Browsing by Subject "CRISPRa"

Mutations in the KCNQ1 gene have been implicated in the onset of hypopituitarism. Regulating KCNQ1 expression would therefore enable future clinical research on the mechanism of the disease. CRISPR offers a flexible toolset for controlling genetic expression via knockout, knock-in, knockdown, and gene activation. Of these approaches, CRISPR activation (CRISPRa) is distinguished by its ability to induce gene overexpression in a cell’s native context, making it a valuable tool in the interrogation of genetic disorder pathogenesis. This thesis therefore tested the efficacy of a CRISPRa subsystem in increasing KCNQ1 expression. The CRISPRa subsystem, VPR, was chosen because of its high activation efficiency and the ease of controlling the activation system of its doxycycline-inducible mode of action. The cell line used for the experiment, HEK293, was similarly chosen because of its ease of culture and transfection. To validate the proper functioning of the activation system, expression rates of the related genes ASCL1 and GHRH were measured as positive controls. The activation system successfully upregulated the expression rates of the two genes. As the dCas9-VPR system is dependent on the Tet-ON operator for inducing activation in a controllable manner, a test for dCas9 leakage was conducted. RT-qPCR analysis showed the upregulation of ASCL1 expression in the uninduced state of the system, confirming the presence of dCas9-VPR leakage. The dCas9-VPR system finally aimed to test the expression rate of KCNQ1. Although one novel guide RNA successfully upregulated KCNQ1 expression, it did so inefficiently and its success was not shared by any of the other tested guide RNAs. Altogether, the dCas9-VPR system was successfully established in HEK293 cells, and the leakage of the inducible system was confirmed, however, KCNQ1 activation by CRISPRa requires further optimization.

Induced pluripotent stem cells (iPSCs) can be derived from somatic cells by transgenically expressing the four transcription factors OCT4, SOX2, KLF4, and C-MYC. This technology has revolutionised the stem cell field, yet cellular reprogramming is still inefficient and slow. To become fully applicable in regenerative medicine, the robust generation of safe and high-quality iPSCs from patient samples is essential. Various methods and potent reprogramming factors have been described to date. Yet, none have been able to circumvent these limitations markedly. The recently published activator-mediated approach (CRISPRa) is considered to be more physiological compared to the forced transgenic expression as the cell’s own genes are activated. Here, guide RNAs (gRNAs) mediate sequence-specific recruitment of non-cutting Cas9 (dCas9) activator proteins to the promoter region. Unlike other methods, it holds great multiplexing capacity and can also target enhancer and non-coding sequences. CRISPRa reprogramming still needs to be optimised since its efficiency is low. Thus, we aimed at enhancing this aspect and the temporal kinetics by targeting the micro RNA (miRNA) clusters 302/367 and miR-371-373, which both have been described as powerful cell fate regulators. We demonstrate successful reprogramming by targeting the miR-302/367 promoter alongside OCT4, SOX2, KLF4, C-MYC, LIN28A, REX1, NANOG, and EEA-motifs with CRISPRa. Activating the miRNA cluster results in a 2.5 fold efficiency increase in human foreskin fibroblast (HFF) reprogramming compared to the published basal CRISPRa system, quantified by staining for alkaline phosphatase. In HFFs, the CRISPRa efficiency is now comparable to the commonly used transgenic approach. Aiming to clarify the molecular mechanisms of these results, we characterised the expression of direct and downstream targets of miR-302/367 at different time points throughout the reprogramming process. Furthermore, validated with immunocytochemical stainings, the generated bona fide iPSCs express pluripotency markers and spontaneously differentiate into the three germ-layers, both signs of high-quality iPSCs. Beyond that, we report that miR-302/367 activation appears to result in earlier iPSC colony formation resulting in faster proliferating stem cell colonies shown with live-cell imaging. Employing a conditionally stabilised activator construct, we further show that with miR-302/367 targeting, the dCas9 activator expression seems to be required for only a short time period, sufficient to induce pluripotency. At the end of the project, the miR-302/367 cluster targeting was optimised and the best-working gRNAs were selected for further studies, which when combined further increase the CRISPRa-induced expression of the miR-302/367 cluster markedly. All in all, this study demonstrates that non-coding genetic elements like the miR-302/367 cluster can be targeted with CRISPRa, and its targeting significantly improves the reprogramming efficiency. Implications of the study for regenerative medicine and future steps are discussed.

A novel method of somatic cell reprogramming employing CRISPR/Cas mediated gene activation (CRISPRa) may lead to improvements in the quality and efficiency of induced pluripotent stem cell (iPSC) generation by directly activating the endogenous factors of the cell. However, this method is yet to be optimized and is inefficient in its current form. Thorough characterization of the molecular events that occur during CRISPRa-mediated reprogramming could permit the fine-tuning of this method to improve iPSC production. B-lymphoblastoid cell lines (LCLs) adhere to culture plates during reprogramming, offering a form of selection for reprogramming cell populations. This thesis aimed to establish a system using LCLs for the characterisation of CRISPRa-mediated pluripotent reprogramming at the single-cell transcriptomic level. In this thesis LCL reprogramming conditions were characterized using alkaline phosphatase staining, immunocytochemistry, embryoid body formation, and live cell imaging. CRISPRa-mediated reprogramming efficiency was greatly increased by the targeting of the miR-302/367 cluster, a group of microRNAs known to improve the efficiency of transgenic reprogramming. Samples were collected for single-cell RNA sequencing (scRNA-seq) at multiple stages of reprogramming, the pluripotency of the iPSC samples was assessed, and a subset of the samples was sequenced. Clustering analysis of the sequencing data showed that the samples clustered apart from one another distinctly based on gene expression. The expression of notable genetic markers of LCLs, pluripotency, and developmental stages was consistent with the loss of somatic cell identity and rise of subpopulations characteristic of reprogramming. These results show that this is a functional system for scRNA-seq sample preparation that can be used to investigate reprogramming kinetics, and the samples collected will be part of a larger study of CRISPRa reprogramming.

Pluripotent stem cells (PSC) can exists in both primed and naïve states. The conventionally derived human PSCs represent the later primed state of pluripotency during embryo development, while the naïve state resembles the inner cell mass (ICM) of pre-implantation blastocyst. Primed human PSCs can be reverted chemically by transient histone deacetylase (HDAC) inhibition back to the naïve state in vitro. The reverted PSCs can then be characterized based on their morphology and expression of selected naïve markers using immunocytochemistry and RT-qPCR assays. Leucine twenty homeobox (LEUTX) is one of the genes expressed during the early stages of embryo development and is capable of activating the transcription of multiple genes, including pluripotency-associated genes, which are upregulated during the human embryonic genome activation (EGA). LEUTX expression could potentially improve the naïve reversion efficiency or the maintenance of naïve PSCs by driving the transcriptome of primed PSCs back towards the earlier cell stages of embryo development, potentially even to cell stages that precede the naïve state. The aim of this thesis was to setup the naïve reversion protocol and study the effects of LEUTX on the reversion by using the generated and tested H9 activator cell line for targeted activation of endogenous LEUTX expression. First, a conditionally stabilized CRISPRa activator cell line was generated for targeted activation of endogenous gene expression in H9 cells. Then sequence-specific guide RNAs (gRNA) targeting LEUTX for activation were introduced to the activator cell line. Using the generated activator cell line during the naïve reversions allows the targeted activation of specific genes, here LEUTX, and thus enables studying the effects of these genes on PSCs during the naïve reversion protocol. The induced activator cells expressing LEUTX managed to form four times as many naïve resembling colonies during the reversion compared to the controls, but most of these were lost after changing the medium conditions towards the end of the protocol. After the reversion was complete, the reverted PSCs were characterized as naïve PSCs based on their domed morphology and the high expression of naïve markers NANOG, KLF17, TFCP2L1 and DNMT3L when compared to the primed PSCs. The naïve reversion protocol was set up and optimized successfully and can now be used as a reliable way of obtaining human naïve PSCs for further experiments studying and modelling the earlier developmental stages during embryo development. Furthermore, the generated H9 activator cell line worked as intended and can be utilized for studying the effects of other targeted genes during the reversion or in the reverted naïve PSCs.