival, which was accompanied by the down-regulation of vitellogenin gene expression. In addition, after silencing the PcAKH, lipid metabolism and carbohydrate homeostasis were disrupted, manifested by increased body width and weight, and fasting phenomenon. Further investigation found that compared with the control, physiological changes in trehalose and triglyceride contents were accompanied by variations in the mRNA expression levels of genes related to lipid metabolism and carbohydrate metabolism. The disorder of lipid and carbohydrate metabolism may affect adult female reproduction, which may lead to insufficient vitellogenin deposition. Moreover, the silencing of PcAKH seriously affected the growth and development of larvae, which was manifested as delayed development period and difficulty in moulting. Conclusively, all these results in current study demonstrated that double-stranded RNA silencing system targeting PcAKH effectively inhibited larval development and female fecundity by disturbing lipid and carbohydrate metabolism, and PcAKH is a specific RNAi target for control of P. citri in the design and development of biopesticide in sustainable agriculture.

An Engineered Citrus Tristeza Virus (T36CA)-Based Vector Induces Gene-Specific RNA Silencing and Is Graft Transmissible to Commercial Citrus Varieties.

A protein-expressing citrus tristeza virus-based vector construct, pT36CA-V1.3, obtained from a California isolate of the T36 strain (T36CA), was retooled into a virus-induced gene silencing system intended for use with studies of California citrus. Virus-induced gene silencing constructs engineered with a truncated Citrus macrophylla PHYTOENE DESATURASE (CmPDS) gene sequence in the sense or antisense orientation worked equally well to silence the endogenous CmPDS gene. In a parallel effort to optimize vector performance, two nonsynonymous nucleotides in open reading frame 1a of pT36CA-V1.3 were replaced with those conserved in the reference sequences from the T36CA cDNA library. The resulting viruses, T36CA-V1.4 (with one amino acid modification: D760N) and T36CA-V1.5 (with two amino acid modifications: D760N and P1174L), along with T36CA-V1.3, were individually propagated in Nicotiana benthamiana and C. macrophylla plants. Enzyme-linked immunosorbent assay (ELISA) measurements of extracts of the newly emerged leaves suggested that all three viruses accumulated to similar levels in N. benthamiana plants at 5 weeks postinoculation. ELISA values of T36CA-V1.4- and -V1.5-infected C. macrophylla samples were significantly higher than that of T36CA-V1.3-infected samples within an 8- to 12-month postinoculation window, suggesting a higher accumulation of T36CA-V1.4 and -V1.5 than T36CA-V1.3. However, at 36 months postinoculation, the ELISA values suggested that all three viruses accumulated to similar levels. When C. macrophylla plants infected with each of the three viruses were grafted to commercial citrus varieties, a limited number of receptor plants became infected, demonstrating a weak but nonetheless (the first) successful delivery of T36CA to California-grown commercial citrus.

Developing and applying a virus-induced gene silencing system for functional genomics in walnut (Juglans regia L.) mediated by tobacco rattle virus

Walnut (Juglans regia L.) is a high-value tree species planted worldwide, but the incomplete less developed genetic transformation system limits its gene function analysis. In this study, virus-induced gene silencing (VIGS) mediated by tobacco rattle virus (TRV) technology was applied to walnut seedlings to degrade the transcript of target gene. Different infiltration methods were used to explore the effects of infection mode, Agrobacterium cell density, silencing fragment length, and walnut cultivars. The results showed that spray infiltration of seedlings resulted in a photobleaching phenotype of the whole plant. Leaf injection was a more effective way of infiltration. The optimal combination was the Agrobacterium cell density at OD600 = 1.1 with target fragment = 255 bp for the treatment of walnut early-fruiting cultivar ’Xiangling.’ This combination can reach up to 48 % of gene silencing efficiency. Based on this optimized VIGS system, silencing a walnut chlorophyll synthesis-related gene, JrPOR (Protochlorophyllide reductase), to further validate the system’s effect. The results showed that the expression of JrPOR was significantly repressed, and the chlorophyll level of the silenced plants was significantly decreased compared with the control. The above results indicate that the walnut TRV-VIGS system has been successfully established and can be used for reverse genetic studies, providing an option for verifying gene function in walnut.

Abscisic acid-, stress-, ripening-induced 2 like protein, TaASR2L, promotes wheat resistance to stripe rust.

Wheat stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is one of the most destructive wheat diseases. The plant hormone abscisic acid (ABA) plays a key regulatory role in plant response to stress. ABA-, stress-, ripening-induced proteins (ASR) have been shown to be abundantly induced in response to biotic and abiotic stresses to protect plants from damage. However, the function of wheat ASR2-like protein (TaASR2L) in plants under biotic stress remains unclear. In this study, transient silencing of TaASR2L using a virus-induced gene silencing system substantially reduced wheat resistance to Pst. TaASR2L interaction with serine/arginine-rich splicing factor SR30-like (TaSR30) was validated mainly in the nucleus. Knockdown of TaSR30 expression substantially reduced wheat resistance to Pst. Overexpression of TaASR2L and TaSR30 demonstrated that they can promote the expression of ABA- and resistance-related genes to enhance wheat resistance to Pst. In addition, the expression levels of TaSR30 and TaASR2L were substantially increased by exogenous ABA, and the resistance of wheat to Pst was increased, and the expression of PR genes was induced. Therefore, these results suggest that TaASR2L interacts with TaSR30 by promoting PR genes expression and enhancing wheat resistance to Pst.

A multifunctional Eu-organic framework for fluorescence sensing properties and the detection of pyrimethanil in real samples

Europium metal–organic frameworks (Eu-MOFs) have unique application prospects in the field of fluorescence sensing, due to the fluorescence characteristics of Eu3+ ion and organic ligands, and the advantages of obvious red luminescence, long emission lifetime, large Stokes shift, and high quantum yield. In this study, a novel Eu-MOF was synthesized hydrothermally using europium acetate hexahydrate (Eu(Ac)3·6H2O), 5-(4′-carboxybenzyloxy) isophthalic acid (5-H3CIA), and 4,4′-bipyridine (4,4′-bipy). Structural analysis revealed that the Eu-MOF exhibited a three-dimensional framework with 4,4′-bipyridine as the guest molecule. Fluorescence sensing experiments revealed that 2,4,6-trinitrophenol, ornidazole, pyrimethanil, and Cr2O72− effectively quenched the fluorescence of the Eu-MOF in aqueous solution, with corresponding limits of detection as low as 0.886, 0.732, 0.569, and 2.800 μM, respectively. Exploiting the ability of Cr2O72− to quench the fluorescence of the Eu-MOF, we devised a Cr2O72−@Eu-MOF fluorescence silencing system that, in the presence of ascorbic acid, gradually recovered its fluorescence to form a unique fluorescence “on–off-on” sensor. In addition, the fluorescence sensing mechanism of Eu-MOF was studied, the fluorescence test was prepared to detect Cr2O72− with an open eye, and the content of pyrimethanil was detected in real samples, including cucumber skin, tomato skin, grape skin, apple and kiwi.

CsNAC17 enhances resistance to Colletotrichum gloeosporioides by interacting with CsbHLH62 in Camellia sinensis

Abstract The pathogen Colletotrichum gloeosporioides causes anthracnose, a serious threat to tea trees around the world, particularly in warm and humid regions. RNA-Seq data have previously indicated NAC transcription factors are involved in anthracnose resistance, but underlying mechanisms remain unclear. The BiFC, Split-LUC, and Co-IP assays validated the interaction between CsbHLH62 and CsNAC17 identified through yeast two-hybrid (Y2H) screening. CsNAC17 or CsbHLH62 overexpression enhanced anthracnose resistance, as well as enhanced levels of H2O2, hypersensitivity, and cell death in Nicotiana benthamiana. The NBS-LRR gene CsRPM1 is regulated by CsNAC17 by binding directly to its promoter (i.e., CACG, CATGTG), while CsbHLH62 facilitates CsNAC17’s binding and increases trascriptional activity of CsRPM1. Additionally, transient silencing of CsNAC17 and CsbHLH62 in tea plant leaves using the virus-induced gene silencing (VIGS) system resulted in decreased resistance to anthracnose. Conversely, transient overexpression of CsNAC17 and CsbHLH62 in tea leaves significantly enhanced the resistance against anthracnose. Based on these results, it appears that CsbHLH62 facilitates the activity of CsNAC17 on CsRPM1, contributing to increased anthracnose resistance.

The TOP-2/condensin II axis silences transcription during germline specification in C. elegans.

In C. elegans, the germline is specified via a preformation mechanism that relies on the PIE-1 protein's ability to globally silence mRNA transcription in germline precursor cells, also known as the P lineage. Recent work from our group has identified additional genome silencing events in C. elegans during oogenesis and in starved L1 larvae, and these require the condensin II complex, topoisomerase II (TOP-2), and components of the H3K9me/heterochromatin pathway. Interestingly, silencing in oocytes also requires PIE-1, but this is not the case in starved L1s. Here, we ask if additional genome silencing components besides PIE-1 are required to repress gene expression in the P lineage of early embryos, and we find that condensin II and TOP-2 are required and the H3K9me/heterochromatin pathway is not. We show that depletion of TOP-2/condensin II activates the normally suppressed RNA polymerase II to inappropriately transcribe somatic genes in the P lineage. We also present evidence that while both PIE-1 and TOP-2/condensin II are required for genome silencing in the P lineage, PIE-1 can silence transcription independently of TOP-2/condensin II when misexpressed in somatic cells. Thus, in oocytes, all three genome silencing systems (TOP-2/condensin II, H3K9me, and PIE-1) are operational while in both early embryos and starved L1s two of the three are active. Our data show that multiple, redundantly acting genome silencing mechanisms act in a mix and match manner to repress transcription at different developmental stages in the C. elegans germline.

Overexpression of GmSRC2 confers resistance to Phytophthora sojae in soybean

Phytophthora root and stem rot caused by Phytophthora sojae (P. sojae) is one of the most destructive diseases to affect soybean (Glycine max (L.) Merr) production. GmSRC2 that encodes a C2 domain-containing protein can respond to various stresses, however, the molecular mechanism of GmSRC2 in resistance of soybean to P. sojae is yet to be fully elucidated. In this study, GmSRC2 was found to be significantly up-regulated under P. sojae treatment; GmSRC2-overexpression (OE) transgenic lines and GmSRC2-silencing transient plants were generated via Agrobacterium tumefaciens mediated transformation and virus-induced gene silencing (VIGS) system, respectively. Infected leaves and cotyledons of OE-GmSRC2–1 and OE-GmSRC2–2 lines showed significant decreases in the disease symptoms and P. sojae biomass than those of wild type (WT); the activities of superoxide dismutase (SOD) and peroxidase (POD) confirmed the accumulation of reactive oxygen species (ROS) in overexpressed transgenic lines. Whereas, silencing of GmSRC2 severely increased the disease symptoms and the biomass of P. sojae. Further, we confirmed that GmSRC2 interacted with the effector PsAvh23 of P. sojae, and the C2 domain was crucial for the interaction. Overexpression of GmSRC2 upregulated the ADA2/GCN5 module upon P. sojae. The aforementioned results demonstrated that GmSRC2 played vital roles in regulating soybean resistance to oomycetes.

A G6P1E isomerase of sugar metabolism is involved in the flower colors of Dianthus chinensis

Dianthus chinensis L. is indigenous to northern China, Korea, Mongolia, Kazakhstan, and southeastern Russia. It is widely cultivated in urban landscapes. Its flower has a great variety of colors and color schemes. Sugars control and induce anthocyanin synthesis and accumulation in plants. In sugar metabolism, many enzymes are specific for their substrate’s α or β anomer. Gaining and characterizing genes involved in sugar metabolism and flower color will be beneficial in clarifying the role of sugar in the flower colors of D. chinensis. Glucose-6-phosphate-1-epimerase (G6P1E, EC 5.1.3.15) catalyzes the α or β change of glucose-6-phosphate at the branch point of glucose metabolism. DchG6P1E1 (MZ292712) was isolated in D. chinensis and characterized using the tobacco rattle virus (TRV)-based virus-induced gene silencing (VIGS) system. Its cDNA full length is 1401 bp, including an open reading frame of 918 bp. In the DchG6P1E1-silenced flowers, the reducing purple was observed, as well as the anthocyanin content, reducing sugar content, G6P1E activity, and DchG6P1E1 expression were significantly decreased. During the development of floral buds and among the three flower colors, the anthocyanin content, reduced sugar content, G6P1E activity, and DchG6P1E1 expression rose dramatically, with pigments increasing in the petals. Among the organs, the flowers had the highest anthocyanin contents and reducing sugar. The highest levels of G6P1E activity and DchG6P1E1 expression were in the roots. The anthocyanin content was positively related to the reducing sugar content at 0.05 levels by correlation analysis. In conclusion, DchG6P1E1 is a root-enriched gene associated with flower colors in D. chinensis.

Systematic single-cell analysis reveals dynamic control of transposable element activity orchestrating the endothelial-to-hematopoietic transition

BackgroundThe endothelial-to-hematopoietic transition (EHT) process during definitive hematopoiesis is highly conserved in vertebrates. Stage-specific expression of transposable elements (TEs) has been detected during zebrafish EHT and may promote hematopoietic stem cell (HSC) formation by activating inflammatory signaling. However, little is known about how TEs contribute to the EHT process in human and mouse.ResultsWe reconstructed the single-cell EHT trajectories of human and mouse and resolved the dynamic expression patterns of TEs during EHT. Most TEs presented a transient co-upregulation pattern along the conserved EHT trajectories, coinciding with the temporal relaxation of epigenetic silencing systems. TE products can be sensed by multiple pattern recognition receptors, triggering inflammatory signaling to facilitate HSC emergence. Interestingly, we observed that hypoxia-related signals were enriched in cells with higher TE expression. Furthermore, we constructed the hematopoietic cis-regulatory network of accessible TEs and identified potential TE-derived enhancers that may boost the expression of specific EHT marker genes.ConclusionsOur study provides a systematic vision of how TEs are dynamically controlled to promote the hematopoietic fate decisions through transcriptional and cis-regulatory networks, and pre-train the immunity of nascent HSCs.

Identification of the Coexisting Virus-Derived siRNA in Maize and Rice Infected by Rice Black-Streaked Dwarf Virus.

Rice black-streaked dwarf virus is transmitted by small brown planthoppers, which causes maize rough dwarf disease and rice black-streaked dwarf disease. This virus leads to slow growth or death of the host plants. During the coevolutionary arms race between viruses and plants, virus-derived small interfering RNAs (vsiRNAs) challenge the plant's defense response and inhibit host immunity through the RNA silencing system. However, it is currently unknown if rice black-streaked dwarf virus can produce the same siRNAs to mediate the RNA silencing in different infected species. In this study, four small RNA libraries and four degradome libraries were constructed by extracting total RNAs from the leaves of the maize (Zea mays) inbred line B73 and japonica rice (Oryza sativa) variety Nipponbare exposed to feeding by viruliferous and nonviruliferous small brown planthoppers. We analyzed the characteristics of small RNAs and explored virus-derived siRNAs in small RNA libraries through high-throughput sequencing. On analyzing the characteristics of small RNA, we noted that the size distributions of small RNAs were mainly 24 nt (19.74 to 62.00%), whereas those of vsiRNAs were mostly 21 nt (41.06 to 41.87%) and 22 nt (39.72 to 42.26%). The 5'-terminal nucleotides of vsiRNAs tended to be adenine or uracil. Exploring the distribution of vsiRNA hot spots on the viral genome segments revealed that the frequency of hotspots in B73 was higher than those in Nipponbare. Meanwhile, hotspots in the S9 and S10 virus genome segments were distributed similarly in both hosts. In addition, the target genes of small RNA were explored by degradome sequencing. Analyses of the regulatory pathway of these target genes unveiled that viral infection affected the ribosome-related target genes in maize and the target genes in the metabolism and biosynthesis pathways in rice. Here, 562 and 703 vsiRNAs were separately obtained in maize and rice and 73 vsiRNAs named as coexisting vsiRNAs (co-vsiRNAs) were detected in both hosts. Stem-loop PCR and real-time quantitative PCR confirmed that co-vsiRNA 3.1 and co-vsiRNA 3.5, derived from genome segment S3, simultaneously play a role in maize and rice and inhibited host gene expression. The study revealed that rice black-streaked dwarf virus can produce the same siRNAs in different species and provides a new direction for developing new antiviral strategies.

Persistent metabolic toxicities following developmental exposure to hexafluoropropylene oxide trimer acid (HFPO-TA): Roles of peroxisome proliferator activated receptor gamma

BackgroundHexafluoropropylene oxide trimer acid (HFPO-TA), a perfluorooctanoic acid (PFOA) substitute, exhibited strong affinity and capability to activate peroxisome proliferator activated receptor gamma (PPARγ), a lipid metabolism regulator, suggesting potential to induce metabolic toxicities. MethodsFertile chicken eggs were exposed to 0, 0.5, 1 or 2 mg/kg (egg weight) HFPO-TA and incubated until hatch. Serum from 0- and 3- month-old chickens were subjected to liquid chromatography ultra-high resolution mass spectrometry for HFPO-TA concentration, while liver, pancreas and adipose tissue samples were collected for histopathological assessments. In ovo PPARγ reporter and silencing system were established with lentivirus microinjection. qRT-PCR and immunohistochemistry were utilized to evaluate the expression levels of PPARγ downstream genes. ResultsIn 3-month-old animals developmentally exposed to HFPO-TA, adipose tissue hyperplasia, hepatic steatosis, pancreas islet hypertrophy and elevated serum free fatty acid / insulin levels were observed. Results of reporter assay and qRT-PCR indicated HFPO-TA-mediated PPARγ transactivation in chicken embryo. Silencing of PPARγ alleviated HFPO-TA-induced changes, while PPARγ agonist rosiglitazone mimicked HFPO-TA-induced effects. qRT-PCR and immunohistochemistry revealed that FASN and GPD1 were upregulated following developmental exposure to HFPO-TA in 3-month-old animals. ConclusionsDevelopmental exposure to HFPO-TA induced persistent metabolic toxicities in chickens, in which PPARγ played a central role.

Establishment of a Homologous Silencing System with Intact-Plant Infiltration and Minimized Operation for Studying Gene Function in Herbaceous Peonies.

Gene function verification is a crucial step in studying the molecular mechanisms regulating various plant life activities. However, a stable and efficient homologous genetic transgenic system for herbaceous peonies has not been established. In this study, using virus-induced gene silencing technology (VIGS), a highly efficient homologous transient verification system with distinctive advantages was proposed, which not only achieves true "intact-plant" infiltration but also minimizes the operation. One-year-old roots of the representative species, Paeonia lactiflora Pall., were used as the materials; prechilling (4 °C) treatment for 3-5 weeks was applied as a critical precondition for P. lactiflora to acquire a certain chilling accumulation. A dormancy-related gene named HOMEOBOX PROTEIN 31 (PlHB31), believed to negatively regulate bud endodormancy release (BER), was chosen as the target gene in this study. GFP fluorescence was detected in directly infiltrated and newly developed roots and buds; the transgenic plantlets exhibited remarkably earlier budbreak, and PlHB31 was significantly downregulated in silenced plantlets. This study established a homologous transient silencing system featuring intact-plant infiltration and minimized manipulation for gene function research, and also offers technical support and serves as a theoretical basis for gene function discovery in numerous other geophytes.

Host-Induced Gene Silencing of Effector AGLIP1 Enhanced Resistance of Rice to Rhizoctonia solani AG1-IA

Rice sheath blight, caused by Rhizoctonia solani AG1-IA, is a major disease in rice-growing areas worldwide. Effectors of phytopathogenic fungi play important roles during the infection process of fungal pathogens onto their host plants. However, the molecular mechanisms by which R. solani effectors regulate rice immunity are not well understood. Through prediction, 78 candidate effector molecules were identified. Using the tobacco rattle virus-host induced gene silencing (TRV-HIGS) system, 45 RNAi constructs of effector genes were infiltrated into Nicotiana benthamiana leaves. The results revealed that eight of these constructs resulted in a significant reduction in necrosis caused by infection with the AG1-IA strain GD-118. Additionally, stable rice transformants carrying the double-stranded RNA construct for one of the effector genes, AGLIP1, were generated to further verify the function of this gene. The suppression of the AGLIP1 gene increased the resistance of both N. benthamiana and rice against GD-118, and also affected the growth rate of GD-118, indicating that AGLIP1 is a key pathogenic factor. Small RNA sequencing showed that the HIGS vectors were processed into siRNAs within the plants and then translocated to the fungi, leading to the silencing of the target genes. As a result, AGLIP1 might be an excellent candidate for HIGS, thereby enhancing crop resistance against the pathogen and contributing to the control of R. solani infection.

Spray-induced and nanocarrier-delivered gene silencing system targeting juvenile hormone receptor components: potential application as fertility inhibitors for Adelphocoris suturalis management.

Adelphocoris suturalis is a destructive pest that attacks > 270 plants, including cotton, maize, soybean, and fruit trees. Adelphocoris suturalis can cause tremendous crop losses when the density exceeds economic thresholds, but because it can be both phytophagous and zoophytophagous it can serve as a natural enemy of other pests when the density is below the economic threshold. Effective control of its population is beneficial for maximizing yield and profits. RNA interference (RNAi) has potential to be a viable alternative to conventional pesticide-based pest management, but the lack of efficient double-stranded RNA (dsRNA) delivery systems and candidate genes are currently limiting factors for field applications. In this study, RNAi of juvenile hormone (JH) receptor components methoprene-tolerant (Met)/Taiman (Tai) in Adelphocoris suturalis reduced fertility. Based on this reproductive role, we targeted Adelphocoris suturalis Met and Tai for knockdown by coupling nanomaterial-dsRNA complexes with a transdermal spray delivery system. Within 12 h of adult emergence, females were sprayed with star polycation (SPc)-dsRNA formulations and the RNAi effects were assessed over time. RNAi knockdown efficiencies of 39-58% were observed at 5 days post-treatment and abnormal ovarian development was apparent by 10 days post-treatment. Our results show that spray-induced and nanocarrier-delivered gene silencing (SI-NDGS) system targeting JH signal genes effectively impaired oviposition, thus developing a novel RNA fertility inhibitor to control Adelphocoris suturalis populations. These results give new perspective on pest management and suggest broad prospects for field applications. © 2024 Society of Chemical Industry.

Targeting S100A9 Prevents β-Adrenergic Activation-Induced Cardiac Injury.

Altered cardiac innate immunity is highly associated with the progression of cardiac disease states and heart failure. S100A8/A9 is an important component of damage-associated molecular patterns (DAMPs) that is critically involved in the pathogenesis of heart failure, thus considered a promising target for pharmacological intervention. In the current study, initially, we validated the role of S100A8/A9 in contributing to cardiac injury and heart failure via the overactivation of the β-adrenergic pathway and tested the potential use of paquinimod as a pharmacological intervention of S100A8/A9 activation in preventing cardiac dysfunction, collagen deposition, inflammation, and immune cell infiltration in β-adrenergic overactivation-mediated heart failure. This finding was further confirmed by the cardiomyocyte-specific silencing of S100A9 via the use of the adeno-associated virus (AAV) 9-mediated short hairpin RNA (shRNA) gene silencing system. Most importantly, in the assessment of the underlying cellular mechanism by which activated S100A8/A9 cause aggravated progression of cardiac fibrosis and heart failure, we discovered that the activated S100A8/A9 can promote fibroblast-macrophage interaction, independent of inflammation, which is likely a key mechanism leading to the enhanced collagen production. Our results revealed that targeting S100A9 provides dual beneficial effects, which is not only a strategy to counteract cardiac inflammation but also preclude cardiac fibroblast-macrophage interactions. The findings of this study also indicate that targeting S100A9 could be a promising strategy for addressing cardiac fibrosis, potentially leading to future drug development.

Rational design of unrestricted pRN1 derivatives and their application in the construction of a dual plasmid vector system for Saccharolobus islandicus.

Saccharolobus islandicus REY15A represents one of the very few archaeal models with versatile genetic tools, which include efficient genome editing, gene silencing, and robust protein expression systems. However, plasmid vectors constructed for this crenarchaeon thus far are based solely on the pRN2 cryptic plasmid. Although this plasmid coexists with pRN1 in its original host, early attempts to test pRN1-based vectors consistently failed to yield any stable host-vector system for Sa. islandicus. We hypothesized that this failure could be due to the occurrence of CRISPR immunity against pRN1 in this archaeon. We identified a putative target sequence in orf904 encoding a putative replicase on pRN1 (target N1). Mutated targets (N1a, N1b, and N1c) were then designed and tested for their capability to escape the host CRISPR immunity by using a plasmid interference assay. The results revealed that the original target triggered CRISPR immunity in this archaeon, whereas all three mutated targets did not, indicating that all the designed target mutations evaded host immunity. These mutated targets were then incorporated into orf904 individually, yielding corresponding mutated pRN1 backbones with which shuttle plasmids were constructed (pN1aSD, pN1bSD, and pN1cSD). Sa. islandicus transformation revealed that pN1aSD and pN1bSD were functional shuttle vectors, but pN1cSD lost the capability for replication. These results indicate that the missense mutations in the conserved helicase domain in pN1c inactivated the replicase. We further showed that pRN1-based and pRN2-based vectors were stably maintained in the archaeal cells either alone or in combination, and this yielded a dual plasmid system for genetic study with this important archaeal model.

A CRISPR interference system for engineering biological nitrogen fixation.

A grand challenge for the next century is in facing a changing climate through bioengineering solutions. Biological nitrogen fixation, the globally consequential, nitrogenase-catalyzed reduction of atmospheric nitrogen to bioavailable ammonia, is a vital area of focus. Nitrogen fixation engineering relies upon extensive understanding of underlying genetics in microbial models, including the broadly utilized gammaproteobacterium, Azotobacter vinelandii (A. vinelandii). Here, we report the first CRISPR interference (CRISPRi) system for targeted gene silencing in A. vinelandii that integrates genomically via site-specific transposon insertion. We demonstrate that CRISPRi can repress transcription of an essential nitrogen fixation gene by ~60%. Further, we show that nitrogenase genes are suitably expressed from the transposon insertion site, indicating that CRISPRi and engineered nitrogen fixation genes can be co-integrated for combinatorial studies of gene expression and engineering. Our established CRISPRi system fills an important gap for engineering microbial nitrogen fixation for desired purposes.IMPORTANCEAll life on Earth requires nitrogen to survive. About 78% of the atmosphere alone is nitrogen, yet humans cannot use it directly. Instead, we obtain the nitrogen we need for our survival through the food we eat. For more than 100 years, a substantial portion of agricultural productivity has relied on industrial methods for nitrogen fertilizer synthesis, which consumes significant amounts of nonrenewable energy resources and exacerbates environmental degradation and human-induced climate change. Promising alternatives to these industrial methods rely on engineering the only biological pathway for generating bioaccessible nitrogen: microbial nitrogen fixation. Bioengineering strategies require an extensive understanding of underlying genetics in nitrogen-fixing microbes, but genetic tools for this critical goal remain lacking. The CRISPRi gene silencing system that we report, developed in the broadly utilized nitrogen-fixing bacterial model, Azotobacter vinelandii, is an important step toward elucidating the complexity of nitrogen fixation genetics and enabling their manipulation.

Robust miniature Cas-based transcriptional modulation by engineering Un1Cas12f1 and tethering Sso7d

The miniature V-F CRISPR-Cas12f system has been repurposed for gene editing and transcription modulation. The small size of Cas12f satisfies the packaging capacity of adeno-associated virus (AAV) for gene therapy. However, the efficiency of Cas12f-mediated transcriptional activation varies among different target sites. Here, we developed a robust miniature Cas-based transcriptional activation or silencing system using Un1Cas12f1. We engineered Un1Cas12f1 and the cognate guide RNA and generated miniCRa, which led to a 1319-fold increase in activation of the ASCL1 gene. The activity can be further increased by tethering DNA binding protein Sso7d to miniCRa and generating SminiCRa, which reached 5628-fold activation of the ASCL1 gene and at least hundreds-fold activation at other genes examined. We adopted these mutations of Un1Cas12f1 for transcriptional repression and generated miniCRi or SminiCRi, which led to the repression ∼80% on average of eight genes. We generated an all-in-one AAV vector AIOminiCRi utilized to silence the disease-related gene SERPINA1. AIOminiCRi AAVs led to the 70% repression of the SERPINA1 gene in the Huh-7 cells. In sum, miniCRa, SminiCRa, miniCRi and SminiCRi are robust miniature transcriptional modulators with high specificity, that expand the toolbox for biomedical research and therapeutic applications.

Optimizing the shape of space-constrained silencers composed of multiple straight/reverse chambers using the simulated annealing method

Abstract To address the issue of hearing health problems caused by the broadband noise emitted from space-constrained marine diesel engines on a ship, an efficient silencer equipped at the engine's venting outlet is necessary. In consideration of the space-constrained situation that often occurs in ships, a reverse silencer with multiple chambers is introduced. A mathematical model of eigenfunction is adopted to consider the acoustic effect at high frequencies. An optimization of the silencer will be performed using the acoustic model of an eigenfunction together with the simulated annealing (SA) method under space-constrained conditions to achieve the best design. To pursue an efficient silencer design, two types of reverse silencers (silencers A and B) are adopted to reduce the engine's venting noise. Acoustical models for straight chamber and reverse chamber silencers are checked before optimization. To assure the reliability of the SA scheme, a pure tone optimization at 250 Hz is exemplified. Results in silencers A and B show that the sound transmission loss is approximately maximized at the desired frequency. Thereafter, a broadband noise elimination of engine noise using silencers A and B is performed. Simulated results show that overall noise abatement can be improved when the number of inverse chambers increases. The optimization of space-constrained reverse silencers with multiple chambers using eigenfunction in cooperation with SA has been accomplished. The overall silencer system's 4-pole matrix is used to predict the silencer's acoustical performance of STL, which is achieved by multiplying individual 4-pole matrices. With the introduction of the reverse silencer, the hearing health of workers can be protected, and noise pollution can be reduced in industrial settings.