Toehold region triggered CRISPR/Cas12a trans-cleavage for detection of uracil-DNA glycosylase activity.

DNA glycosylases are a group of enzymes that play a crucial role in the DNA repair process by recognizing and removing damaged or incorrect bases from DNA molecules, which maintains the integrity of the genetic information. The abnormal expression of uracil-DNA glycosylase (UDG), one of significant DNA glycosylases in the base-excision repair pathway, is linked to numerous diseases. Here, we proposed a simple UDG activity detection method based on toehold region triggered CRISPR/Cas12a trans-cleavage. The toehold region on hairpin DNA probe (HP) produced by UDG could induce the trans-cleavage of ssDNA with fluorophore and quencher, generating an obvious fluorescence signal. This protospacer adjacent motif (PAM)-free approach achieves remarkable sensitivity and specificity in detecting UDG, with a detection limit as low as 0.000368 U mL-1. Moreover, this method is able to screen inhibitors and measure UDG in complex biological samples. These advantages render it highly promising for applications in clinical diagnosis and drug discovery.

Spectrally engineered textile for radiative cooling against urban heat islands.

Radiative cooling textiles hold promise for achieving personal thermal comfort under increasing global temperature. However, urban areas have heat island effects that largely diminish the effectiveness of cooling textiles as wearable fabrics because they absorb emitted radiation from the ground and nearby buildings. We developed a mid-infrared spectrally selective hierarchical fabric (SSHF) with emissivity greatly dominant in the atmospheric transmission window through molecular design, minimizing the net heat gain from the surroundings. The SSHF features a high solar spectrum reflectivity of 0.97 owing to strong Mie scattering from the nano-micro hybrid fibrous structure. The SSHF is 2.3°C cooler than a solar-reflecting broadband emitter when placed vertically in simulated outdoor urban scenarios during the day and also has excellent wearable properties.

Microfluidic particle counter visualizing mucosal antibodies against SARS-CoV-2 in the upper respiratory tract for rapid evaluation of immune protection.

Mucosal antibodies in the upper respiratory tract are the earliest and most critical responders to prevent respiratory infections, providing an indication for the rapid evaluation of immune protection. Here, we report a microfluidic particle counter that directly visualizes mucosal antibody levels in nasal mucus. The mucosal anti-SARS-CoV-2 spike receptor binding domain (RBD) antibodies in nasal secretions first react with magnetic microparticles (MMPs) and polystyrene microparticles (PMPs) that are surface-modified to form a "MMPs-anti-spike RBD IgG-PMPs" complex when RBD is present. After magnetic separation and loading into the microfluidic particle counter, the free PMPs, which are reduced with increasing anti-spike RBD IgG antibody levels, are trapped by a microfluidic particle dam and accumulate in the trapping channel. A sensitive mode [limit of detection (LOD): 14.0 ng mL-1; sample-to-answer time: 70 min] and an equipment-free rapid mode (LOD: 37.4 ng mL-1; sample-to-answer time: 20 min) were achieved. Eighty-seven nasal secretion (NS) samples from vaccinees were analyzed using our microfluidic particle counter, and the results closely resemble those of the gold-standard enzyme-linked immunosorbent assay (ELISA). The analysis shows that higher antibody levels were found in convalescent volunteers compared to noninfected volunteers. Together, we demonstrate a rapid kit that directly indicates immune status, which can guide vaccine strategy for individuals and the government.

On-Site Melanoma Diagnosis Utilizing a Swellable Microneedle-Assisted Skin Interstitial Fluid Sampling and a Microfluidic Particle Dam for Visual Quantification of S100A1.

Malignant melanoma (MM) is the most aggressive form of skin cancer. The delay in treatment will induce metastasis, resulting in a poor prognosis and even death. Here, a two-step strategy for on-site diagnosis of MM is developed based on the extraction and direct visual quantification of S100A1, a biomarker for melanoma. First, a swellable microneedle is utilized to extract S100A1 in skin interstitial fluid (ISF) with minimal invasion. After elution, antibody-conjugated magnetic microparticles (MMPs) and polystyrene microparticles (PMPs) are introduced. A high expression level of S100A1 gives rise to a robust binding between MMPs and PMPs and reduces the number of free PMPs. By loading the reacted solution into the device with a microfluidic particle dam, the quantity of free PMPs after magnetic separation is displayed with their accumulation length inversely proportional to S100A1 levels. A limit of detection of 18.7 ng mL-1 for S100A1 is achieved. The animal experiment indicates that ISF-based S100A1 quantification using the proposed strategy exhibits a significantly higher sensitivity compared with conventional serum-based detection. In addition, the result is highly comparable with the gold standard enzyme-linked immunosorbent assay based on Lin's concordance correlation coefficient, suggesting the high practicality for routine monitoring of melanoma.

Theranostic DNA nanostructure based on phenotype-specific activation of antisense oligonucleotides.

Antisense oligonucleotide (ASO) is a powerful agent for gene therapy, designed to form complementary pairs with specific mRNA to inhibit gene expression. However, low specificity limits its potential. To overcome this challenge, we developed a Y-shape DNA nanostructure that enhances the specificity in ASO-based treatment by introducing a detection trigger. The design incorporates the phenotype-specific miR21 activation and the sequential release of Bcl2 ASO. As a result, our Y-shape DNA nanostructure downregulates >50 % Bcl2 mRNA expression and induces >60 % cell death in breast cancer cells. Meanwhile, this approach shows no obvious damage to the non-cancerous cells, indicating the therapeutic potential as a theranostics agent in precision medicine with the combination of biomarker sensing and treatment. Overall, our Y-shape DNA nanostructure serves as a promising strategy providing potential in customized conformation design with specific target sequences in gene therapy.

CRISPR/Cas12a trans-cleavage triggered by cleavage ligation of dumbbell DNA for specific detection of human 8-oxoguanine DNA glycosylase activity.

Human 8-oxoguanine DNA glycosylase (hOGG1) is an essential enzyme that recognizes and removes 8-oxoguanine (8-oxoG), a common DNA oxidative damage caused by reactive oxygen species, to maintain genomic integrity of living organisms. Abnormal expression of hOGG1 has been proved to be associated with different diseases such as cancer and neurogenerative disorders, making it a potential biomarker and therapeutic target. In this study, we report the development of  a novel strategy for detecting hOGG1 activity based on CRISPR/Cas12a trans-cleavage triggered by cleavage ligation of a dumbbell DNA probe (DBP) designed with a 3' overhang and an 8-oxoG modification. When hOGG1 is present, it cleaves the DBP at the 8-oxoG site, forming a 5' phosphate termini and exposing a single-strand region allowing complementary to the 3' overhang. After hybridization, the 3' and 5' termini in the juxtaposition are ligated by T4 DNA ligase, leading to a closed DBP for CRISPR/Cas12a-crRNA to recognize and initiate the trans-cleavage of the surrounding ssDNAs with fluorophore and quencher. The method achieves a limit of detection (LOD) with 370 µU/mL and high selectivity. Furthermore, it demonstrates a good compatibility for detecting hOGG1 activity in cell lysates, suggesting a good performance for further application in disease diagnosis and scientific research.

Single-Cell Micropatterning by Non-fouling Hydrogels.

Micropatterned substrate is a unique method for studying cell biology at the single-cell level. Using photolithography to create binary patterns of cell-adherent peptide surrounding by non-fouling cell-repellent poly(ethylene glycol) (PEG) hydrogel, this patterning method allows for controlling cell attachment with desired sizes and shapes up to 19 days. Here we provide the detailed procedure of fabrication for such patterns. This method will allow monitoring of prolonged reaction of single cells such as cell differentiation upon induction or time-resolved apoptosis stimulated by drug molecules for cancer treatment.

A kirigami-enabled electrochromic wearable variable-emittance device for energy-efficient adaptive personal thermoregulation.

For centuries, people have put effort to improve the thermal performance of clothing to adapt to varying temperatures. However, most clothing we wear today only offers a single-mode insulation. The adoption of active thermal management devices, such as resistive heaters, Peltier coolers, and water recirculation, is limited by their excessive energy consumption and form factor for long-term, continuous, and personalized thermal comfort. In this paper, we developed a wearable variable-emittance (WeaVE) device, enabling the tunable radiative heat transfer coefficient to fill the missing gap between thermoregulation energy efficiency and controllability. WeaVE is an electrically driven, kirigami-enabled electrochromic thin-film device that can effectively tune the midinfrared thermal radiation heat loss of the human body. The kirigami design provides stretchability and conformal deformation under various modes and exhibits excellent mechanical stability after 1,000 cycles. The electronic control enables programmable personalized thermoregulation. With less than 5.58 mJ/cm2 energy input per switching, WeaVE provides 4.9°C expansion of the thermal comfort zone, which is equivalent to a continuous power input of 33.9 W/m2. This nonvolatile characteristic substantially decreases the required energy while maintaining the on-demand controllability, thereby providing vast opportunities for the next generation of smart personal thermal managing fabrics and wearable technologies.

Scalable pattern formation of skeletal myotubes by synergizing microtopographic cues and chiral nematics of cells.

Topographical cues have been widely used to facilitate cell fusion in skeletal muscle formation. However, an unexpected yet consistent chiral orientation of myotubes deviating from the groove boundaries is commonly observed but has long been unattended. In this study, we report a method to guide the formation of skeletal myotubes into scalable and controlled patterns. By inducing C2C12 myoblasts onto grooved patterns with different widths (from 0.4 to 200µm), we observed an enhanced chiral orientation of cells developing on wide grooves (50 and 100µm width) since the first day of induction. Active chiral nematics of cells involving cell migration and chiral rotation of the cell nucleus subsequently led to a unified chiral orientation of the myotubes. Importantly, these chiral myotubes were formed with enhanced length, diameter, and contractility on wide grooves. Treatment of latrunculin A (Lat A) suppressed the chiral rotation and migration of cells as well as the myotube formation, suggesting the essence of chiral nematics of cells for myogenesis. Finally, by arranging wide grooved/striped patterns with corresponding compensation angles to synergize microtopographic cues and chiral nematics of cells, intricate and scalable patterns of myotubes were formed, providing a strategy for engineering skeletal muscle tissue formation.

Multimodal detection of flap endonuclease 1 activity through CRISPR/Cas12a trans-cleavage of single-strand DNA oligonucleotides.

Flap endonuclease 1 (FEN1) is an endonuclease that specially removes 5' single-stranded overhang of branched duplex DNA (5' flap). While FEN1 is essential in various DNA metabolism pathways for preventing the malignant transformation of cells, an unusual expression of FEN1 is often associated with tumor progression, making it a potential biomarker for cancer diagnosis and treatment. Here we report a multimodal detection of FEN1 activity based on CRISPR/Cas12a trans-cleavage of single-strand DNA oligonucleotides (ssDNA). A dumbbell DNA structure with a 5' flap was designed, which can be cleaved by the FEN1 and the dumbbell DNA is subsequently ligated by T4 DNA ligase. The resulting closed duplex DNA contains a specific protospacer adjacent motif (PAM) that activates trans-cleavage of ssDNA after binding to CRISPR/Cas12a-crRNA. The trans-cleavage is activated only once and is independent to length or sequence of the ssDNA, which allows efficient signal amplification and multimodal signals such as fluorescence or cleaved connection between magnetic microparticles (MMPs) and polystyrene microparticles (PMPs) that alters solution turbidity after magnetic separation. In addition, by loading the particle solution into a microfluidic chip, unconnected PMPs escaping from a magnetic separator are amassed at the particle dam, enabling a visible PMP accumulation length proportional to the FEN1 activity. This multimodal detection is selective to FEN1 and achieves a low limit of detection (LOD) with only 40 min of reaction time. Applying to cell lysates, higher FEN1 activity was detected in breast cancer cells, suggesting a great potential for cancer diagnosis.

A Fully Integrated, Ready-to-Use Distance-Based Chemosensor for Visual Quantification of Multiple Heavy Metal Ions.

Point-of-care devices offering quantitative results with simple steps would allow great useability for untrained end-users. Here, we report a ready-to-use chemosensor integrating automatic sample metering, on-chip reaction, gravitational-magnetic separation, and a distance-based readout for visual quantification of multiple heavy metal ions. Deoxyribozymes (DNAzymes), probe-modified magnetic microparticles (MMPs), and polystyrene microparticles (PMPs) are preloaded into a microfluidic chip and freeze-dried. After the water sample is collected with automatic volume metering, the particles are resuspended, and the MMPs and PMPs hybridize with DNAzyme at its two termini, forming the "MMPs-DNAzyme-PMPs" structure. When target metal ions are present, the DNAzymes are cleaved, yielding an increased number of free PMPs. All on-chip reactions are controlled by stopping the liquid flow at capillary valves and bursting it with hand-controlled tilting. Using the chip with a gravitational-magnetic separator, the free PMPs are separated from "MMPs-DNAzyme-PMPs" and accumulate into the trapping channel with a nozzle, forming a visual bar with growing distances proportional to the concentration of target metal ions. The achieved limit of detection (LOD) values for Cu2+ (103.1 nM), Pb2+ (69.5 nM), and Ag+ (793.6 nM) are below the maximum contamination levels. High selectivity of 100-fold, 200-fold, and 20-fold against interference is obtained. Moreover, by integrating three identical channels in parallel, simultaneous detection of the above-mentioned heavy metal ions in fresh and tap water samples is also achieved with high accuracy. Together, this fully integrated and easily operated platform embodies excellent potential for rapid, on-site sensing by unskilled users.

Detection of intracellular sodium ions based on phenotype-specific activation of NaA43 DNAzyme.

The intracellular sodium ion is one of the crucial elements for regulating physiological functions such as action potential and muscle contractions. However, detecting sodium ions in live cells is challenging because false signals may arise from the abundant sodium ions in the extracellular environment when introducing the detection agents. To minimize it, we report a DNAzyme-based detection of sodium ions in live cells via activation by endogenous mRNA. The substrate strand of DNAzyme first hybridizes to a blocking strand that prevents undesired cleavage of DNAzyme when delivered. Once entering cells, an endogenous mRNA biomarker binds to the toehold region of the blocking strand and displaces it, allowing the proper formation of the DNAzyme, which specifically catalyzes the cleavage of the substrate strand in the presence of intracellular Na+ and produces fluorescence signals. Using differentiating skeletal muscle cells as the model system, we demonstrated the successful delivery and phenotype-specific detection of intracellular sodium ions only in differentiated myotubes with highly-expressed myosin heavy chain mRNA. Moreover, using a drug cocktail to increase the permeability of the cell membrane, elevated levels of intracellular sodium ion was observed. This platform offers a broad and promising strategy for detecting intracellular metal ions, suggesting a great potential for understanding its role in cell/tissue physiology.

Microfluidic particle dam for direct visualization of SARS-CoV-2 antibody levels in COVID-19 vaccinees.

Various COVID-19 vaccines are currently deployed, but their immunization varies and decays with time. Antibody level is a potent correlate to immune protection, but its quantitation relies on intensive laboratory techniques. Here, we report a decentralized, instrument-free microfluidic device that directly visualizes SARS-CoV-2 antibody levels. Magnetic microparticles (MMPs) and polystyrene microparticles (PMPs) can bind to SARS-CoV-2 antibodies simultaneously. In a microfluidic chip, this binding reduces the incidence of free PMPs escaping from magnetic separation and shortens PMP accumulation length at a particle dam. This visual quantitative result enables use in either sensitive mode [limit of detection (LOD): 13.3 ng/ml; sample-to-answer time: 70 min] or rapid mode (LOD: 57.8 ng/ml; sample-to-answer time: 20 min) and closely agrees with the gold standard enzyme-linked immunosorbent assay. Trials on 91 vaccinees revealed higher antibody levels in mRNA vaccinees than in inactivated vaccinees and their decay in 45 days, demonstrating the need for point-of-care devices to monitor immune protection.

Neuraminidase (NA) 370-Loop Mutations of the 2009 Pandemic H1N1 Viruses Affect NA Enzyme Activity, Hemagglutination Titer, Mouse Virulence, and Inactivated-Virus Immunogenicity.

Hemagglutinin (HA) and neuraminidase (NA) are the two major envelope proteins of influenza viruses. The spatial organization of HA and NA on the virus surface needs to be optimized to promote viral fitness, host specificity, transmissibility, infectivity, and virulence. We previously demonstrated that the recombinant NA protein of the 2009 pandemic H1N1 (pH1N1) with the I365T/S366N mutation in the NA 370-loop elicited higher NA-inhibition antibody titers against the homologous pH1N1 virus and three heterologous H5N1, H3N2, and H7N9 viruses in mice. In this study, we used PR8-based reverse genetics (RG) by replacing the HA and NA genes of A/Texas/05/2009 pH1N1 virus to obtain the wild-type pH1N1 and three NA 370-loop mutant viruses of pH1N1 (I365T/S366N), RG pH1N1 (I365E/S366D), and RG pH1N1 (I365T/S366A). Our results revealed that the viral NA enzyme activity increased for the RG pH1N1(I365T/S366N) and RG pH1N1 (I365E/S366D) viruses but reduced for the RG pH1N1 (I365T/S366A) virus. The increased or decreased NA enzyme activity was found to correlate with the increase or decrease in HA titers of these NA 370-loop mutant viruses. All of these three NA 370-loop mutant RG pH1N1 viruses were less virulent than the wild-type RG pH1N1 virus in mice. Immunizations with the inactivated viruses carrying the three NA 370-loop mutations and the wild-type RG pH1N1 virus were found to elicit approximately the same titers of NA-inhibition antibodies against H1N1 and H5N1 viruses. These results may provide information for developing NA-based influenza virus vaccines.

Comprehensive analysis of both long and short read transcriptomes of a clonal and a seed-propagated model species reveal the prerequisites for transcriptional activation of autonomous and non-autonomous transposons in plants.

BACKGROUND: Transposable element (TE) transcription is a precursor to its mobilisation in host genomes. However, the characteristics of expressed TE loci, the identification of self-competent transposon loci contributing to new insertions, and the genomic conditions permitting their mobilisation remain largely unknown. RESULTS: Using Vitis vinifera embryogenic callus, we explored the impact of biotic stressors on transposon transcription through the exposure of the callus to live cultures of an endemic grapevine yeast, Hanseniaspora uvarum. We found that only 1.7-2.5% of total annotated TE loci were transcribed, of which 5-10% of these were full-length, and the expressed TE loci exhibited a strong location bias towards expressed genes. These trends in transposon transcription were also observed in RNA-seq data from Arabidopsis thaliana wild-type plants but not in epigenetically compromised Arabidopsis ddm1 mutants. Moreover, differentially expressed TE loci in the grapevine tended to share expression patterns with co-localised differentially expressed genes. Utilising nanopore cDNA sequencing, we found a strong correlation between the inclusion of intronic TEs in gene transcripts and the presence of premature termination codons in these transcripts. Finally, we identified low levels of full-length transcripts deriving from structurally intact TE loci in the grapevine model. CONCLUSION: Our observations in two disparate plant models representing clonally and seed propagated plant species reveal a closely connected transcriptional relationship between TEs and co-localised genes, particularly when epigenetic silencing is not compromised. We found that the stress treatment alone was insufficient to induce large-scale full-length transcription from structurally intact TE loci, a necessity for non-autonomous and autonomous mobilisation.

Effects of thoracic sympathetic stimulation on palmar perfusion: a preliminary study in pigs.

OBJECTIVE: Ablation of the upper thoracic sympathetic ganglia that innervates the hands is the most effective and permanent cure of palmar hyperhidrosis. However, this type of sympathectomy causes irreversible neural damage and may result in severe compensatory hyperhidrosis. This experiment is designed to confirm the hypothesis, in which the stimulation of T2 sympathetic chain leads to increased palmar microcirculation, and thus results in treating hyperhidrosis. METHODS: In this study, we used electric stimulation to induce reversible blockade of the sympathetic ganglion in pigs and investigated its effect on palmar perfusion. An electrode was inserted to the T2 sympathetic ganglion of the pig through three different approaches: open dorsal, thoracoscopic, and fluoroscopy-guided approaches. Electric stimulation was delivered through the electrode using clinically available pulse generators. Palmar microcirculation was evaluated by laser speckle contrast imaging. RESULTS: The T2 sympathetic ganglion of the pig was successfully accessed by all the three approaches, as confirmed by changes in palmar microcirculation during electric stimulation. Similar effects were not observed when the electrode was placed on the T4 sympathetic ganglion or off the sympathetic trunk. CONCLUSION: We established a large animal model to verify the effect of thoracic sympathetic stimulation. Electric stimulation can be used for sympathetic blockade, as confirmed by increased blood perfusion of the palm. Our work suggests that sympathetic stimulation is a potential solution for palmar hyperhidrosis.

Visual Quantitation of Copper Ions Based on a Microfluidic Particle Dam Reflecting the Cu(II)-Catalyzed Oxidative Damage of DNA.

Due to the use of copper water pipes and the discharge of industrial wastewater, contamination of copper ions in drinking water has become a severe hazard globally. To routinely check water safety on a daily basis, easy-to-use platforms for quantitative analysis of trace amounts of copper ions (Cu2+) in drinking water is needed. Here, we report microfluidic particle accumulation integrated with a Cu(II)-catalyzed Fenton reaction for visual and quantitative copper ion detection. Microparticles (MMPs) and polystyrene microparticles (PMPs) are connected via a single strand DNA, MB155. However, when Cu2+ is present, MB155 is cleaved by hydroxyl free radicals (•OH) produced from Cu2+/hydrogen peroxide (H2O2) Fenton reactions, causing an increased amount of free PMPs. To visually count them, the particle solution is loaded onto a microfluidic chip where free MMPs and MMPs-MB155-PMPs can be collected by the magnetic separator, while the free PMPs continue flowing until being accumulated at the particle dam. The results showed a good linear relationship between the trapping length of PMP accumulation and the Cu2+ concentration from 0 to 300 nM. A limit of detection (LOD) of 70.1 nM was achieved, which is approximately 449 times lower than the 2 × 103 µg·L-1 (~31.5 µM) required by the World Health Organization (WHO). Moreover, the results showed high selectivity and good tolerance to pH and hardness, indicating compatibility for detection in tap water, suggesting a potential platform for the routine monitoring of copper contamination in drinking water.

Visual quantitation of silver contamination in fresh water via accumulative length of microparticles in capillary-driven microfluidic devices.

Silver is a heavy metal commonly used as bacteriostatic agents or disinfectants. However, excess amount of silver ion (Ag+) could lead to adverse biological effects on human health. To monitor silver ions in environmental samples, we report a visual quantitative method for analyzing the trace amount of Ag+. A sliver-specific RNA-cleaving DNAzyme Ag10C firstly makes the connection between magnetic microparticles (MMPs) and polystyrene microparticles (PMPs) forming a complex as "MMPs-Ag10C-PMPs". When Ag+ is present, the Ag10C is cleaved, resulting in an increase of free PMPs. By dropping 3 µL of reacted particle solution to a capillary-driven microfluidic chip, MMPs and MMPs-Ag10C-PMPs are removed by a magnetic separator during the flow, while free PMPs can continue flowing until being trapped and accumulating at a particle dam with a narrow nozzle. The accumulation length of PMPs linearly increases with the increment of Ag+ concentrations in the range of 0-10 µM, and readable by the naked eye. We have achieved a limit of detection (LOD) down to 453.7 nM, which is significantly lower than the maximum contaminant level of 926 nM set by World Health Organization (WHO). More importantly, after validating the high selectivity against other metal ions and stable performance in different pH and water hardness, we demonstrate recovery rate >96.8% for tests of multiple fresh water sources, manifesting the feasibility in practical detection in real water samples.

Site-Specific Glycan-Masking/Unmasking Hemagglutinin Antigen Design to Elicit Broadly Neutralizing and Stem-Binding Antibodies Against Highly Pathogenic Avian Influenza H5N1 Virus Infections.

The highly pathogenic avian influenza (HPAI) H5N1 viruses with the capability of transmission from birds to humans have a serious impact on public health. To date, HPAI H5N1 viruses have evolved into ten antigenically distinct clades that could cause a mismatch of vaccine strains and reduce vaccine efficacy. In this study, the glycan masking and unmasking strategies on hemagglutinin antigen were used for designing two antigens: H5-dm/st2 and H5-tm/st2, and investigated for their elicited immunity using two-dose recombinant H5 (rH5) immunization and a first-dose adenovirus vector prime, followed by a second-dose rH5 protein booster immunization. The H5-dm/st2 antigen was found to elicit broadly neutralizing antibodies against different H5N1 clade/subclade viruses, as well as more stem-binding antibodies to inhibit HA-facilitated membrane fusion activity. Mice immunized with the H5-dm/st2 antigen had a higher survival rate when challenged with homologous and heterologous clades of H5N1 viruses. Mutant influenza virus replaced with the H5-dm/st2 gene generated by reverse genetics (RG) technology amplified well in MDCK cells and embryonated chicken eggs. Again, the inactivated H5N1-dm/st2 RG virus elicited more potent cross-clade neutralizing and anti-fusion antibodies in sera. Therefore, the H5N1-dm/st2 RG virus with the site-specific glycan-masking on the globular head and the glycan-unmasking on the stem region of H5 antigen can be used for further development of cross-protective H5N1 vaccines.