Extracellular Vesicle-Liposome Hybrid Nanoparticles Delivery of CRISPR/Cas9 Induces a Unique DNA Repair Pattern in the HGF Gene of Stem Cells from Apical Papilla Razieh Yazdani, Mohammad Hossein Nasr Esfahani, Shahin Eghbalsaied, Fereshteh Karamali DNA and Cell Biology, 2025 Extracellular vesicles (EVs) have been investigated due to their natural biocompatibility and targeting capabilities. The specific approach of combining EVs with liposomes to create hybrid nanoparticles (ELNPs) for the delivery of the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas9) system for deletion of the HGF gene in stem cells, but their effectiveness in encapsulating large nucleic acids is limited due to their small size. This study aimed to knock out the HGF gene by the CRISPR/Cas9 system by ELNPs, and it was expected that the efficiency of the CRISPR/Cas9 system transfer would increase compared to the usual methods of using lipofectamine in stem cells from apical papilla (SCAPs). In this study, gRNA suitable for the HGF gene is designed first, and after insertion into the CRISPR/Cas9 vector, it enters Lipofectamine 2000. In the next step, ELNPs are prepared after collecting EVs and hybridizing them with liposomes containing CRISPR/Cas9 vector. Then, these integrated nanoparticles were presented to SCAPs, and the removal of HGF gene expression was evaluated at the level of RNA and protein. This study showed that the CRISPR/Cas9 system can be efficiently transferred to SCAP cells using ELNPs. Genomic DNA sequencing analyses of SCAP cells showed a unique pattern of mutation, highly likely mediated through EVs. Quantitative PCR and protein staining further showed a decrease in HGF gene expression in the knockout cells. Moreover, cell proliferation analysis showed a decrease in cell proliferation in KO-HGF adipose cells compared to the nonedited counterpart. In summary, this study highlights the supportive role of EVs in facilitating cell transfection and promoting a dominant DNA repair pattern, likely through an RNA-mediated mechanism, rather than the random insertions and deletions typically induced during CRISPR editing of the HGF gene in SCAPs.
Detection and phylogenetic analysis of new iranian isolates of cucumber mosaic virus on achillea species Journal of Crop Protection, 2021
Production and characterization of virus-like particles of grapevine fanleaf virus presenting L2 epitope of human papillomavirus minor capsid protein Razieh Yazdani, Masoud Shams-Bakhsh, Afshin Hassani-Mehraban, Seyed Shahriar Arab, Nicolas Thelen, et al. BMC Biotechnology, 2019 Background Virus-like particle (VLP) platform represents a promising approach for the generation of efficient and immunogenic subunit vaccines. Here, the feasibility of using grapevine fanleaf virus (GFLV) VLPs as a new carrier for the presentation of human papillomavirus (HPV) L2 epitope was studied. To achieve this goal, a model of the HPV L2 epitope secondary structure was predicted and its insertion within 5 external loops in the GFLV capsid protein (CP) was evaluated. Results The epitope sequence was genetically inserted in the αB-αB” domain C of the GFLV CP, which was then over-expressed in Pichia pastoris and Escherichia coli. The highest expression yield was obtained in E. coli. Using this system, VLP formation requires a denaturation-refolding step, whereas VLPs with lower production yield were directly formed using P. pastoris, as confirmed by electron microscopy and immunostaining electron microscopy. Since the GFLV L2 VLPs were found to interact with the HPV L2 antibody under native conditions in capillary electrophoresis and in ELISA, it can be assumed that the inserted epitope is located at the VLP surface with its proper ternary structure. Conclusions The results demonstrate that GFLV VLPs constitute a potential scaffold for surface display of the epitope of interest.
Association of Pantoea ananatis and Pantoea agglomerans with leaf spot disease on ornamental plants of Araceae Family R. Yazdani, N. Safaie, M. Shams-Bakhsh European Journal of Plant Pathology, 2018 During a survey in 2011–2012, three ornamental plants of Araceae namely Aglaonema nitidum, Syngonium podophyllum and Dieffenbachia amoena showing foliar disease symptoms were collected from central region of Iran. Infected plants exhibited spots on their leaves which appeared as yellow and water-soaked with chlorotic haloes and necrotic center. To investigate the etiology of this disorder, symptomatic leaves were collected from affected plants and six bacterial strains (B2Y, J3Y, SY, E60Y, E68Y and E5MM) were isolated and identified as Pantoea ananatis or P. agglomerans based on morphological, physiological, biochemical and molecular characters. The pathogenicity tests of the isolates demonstrated that they were not host specific. Furthermore, 16S rRNA gene sequencing revealed that the strains were phylogenetically closely related to genus Pantoea. Multilocus sequence analysis (MLSA) of concatenated partial atpD, gyrB and rpoB gene sequences of the six isolates showed a high similarity of B2Y, J3Y, and SY strains to P. ananatis and also of E60Y, E5MM and E68Y strains to P. agglomerans. These results were confirmed by phylogenetic analysis. To the best of our knowledge, this is the first report of leaf spot and necrosis of A. nitidum, S. podophyllum and D. amoena caused by the genus Pantoea.
Publications
1-Yazdani, R., N. Safaie, and M. Shams-Bakhsh. "Association of Pantoea ananatis and Pantoea agglomerans with leaf spot disease on ornamental plants of Araceae European Journal of Plant Pathology. (2018), 150(1), 167-178.
2- Abtahi, Faezehossadat, Mehrnaz Hatami, Hossein Salehi-Arjmand, Majid Mahdiyeh, and Razieh Yazdani. "Detection and phylogenetic analysis of new Iranian isolates of Cucumber mosaic virus on Achillea Journal of Crop Protection 10, no. 2 (2021): 309-317.
3-Yazdani, Razieh, Masoud Shams-Bakhsh, Afshin Hassani-Mehraban, Seyed Shahriar Arab, Nicolas Thelen, Marc Thiry, Jacques Crommen et al. "Production and characterization of virus-like particles of grapevine fanleaf virus presenting L2 epitope of human papillomavirus minor capsid BMC biotechnology 19, no. 1 (2019): 1-12.
