Autoimmune disease, such as type 1 diabetes, often arrise as a result of a defect in one or mechanisms of immune tolerance. An important part of immune tolerance are a subpopulation of T cells, called regulatory T cells (Treg), which prevent the autoimmune activity of aggresive immune cells, and whose depletion or inactivity can lead to severe autoimmune reactions. Stable expression of the master regulator transcription factor FOXP3 is key for their development from thymocytes. Stable induction of FOXP3 expression could potentially drive the differentiation of other T cell population, as well as other cells, towards a Treg lineage and an acquisition of an immosupresive phenotype. In my masters thesis, we aimed at developing and testing transcription activators, bound to modified Cas9 proteins, for the specific induction of FOXP3 expression. We have designed novel short guide RNA (sgRNA), that direct the Cas9 to specific sites on key regulatory regions of the FOXP3 gene. We have achieved high in vivo expression induction of FOXP3 with the use of a tripartite transcription activator VPR, bound to a nuclease-null mutant of Cas9 protein (dCas9:VPR). The highest induction of expression was obtained when we used dCas9:VPR in combination with sgRNA which target the core promotor of FOXP3 and a regulatory region, which we termed Cage1. After the success in achieving transcription activation, we wanted to check the expression profile of key FOXP3 target genes in cells, which normally do not express FOXP3. Initial experiments in HEK293T cell line showed a certain degree of correlation of gene expression to that of Treg cells, after the activation of FOXP3 expression. With our work we have presented a powerfull tool for targeted gene expression, as well as the imporatance of the Cage1 region for the activation of FOXP3 expression. Our tool could be used in further experiments which aim at the generation of Treg cells, which could potentially be used to treat different autoimmune diseases.
B.06 Other
COBISS.SI-ID: 8723833One of the main current areas of research in synthetic biology is the development of artificially designed DNA binding proteins for use in transcription regulation of endogenous genes. Currently used systems of artificial transcription regulation rely on mechanisms of binding site competition and on the use of effector domains for activation of repression of transcription. In this work, we examined the effects of proximal binding of TALE (transcription activator-like effector) proteins, to zinc-finger transcription factors Zif268 and Gal4. Binding of TALE proteins in the vicinity of Zif268 and Gal4 was found to have an effect on their transcriptional activation, dependent of the position and distance between binding sites. As TALE proteins can be designed to bind in the vicinity of endogenous transcription factors, this method could be employed as a novel approach to transcriptional regulation.
B.06 Other
COBISS.SI-ID: 4857208Immune tolerance is a group of mechanisms, responsible for preventing activation of immune system in the presence of self-antigens and is thus preventing the development of autoimune diseases. Regulatory T cells (Treg) present a specialised subpopulation of T cells that supresse or downregulate activation and proliferation of effector T cells and other immune cells, including B lymphocyte and antigen presenting cells. Treg develop from naive T cells in the thymus or in periphery, when T cell receptor binds to a peptide presented in MHC II in the presence of a combination of cytokines in the specific cellular microenvironment. Differentiation of naive T cells into Treg is triggered by expression of master transcription regulator, responsible for the control of Treg differentiation, FOXP3. FOXP3, in a complex with other proteins, acts as an activator or repressor of transcription of over 700 genes, responsible for establishment of a specific Treg expression profile. Mutations in FOXP3 and other dysfunctions, that lead to diminished number or inefficiency of Treg, can cause the development of autoimmune diseases, chronic inflammation and allergies.However, research in animal models showed, that the re-introduction of active Treg cells can improve the above mentioned conditions. Because of its therapeutic potential, methods for expanding the number of regulatory T cells are under development, including ex vivo expansion of Treg and differentiation of naive T cells into Treg. Modification of the CRISPR/Cas9 system, that uses mutated Cas9 without nuclease activity (dCas9), fused with synthetic transcriptional activator VPR, enables strong activation of one or multiple genes at the same time. In a combination with target-specific sgRNA it thus presents a simple and accurate approach to activation or enhanced expression of target genes. In this master thesis project we aimed to activate or increase expression of selected target genes important in the differentiation of naive T cells into Treg. We used dCas9-VPR and a combination of sgRNA molecules, that bind to regulatory regions of target genes to activate their expression in primary T cells and a cell-culture line. We also determined the effect of FOXP3 activation on its downstream targets, IL2RA, CTLA-4, SATB-1 and TIGT in primary cells. To improve the efficiency of activation we then optimised the electroporation parameters for both primary cells and Jurkat cell line. In Jurkat cells we optimised the combination of sgRNA that targeted different regulatory regions in the vicinity of FOXP3 and achieved an improved level of activation. Further on, we analyzed FOXP3 expresion at the mRNA and protein levels during different time points of incubation . We demonstrated that FOXP3 expression was increased even after four days of incubation.Additionaly, we constructed sgRNA molecules, that targeted promotor regions of other genes that have a role in Treg differentiation. We managed to activate EOS expression and coactivate FOXP3 and EOS. Coactivation of these two genes showed, that in the presence of EOS, expression level of FOXP3 is additionaly increased. To conclude, in this master thesis project we managed to show, that by using the system of dCas9-VPR along with sgRNAs targeted to different regulatory elemnts of target genes, we can activate the expression of one or more genes important for Treg differentiation, which may help to develop future effective therapies for treatment of autoimune diseases.
E.01 National awards
COBISS.SI-ID: 1538039747Nowadays, obesity and type 2 diabetes, often associated with it, are amongst the most widespread health disorders. According to the World Health Organisation, 13 % of the world adult population is obese and 39 % is overweight; 8 % suffer from type 2 diabetes. Currently, treatment that would efficiently cure or even prevent both diseases without having systemic and/or central effects simultaneously does not exist. Being one of a few genes with the proven role in maintaining metabolic health, Tst manifests a very interesting potential therapeutic target. High Tst expression has been proven to correlate with low fat mass and with low blood glucose levels in mouse as well as in human. Based on this correlation, we tried to activate Tst expression in mouse cell line NIH3T3 by using CRISPR/dCas9-VPR system. Firstly, target DNA sequence for sgRNA binding was chosen based on the regulatory regions in Tst locus. According to the chosen target sequence, sgRNA was designed. By successful cleavage of the target DNA in vitro and in the cellular environment, we showed that designed sgRNA is able to bind target DNA sequence. By using activation complex dCas9-VPR, expressed from vector pCMV-dCas9:NLS:VPR, we successfully activated Oct4 transcription and therefore proved that this activation complex works well in NIH3T3 cell line. Having both components tested, we tried to activate transcription of Tst by transfecting NIH3T3 cells with the designed sgRNA and vector pCMV-dCas9:NLS:VPR. Unfortunately, no significant change in gene expression was observed. Lack of activation could have been the consequence of very poor transfection efficiency and/or wrong choice of the target DNA region and/or lack of constitutive expression of sgRNA, since the cells were transfected with sgRNA in synthetic form. Despite unsuccessful Tst activation, we believe that our work provides good basis for additional attempts of Tst – and potentially some other gene – expression activation using CRISPR/dCas9 system and therefore for the development of treatments for obesity, diabetes and potentially some other diseases that could be treated by genetic manipulations.
B.06 Other
COBISS.SI-ID: 1538397123Background: The CRISPR/Cas system is a highly potent tool, which has revolutionized genome engineering and regulation of gene transcription in various cells and organisms. This gene-editing tool consists of a guide RNA (gRNA), which targets the Cas9 endonuclease to the desired genomic site. Cas9 catalyzes the formation of double-strand DNA breaks, which are then repaired by different cell mechanisms. Depending on the size (tens of base pairs) of indel mutations, higher rates of “knock-out” can be achieved. To achieve greater indel mutations, CRISPR system can be coexpressed in cells with DNA exonucleases, which cause increased recessions of DNA following DNA breaks. We show that joint action of the CRISPR system with different exonucleases significantly increases the percentage of indel mutations at various targeted genes. Of the different exonucleases tested, the E.coli-derived exonuclease III (EXOIII) exhibited the best performance in terms of indel formation. Material and methods: K562 cells, model for Philadelphia chromosome positive cells and chronic myelogenous leukemia (CML) patient cells were used. Constructs, expressing BCR- ABL1 targeting gRNA and Cas9, tethered via coiled-coil forming peptides to E.coli exonuclease EXOIII, were nucleofected into target cells. T7E1 assay to detect genome modifications was carried out. TUNEL assay, FACS analysis with bioluminescence measurement were used for cell death determination. SCID mice were used for a subcutaneous K562 cancer model. Results: Of the different exonucleases tested, the EXOIII exhibited the best performance in terms of indel formation. To improve the rate of indel mutations, we connected Cas9 and EXOIII via coiled-coil forming peptides, bringing the two enzymes into close proximity. This resulted in increased indel formation compared to the classical CRISPR/Cas system. We performed a case study for the use of our novel CRISPR system as a potential anti-cancer therapeutic tool. In the case of our new system, we showed significant increase in cell death due to higher genome modification in BCR-ABL1 region. Later, these findings were confirmed also in animal cancer model, where animals with tumors, electroporated with CRISPR-EXO system showed 100% survival and drastic reduction in tumor size. Conclusion: Our de novo upgraded CRISPR system by tethering Cas9 protein to exonuclease EXOIII by heterodimeric coiled-coil forming peptides, resulted in higher editing of BCR- ABL1 fusion gene, leading to enhanced death of CML cancer cells.
B.03 Paper at an international scientific conference
COBISS.SI-ID: 44496899