|
|
Study the optimal condition to knockout metallothionein by CRISPR-Cas9 in neuroblastoma
Májková, Klára
Cisplatin-based treatment strategies are commonly employed for the treatment of Neuroblastoma (Nbl), an embryonal tumour that most commonly affects infants and children under the age of five. However, the efficacy of these strategies has been found to be lower than 50 % due to the frequent chemoresistance developed by the tumour cells. Among others, this resistance has been linked to the increased expression of metallothionein (MTs) by the Nbl cells. Human MT-3, a protein primarily expressed in central nervous system cells, plays a vital role in metal detoxification and the maintenance of homeostasis during oxidative stress. Therefore, knockout of MT-3 using CRISPR/Cas9 could make the cells sensitive to cytotoxic drugs. This study is aimed to optimize the conditions for successful knockout of MT-3 from Nbl cells. The MT3-CRISPR/Cas9 plasmid was transfected into Nbl using Lipofectamine 2000 in three different plasmid concentrations (250 ng/μL, 500 ng/μL, and 1000 ng/μL). The optimal conditions were achieved using 0.75 μl of Lipofectamine, 1000 ng/μl of plasmid, and 0.5 ng/μl of puromycin.
|
|
|
Targeted editing of the Arabidopsis CYP98A3 gene using the CRISPR/Cas9 method
Šamaj, Matej
Lignin is an abundant biopolymer, which fulfils several important functions including facilitation of polar water transport and mechanical strengthening of plant body. It is required for proper growth and development of plants but also for plant defence responses. Complex lignin biosynthesis involves the activity of numerous enzymes catalysing diverse chemical reactions that are stepwise generating various precursors for lignin formation. One such enzyme of lignin biosynthetic pathway, P‐COUMAROYL SHIKIMATE 3′‐HYDROXYLASE (C3′H), is encoded by the gene CYP98A3/ C3′H and convert p-coumaroyl shikimate to caffeoyl shikimate. CRISPR/Cas systems adapted from prokaryotes, where they provide adaptive immunity, have very quickly evolved to unique and powerful tool for precise gene editing. The CRISPR/Cas9 technology allows to specifically target desired sites within the genomes to subsequently perform very precise mutagenesis of selected gene(s). The purpose of this thesis was to employ the CRISPR/Cas9 method to perform targeted editing of the CYP98A3/C3′H gene in transformed Arabidopsis thaliana plants. Selected transformed plants were verified to confirm the presence of CRISPR/Cas9-mediated gene editing. The verified T2 generation putative mutant plant shows altered phenotypes of leaves, leaf rosettes, stems and inflorescences accompanied by lower deposition of lignin as revealed by histochemical staining by basic fuchsin and microscopic observations.
|
|
|
CRISPR/Cas9 gene editing in Drosophila melanogaster
KALTENBÖCK, Konstantin
The aim of this thesis was to predict functional sites in the timeless gene of Drosophila melanogaster, to design fitting gRNAs for the identified sites to create transformation vectors for fly transformations and to induce CRISPR/Cas9 mediated target mutations by crossing gRNA expressing flies with Cas9 expressing flies.
|
|
|
Optimization of CRISPR/Cas9 technique for the Ixodes ticks genome editing
KITZBERGER, Daniel
CRISPR/Cas9 technique was used for genome editing of several arthropod species, but it has not yet been used on Ixodes spp. tick species. Therefore, in my work, I performed a bioinformatic analysis of the I. ricinus transcriptome and I. scapularis genome to reveal the sequence of the target gene for the CRISPR/Cas9-mediated knock-out. We prepared single guide RNA and mixed it with the Cas9 endonuclease to produce the Ribonucleoprotein complex and cleave the target sequence of the I. ricinus gene beta-galactoside alpha-2,6-sialyltransferase.
|
|
|
CRISPR/Cas9 Genome editing in Pyrrhocoris apterus
BERTOLUTTI, Maly
The CRISPR/Cas9 technology is a cutting edge method to genetically modify specific targets in the genome, which allows to create mutants in non-model organisms. The aim of this project was to genetically modify the Pyrrhocoris apterus genomic sequence of the cryptochrome2 gene using the CRISPR/Cas9 technology. Specifically, this project aimed to create a mutant with removed or modified C-terminal part of the CRY2 protein, which should later allow for studying the role of this sequence in CRY2 function.
|
|
|
Functional analysis of circadian clock gene timeless in temperature compensation mechanism
SINGH, Samarjeet
This thesis is focussed on investigating the role of the circadian clock gene timeless in the temperature compensation mechanism in Drosophila melanogaster. To conduct the studies presented in this thesis advanced gene editing technique, CRISPR/Cas9, was used to target timeless and to create a variety of mutations. Application of various reverse genetics tools and behavioural methods described here contributed to the elucidation of timeless role in temperature compensation of the circadian clock.
|
|
|
Czech public opinion on CRISPR/Cas9 technology – Food 2021
Hanzlová, Radka
The majority (84%) of the Czech Republic has never heard of the CRISPR/Cas9 method. A more than one-half majority (60%) of the Czech public agrees with the possibility of using the CRISPR/Cas9 method for medical purposes. The Czech public least agrees with the possibility oof using this method to improve the performance of athletes (66% do not agree with this idea). On the matter of treating hereditary diseases, most respondents (37%) agreed with using the CRISPR/Cas9 method only in medically justified cases and on the condition that none of the patient’s altered genetic information be passed on to any offspring.
|
|
|
Využití metod genetického inženýrství při šlechtění plodin na rezistenci vůči patogenům
Baránková, Simona
Strategies for plant-pathogen resistance breeding are an important field of research; which in an era of increasing demand for human feed is more and more significant. The topic of this thesis is the use of genetic engineering in case of enhancing plant disease resistance. Resistance is a complex trait, which is based on many conditions, either in an organism and in the environment. In this work, the principles of these mechanisms are being described, together with their possible use in breeding for plant resistance trait. You can find here several approaches to do so, also some links for academic papers, which are solving this problem. Further, the relevant methods of genetic engineering, which are possible to use as a tool for plant genome editing are being described here.
|
|
|
Genová terapie, její principy, mechanizmy a typy vektorů
Dobešová, Lucie
In the beginning chapters, the thesis deals with general information about gene therapy and its history. The following chapters provide information on its principles (both classical and gene therapy in conjunction with CRISPR/Cas9) and the vectors that enable the gene therapy process to take place. In the final chapters, the thesis deals with model organisms that are used for clinical trials, the use of gene therapy in human patients and the ethical and safety circumstances associated with its use.
|
|
|
Epigenetické mechanismy vs. RNA řízená editace genů v jednobuněčných zelených mikrořasách =: Epigenetic mechanisms vs. RNA directed gene editing in unicellular green microalgae /
Bačová, Romana
The first part of the thesis is devoted to the study of an epigenetic regulation of genes in unicellular green microalgae. The influence of environment on DNA methylation level, histones modifications, chromatin structure and RNA interferences are described as well as their effect on metabolome. The work focuses more closely on the effect of the demethylation reagent 5-azacytidine and CdCl2 on the amount of 5-mC and metabolites of the methionine cycle in Chlamydomonas reinhardtii and Scenedesmus quadricauda in the context of the production of secondary metabolites. The second part of the thesis is focused on genetic engineering. Specifically, the CRISPR/Cas9 method was used as RNA-directed gene editing in the model organism, C. reinhardtii. An optimization of the cpFTSY chloroplast gene was performed, demonstrating phenotypic change in mutated colonies. Furthermore, CRISPR/Cas9 was used to edit the adiposuppressor gene WDTC1. For both genes we obtained mutants whose DNA was repaired using both NHEJ and HDR. Using the fluorescence NileRed analysis, two ?WDTC1 colonies demonstrated an increased TAG content. Epigenetic mechanisms should be studied more to understand the adaptation of microalgae to stress and the environment, providing valuable information for knowledge of metabolic pathways, transcription factors. This information can be used for controlled gene editing, resulting in increased amounts of product for the same or better biomass growth.
|
guest ::
