Browsing by Subject "CRISPR-Cas9"

Renewal of the intestinal epithelium is driven by the actively dividing and strictly regulated Lgr5-expressing intestinal stem cells (ISCs). As uncontrolled proliferation may lead to colorectal cancer (CRC), ISCs and the regulatory circuit around them could elucidate new targets for colorectal cancer therapy. The regulatory crosstalk between the stem cells and surrounding stroma is under intensive investigation, but little is known about the neuronal control of the intestinal epithelium. Adrenergic signals from sympathetic nervous system regulate hematopoietic and melanocyte stem cells and promote tumorigenesis in e.g., pancreatic, and prostate cancer. Adra2a, one of the nine different adrenergic receptors, is expressed in the ISCs, and adrenergic neurons project neurites to the stem cell niche suggesting a paracrine signaling role. However, whether the adrenergic signaling plays a role in ISC regulation and/or in colorectal cancer remains unknown. The aim of this study was to develop tools to investigate the role of adrenergic signaling in ISC regulation. First, I set up a protocol to delete Adra2a and other genes in intestinal organoids recapitulating the stem cell hierarchy of the intestinal epithelium in vitro, using CRISPR-Cas9 technology. This led to successful deletion of the Ret proto-oncogene while an Adra2a-deficient organoid pool could not be established with these efforts. I differentiated neuroblastoma cells to a catecholaminergic phenotype and cultured them together with intestinal organoids to address the effect of catecholaminergic signaling on the intestinal epithelium in organoid cocultures. The coculture with catecholaminergic cells induced upregulation of the regeneration markers Ly6a and Clu in intestinal organoids, while Reg3b as well as the stem cell markers Lgr5, Olfm4, and Adra2a and the Paneth cell marker Lyz1 were reduced. Third, I assessed the direct effect of a 3-hour norepinephrine (NE) pulse on wild-type organoids with 3’RNA sequencing, however, this did not induce significant changes in the expression levels of the respective regeneration marker genes. Altogether, my work established a CRISPR-Cas9-based method to delete genes of interest in primary intestinal organoids. Further investigation is needed to verify if NE contributes to the regulation of intestinal regeneration and stem cell maintenance.

Spinal muscular atrophy of Jokela type (SMAJ) is an autosomal dominant motor-neuron disease caused by a missense mutation c.197G>T, p.G66V in the gene CHCHD10. Coiled-coil-helix-coiled-coil-helix domain-containing protein 10 (CHCHD10) is a nuclear-encoded mitochondrial protein located in the intermembrane space (IMS) of mitochondria with an unknown exact function and disease-causing mechanism. In this project, the overarching aim was to correct a heterozygous SMAJ-causing mutation in patient myoblast cells with CRISPR-Cas9 genome editing. The goal was to create a genetically identical, isogenic, cell line to study only the effects of the mutation on cellular phenotype in vitro. Human myoblast cells isolated from patient biopsies provide the most pertinent experimental model to study neuromuscular atrophy-associated mutations in their natural genomic environment. More specific aims included genome editing optimization with myoblast cells, since it is not as widely conducted as with some other cell types, such as iPSCs. CRISPR-Cas9 ribonucleoprotein (RNP) complex and associated donor template were used to induce homology-directed repair (HDR) in the genome of patient-derived myoblast cells and correct the mutation. After optimization of electroporation conditions for myoblast cells, guide RNAs were designed and transfected into patient myoblasts. Clonal cell lines were made by utilizing techniques such as fluorescence adjusted cell sorting (FACS) and manual colony picking. The success and precision of genome editing were analyzed by Sanger sequencing, comparing the performance of the different guide RNAs with restriction enzyme analysis and Synthego ICE CRISPR web tool, and screening regions of potential off-target genome editing. A genome-edited myoblast cell line with the CHCHD10 c.197G>T mutation corrected, was successfully generated to provide an isogenic control for the patient myoblast cell line. Optimization of myoblast electroporation was successful and conditions used proved to be effective. Clonal cell line creation proved to be challenging with myoblast cells and work is still needed to improve the viability of single-cell clones after FACS. Nevertheless, the advances taken here regarding myoblast genome editing with CRISPR-Cas9 offer a fertile avenue for future research of myoblasts genome manipulation, myogenic disorders, and the role of CHCHD10 in skeletal muscle and SMAJ. Comparing the CHCHD10 protein level and mRNA expression between patient cells, corrected myoblasts, and differentiated myotubes is an area of future research. Future work also includes measuring the mitochondrial integrated stress response in both cell lines and co-culturing myotubes and iPSC derived motor neurons to study the effects of p.G66V on neuromuscular junction (NMJ) formation.

Mitochondrial aminoacyl tRNA-synthetases (mt-aaRS) catalyse the charging of tRNAs with their cognate amino acids in mitochondria. Mutations in mt-aaRS cause tissue-specific mitochondrial diseases, especially affecting tissues with high energy expenditure like the nervous system, heart, and kidneys. However, disease mechanisms for the heterogeneous group of diseases have not yet been fully elucidated. Harnessing CRISPR-Cas9 genome editing in induced pluripotent stem cells (iPSC) provides an opportunity to model mt-aaRS mutations in vitro and investigate the effects of individual mutations on cellular phenotype. SARS2 encodes mitochondrial seryl tRNA-synthetase, and its c.1347 G>A mutation causes severe childhood-onset progressive spastic paresis. Here, CRISPR-Cas9 ribonucleoprotein (RNP) complex and associated donor template were used to induce homology directed repair (HDR) the genome of iPSC and knock-in the patient mutation. Guide RNAs were designed and tested for efficiency before electroporation into wild type iPSC. Clonal cell lines were made by low-density seeding and manual colony picking. The expression of pluripotency markers was measured by RT-qPCR. RT-qPCR and Western blot measured SARS2 mRNA expression and protein level respectively. The success and precision of genome editing were analysed by Sanger sequencing, comparing the performance of the different guide RNAs, and screening regions of potential off-target genome editing. Two genome-edited iPSC lines with the SARS2 c.1347 G>A mutation were successfully generated to model the patient mutation. The iPSC lines expressed pluripotency markers and contained no off-target genome editing and modelled the patient’s decrease in SARS2 protein level and mRNA expression. More evidence of differentiation ability is needed before differentiation into the affected cell type (motor neurons) and further disease modelling. The efficiency of CRISPR-Cas9 for genome editing, especially harnessing HDR in iPSC, is an area of future research.

CRISPR-Cas9 is one variant of newly emerging technologies utilizing targeted mutagenesis based on Cas family proteins and guide RNA that enable binding and modifying selected target sequence. The aim of the master’s thesis was to compare different methods of CRISPR-Cas9 induced gene editing in the genus Nicotiana and other secondary protocols necessary to identify successful mutations. PDS1 and PDS2 genes coding phytoene desaturase in plants were selected as target genes as mutant genotype produce visually identifiable photobleaching phenotype. CRISPR-Cas9 ribonucleoprotein complex mediated transformation uses separately produced Cas9 protein and guide RNA that when combined perform transient gene editing in cell. This method was planned to be used but Cas9 protein was challenging to produce in soluble form and final transformation was not achieved. This study suggests that acquiring ready-to-use Cas9 protein might be preferable choice when targeting only few transformations with CRISPR-Cas9 RNP-complex. Agrotransformation is well established method for genus Nicotiana and using Single Transcriptional Unit CRISPR-Cas9 system it is straightforward procedure from plasmid design to transformation. Successfully transformed plants were redeemed from transient agroinfiltration and stable agrotransformation experiments. Off-target mutations are possible and selective outbreeding may be needed. This method lacks the several advantages of CRISPR-Cas9 RNP-complex such as instant gene editing in cell, avoiding RNA interference and transformation over species boundaries, but is simple and functional in genus Nicotiana. Successful mutations were detected using commercial T7E1 and with natural CEL I endonuclease from celery extract. Celery extract can be used as cost-effective alternative to T7E1 for verifying or replicating previously confirmed results.

Uterine leiomyomas (ULs) are common benign tumors that originate from the smooth muscle cells of the uterine wall known as the myometrium. Around 70% of pre-menopausal women are affected by these tumors. The high prevalence of ULs is a significant public health issue and ULs are the leading cause for hysterectomy. Many tumors remain asymptomatic, but 15-30% of affected women develop symptoms ranging from pain and heavy menstrual bleeding to pregnancy complications and infertility. Despite their common occurrence, the underlying mechanisms of UL genesis are still largely unknown. Based on mutually exclusive recurring genetic alterations, ULs can be divided into molecular subclasses. Three main molecular subclasses have been established: MED12 mutated tumors, HMGA2 overexpressing tumors and tumors with biallelic FH inactivation. Combined, these three subclasses represent around 90% of ULs, indicating that additional smaller molecular subclasses also exist. Recently, novel mutations associated with ULs have been identified in genes encoding for subunits of the SRCAP chromatin remodeling complex that deposits histone variant H2A.Z onto chromatin. These included loss-of-function mutations in YEATS4, DMAP1 and ZNHIT1, and resulted in deficient H2A.Z loading in the tumors. The detailed functional consequences of these driver mutations need to be further investigated to fully understand their significance in UL genesis. This work aimed to elucidate the effects of YEATS4 mutations by characterizing previously established CRISPR-Cas9 edited immortalized human myometrial cell models carrying heterozygous mutations in YEATS4 using various molecular biology methods. Subcellular fractionation and western blot analysis was used to detect chromatin bound H2A.Z from cell lysates. Quantitative PCR was performed to determine relative YEATS4 expression levels in mutated and wild-type cells. No significant reduction of chromatin bound H2A.Z or YEATS4 expression was observed in the studied heterozygous mutants when compared to wild-type immortalized myometrial smooth muscle cells. Additional myometrial cell models were created by CRISPR-Cas9 gene editing. Objective was to achieve homozygous YEATS4 mutations to better reflect the changes previously reported in ULs. One homozygous YEATS4 mutant cell line was achieved. Understanding the detailed molecular mechanisms behind UL genesis will be instrumental for developing curative non-invasive therapies in the future. Insight into dysregulated pathways and identification of UL biomarkers could improve diagnostic accuracy and help design personalized targeted therapies effective for specific UL subclasses. Characterization of each molecular subclass offers a unique opportunity to understand UL genesis.

Nuclear receptor subfamily 5 group A member 1 (NR5A1) is a master regulator of both steroidogenesis and gonadal development. Disruptions of NR5A1 can result in differences in sexual development (DSD). With proven interspecies differences in NR5A1 functioning and human material not being available, human stem cells are one of the most achievable, ethical, and accurate models to study the earliest developmental stages of foetal life. However, in currently existing human stem cell-derived gonadal models the expression of NR5A1 has been insufficient without artificial induction due to the lack of knowledge of its distinct biological mechanisms, endogenous ligands, and co-factors. A functional reporter cell line would enable high throughput microscope screening of differentiation protocols with expressed NR5A1. The aim of this thesis was to generate a functional monoclonal human embryonic stem cell (hESC) reporter line for the gene NR5A1 with Alt-R CRISPR-Cas9 ribonucleoprotein (RNP) complex. Firstly, an efficient guide RNA was determined for NR5A1 by T7 assay, and a homology-directed repair (HDR) donor plasmid was designed based on it. Secondly, monoclonal hESC lines were generated with the Alt-R CRISPR-Cas9 RNP complex knock-in method and HDR donor plasmid via electroporation and single-cell sorting. Finally, monoclonal hESC reporter lines were screened with Touchdown PCR and a functionality analysis based on fluorescence and mRNA expression was performed. Two monoclonal hESC reporter lines H9-NR5A1-eGFP cl. 1 and dual-inducible H9-NR5A1-DDdCas9VP192-eGFP cl. 28 were established by using Alt-R CRISPR-Cas9 RNP complex. However, a functional validation performed on H9-NR5A1-DDdCas9VP192-eGFP cl. 28 cells showed the cell line to be non-functional upon NR5A1 upregulation regardless of the expressed eGFP mRNA detected with RT-qPCR.

The Clustered Regularly-Interspaced Short Palindromic Repeats (CRISPR) and CRISPR associated protein (Cas9) (CRISPR-Cas9) system is a widely used gene editing technology due to its potential to alter the genome precisely in desired locations. Due to the potential of the CRISPR-Cas9 system, the objective of the thesis is to improve the precise editing of genes by modifying the CRISPR-Cas9 platform. Ultimately, the aim is to develop a platform that can edit any mutation and repair it to a normal, functional gene in patient cells. In general, CRISPR-Cas9 provides opportunities in treating monogenic diseases, for example by modifying long-term hematopoietic stem cells in immunodeficiencies. CRISPR-Cas9 can target disease-causing mutation sites and introduce double-strand breaks. Afterwards, the native DNA repair machinery of a cell repairs the cut site either by more efficient, error-prone non-homologous end joining (NHEJ) or precise homology-directed recombination (HDR). In most clinically oriented genome editing studies, the desired repair outcome is the latter because it allows precise repair of the mutation according to the exogenous repair template. Despite all its positive features, the optimization of CRISPR-based editing system is crucial before medical use; CRISPR-Cas9 induces a p53-mediated DNA damage response, which leads to a transient G1 cell cycle arrest and hampers HDR-based precision genome editing. Other problems include the repair pathway depending on the cell cycle phase, repair template proximity, and off-target activity. This thesis demonstrates that Cas9 fusions allow addressing the problems mentioned above. Cas9 fusions with DNA repair proteins ensure improved editing efficiency at the close proximity to the target site in HEK293T, BJ5-ta and RPE reporter cell lines. In addition, Cas9 coupled with the engineered cell cycle timer, AcrⅡA2-cdt1, favors the editing at the S/G2 cell cycle phases avoiding the p53-mediated response. AcrⅡA2-cdt1 is a reversible, phage-derived CRISPR inhibitor that selectively inhibit CRISPR-Cas9 at the G1 cell cycle phase and releasing it at the S phase. This thesis provides extensive look on the CRISPR-Cas9 editing and its challenges in immortalized cell lines and primary cells. In the thesis, the generation of reporter cell lines is prior to the validation of the novel Cas9-fusions. Furthermore, the optimization of primary T cell and CD34+ hematopoietic stem cell electroporation with different electroporation systems brings the study closer to clinical applications. The thesis provides insights about the effect of the target site and the cell type for genome editing outcomes. The editing efficiencies depend on the Cas9 fusion protein, cell type and its proliferation rate. The editing efficiency in primary T cells and CD34+ hematopoietic stem cells can significantly improve by optimizing transfection and culturing conditions, such as concentration of the CRISPR-Cas9 complex, cell culturing time and electroporation program. Cas9 fusions improve the safety and efficiency of the CRISPR-Cas9 system depending the cell type and the proliferation rate of the cell. Timing the induction of double-strand breaks also improves the editing efficiency. Overall, the methods used in the thesis give useful tools for eventual translational applications.

In biotechnological protein production and metabolic engineering, regulating the expression of genes is essential. For this, expression systems composed of promoters, terminators and transcription factors are essential. So far, majority of these systems use native promoters and transcription factors. That however rises two problems: 1) these systems usually work in only a set of closely related species, 2) native regulatory components can cause unintended expression levels due to the complexity of cellular regulation. Recently, a synthetic expression system (SES) was established for a wide range of fungal species. The transcription factor used in this system comprises an activation domain that originates from a virus. However, in the field of biotechnology and especially food industry, viral DNA constructs are not favorable because of customer concerns. In this paper, plant-derived activation domains were screened in Trichoderma reesei and Pichia pastoris using mCherry as a target gene for measuring the expression levels. The best expression systems were also tested for protein production in T. reesei and P. pastoris. We tested the production of two different proteins – a bacterial xylanase and a phytase. Two of the novel activation domains provided similar expression levels to the viral activation domain in both fungi. In addition, we developed optimized expression systems for an unconventional yeast from Zygosaccharomyces spp. using the novel transcription factors. The best SES version was used for secretion signal sequence screening for xylanase protein production. To further improve the use of T. reesei as a production host, the CRISPR-Cas9 system with the Cas9 D10A nickase version was tested for transformation of T. reesei. Here, we demonstrated the genomic integration and expression of Cas9 D10A nickase in T. reesei using the SES system with the novel plant-derived activation domain. Furthermore, we successfully transformed the T. reesei Cas9 D10A nickase expressing strain using only guide-RNAs and a donor DNA.

Filamentous fungus Trichoderma reesei (teleomorph Hypocrea jecorina) is a crucial production organism for enzymes used in industrial applications, such as in feed, food, textile, and biofuel production, due to its ability to secrete high amounts of homologous and heterologous enzymes. Therefore, development of genetic tools to improve the properties of industrial T. reesei strains for even better production yields is essential. In this study, a polyethylene glycol mediated CRISPR-Cas9 transformation method for industrial T. reesei production strains was aimed to be optimised by testing an alternative Cas9 enzyme and varying the stoichiometry and total amount of Cas9 enzyme and single guide RNA in the ribonucleoprotein complex. Correct integration of the gene constructions in the obtained transformants was determined by colony PCR and Southern blot analysis. In addition, two selected background activity encoding genes, endoglucanase 6 and α-glucuronidase 1, were individually deleted from T. reesei xylanase production strain utilising the improved CRISPR-Cas9 transformation protocol. The effect of background activity deletions on the strain growth and protein production were analysed from culture supernatants by pH measurement, Bradford protein assay, sodium dodecyl sulfate polyacrylamide gel electrophoresis, and enzyme activity assays. An improved CRISPR-Cas9 transformation protocol for T. reesei was successfully established basing on high number of transformants and improved DNA integration fidelity. No negative effects were observed in the growth or protein production properties of the background activity deletion strains compared to the xylanase production strain. Thus, further cleansing of T. reesei secretome can be continued to refine the industrial production strains.

Charcot-Marie-Tooth (CMT) on yhteinen nimitys perinnöllisille ääreishermosoluja vaurioittaville neuropatioille. Kyseessä on yksi yleisimmistä neurologisista perinnöllisistä sairauksista esiintyvyyden ollessa noin 1:2500. Monimutkaisten tautimekanismien ja riittämättömien hoitomuotojen vuoksi kyseinen tauti on mielenkiintoinen tutkimuksen kohde. Tutkimusryhmämme on selvittänyt CMT-taudin geenitaustaa Suomessa. Sairauden kliiniseen kuvaan kuuluu vaihtelevin vaikeusastein motosensoriset häiriöt, jotka lievimmillään rajoittuvat distaalisten lihasten heikkouteen. Kuitenkin vakavimmissa tapauksissa sairaus voi viedä potilaan liikuntakyvyn. Tällä hetkellä mitään spesifisiä hoitoja ei ole tarjolla, mutta alati lisääntyvän molekyylitason tiedon myötä muutamia lääkekandidaatteja on otettu kliinisiin kokeisiin. Hermosolujen välikokoista filamenttia koodaava geeni NEFL (neurofilament light) on yksi CMT-tautigeeneistä (CMT1F/2E muoto). Tässä tutkimuksessa pyrin luomaan tautimallin NEFL-proteiinin puutokselle. Muokkasin indusoituja pluripotentteja kantasoluja CRISPR-Cas9 - teknologialla NEFL-geenin poistamiseksi. Sen jälkeen erilaistin muokatut kantasolut alemmiksi motoneuroneiksi. Mallin avulla pyrin selvittämään, miten muiden välikokoisten filamenttien ja sairauteen liitettyjen geenien ilmentyminen muuttuu NEFL-poistogeenisissä neuroneissa. Sain erilaistettua kolme muokkaamaamme solulinjaa alemmiksi motoneuroneiksi. Täydellinen NEFL-proteiinin poisto oli tapahtunut kahdessa näistä linjoista. Käytin tutkimuksen analyyttisessä osassa avuksi RNA- ja proteiinitason tutkimusmenetelmiä (qPCR, Western Blot, immunosytokemia). Tuloksista selvisi, että periferiinin määrä oli noussut western blotissa, kun NEFL puuttuu soluissa. Lisäksi SLITRK2 ja LYNX1 mRNA määrä oli qPCR-analyysin mukaan vähentynyt ja GRM3 mRNA oli lisääntynyt muokatuissa soluissa. Näiden tulosten perusteella CMT2E-taudin patofysiologiaan liittynee muutoksia monissa erilaisissa solunsisäisissä kompensatorisissa mekanismeissa. (202 sanaa)