BackgroundThe continuing spread of the newly emerged H7N9 virus among poultry in China, as well as the possibility of human-to-human transmission, has attracted numerous efforts to develop an effective vaccine against H7N9. The use of nanoparticles in vaccinology is inspired by the fact that most pathogens have a dimension within the nano-size range and therefore can be processed efficiently by the immune system, which leads to a potent immune response. Herein, we report a facile approach to increase antigen size to achieve not only fast but also effective responses against the recombinant HA/H7N9 protein via a simple conjugation of the protein onto the surface of nanodiamond particles.ResultsIn this study, trimeric Haemagglutinin (H7) that is transiently expressed in N. benthamiana was purified using affinity chromatography, and its trimeric state was revealed successfully by the cross-linking reaction. The trimeric H7 solution was subsequently mixed with a nanodiamond suspension in different ratios. The successful conjugation of the trimeric H7 onto the surface of nanodiamond particles was demonstrated by the changes in size and Zeta-potential of the particles before and after protein coating, Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and Western-blot analysis. Next, biofunction of the protein-nanodiamond conjugates was screened using a haemagglutination assay. A mixture containing 5 µg of trimeric H7 and 60 µg of nanodiamond corresponds to a ratio of 1:12 (w/w) of agglutinated chicken red blood cells at HA titer of 1024, which is 512-fold higher than the HA titer of free trimeric H7. After the 2nd and 3rd immunization in mice, ELISA and Western blot analyses demonstrated that the physical mixture of trimeric H7 protein and nanodiamond (1:12, w/w) elicited statistically significant stronger H7-specific-IgG response demonstrated by higher amounts of H7N9-specific IgG (over 15.4-fold with P < 0.05 after the second immunization).ConclusionsThese results indicated a potential effect inherent to nanodiamond towards modulating immune systems, which should be further evaluated and broadly applied in nanovaccine development.

Raffinose family oligosaccharides (RFOs) are major soluble carbohydrates in soybean seeds that cannot be digested by human and other monogastric animals. Hence, a major goal is to reduce RFO levels to improve the nutritional quality of soybean. In this study, we utilized a dual gRNAs CRISPR/Cas9 system to induce knockouts in two soybean galactinol synthase (GOLS) genes, GmGOLS1A and its homeolog GmGOLS1B. Genotyping of T0 plants showed that the construct design was efficient in inducing various deletions in the target sites or sequences spanning the two target sites of both GmGOLS1A and GmGOLS1B genes. A subset of induced alleles was successfully transferred to progeny and, at the T2 generation, we identified null segregants of single and double mutant genotypes without off-target induced mutations. The seed carbohydrate analysis of double mutant lines showed a reduction in the total RFO content of soybean seed from 64.7 mg/g dry weight to 41.95 mg/g dry weight, a 35.2% decrease. On average, the stachyose content, the most predominant RFO in soybean seeds, decreased by 35.4% in double mutant soybean, while the raffinose content increased by 41.7%. A slight decrease in verbascose content was also observed in mutant lines. Aside from changes in soluble carbohydrate content, some mutant lines also exhibited increased protein and fat contents. Otherwise, no difference in seed weight, seed germination, plant development and morphology was observed in the mutants. Our findings indicate that GmGOLS1A and GmGOLS1B contribute to the soybean oligosaccharide profile through RFO biosynthesis pathways, and are promising targets for future investigation, as well as crop improvement efforts. Our results also demonstrate the potential in using elite soybean cultivars for transformation and targeted genome editing.

Hairy root induction system has been applied in various plant species as an effective method to study gene expression and function due to its fast-growing and high genetic stability. Recently, these systems have shown to be an effective tool to evaluate activities of CRISPR/Cas9 systems for genome editing. In this study, Rhizobium rhizogenes mediated hairy root induction was optimized to provide an effective tool for validation of plant transformation vector, CRISPR/Cas9 construct activities as well as selection of targeted gRNAs for gene editing in cucumber (Cucumis sativus L.). Under the optimized conditions including OD650 at 0.4 for infection and 5 days of co-cultivation, the highest hairy root induction frequency reached 100% for the cucumber variety Choka F1. This procedure was successfully utilized to overexpress a reporter gene (gus) and induce mutations in two Lotus japonicus ROOTHAIRLESS1 homolog genes CsbHLH66 and CsbHLH82 using CRISPR/Cas9 system. For induced mutation, about 78% of transgenic hairy roots exhibited mutant phenotypes including sparse root hair and root hair-less. The targeted mutations were obtained in individual CsbHLH66, CsbHLH82, or both CsbHLH66 and CsbHLH82 genes by heteroduplex analysis and sequencing. The hairy root transformation system established in this study is sufficient and potential for further research in genome editing of cucumber as well as other cucumis plants.

Tobacco is an important commercial crop and a rich source of alkaloids for pharmaceutical and agricultural applications. However, its yield can be reduced by up to 70% due to virus infections, especially by a potyvirus Potato virus Y (PVY). The replication of PVY relies on host factors, and eukaryotic translation initiation factor 4Es (eIF4Es) have already been identified as recessive resistance genes against potyviruses in many plant species. To investigate the molecular basis of PVY resistance in the widely cultivated allotetraploid tobacco variety K326, we developed a dual guide RNA CRISPR/Cas9 system for combinatorial gene editing of two clades, eIF4E1 (eIF4E1-S and eIF4E1-T) and eIF4E2 (eIF4E2-S and eIF4E2-T) in the eIF4E gene family comprising six members in tobacco. We screened for CRISPR/Cas9-induced mutations by heteroduplex analysis and Sanger sequencing, and monitored PVYO accumulation in virus challenged regenerated plants by DAS-ELISA both in T0 and T1 generations. We found that all T0 lines carrying targeted mutations in the eIF4E1-S gene displayed enhanced resistance to PVYO confirming previous reports. More importantly, our combinatorial approach revealed that eIF4E1-S is necessary but not sufficient for complete PVY resistance. Only the quadruple mutants harboring loss-of-function mutations in eIF4E1-S, eIF4E1-T, eIF4E2-S and eIF4E2-T showed heritable high-level resistance to PVYO in tobacco. Our work highlights the importance of understanding host factor redundancy in virus replication and provides a roadmap to generate virus resistance by combinatorial CRISPR/Cas9-mediated editing in non-model crop plants with complex genomes.

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Tóm tắt: Việc tăng năng suất tinh bột sử dụng công nghệ gen là một trong những hướng nghiên cứu quan trọng và luôn được các nhà khoa học quan tâm hàng đầu. Do vậy, các nghiên cứu tìm hiểu về con đường tổng hợp và phân hủy tinh bột ở cây trồng nói chung và đối với sắn nói riêng sẽ góp phần thúc đẩy mục tiêu cải tạo năng suất tinh bột. Các starch synthase (SS) của thực vật bậc cao mã hóa bởi 5 nhóm gen ký hiệu là GBSS (granule-bound starch synthase), SSI, SSII, SSIII, và SSVI. Trong đó, mỗi biến thể enzyme SS có các cấu thành khác nhau và vai trò nhất định trong tổng hợp amylopectin. Ở nghiên cứu này chúng tôi đã phân lập được gen ssiv từ giống sắn KM140. Cấu trúc này được chèn vào vector pK7WG2D-35S:SSIV:T35S và biến nạp vào cây thuốc lá bằng phương pháp chuyển gen thông qua A. tumefaciens. S a u đ ó , cây chuyển gen được kiểm tra bằng phương pháp PCR và đánh giá sự biểu hiện của gen qua phương pháp phân tích hàm lượng tinh bột. Kết quả cho thấy hàm lượng tinh bột tích lũy trong cây chuyển gen vượt trội hơn các cây không chuyển gen (26,9 -67,9%; rễ 6,8 -17,6%) ở cùng điều kiện sinh trưởng. Nghiên cứu này đã tạo ra một hướng mới trong việc tạo cây trồng biến đổi gen có khả năng tăng sự tích lũy tinh bột.

Papaya is one of the most important fruits in tropical and subtropical countries. However, genetic improvement has had limited success to date due to time-consuming and complex transformation and regeneration technologies, as well as a lack of reproducible and efficient transient gene expression assays. Here, we report the development of a highly efficient Rhizobium rhizogenes-based in vivo hairy root system for evaluating transgene expression and activity including CRISPR/Cas gene editing reagents in the Vietnamese papaya cultivar Linhan.To optimize the papaya transformation parameters, we introduced the R. rhizogenes strain K599 into papaya hypocotyls at 1-, 5-and 10-mm below the cotyledon nodes by a needle using 5-, 7-and 10-day old seedlings and then monitored the frequency of hairy root formation at 18 days post infection. We found that the age of the seedlings and the distance of the infection site from the cotyledon node were inversely correlated with the efficacy of hairy root induction, being 5-day-old plants and 1-mm distance the best parameters. The etablished protocol was then employed to investigate transformation frequency using the GUS reporter gene. Of the tested hairy roots, 47.22% were positive for GUS staining, which indicates high level of transgene transfer and stability. Finally, we introduced a dual guide RNA CRISPR/Cas9 cassette targeting eukaryotic translation initiation factor isoform 4E (eIF(iso)4E) gene into papaya by R. rhizogenes and then screened for gene editing events by heteroduplex analysis and Sanger sequencing. Our analysis revealed that 50% of induced roots contained the expected mutations in the eIF(iso)4E gene, which makes our system ideal for testing transgene activity prior making stable transgenic papaya lines. Key messageWe developed an efficient procedure for papaya in vivo hairy root induction which may be used to validate transgene expression and accelerate CRISPR/Cas-based genome editing studies in papaya.

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