Amphipathic Cell-Penetrating Peptide-Aided Delivery of Cas9 RNP for In Vitro Gene Editing and Correction.

The therapeutic potential of the CRISPR-Cas9 gene editing system in treating numerous genetic disorders is immense. To fully realize this potential, it is crucial to achieve safe and efficient delivery of CRISPR-Cas9 components into the nuclei of target cells. In this study, we investigated the applicability of the amphipathic cell-penetrating peptide LAH5, previously employed for DNA delivery, in the intracellular delivery of spCas9:sgRNA ribonucleoprotein (RNP) and the RNP/single-stranded homology-directed repair (HDR) template. Our findings reveal that the LAH5 peptide effectively formed nanocomplexes with both RNP and RNP/HDR cargo, and these nanocomplexes demonstrated successful cellular uptake and cargo delivery. The loading of all RNP/HDR components into LAH5 nanocomplexes was confirmed using an electrophoretic mobility shift assay. Functional screening of various ratios of peptide/RNP nanocomplexes was performed on fluorescent reporter cell lines to assess gene editing and HDR-mediated gene correction. Moreover, targeted gene editing of the CCR5 gene was successfully demonstrated across diverse cell lines. This LAH5-based delivery strategy represents a significant advancement toward the development of therapeutic delivery systems for CRISPR-Cas-based genetic engineering in in vitro and ex vivo applications.

Impact of Formulation Conditions on Lipid Nanoparticle Characteristics and Functional Delivery of CRISPR RNP for Gene Knock-Out and Correction.

The CRISPR-Cas9 system is an emerging therapeutic tool with the potential to correct diverse genetic disorders. However, for gene therapy applications, an efficient delivery vehicle is required, capable of delivering the CRISPR-Cas9 components into the cytosol of the intended target cell population. In this study, we optimized the formulation conditions of lipid nanoparticles (LNP) for delivery of ready-made CRISPR-Cas9 ribonucleic protein (RNP). The buffer composition during complexation and relative DOTAP concentrations were varied for LNP encapsulating in-house produced Cas9 RNP alone or Cas9 RNP with additional template DNA for gene correction. The LNP were characterized for size, surface charge, and plasma interaction through asymmetric flow field flow fractionation (AF4). Particles were functionally screened on fluorescent reporter cell lines for gene knock-out and gene correction. This revealed incompatibility of RNP with citrate buffer and PBS. We demonstrated that LNP for gene knock-out did not necessarily require DOTAP, while LNP for gene correction were only active with a low concentration of DOTAP. The AF4 studies additionally revealed that LNP interact with plasma, however, remain stable, whereby HDR template seems to favor stability of LNP. Under optimal formulation conditions, we achieved gene knock-out and gene correction efficiencies as high as 80% and 20%, respectively, at nanomolar concentrations of the CRISPR-Cas9 RNP.

The role of silver ions in the regulation of the senescence process in Triticum aestivum.

The control of senescence has economic importance due to its effects on parameters such as herbal product quality and shelf life. This study is on the control of induced senescence in Triticum aestivum L. 'Gün-91' plants with silver nitrate (AgNO3) treatments. It was observed that some changes that occurred with dark and indole-1-acetic acid (IAA) treatments could be reduced with AgNO3 treatments. After dark-induced senescence, it was observed in plants that seedling length, relative water content (RWC), chlorophyll, ß-carotene, xanthophylls, total antioxidant capacity, soluble phenol, total soluble protein, catalase (CAT), total superoxide dismutase (SOD), copper-zinc superoxide dismutase (Cu/Zn-SOD) activities, and expression of genes encoding these enzymes declined. After IAA treatments, seedling length, RWC, chlorophyll, ß-carotene, xanthophylls, total antioxidant capacity, soluble phenolics, and soluble protein levels declined, whereas activities of CAT, total SOD, and Cu/Zn-SOD enzymes and expression of Cu/Zn-SOD and CAT genes increased. AgNO3 (200 mg L-1 ) applied by spraying onto leaves led to an increase in seedling length, RWC, chlorophyll, ß-carotene, xanthophylls, total antioxidant capacity, soluble phenolics, soluble protein levels, and expression of Cu/Zn-SOD, CAT genes, CAT, SOD, and Cu/Zn-SOD enzyme activities compared to controls. Findings obtained from this study showed that the senescence process was related to changes in the levels of antioxidant compounds and enzymes. It was defined that the role of silver ions in slowing senescence was related to antioxidant defense capacity.

Canine Infections and Partial S Segment Sequence Analysis of Toscana Virus in Turkey.

INTRODUCTION: Toscana virus (TOSV) is a sandfly-borne bunyavirus with a significant public health impact. Preliminary studies have revealed TOSV exposure in dogs and they were suggested as potential reservoirs. This study was performed to characterize canine TOSV infections in an endemic region. Sequencing of TOSV small (S) segment in several previously identified specimens was also undertaken to reveal viral genealogy. MATERIALS AND METHODS: Canine and feline plasma were collected in several districts of Mersin province, Mediterranean Anatolia, Turkey, during May-September, 2015. Phlebovirus RNA was screened through two nested polymerase chain reaction (PCR) assays, targeting S and large (L) segments of the viral genome. A kinetoplast minicircle nested PCR was employed for Leishmania DNA detection and typing. Previously collected TOSV-positive specimens from humans, dogs, cats, and sandflies from various regions in Turkey and Cyprus were further evaluated through the S segment PCR. All amplicons were characterized through sequencing. RESULTS: A total of 210 specimens that comprise canine (76.2%) and feline (23.8%) plasma were screened. In three (1.9%) and two (1.3%) canine specimens, TOSV and Leishmania nucleic acids were detected, respectively. The TOSV strains were characterized as genotype B, and Leishmania infantum was identified in positive specimens. Twenty-four partial S segment sequences were amplified, which demonstrated a maximum intramural diversity of 3.88% in the nucleotide level. Sequence comparisons revealed significant similarities to particular genotype B strains characterized in Spain and France, whereas a notable divergence was observed among several TOSV strains. Single or recurrent amino acid substitutions were noted in eight residues of the viral nucleocapsid. DISCUSSION: Canine infections of TOSV genotype B, with temporal and spatial association with L. infantum, were detected. Divergent TOSV S segment sequences with amino acid substitutions, presumably associated with host adaptation, were observed.