Browsing by Subject "CRISPR"

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Now showing items 1-13 of 13
  • Balboa, Diego; Weltner, Jere; Eurola, Solja; Trokovic, Ras; Wartiovaara, Kirmo; Otonkoski, Timo (2015)
    CRISPR/Cas9 protein fused to transactivation domains can be used to control gene expression in human cells. In this study, we demonstrate that a dCas9 fusion with repeats of VP16 activator domains can efficiently activate human genes involved in pluripotency in various cell types. This activator in combination with guide RNAs targeted to the OCT4 promoter can be used to completely replace transgenic OCT4 in human cell reprogramming. Furthermore, we generated a chemically controllable dCas9 activator version by fusion with the dihydrofolate reductase (DHFR) destabilization domain. Finally, we show that the destabilized dCas9 activator can be used to control human pluripotent stem cell differentiation into endodermal lineages.
  • Hoikkala, Ville; Ravantti, Janne; Diez-Villasenor, Cesar; Tiirola, Marja; Conrad, Rachel A.; McBride, Mark J.; Moineau, Sylvain; Sundberg, Lotta-Riina (2021)
    CRISPR-Cas immune systems adapt to new threats by acquiring new spacers from invading nucleic acids such as phage genomes. However, some CRISPR-Cas loci lack genes necessary for spacer acquisition despite variation in spacer content between microbial strains. It has been suggested that such loci may use acquisition machinery from cooccurring CRISPR-Cas systems within the same strain. Here, following infection by a virulent phage with a double-stranded DNA (dsDNA) genome, we observed spacer acquisition in the native host Flavobacterium columnare that carries an acquisition-deficient CRISPR-Cas subtype VI-B system and a complete subtype II-C system. We show that the VI-B locus acquires spacers from both the bacterial and phage genomes, while the newly acquired II-C spacers mainly target the viral genome. Both loci preferably target the terminal end of the phage genome, with priming-like patterns around a preexisting II-C protospacer. Through gene deletion, we show that the RNA-cleaving VI-B system acquires spacers in trans using acquisition machinery from the DNA-cleaving II-C system. Our observations support the concept of cross talk between CRISPR-Cas systems and raise further questions regarding the plasticity of adaptation modules. IMPORTANCE CRISPR-Cas systems are immune systems that protect bacteria and archaea against their viruses, bacteriophages. Immunity is achieved through the acquisition of short DNA fragments from the viral invader's genome. These fragments, called spacers, are integrated into a memory bank on the bacterial genome called the CRISPR array. The spacers allow for the recognition of the same invader upon subsequent infection. Most CRISPR-Cas systems target DNA, but recently, systems that exclusively target RNA have been discovered. RNA-targeting CRISPR-Cas systems often lack genes necessary for spacer acquisition, and it is thus unknown how new spacers are acquired and if they can be acquired from DNA phages. Here, we show that an RNA-targeting system "borrows" acquisition machinery from another CRISPR-Cas locus in the genome. Most new spacers in this locus are unable to target phage mRNA and are therefore likely redundant. Our results reveal collaboration between distinct CRISPR-Cas types and raise further questions on how other CRISPR-Cas loci may cooperate.
  • Keskinen, Timo (Helsingin yliopisto, 2020)
    Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is an inherited autosomal dominant disease that leads to cognitive impairment, vascular dementia and ischemic strokes. In CADASIL, vascular smooth muscle cells (VSMCs) degrade gradually and are replaced by connective tissue in the small and mid-sized arteries in the brain. Extracellular granular osmiophilic material (GOM) that surround the VSMCs are a unique feature in CADASIL. The causal gene behind CADASIL is Notch3, which encodes a transmembrane protein with a signaling function. There are over 200 cysteine-altering mutations that cause CADASIL in Notch3. The potential pathology causing mechanism is still unclear, but most likely the mechanism is linked to the aggregation of GOM deposits that are potentially toxic to VSMCs. This thesis project aimed to correct CADASIL causing c.475C>T mutation in Notch3 in different CADASIL cell lines with different CRISPR base editor systems. Another aim was to create induced pluripotent stem cell (iPSC) lines from a CADASIL patient-derived skin biopsy sample to be used in the creation of an in vitro disease model for CADASIL. RNA-based ABEmax base editor system was used to correct immortalized- and primary- CADASIL cell lines. DNA-based ABEmax base editor system was used as a positive control. Simultaneous pluripotent reprogramming and pathogenic CADASIL mutation correction were done in the same transfection during this project. The editing efficiencies were evaluated by Sanger sequencing the genomic target region before and after the transfection. The editing efficiencies were good in general compared to literature. They ranged from 27 % to 73 % target base editing efficiency depending on the editing system-, guide-RNAs - and electroporation parameters used. Confirmed proximal off-target effects were not detected, and distal off-target effects were not evaluated.
  • Lohva, Henri (Helsingfors universitet, 2016)
    Saccharomyces cerevisiae is a popular organism in the production of biofuels, chemicals and pharmaceuticals. This is thanks to a good understanding of its metabolism, GRAS status and the ease of modification. Traditionally its genetic modification has been based on the use of selectable markers. Modifying multi gene pathways has required a sequential process consisting of multiple single gene disruptions together with marker recycling. Additionally, many industrial S. cerevisiae strains are polyploid and lack the same tools for their modification as laboratory strains. In this study we sought to develop CRISPR/Cas9 based genetic engineering method for the modification of industrial S. cerevisiae strains. The CRISPR/Cas9 system is based on the adaptive immunity system of bacteria. It makes use of the Cas9 endonuclease which produces double stranded DNA brake to any location determined by a gRNA molecule. This causes the activation of DNA repair mechanisms which can be utilized to for the genomic integration of a template DNA. This makes transformation events much more likely and thus enables producing multiple modifications at once and removes the need for the of use selectable markers. In our approach Cas9 and gRNA were transformed into the cell in a plasmid together with a separate template DNA molecule. We used this method to remove lyp1, ura3 and can1 genes from diploid and polyploid industrial S. cerevisiae strains multiple genes at a time. Simultaneously we evaluated the effect of the NHEJ repair mechanism on CRISPR/Cas9 by repeating the tests with a deletion strain missing the ku70 gene required by NHEJ. Finally the method was used for the metabolic engineering by integrating the five gene violacein metabolic pathway into two loci in a single transformation event. This study demonstrated the CRISPR/Cas9 method is well suited for the modification of industrial S. cerevisiae strains and is capable of modifying up to three loci at a time in a polyploid yeast strain.
  • Schulman, Alan; Oksman-Caldentey, Kirsi-Marja; Teeri, Teemu (2020)
  • Duru, Ilhan Cem; Andreevskaya, Margarita; Laine, Pia; Rode, Tone Mari; Ylinen, Anne; Lovdal, Trond; Bar, Nadav; Crauwels, Peter; Riedel, Christian U.; Bucur, Florentina Ionela; Nicolau, Anca Ioana; Auvinen, Petri (2020)
    BackgroundHigh pressure processing (HPP; i.e. 100-600MPa pressure depending on product) is a non-thermal preservation technique adopted by the food industry to decrease significantly foodborne pathogens, including Listeria monocytogenes, from food. However, susceptibility towards pressure differs among diverse strains of L. monocytogenes and it is unclear if this is due to their intrinsic characteristics related to genomic content. Here, we tested the barotolerance of 10 different L. monocytogenes strains, from food and food processing environments and widely used reference strains including clinical isolate, to pressure treatments with 400 and 600MPa. Genome sequencing and genome comparison of the tested L. monocytogenes strains were performed to investigate the relation between genomic profile and pressure tolerance.ResultsNone of the tested strains were tolerant to 600MPa. A reduction of more than 5 log(10) was observed for all strains after 1min 600MPa pressure treatment. L. monocytogenes strain RO15 showed no significant reduction in viable cell counts after 400MPa for 1min and was therefore defined as barotolerant. Genome analysis of so far unsequenced L. monocytogenes strain RO15, 2HF33, MB5, AB199, AB120, C7, and RO4 allowed us to compare the gene content of all strains tested. This revealed that the three most pressure tolerant strains had more than one CRISPR system with self-targeting spacers. Furthermore, several anti-CRISPR genes were detected in these strains. Pan-genome analysis showed that 10 prophage genes were significantly associated with the three most barotolerant strains.ConclusionsL. monocytogenes strain RO15 was the most pressure tolerant among the selected strains. Genome comparison suggests that there might be a relationship between prophages and pressure tolerance in L. monocytogenes.
  • Duru, Ilhan C; Andreevskaya, Margarita; Laine, Pia; Rode, Tone M; Ylinen, Anne; Løvdal, Trond; Bar, Nadav; Crauwels, Peter; Riedel, Christian U; Bucur, Florentina I; Nicolau, Anca I; Auvinen, Petri (BioMed Central, 2020)
    Abstract Background High pressure processing (HPP; i.e. 100–600 MPa pressure depending on product) is a non-thermal preservation technique adopted by the food industry to decrease significantly foodborne pathogens, including Listeria monocytogenes, from food. However, susceptibility towards pressure differs among diverse strains of L. monocytogenes and it is unclear if this is due to their intrinsic characteristics related to genomic content. Here, we tested the barotolerance of 10 different L. monocytogenes strains, from food and food processing environments and widely used reference strains including clinical isolate, to pressure treatments with 400 and 600 MPa. Genome sequencing and genome comparison of the tested L. monocytogenes strains were performed to investigate the relation between genomic profile and pressure tolerance. Results None of the tested strains were tolerant to 600 MPa. A reduction of more than 5 log10 was observed for all strains after 1 min 600 MPa pressure treatment. L. monocytogenes strain RO15 showed no significant reduction in viable cell counts after 400 MPa for 1 min and was therefore defined as barotolerant. Genome analysis of so far unsequenced L. monocytogenes strain RO15, 2HF33, MB5, AB199, AB120, C7, and RO4 allowed us to compare the gene content of all strains tested. This revealed that the three most pressure tolerant strains had more than one CRISPR system with self-targeting spacers. Furthermore, several anti-CRISPR genes were detected in these strains. Pan-genome analysis showed that 10 prophage genes were significantly associated with the three most barotolerant strains. Conclusions L. monocytogenes strain RO15 was the most pressure tolerant among the selected strains. Genome comparison suggests that there might be a relationship between prophages and pressure tolerance in L. monocytogenes.
  • Elbasani, Endrit; Falasco, Francesca; Gramolelli, Silvia; Nurminen, Veijo; Günther, Thomas; Weltner, Jere; Balboa, Diego; Grundhoff, Adam; Otonkoski, Timo; Ojala, Päivi M. (2020)
    CRISPR activation (CRISPRa) has revealed great potential as a tool to modulate the expression of targeted cellular genes. Here, we successfully applied the CRISPRa system to trigger the Kaposi's sarcoma-associated herpesvirus (KSHV) reactivation in latently infected cells by selectively activating ORF50 gene directly from the virus genome. We found that a nuclease-deficient Cas9 (dCas9) fused to a destabilization domain (DD) and 12 copies of the VP16 activation domain (VP192) triggered a more efficient KSHV lytic cycle and virus production when guided to two different sites on the ORF50 promoter, instead of only a single site. To our surprise, the virus reactivation induced by binding of the stable DD-dCas9-VP192 on the ORF50 promoter was even more efficient than reactivation induced by ectopic expression of ORF50. This suggests that recruitment of additional transcriptional activators to the ORF50 promoter, in addition to ORF50 itself, are needed for the efficient virus production. Further, we show that CRISPRa can be applied to selectively express the early lytic gene, ORF57, without disturbing the viral latency. Therefore, CRISPRa-based systems can be utilized to facilitate virus-host interaction studies by controlling the expression of not only cellular but also of specific KSHV genes.
  • Culebro, Alejandra (Helsingfors universitet, 2013)
    Campylobacteriosis, the most common bacterial food-borne disease worldwide, is mainly caused by Campylobacter jejuni and C. coli. Most studies have focused on the genetic diversity of C. jejuni, but little is known about C. coli. The aim of this work was to characterize C. coli from different sources, by evaluating the distribution and/or diversity of certain genetic markers. A total of 145 C. coli isolates from different sources (2 goose, 18 poultry, 35 human and 90 swine) were screened for fucP, ggt, cytC, sialyltransferases genes and CRISPRs. Additionally, the diversity of the LOS loci and of the CRISPRs, were assessed. A frequency of 90.34% was observed for fucP and CRISPRs among C. coli. Conversely, the frequency of GGT phenotype, and cytC, and cst-I genotype was 1.38%, while cst-V genotype was 0.69%. Only one isolate was positive for all markers except fucP; no source association was observed. LOS and CRISPRs exhibited a wide diversity. In conclusion, ggt, fucP, and cytC seem to be lineage related in C. coli, and not host associated. CRISPRs were too discriminatory to be of use in epidemiological investigations. Results suggest a high diversity of the LOS, and there may exist more classes than those previously described.
  • Jämsä, Joonas (Helsingin yliopisto, 2020)
    Tämän lainopillisen tutkielman keskeisenä tarkoituksena on selventää patenttipoolien muodostamiseen liittyviä kilpailuoikeudellisia kysymyksiä. Lisäksi erityistarkastelussa ovat biolääketieteen alalle syntyvät patenttipoolit ja niihin kohdistuvat mahdolliset erityisvaatimukset. Patenttipoolien hyötyjä ja haittoja on pohdittu niin oikeudellisista kuin taloustieteellisistä näkökulmista. Nämä tutkimukset ovat kuitenkin pääosin lähtöisin Yhdysvalloista. Euroopassa patenttipoolien hyötyihin ja haittoihin kilpailuoikeudellisesta näkökulmasta ei olla paneuduttu yhtä laajasti. Parhaimmillaan patenttipoolit ovat innovaatioita edistäviä sopimusoikeudellisia konstruktioita, jotka mahdollistavat patenttien lisensoinnin keskitetysti yhden tahon kautta. Lisäksi patenttipoolit voivat vähentää patenttien päällekkäisyyksistä aiheutuvia ongelmia, joissa teknologian tehokas hyödyntäminen on riippuvainen useasta eri oikeudenhaltijasta. On kuitenkin mahdollista, etteivät patenttipoolit kaikissa tapauksissa täytä EU-kilpailuoikeuden asettamia vaatimuksia. Tämän jännitteen johdosta aiheen tarkastelun olennaisuus korostuu. Keskeiseksi kysymykseksi patenttipoolien tehokkaan hyödyntämisen kohdalla nousevat patentinhaltijoiden väliseen suhteeseen ja lisensointiin liittyvät kilpailuoikeudelliset seikat. Tähän liittyy esimerkiksi se, kuinka kilpailijoiden yhteistoiminta voidaan järjestää tavalla, joka ei nosta lisensoinnista aiheutuvia kustannuksia. Bioteknologiapoolit on otettu tutkielmassa erityistarkasteluun, koska ala on kehittynyt huomattavasti viime vuosikymmenien aikana ja sen potentiaali yleisen terveyden ja hyvinvoinnin edistämiseksi merkittävä. Lisäksi vuosituhannen merkittävimmäksi bioteknologian keksinnöksi tituleerattua CRISPR/Cas9-geenitekniikaa koskevien patenttien ympärille on yritetty muodostaa patenttipoolia. Täten aiheen käsittely tarkemmin on ajankohtaista. Patenttipoolit voivat siis osoittautua tehokkaaksi tavaksi lisensoida myös bioteknologian sovelluksia. Nykyinen patenttipooleja koskeva kilpailuoikeudellinen kehys on kuitenkin laadittu pääosin vastaamaan informaatioteknologian ja kuluttajateknologioiden tarpeita. Bioteknologia eroaa merkittävästi näistä teknologian aloista. Tästä johtuen nykyinen regulaatio ei välttämättä sovi yhtä hyvin bioteknologian keksintöjen lisensointiin, vaikka tarve patenttipooleille joillain bioteknologian osa-alueilla saattaa osoittautua ilmeiseksi. Komission antamat suuntaviivat ovat keskeisin oikeuslähde patenttipoolien syvällisemmän kilpailuoikeudellisen arvioinnin kannalta tällä hetkellä. Vaikka suuntaviivojen oikeuslähdeopillinen asema on jokseenkin epävarma, antavat ne selkeät puitteet arvioida keskeisiä kilpailuoikeudellisia kysymyksiä liittyen patenttipoolien perustamiseen. Poolin perustamisen osalta erityisen selviä ovat tilanteet, joissa teknologia perustuu standardeihin. Standardoimattomat teknologiat, joiden osalta pooli saattaa sisältää muitakin kuin teknologialle keskeisiä patentteja osoittautui monimutkaisemmaksi. Tutkimuksessa selvisi, etteivät bioteknologian patentit aina perustu standardoituihin teknologioihin ja esimerkkinä tästä toimii muun muassa perusteilla oleva CRISPR/Cas9-patenttipooli. Komission käytännössä ei ole toistaiseksi arvioitu standardoimattomiin teknologioihin perustuvia patenttipooleja ja niiden asettamia kysymyksiä. Näin ollen mahdollinen CRISPR/Cas9-patenttipooli saattaa kiinnittää mahdollisten kilpailijoiden tai lisenssinsaajien huomion siinä määrin, että poolin lainmukaisuus saattaa tulla komission arvioitavaksi.
  • Mansnérus, Juli; Lahti, Raimo; Blick, Amanda (Faculty of Law, University of Helsinki, 2020)
    Forum Iuris
    The paradigm of personalized medicine is an emerging topic, triggering some specific legal and ethical challenges as regards data collection, sharing and use, informed consent, privacy and public trust, and the changing status of patients and social equality. These legislative developments and challenges have been discussed in light of the Finnish and the common-European experiences. During recent years, the Finnish legislative processes aiming at generating ‘innovation-friendly’ legislation for scientific research purposes as well as integrating genomic research results into the clinical setting have been heavily challenged by rapid developments in technology and medicine. In particular, there is a need to pursue the right balance between scientific and commercial interests, public health, and individual rights. We aim at providing insights into the legislative processes surrounding personalized medicine with a special focus on how the freedom of science, equitable access to healthcare, public health, and commercial issues that must be balanced with individual rights as expressed in the EU Charter and the Council of Europe’s Oviedo Convention on Human Rights and Biomedicine. A wide spectrum of different types of challenges arises; among other things, there is a need to discuss the Finnish and international legislation of genome testing in terms of consent on behalf of a young child. Also the legal and ethical aspects of disruptive gene-editing technologies need to be analysed: How should we interpret the concept of human dignity in the bioethical discussion surrounding germline editing? Furthermore, an overview of ongoing initiatives to accelerate the market-entry of advanced therapy medicinal products will be provided. The European regulators are now taking measures to create a facilitative regulatory environment that encourages innovation, protects public health, and enables timely patient access to innovative, new therapies whilst ensuring patient safety. The role of risk-proportionate adaptations to clinical trials and GMP manufacture along with the European Medicines Agency’s early-access incentives and initiatives are presented as potential facilitators of market entry. Furthermore, in this context, the role of conditional reimbursement schemes and risk sharing-agreements is also discussed in light of the newest Finnish experiences. Furthermore, in terms of patent law, some specific challenges arise; in this anthology, attention is paid to the recent transatlantic legal dispute over a patent concerning the use of the CRISPR/Cas9 system in eukaryotic cells. Finally, some considerations beyond legal or ethical aspects of personalised medicine are presented. How can machine learning be used to support personalized care that addresses the patient’s needs?
  • Seecharran, Tristan; Kalin-Manttari, Laura; Koskela, Katja; Nikkari, Simo; Dickins, Benjamin; Corander, Jukka; Skurnik, Mikael; McNally, Alan (2017)
    Yersinia pseudotuberculosis is a Gram-negative intestinal pathogen of humans and has been responsible for several nationwide gastrointestinal outbreaks. Large-scale population genomic studies have been performed on the other human pathogenic species of the genus Yersinia, Yersinia pestis and Yersinia enterocolitica allowing a high-resolution understanding of the ecology, evolution and dissemination of these pathogens. However, to date no purpose-designed large-scale global population genomic analysis of Y. pseudotuberculosis has been performed. Here we present analyses of the genomes of 134 strains of Y. pseudotuberculosis isolated from around the world, from multiple ecosystems since the 1960s. Our data display a phylogeographic split within the population, with an Asian ancestry and subsequent dispersal of successful clonal lineages into Europe and the rest of the world. These lineages can be differentiated by CRISPR cluster arrays, and we show that the lineages are limited with respect to inter-lineage genetic exchange. This restriction of genetic exchange maintains the discrete lineage structure in the population despite co-existence of lineages for thousands of years in multiple countries. Our data highlights how CRISPR can be informative of the evolutionary trajectory of bacterial lineages, and merits further study across bacteria.
  • Vesikansa, Aino (2018)
    The complex structure and highly variable gene expression profile of the brain makes it among the most challenging fields to study in both basic and translational biological research. Most of the brain diseases are multifactorial and despite the rapidly increasing genomic data, molecular pathways and causal links between genes and central nervous system (CNS) diseases are largely unknown. The advent of an easy and flexible CRISPR-Cas genome editing technology has rapidly revolutionized the field of functional genomics and opened unprecedented possibilities to dissect the mechanisms of CNS disease. CRISPR-Cas allows a plenitude of applications for both gene-focused and genome-wide approaches, ranging from original "gene scissors" making permanent modifications in the genome to the regulation of gene expression and epigenetics. CRISPR technology provides a unique opportunity to establish new cellular and animal models of CNS diseases and holds potential for breakthroughs in the CNS research and drug development.