Synthetic and Systems Biotechnology
·
2024
·
IF 4.4
Bing Fu,Meng Chen,Xianfeng Bao,Jiajie Lu,Zhiwen Zhu,Fuyao Guan,Chuyang Yan,Peize Wang,Linglin Fu,Ping Yu
Vitamin B 2 is an essential water-soluble vitamin. For most prokaryotes, a bifunctional enzyme called FAD synthase catalyzes the successive conversion of riboflavin to FMN and FAD. In this study, the plasmid pNEW-AZ containing six key genes for the riboflavin synthesis was transformed into strain R2 with the deleted FMN riboswitch, yielding strain R5. The R5 strain could produce 540.23 ± 5.40 mg/L riboflavin, which was 10.61 % higher than the R4 strain containing plasmids pET-AE and pAC-Z harboring six key genes. To further enhance the production of riboflavin, homology matching and molecular docking were performed to identify key amino acid residues of FAD synthase. Nine point mutation sites were identified. By comparing riboflavin kinase activity, mutations of T203D and N210D, which respectively decreased by 29.90 % and 89.32 % compared to wild-type FAD synthase, were selected for CRISPR/Cas9 gene editing of the genome, generating engineered strains R203 and R210. pNEW-AZ was transformed into R203, generating R6. R6 produced 657.38 ± 47.48 mg/L riboflavin, a 21.69 % increase compared to R5. This study contributes to the high production of riboflavin in recombinant E. coli BL21.
研究领域:
生物工程
·
分子生物学
✓ 已验证
DOI
PubMed
高影响因子
Pharmaceutics
·
2024
·Vol. 16
·期 2
·213
·
IF 4.9
Jiahui Ding,Handan Zhang,Tianli Dai,Xueqin Gao,Zhongyuan Yin,Qiong Wang,Mengqi Long,Songwei Tan
Poly (β-amino ester) (PBAE) is an exceptional non-viral vector that is widely used in gene delivery, owing to its exceptional biocompatibility, easy synthesis, and cost-effectiveness. However, it carries a high surface positive charge that may cause cytotoxicity. Therefore, hydrophilic d-α-tocopherol polyethylene glycol succinate (TPGS) was copolymerised with PBAE to increase the biocompatibility and to decrease the potential cytotoxicity of the cationic polymer-DNA plasmid polyplex nanoparticles (NPs) formed through electrostatic forces between the polymer and DNA. TPGS-b-PBAE (TBP) copolymers with varying feeding molar ratios were synthesised to obtain products of different molecular weights. Their gene transfection efficiency was subsequently evaluated in HEK 293T cells using green fluorescent protein plasmid (GFP) as the model because free GFP is unable to easily pass through the cell membrane and then express as a protein. The particle size, ζ-potential, and morphology of the TBP2-GFP polyplex NPs were characterised, and plasmid incorporation was confirmed through gel retardation assays. The TBP2-GFP polyplex NPs effectively transfected multiple cells with low cytotoxicity, including HEK 293T, HeLa, Me180, SiHa, SCC-7 and C666-1 cells. We constructed aMUC2(Mucin2)-targeting CRISPR/cas9 gene editing system in HEK 293T cells, with gene disruption supported by oligodeoxynucleotide (ODN) insertion in vitro. Additionally, we developed anLMP1(latent membrane protein 1)-targeting CRISPR/cas9 gene editing system in LMP1-overexpressing SCC7 cells, which was designed to cleave fragments expressing the LMP1 protein (related to Epstein–Barr virus infection) and thus to inhibit the growth of the cells in vivo. As evidenced by in vitro and in vivo experiments, this system has great potential for gene therapy applications.Keywords:poly (β-amino ester);gene transfection;D-α-tocopherol polyethylene glycol succinate;copolymer;CRISPR/cas9 gene editing;gene therapy
研究领域:
生物技术
·
基因治疗
·
药学
·
分子生物学
✓ 已验证
DOI
PubMed
高影响因子
Science Advances
·
2019
·Vol. 5
·期 6
·
IF 12.804
Chongyi Liu,Tao Wan,Hui Wang,Song Zhang,Yuan Ping,Yiyun Cheng
Cytosolic protein delivery is of central importance for the development of protein-based biotechnologies and therapeutics; however, efficient intracellular delivery of native proteins remains a challenge. Here, we reported a boronic acid–rich dendrimer with unprecedented efficiency for cytosolic delivery of native proteins. The dendrimer could bind with both negatively and positively charged proteins and efficiently delivered 13 cargo proteins into the cytosol of living cells. All the delivered proteins kept their bioactivities after cytosolic delivery. The dendrimer ensures efficient intracellular delivery of Cas9 protein into various cell lines and showed high efficiency in CRISPR-Cas9 genome editing. The rationally designed boronic acid–rich dendrimer permits the development of an efficient platform with high generality for the delivery of native proteins.
研究领域:
生物技术
·
药学
·
分子生物学
·
基因组编辑
✓ 已验证
产品关联
DOI
PubMed
高影响因子
Nature Communications
·
2018
·Vol. 9
·期 1
·1-15
·
IF 12.353
Usman Waqas Muhammad,Pham Tin Chanh,Kwok Yuk Yan,Vu Luyen Tien,Ma Victor,Peng Boya,Chan Yuen San,Wei Likun,Chin Siew Mei,Azad Ajijur,He Alex Bai-Liang,Leung Anskar Y. H.,Yang Mengsu,Shyh-Chang Ng,Cho William C.,Shi Jiahai,Le Minh T. N.
Most of the current methods for programmable RNA drug therapies are unsuitable for the clinic due to low uptake efficiency and high cytotoxicity. Extracellular vesicles (EVs) could solve these problems because they represent a natural mode of intercellular communication. However, current cellular sources for EV production are limited in availability and safety in terms of horizontal gene transfer. One potentially ideal source could be human red blood cells (RBCs). Group O-RBCs can be used as universal donors for large-scale EV production since they are readily available in blood banks and they are devoid of DNA. Here, we describe and validate a new strategy to generate large-scale amounts of RBC-derived EVs for the delivery of RNA drugs, including antisense oligonucleotides, Cas9 mRNA, and guide RNAs. RNA drug delivery with RBCEVs shows highly robust microRNA inhibition and CRISPR–Cas9 genome editing in both human cells and xenograft mouse models, with no observable cytotoxicity.
研究领域:
药理学
·
分子生物学
·
基因治疗
·
生物技术
✓ 已验证
产品关联
DOI
PubMed
高影响因子
ACS Applied Materials & Interfaces
·
2024
·
IF 8.3
Junyi Zeng,Xinbo Huang,Yajie Yang,Jieyi Wang,Yuanchao Shi,Hui Li,Ning Hu,Bo Yu,Jing Mu
Controlling CRISPR/Cas9 gene editing at the spatiotemporal resolution level, especially for in vivo applications, remains a great challenge. Here, we developed a near-infrared (NIR) light-activated nanophotonic system (UCPP) for controlled CRISPR-Cas9 gene editing and synergistic photodynamic therapy (PDT). Lanthanide-doped upconversion nanoparticles are not only employed as carriers for intracellular plasmid delivery but also serve as the nanotransducers to convert NIR light (980 nm) into visible light with emission at 460 and 650 nm, which could result in simultaneous activation of gene editing and PDT processes, respectively. Such unique design not only achieves light-controlled precise gene editing of hypoxia-inducible factor 1α with minimal off-target effect, which effectively ameliorates the hypoxic state at tumor sites, but also facilitates the deep-seated PDT process with synergistic antitumor effect. This optogenetically activatable CRISPR-Cas9 nanosystem holds great potential for spatially controlled in vivo gene editing and targeted cancer therapy.
研究领域:
生物医学工程
·
分子生物学
·
基因治疗
·
癌症研究
·
纳米技术
✓ 已验证
产品关联
DOI
PubMed
高影响因子
危险品化学品经营许可证（带存储）