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

Breaching Barriers in Gene Editing: Getting CRISPR/Cas9 Ribonucleoproteins into the Cell with Cell Penetrating Peptides

The CRISPR/Cas system is a powerful tool for both scientific research and potential therapeutic strategies for inherited diseases. This PhD thesis investigated the feasibility of cell-penetrating peptide-mediated direct delivery of CRISPR/Cas components to achieve various CRISPR/Cas-based gene editing applications. In order to reduce off-target effects that result from long-term high expression and activity of CRISPR/Cas9, rather than employing mRNA or plasmid platforms of Cas9 as delivery format, this work focused on the direct delivery of the Cas9 ribonucleoprotein (RNP) complex. After screening a variety of cell-penetrating peptide sequences, we encovered a highly efficient delivery of Cas9 using the amphipathic LAH5 peptide. This peptide sequence showed a robust capacity to form complexes with Cas9 ribonucleoprotein, even with an additional single-stranded DNA homology-directed repair (HDR) template which can be used to generate specific genetics repairs or mutations. The nanocomplexes formed with the Cas9 ribonucleoprotein and the LAH5 peptides were effectively taken up by cells, resulting in effective gene editing and correction in a variety of human cell types. Whereas cell-penetrating peptides have been shown to facilitate efficient transfected of various cargos in an in vitro setting, these systems have various limitations that may hamper therapeutic applications, such as decreased stability in the presence of serum. To address these limitations, we studied the effects of N-terminal acylation with various fatty acids on the capacity of LAH5 peptides to form nanocomplexes with Cas9 ribonucleoprotein. Furthermore, we studied the overall stability of such nanocomplexes, as well as their protective capabilities against protein-degrading enzymes, their capability of disrupting cell membranes, and their efficiency in Cas9 delivery. Fatty acid modifications, especially Oleic acid provided increased durability against proteases and increased stability in serum-rich conditions and, more importantly, Oleic acid-LAH5/Cas9 nanocomplexes showed increased capability of facilitating effective gene editing and correction in various cell types. To further study the potential therapeutic applications of these peptides, their potential to correct pathogenic mutations was studied. Using the described Oleic acid-LAH5 peptides, a CRISPR/Cas9 mediated editing strategy was used to eliminate multiple splicing defects that have been shown to cause Stargardt disease (STGD1). Deep intronic (DI) variants cause this rare genetic disease that results in vision loss within the same intron of the ABCA4 gene. Ultimately, our strategy led to the restoration of normal ABCA4 gene splicing and expression levels in derived pluripotent stem cell (iPSC) cell models. In a further study, nanocomplexes were tested in vivo and successfully delivered Cas9 to muscle tissue, achieving effective gene editing in skeletal muscle. Altogether, our findings demonstrate the potential of LAH5 peptide-mediated efficient gene editing in a wide variety of cell types under in vitro conditions, as well as local in vivo gene editing. Additionally, the findings in this thesis provide a versatile, highly efficient delivery method for Cas9 RNP, which could be utilized as a robust tool for designing and applying novel delivery vectors for future therapeutic strategies to treat genetic diseases

Evaluation of the genetic diversity of pomegranate accessions from Turkey using new microsatellite markers

Turkey has many valuable genetic resources for pomegranate; however, there have not been many studies on the identification and characterization of these important genetic resources. New microsatellite markers were used to characterize a set of 78 pomegranate accessions from Turkey. Using six SSR primers, a total of 41 alleles were characterized with an average of 4.6 alleles per locus and mean probability of identity (PI) value of 0.366. These data indicated a high level of polymorphism in pomegranate germplasm. Five synonymous groups could be detected among 30 accessions. This microsatellite-based key is a first step towards a database for markerassisted identification of pomegranate accessions in Turkey. The present study provides essential information to devise a pomegranate core germplasm collection without duplication of plant material, to sustainably manage pomegranate breeding programs, and to establish conservation strategies for preserving local pomegranate genetic resources.Turkey has many valuable genetic resources for pomegranate; however, there have not been many studies on the identification and characterization of these important genetic resources. New microsatellite markers were used to characterize a set of 78 pomegranate accessions from Turkey. Using six SSR primers, a total of 41 alleles were characterized with an average of 4.6 alleles per locus and mean probability of identity (PI) value of 0.366. These data indicated a high level of polymorphism in pomegranate germplasm. Five synonymous groups could be detected among 30 accessions. This microsatellite-based key is a first step towards a database for markerassisted identification of pomegranate accessions in Turkey. The present study provides essential information to devise a pomegranate core germplasm collection without duplication of plant material, to sustainably manage pomegranate breeding programs, and to establish conservation strategies for preserving local pomegranate genetic resources

Evaluation of the genetic diversity of pomegranate accessions from Turkey using new microsatellite markers

Turkey has many valuable genetic resources for pomegranate; however, there have not been many studies on the identification and characterization of these important genetic resources. New microsatellite markers were used to characterize a set of 78 pomegranate accessions from Turkey. Using six SSR primers, a total of 41 alleles were characterized with an average of 4.6 alleles per locus and mean probability of identity (PI) value of 0.366. These data indicated a high level of polymorphism in pomegranate germplasm. Five synonymous groups could be detected among 30 accessions. This microsatellite-based key is a first step towards a database for markerassisted identification of pomegranate accessions in Turkey. The present study provides essential information to devise a pomegranate core germplasm collection without duplication of plant material, to sustainably manage pomegranate breeding programs, and to establish conservation strategies for preserving local pomegranate genetic resources

Ratiometric fluorescence detection of an anthrax biomarker with Eu3+-chelated chitosan biopolymers

A novel chitosan-based ratiometric fluorescent probe incorporating an EDTA-Eu3+ complex as the sensing unit and fluorescein dye as the internal standard was designed to detect dipicolinic acid (DPA) as an anthrax bio-marker with high sensitivity and selectivity. The fluorescence intensity of fluorescein dye attached to the chitosan backbone remains constant as an internal reference, while the Eu3+ emission increased linearly upon the consecutive addition of DPA. The selectivity studies were performed by adding different competitive aromatic ligands to the sensing environment and no signifacant fluorescence response was observed. The results demonstrated the superior selectivity of the system to DPA. Overall, this novel chitosan-based ratiometric fluorescent probe enables ratiometric and sensitive DPA detection over nanomolar concentrations (as low as 10 nM) and displays straightforward selectivity over other competitive aromatic ligands

Growth of microalgae Chlorella vulgaris at the photobioreactor for biodiesel production

Microalga is known to have higher lipid contents and biodiesel efficiency than most plant oil sources e.g. palm oil. We studied the growth of Chlorella vulgaris at the photobioreactor in our laboratory. We aimed to use this photobioreactor of laboratory scale as feed stream to an open pond larger scale bioreactor for future work. Photobioreactor had three compartments which had separate controls for light and air circulation. Temperature was kept at 22°C-26°C. The circulation rate was 180 L/h. The light intensity was set at 16 hours on and 8 h off. The nutrient powder was dissolved in sterile water and the pH of the solution was 6.5-6.7. Inoculation of culture was performed aseptically. The algae culture was an original strain of Chlorella vulgaris. This specific culture was proposed for use as bioenergy due to high lipid content. Culture growth was maintained aseptically and samples were taken from the reactor periodically for microbial analysis. The continuous growth was achieved at the bioreactor without contamination for more than 9 months. Slurry was centrifuged, dried and algae biomass was obtained. Extraction of lipids of the dried algae was performed by Bligh and Dyer method Extracted lipid was subject to transesterification reaction for production of fatty acid methylesters (FAMES).The lipid contents of sample was analysed by GC-FID. The results for the lipid contents were: Palmitic acid: 28%, Linoleic acid: 26%, Heptadecanoic acid: 12%, Oleic acid: 10%, Palmitoleic acid: 3%, stearic acid: 5% and the rest is arachidic acid, myristic acid et

The role of biochemical regulation on the adaptation of gypsophile and gypsovag species

WOS: 000452565100002There are two groups of plant species that spread in gypsiferous soils. While gypsophytes only grow on gypsi-ferous soils, gypsovags can grow on both gypsum and non-gypsum soil. Adaptation of plants to gypsum soils requires biochemical arrangements in addition to proper morphological and physiological characteristics. In this study, three gypsophyte species and on-gypsum and non-gypsum speciments of three gypsovag species were examined for antioxidant capacities. The average phenolic substance contents were 126.5, 30.5 and 37.6 mu g g(-1)W in gypsophile, on-gypsum and in non-gypsum gypsovag species respectively. Gypsophyte, Thymus leucos-tomus var. gypsaceus species was identified as having seven different phenolic compounds and the highest phenolic substance content. However, phenolic substance compositions of gypsophyte and gypsovag plant species do not have common properties and show specific differences for each species. The total antioxidant capacity and carotenoid levels of gypsophyte were found to be quite high even though there was no significant difference between the chlorophyll values of the plant groups under investigation. Differences in car/chl and aox/chl ratios of gypsophiles and gypsovag species suggest that antioxidant compounds have a role in the adaptation of these plants. On the other hand, the values observed in on-gypsum and non-gypsum gypsovag species should be assessed as having no specific role in the formation of oxidative stress in gypsum soils