Plasmid DNA Complexes Research Articles

The repeated 36 amino acid motif of Chlamydia trachomatis Hc2 protein binds to the major groove of DNA

The gram-negative, obligate intracellular human pathogen, Chlamydia trachomatis has a bi-phasic developmental cycle. The histone H1-like C. trachomatis DNA binding protein, Hc2, is produced late during the developmental cycle when the dividing reticulate body transforms into the smaller, metabolically inactive elementary body. Together with Hc1, the two proteins compact the chlamydial chromosome and arrest replication and transcription. Hc2 is heterogeneous in length due to variation in the number of lysine rich pentamers. Six pentamers and one hexamer constitute a 36 amino acid long repetitive unit that, in spite of variations, is unique for Chlamydiaceae.Using synthetic peptides, the DNA-binding capacity of the 36 amino acid peptide and that of a randomized peptide was analyzed. Both peptides bound and compacted plasmid DNA, however, electron microscopy of peptide/DNA complexes showed major differences in the resulting aggregated structures. Fluorescence spectroscopy was used to analyze the binding. After complexing plasmid DNA with each of three different intercalating dyes, increasing amounts of peptides were added and fluorescence spectroscopy performed. The major groove binder, methyl green, was displaced by both peptides at low concentrations, while the minor groove binder, Hoechts, and the intercalating dye, Ethidium Bromide, were displaced only at high concentrations of peptides.

Effect of lipopolysaccharide addition on the gene transfection of spermine-introduced pullulan-plasmid DNA complexes for human mesenchymal stem cells

The objective of this study is to investigate the effect of lipopolysaccharide (LPS) addition on the gene transfection of human mesenchymal stem cells (hMSC). hMSC were treated with the LPS at different concentrations and the complex of spermine-introduced pullulan and luciferase plasmid DNA for 3 h. The maximum level of gene expression was observed for hMSC treated with a certain concentration range of LPS. In addition, the cytotoxicity, cellular internalization of complexes, and cell cycle after LPS treatment were investigated. The cytotoxicity increased with an increase in the LPS concentration treated. On the other hand, the cellular internalization of complexes increased with the increased LPS concentration, although the internalization was sharply reduced at the high concentration. The LPS treatment increased the actin polymerization of cells to allow to spread more. The enhanced cells spreading would enhance the cellular internalization of complexes. In addition, the LPS treatment increased the rate of cell cycle. It is possible that the balance of cytotoxicity, cellular internalization, and cell cycle caused by the LPS addition results in the enhanced gene transfection at a certain LPS concentration. It is concluded that LPS treatment positively modified the cellular internalization and the cell cycle, resulting in the enhanced gene transfection.

Gene delivery into hepatic cells with ternary complexes of plasmid DNA, cationic liposomes and apolipoprotein E-derived peptide.

Cationic liposomes containing a cationic lipid, such as 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), have often been used for the transduction of plasmid DNA (pDNA) in vivo. However, such liposomes induce gene expression primarily in the lungs after intravenous injection. To improve the delivery of cationic liposomes/pDNA complexes (pDNA lipoplexes) to the liver by intravenous administration, the current study synthesized two apolipoprotein E (ApoE)-derived peptides, dApoE-R9 and ApoE-F-R9, for liver targeting via certain ApoE receptors, including the low-density lipoprotein receptor. Ternary complexes of pDNA, cationic liposomes and ApoE-R9 peptide were also prepared. After in vitro transfection, ternary complexes with DOTAP/1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) liposomes exhibited high transfection activity in HepG2 cells compared with DOTAP/cholesterol (Chol) liposomes. In particular, ternary complexes with dApoE-R9 exhibited high transfection activity in cells compared with ApoE-F-R9. However, in vivo transfection studies revealed that ternary complexes with DOTAP/DOPE liposomes and dApoE-R9 did not increase gene expression in the liver compared with DOTAP/DOPE lipoplexes. In contrast, ternary complexes with DOTAP/Chol liposomes and dApoE-R9 increased gene expression in the liver compared with DOTAP/Chol lipoplexes. The results demonstrated that the in vivo optimal liposomal formulation in ternary complexes with ApoE-R9 peptide for liver delivery were different from those that were in vitro.

Less is more: Investigating the influence of cellular nanoparticle load on transfection outcomes in neural cells.

Genetic engineering of cell transplant populations offers potential for delivery of neurotherapeutic factors to modify the regenerative microenvironment of the injured spinal cord. The use of magnetic nanoparticle (MNP)-based vectors has reduced the traditional reliance on viral methods and their associated obstacles in terms of scale up and safety. Studies utilizing magnetic assistive platforms for MNP-mediated gene delivery have found transfection efficiency in astrocytes (a major transplant and homeostatic neural cell type) to be both frequency- and amplitude-dependent. It is widely assumed that increased intracellular particle load will enhance transfection efficiency in a cell population. Therefore, we tested repeat delivery of MNP:plasmid complexes in conjunction with oscillating magnetic field parameters-a process termed "magneto-multifection"-in astrocytes of primary origin in an attempt to enhance transfection levels. We show (a) levels of transfection using magneto-multifection equal that seen with viral methods; (b) reporter protein expression using two reporter plasmids shows a diverse profile of single/dual transfected cells with implications for delivery of a "cocktail" of neurotherapeutic proteins; and (c) contrary to expectation, an inverse relationship exists between particle load and reporter protein expression.

In Vitro Evaluation of Chitosan-DNA Plasmid Complex Encoding Jembrana Disease Virus Env-TM Protein as a Vaccine Candidate.

IntroductionThe development of Jembrana disease vaccine is an important effort to prevent losses in the Bali cattle industry in Indonesia. This study aims to prepare a Jembrana DNA vaccine encoding the transmembrane portion of the envelope protein in pEGFP-C1 and test the success of its delivery in culture cells using a chitosan-DNA complex.Material and MethodsCloning of the DNA vaccine was successfully performed on E. coli DH5α and confirmed by colony PCR, restriction analysis and sequencing. The plasmids were prepared as a chitosan complex using the complex coacervation method and physicochemically characterised using a particle size analyser. A transfection assay was performed in HeLa cells with 4 h exposure, and mRNA expression was assessed at 24 h post transfection.ResultsWith a 1:2 (wt./wt.) ratio of DNA and chitosan, the complexes have a mean diameter of 236 nm, zeta potential value of + 17.9 mV, and showed no high toxicity potential in the HeLa cells. This complex successfully delivered the DNA into cells, as shown by the presence of a specific RT-PCR product (336 bp). However, the real-time PCR analysis showed that the delivery with chitosan complex resulted in lower target mRNA expression when compared with a commercial transfecting agent.ConclusionpEGFP-env-tm JDV as a candidate vaccine can be delivered as the chitosan-DNA complex and be expressed at the transcription level in vitro. This initial study will be used for further improvement and evaluation in vivo.

Powerful Anticolon Tumor Effect of Targeted Gene Immunotherapy Using Folate-Modified Nanoparticle Delivery of CCL19 To Activate the Immune System

Targeted gene delivery systems have recently shown potential clinical benefits in cancer treatment. Recently, the immunologic therapies application in cancer therapy also showed a continuously increase. CCL19 has shown its great potential as a candidate immunomodulator for colon cancer therapy by increasing the possibility of interaction among dendritic cells, T and B cells in secondary lymphatic tissue, thus regulating the primary (or secondary) adaptive immune responses. In this work, a folic acid modified targeted gene-delivery system consisting of DOTAP, MPEG-PLA, and Fa-PEG-PLA (F-DMA) was developed successfully through a self-assembly approach. We proved that CCL19 expression was much higher in cancer cells after transfection with F-DMA/CCL19 than after transfection with DMA/CCL19. The supernatant from cancer cells transfected with both F-DMA/CCL19 and DMA/CCL19 stimulated the activation and cytotoxicity of T lymphocytes, the maturation of DCs, and the polarization of macrophages in vitro. Moreover, the administration of F-DMA/CCL19 complex to treat tumor-bearing mice has shown significant cancer growth repression in both subcutaneous and peritoneal models. The underling antitumor mechanism is established through repressing neovascularization, promoting apoptosis, as well as reducing proliferation by activating the immune system. The CCL19 plasmid and F-DMA complex may be used as a novel method for colorectal cancer therapy in the clinic.

Vacuum/Compression Infiltration-mediated Permeation Pathway of a Peptide-pDNA Complex as a Non-Viral Carrier for Gene Delivery in Planta

Non-viral gene carriers have been extensively investigated as alternatives to viral vectors for gene delivery systems into animal and plant cells. A non-viral gene carrier containing a cell-penetrating peptide and a cationic sequence was previously developed for use in intact plants and plant cells; however, the permeation pathway of the gene carrier into plant cells is yet to be elucidated, which would facilitate the improvement of the gene delivery efficiency. Here, we identified the vacuum/compression infiltration-mediated permeation pathway of a non-viral gene carrier into plant tissues and cells using a complex of plasmid DNA and a peptide-based gene carrier. This complex was taken up via the hydathodes in Arabidopsis thaliana, and from root hairs in Nicotiana benthamiana. Remarkably, these structurally weak tissues are also routes of bacterial invasion in nature, suggesting that peptide-pDNA complexes invade intact plants through similar pathways as bacterial pathogens.

Efficient Delivery of Plasmid DNA Using Incorporated Nucleotides for Precise Conjugation of Targeted Nanoparticles.

Many obstacles restrict development of DNA plasmid-based therapeutic delivery, involving but not limited to poor cellular uptake, premature material dissociation, and inefficient response. Additionally, lack of precision loading of the plasmids on the carrier nanoparticle may affect the overall nonspecificity in terms of loading as well as the site of loading. Here we report a strategy using the incorporation of a biotin-modified nucleotide into a 4.7 kb plasmid sequence for the site-specific nanoparticle conjugation as an improvement on targeted DNA plasmid delivery. Initially, a designed 80-nucleotide sequence was elongated by incorporating biotin-16-aminoallyl-2'-dCTP that facilitated streptavidin binding as determined via polyacrylamide gel electrophoresis (PAGE). This modified sequence was ligated into a specific location of the EGFP plasmid to avoid possible interference with important functional elements and gene expression off of the plasmid. In parallel, a gold nanoparticle complex comprising of either a CD44 or mutant DNA conjugated aptamer, a PEGylated streptavidin, and a derivatized hyaluronic acid stabilizing polymer was synthesized. To delineate the ability of this nanoparticle-plasmid complex to exhibit an improved cellular delivery, MDA-MB-231 cells were treated with a set of plasmid and plasmid-nanoparticle complexes. Successful expression of EGFP was only observed in cells treated with the biotin-modified EGFP plasmid and a streptavidin-CD44 aptamer-nanoparticle. This demonstrated the need for the specific biotin-streptavidin binding to avoid nanoparticle-plasmid dissociation for improved efficacy. This proof-of-principle concept creates a flexible scaffold that can be assimilated into any plasmid and can produce small RNAs or encoding a therapeutic gene via an installation of a design that uses incorporated modified nucleotides as tethering points for nanoparticles which can play host to stabilizing ligands, additional therapeutic molecules and antibody conjugates among other possibilities. In our system, the nanoparticles are vehicles for the addition of targeting ligands that were essential for cell specificity and enhanced cellular uptake.

Cationized Bombyx mori silk fibroin as a delivery carrier of the VEGF165-Ang-1 coexpression plasmid for dermal tissue regeneration.

The angiogenesis of an implanted construct is among the most important issues in tissue engineering. In this study, spermine was used to modify Bombyx mori silk fibroin (BSF) to synthesize cationized BSF (CBSF). BSF and CBSF were coated in sequence on the surface of polyethyleneimine (PEI)/vascular endothelial growth factor 165/angiopoietin-1 coexpression plasmid DNA (pDNA) complexes to form CBSF/BSF/PEI/pDNA quaternary complexes. BSF scaffolds loaded with carrier/pDNA complexes were prepared as dermal regeneration scaffolds by freeze-drying. In one set of experiments, scaffolds were used to cover a chick embryo chorioallantoic membrane (CAM) to investigate the influence of carrier/pDNA complexes on angiogenesis; in another set of experiments, scaffolds were implanted into dorsal full-thickness wounds in Sprague-Dawley rats to evaluate the effect of carrier/pDNA complex-loaded BSF scaffolds on neovascularization and dermal tissue regeneration. After modification with spermine, the surface zeta potential value of BSF rose to +11 mV from an initial value of -9 mV, and the isoelectric point of BSF increased from 4.20 to 9.04. The in vitro transfection of human umbilical vein endothelial cells (EA.hy926) with quaternary complexes revealed that the CBSF/BSF/PEI/pDNA complexes clearly exhibited lower cytotoxicity and higher transfection efficiency than the PEI/pDNA complexes. The CAM assay showed a more abundant branching pattern of blood vessels in BSF scaffolds loaded with CBSF/BSF/PEI/pDNA complexes than in BSF scaffolds without complexes or loaded with PEI/pDNA complexes. The in vivo experimental results demonstrated that the incorporation of CBSF/BSF/PEI/pDNA complexes could effectively enhance angiogenesis in the implanted BSF scaffolds, thereby promoting the regeneration of dermal tissue, providing a new scaffold for the regeneration of dermal tissue and other tissues containing blood vessels.

Influence of Cell Morphology on Mesenchymal Stem Cell Transfection.

Gene transfection has broad applications in bioengineering and biomedical fields. Although many gene carrier materials and transfection methods have been developed, it remains unclear how cell morphology including cell spreading and elongation affects gene transfection. In this study, human bone marrow-derived mesenchymal stem cells (hMSCs) were cultured on micropatterns and transfected with cationic pAcGFP1-N1 plasmid complexes. The relationship between the cell morphology of hMSCs and gene transfection was investigated using micropatterning techniques. Spreading and elongation of hMSCs were precisely controlled by micropatterned surfaces. The results showed that well-spread and elongated hMSCs had high transfection efficiency. Analysis of the uptake of exogenous genes and DNA synthesis activity indicated that the well-spread and elongated cell morphology promoted gene transfection through enhanced uptake of the cationic complexes and accelerated DNA synthesis. The results should provide useful information for understanding of cell morphology on gene transfection and development of efficient gene transfection methods.

The effect of glucose modification of hydroxyapatite nanoparticles on gene delivery.

Surface modification techniques have been employed for the use of biocompatible and bioresorbable hydroxyapatite (HAp) nanoparticles in cell biology and medicine for the delivery of bioactive molecules. We demonstrated the effects of glucose modification of HAp (GlcHAp) on the transfection efficiency in endothelial cells. After preparing homogeneous HAp nanoparticles with a microemulsion technique, the particles with or without glucose modification and plasmid DNA (pDNA) complexes were transfected into endothelial cells. The transfection efficiency of GlcHAp/pDNA was higher than that of HAp/pDNA. To elucidate the mechanism underlying the improvement in the transfection efficiency following glucose modification, the uptake route into the cells and the inhibition of DNA degradation were investigated. GlcHAp/pDNA enhanced the transfection efficiency after interacting with the glucose transporter 1, as observed by the selective inhibitor assay. In addition, GlcHAp/pDNA was more stable than HAp/pDNA in the DNA degradation assay. Our results suggest that the glucose modification could promote the uptake of HAp nanoparticles by cells and protect the internalized DNA; properties essential for non-viral transfection carriers. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 61-66, 2019.

Peptide T7-modified polypeptide with disulfide bonds for targeted delivery of plasmid DNA for gene therapy of prostate cancer.

BackgroundVectors are essential for successful gene delivery. In the present study, a tumor-targeting cationic gene vector, known as the disulfide cross-linked arginine-aspartic acid peptide modified by HAIYPRH (T7) peptide (CRD-PEG-T7), was designed for targeted delivery of plasmid DNA (pDNA) for gene therapy of prostate cancer (PCa).MethodsThe structure of CRD-PEG-T7 was determined and the cellular uptake efficacy, gene transfection efficacy, cytotoxicity, and the targeting effect of the CRD-PEG-T7–plasmid DNA complex were examined.ResultsThe results demonstrated that the CRD-PEG-T7–plasmid DNA complex was nanosized and had a positively charged surface, good cellular uptake efficacy, minimal cytotoxicity, and a dual-targeting effect as compared with the CRD-PEG–plasmid DNA complex. The peptide T7-modifed new delivery system was able to target the highly expressed transferrin receptor (TfR) on tumor cells with an efficiency four-fold higher than that of the non-modified system.ConclusionThe results above indicatd that the CRD-PEG-T7–plasmid DNA complex may prove to be a promising gene delivery system targeting bone-metastatic tumor.

Versatile Redox-Responsive Polyplexes for the Delivery of Plasmid DNA, Messenger RNA, and CRISPR-Cas9 Genome-Editing Machinery.

Gene therapy holds great promise for the treatment of many diseases, but clinical translation of gene therapies has been slowed down by the lack of safe and efficient gene delivery systems. Here, we report two versatile redox-responsive polyplexes (i.e., cross-linked and non-crosslinked) capable of efficiently delivering a variety of negatively charged payloads including plasmid DNA (DNA), messenger RNA, Cas9/sgRNA ribonucleoprotein (RNP), and RNP-donor DNA complexes (S1mplex) without any detectable cytotoxicity. The key component of both types of polyplexes is a cationic poly( N, N'-bis(acryloyl)cystamine- co-triethylenetetramine) polymer [a type of poly( N, N'-bis(acryloyl)cystamine-poly(aminoalkyl)) (PBAP) polymer] containing disulfide bonds in the backbone and bearing imidazole groups. This composition enables efficient encapsulation, cellular uptake, controlled endo/lysosomal escape, and cytosolic unpacking of negatively charged payloads. To further enhance the stability of non-crosslinked PBAP polyplexes, adamantane (AD) and β-cyclodextrin (β-CD) were conjugated to the PBAP-based polymers. The cross-linked PBAP polyplexes formed by host-guest interaction between β-CD and AD were more stable than non-crosslinked PBAP polyplexes in the presence of polyanionic polymers such as serum albumin, suggesting enhanced stability in physiological conditions. Both cross-linked and non-crosslinked polyplexes demonstrated either similar or better transfection and genome-editing efficiencies, and significantly better biocompatibility than Lipofectamine 2000, a commercially available state-of-the-art transfection agent that exhibits cytotoxicity.

Packaging pDNA by Polymeric ABC Micelles Simultaneously Achieves Colloidal Stability and Structural Control.

Compaction of DNA by oppositely charged nanoparticles is a fundamental phenomenon in nature and of great interest to developing therapeutics. In addition, the ability to orthogonally control the composition and structure of interpolyelectrolyte complexes is needed to develop materials for diverse applications. Herein, we systematically investigate the complexation of plasmid DNA and polymeric cationic AB and ABC micelles to explore the influence of micelle outer nonionic corona length on the colloidal stability, size, composition, and structure of the resulting "micelleplexes". The micelles were self-assembled from amphiphilic block polymers, poly(ethylene glycol)- block-poly((2-dimethylamino)ethyl methacrylate)- block-poly( n-butyl methacrylate) (PEG- b-PDMAEMA- b-PnBMA), and PDMAEMA- b-PnBMA with the same Mn of PDMAEMA. These spherical micelles have similar hydrodynamic radii and core sizes, but the Mn of the outer PEG block ranged from 0 to 10 kDa. The colloidal stability of micelleplexes as a function of stoichiometric charge ratio was assessed by turbidimetric titration and was found to dramatically improve with the addition of an outer PEG corona, even as short as 2 kDa. With the use of a combination of dynamic and static light scattering, ζ-potential, and cryogenic transmission electron microscopy, it was found that the size, composition, and structure of micelleplexes are closely correlated with the Mn of the PEG block. Indeed, these micelleplexes were found to adopt beads-on-a-string morphologies that resemble the general structure of chromatin, and the number of micelles per micelleplex systematically decreased with increasing PEG length. These findings demonstrate the power of polycationic micelles to condense DNA into biomimetic structures and provide a mechanistic understanding of nucleic acid complexation and of how micelle architecture affects the properties of micelleplexes, while offering an appealing strategy to control the properties of micelleplexes by tuning a single parameter.

Recombinant Histones as an Instrument for the Delivery of Nucleic Acids into Eukaryotic Cells

Naturally occurring positively charged proteins can be promising carriers for nucleic acid transport in gene therapy. The most attractive alternative among them is histones. In this work, we describe expression and purification of recombinant human histones H2A and H2B and of chimeric histone H2A with HIV-1 TAT fragment (TAT-peptide). The proposed method of purification of histone proteins can significantly reduce the content of bacterial endotoxins in the target preparation, which makes it possible to use these proteins in in vivo experiments. The transfection ability of plasmid DNA complexes with core histones H2A and H2B and the chimeric histone was demonstrated. A highly specific and efficient transfection of human HT1080 cell line with the use of histones H2A and H2B was detected, whereas transfection by plasmid DNA complexes with chimeric H2A-TAT protein was observed for many cell lines.

Delivery of a Modified mRNA Encoding IL-22 Binding Protein (IL-22BP) for Colon Cancer Gene Therapy

As an alternative form of genetic material, mRNA lacks a CpG island and can function without crossing the nuclear membrane. These properties make mRNA less of a potential immune stimulant than plasmid DNA. Therefore, chemically modified mRNA is an effective alternative to plasmid DNA for gene therapy. In this study, cationic liposomes were used as a vector to transport mRNA complexes that had been compressed using protamine and to obtain high mRNA transport and expression efficiency. The liposome-protamine-IL-22BP mRNA complex can strongly inhibit the growth of C26 tumour cells by inducing apoptosis, inhibiting tumour angiogenesis and increasing the number of immune cells that infiltrate the tumour microenvironment. The obvious tumour regression and safety of this approach were observed in peritoneal metastasis and subcutaneous xenograft models. The antitumour effect of the liposome protamine mRNA complex was as good as that for a plasmid DNA complex, and demonstrated the potential for mRNA-based gene therapy.

Complexation of plasmid DNA and poly(ethylene oxide)/poly(propylene oxide) polymers for safe gene delivery

Gene delivery is the process of introducing foreign genetic material, such as DNA or RNA, into host cells. Gene therapy utilizes gene delivery to deliver genetic material with the goal of treating a disease or condition in the cell. Actual viral vectors may have side effects, while actual systems using metal nanoparticles for gene delivery are toxic. Therefore, we designed here a biocompatible tri-block copolymer PEO20–PPO69–PEO20 as a gene delivery vector [PEO: poly(ethylene oxide); PPO: poly(propylene oxide)]. We studied the conjugation of PEO20–PPO69–PEO20 and DNA using various techniques. Results of gel retardation assay along with zeta potential and dynamic light scattering provide evidence of DNA sequestration. Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy show that the PO43− groups of plasmid DNA are primarily involved during nanoconjugate construction. The integrity and functionality of plasmid DNA within the cellular environment is further demonstrated by the expression of green fluorescent protein gene in Escherichia coli. Overall, our findings support the use of block copolymers as delivery systems for mammalian and plant cells.

Beyond Gene Transfection with Methacrylate-Based Polyplexes-The Influence of the Amino Substitution Pattern.

Methacrylate-based polymers represent promising nonviral gene delivery vectors, since they offer a large variety of polymer architectures and functionalities, which are beneficial for specific demands in gene delivery. In combination with controlled radical polymerization techniques, such as the reversible addition-fragmentation chain transfer polymerization, the synthesis of well-defined polymers is possible. In this study we prepared a library of defined linear polymers based on (2-aminoethyl)-methacrylate (AEMA), N-methyl-(2-aminoethyl)-methacrylate (MAEMA), and N,N-dimethyl-(2-aminoethyl)-methacrylate (DMAEMA) monomers, bearing pendant primary, secondary, and tertiary amino groups, and investigated the influence of the substitution pattern on their gene delivery capability. The polymers and the corresponding plasmid DNA complexes were investigated regarding their physicochemical characteristics, cytocompatibility, and transfection performance. The nonviral transfection by methacrylate-based polyplexes differs significantly from poly(ethylene imine)-based polyplexes, as a successful transfection is not affected by the buffer capacity. We observed that polyplexes containing a high content of primary amino groups (AEMA) offered the highest transfection efficiency, whereas polyplexes bearing tertiary amino groups (DMAEMA) exhibited the lowest transfection efficiency. Further insights into the uptake and release mechanisms could be identified by fluorescence and transmission electron microscopy, emphasizing the theory of membrane-pore formation for the time-efficient endosomal release of methacrylate-based vectors.

Engineered CRISPR/Cas9 system for multiplex genome engineering of polyploid industrial yeast strains

The CRISPR/Cas9 system has been widely used for multiplex genome engineering of Saccharomyces cerevisiae. However, its application in manipulating industrial yeast strains is less successful, probably due to the genome complexity and low copy numbers of gRNA expression plasmids. Here we developed an efficient CRISPR/Cas9 system for industrial yeast strain engineering by using our previously engineered plasmids with increased copy numbers. Four genes in both a diploid strain (Ethanol Red, 8 alleles in total) and a triploid strain (ATCC 4124, 12 alleles in total) were knocked out in a single step with 100% efficiency. This system was used to construct xylose-fermenting, lactate-producing industrial yeast strains, in which ALD6, PHO13, LEU2, and URA3 were disrupted in a single step followed by the introduction of a xylose utilization pathway and a lactate biosynthetic pathway on auxotrophic marker plasmids. The optimized CRISPR/Cas9 system provides a powerful tool for the development of industrial yeast based microbial cell factories.

Plasmid-Templated Control of DNA-Cyclodextrin Nanoparticle Morphology through Molecular Vector Design for Effective Gene Delivery.

Engineering self-assembled superstructures through complexation of plasmid DNA (pDNA) and single-isomer nanometric size macromolecules (molecular nanoparticles) is a promising strategy for gene delivery. Notably, the functionality and overall architecture of the vector can be precisely molded at the atomic level by chemical tailoring, thereby enabling unprecedented opportunities for structure/self-assembling/pDNA delivery relationship studies. Beyond this notion, by judiciously preorganizing the functional elements in cyclodextrin (CD)-based molecular nanoparticles through covalent dimerization, here we demonstrate that the morphology of the resulting nanocomplexes (CDplexes) can be tuned, from spherical to ellipsoidal, rod-type, or worm-like nanoparticles, which makes it possible to gain understanding of their shape-dependent transfection properties. The experimental findings are in agreement with a shift from chelate to cross-linking interactions on going from primary-face- to secondary-face-linked CD dimers, the pDNA partner acting as an active payload and as a template. Most interestingly, the transfection efficiency in different cells was shown to be differently impacted by modifications of the CDplex morphology, which has led to the identification of an optimal prototype for tissue-selective DNA delivery to the spleen in vivo.