Advances in biotechnology: genomics and genome editing

Genomics, the study of genes, their functions and related techniques has become a crucial science for developing understanding of life processes and how they evolve. Since the advent of the human genome project, huge strides have been made in developing understanding of DNA and RNA sequence information and how it can be put to good use in the biotechnology sector. Newly derived sequencing and bioinformatics tools have added to the torrent of new insights gained, so that 'sequence once and query often' type DNA apps are now becoming reality. Genome editing, using tools such as CRISPR/Cas9 nuclease or Cpf1 nuclease, provide rapid methods for inserting, deleting or modifying DNA sequences in highly precise ways, in virtually any animal, plant or microbial system. Recent international discussions have considered human germline gene editing, amongst other aspects of this technology. Whether or not gene edited plants will be considered as genetically modified remains an important question. This will determine the regulatory processes adopted by different groups of nations and applicability to feeding the world's ever growing population. Questions surrounding the intellectual property rights associated with gene editing must also be resolved. Mitochondrial replacement therapy leading to '3-Parent Babies' has been successfully carried out in Mexico, by an international team, to correct mother to child mitochondrial disease transmission. The UK has become the first country to legally allow 'cautious use' of mitochondrial donation in treatment. Genomics and genome editing will continue to advance what can be achieved technically, whilst society determines whether or not what can be done should be applied

Health Biotechnology Innovation for Social Sustainability -A Perspective from China

China is not only becoming a significant player in the production of high-tech products, but also an increasingly important contributor of ideas and influence in the global knowledge economy. This paper identifies the promises and the pathologies of the biotech innovation system from the perspective of social sustainability in China, looking at the governance of the system and beyond. Based on The STEPS Centre’s ‘Innovation, Sustainability, Development: A New Manifesto’, a ‘3D’ approach has been adopted, bringing together social, technological and policy dynamics, and focusing on the directions of biotechnological innovation, the distribution of its benefits, costs and risks and the diversity of innovations evolving within it and alongside it

Creating CRISPR-Cas9 genome edited iPSC lines to model a patient-specific mutation in mitochondrial disease

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

For a learnable mathematics in the digital cultures

I begin with some general remarks concerning the co-evolution of representational forms and mathematical meanings. I then discuss the changed roles of mathematics and novel representations that emerge from the ubiquity of computational models, and briefly consider the implications for learning mathematics. I contend that a central component of knowledge required in modern societies involves the development of a meta-epistemological stance – i.e. developing a sense of mechanism for the models that underpin social and professional discourses. I illustrate this point in relation to recent research in which I am investigating the mathematical epistemology of engineering practice. Finally, I map out one implication for the design of future mathematical learning environments with reference to some data from the "Playground Project"

Targeting FGF8a Promoter for Gene Expression

In this paper, I will be assisting in developing a new method of characterizing communication between the enhancers and promoter within the fgf8a gene in vertebrates, specifically in zebrafish. The fgf8a gene controls important growth functions and regulates developmental processes in zebrafish. Currently, there is no understanding on how the enhancers function within the fgf8a gene, thus my research will further describe gene regulation and the role of these enhancers. My expected outcomes are to create specific engineered CRISPR RNA that will target the gene promoter and to potentially visualize the gene promoter within the nucleus. Previous research studied the role of enhancers in deceased samples and our research will develop an approach for imaging the promoter portion of the gene in real-time. Targeting the promoter is the first step to identifying the interaction between the promoter and enhancers in the fgf8a gene. Two CRISPR RNA sequences developed to target the fgf8a promoter were inserted into zebrafish embryos to test against a control. We conducted an inexpensive, rapid genomic extraction method with a high-resolution melt assay that is more sensitive and allows early detection of CRISPR-induced indels in zebrafish. The results showed that the CRISPR RNA sequences caused a mutation in genetic expression and confirmed targeting of the fgf8a promoter. These findings will lead us to a step closer in visualizing the interaction of the enhancers and promoters of the gene to further understand its regulation. Demonstrating and defining how this gene expresses itself in zebrafish can advance our understanding of gene expression in human development

SMAJ-tautia aiheuttavan mutaation korjaus potilaan lihaksen kantasoluissa CRISPR-Cas9 geenieditointitekniikalla

Vuonna 2011 Suomesta löydettiin uusi perinnöllinen sairaus, jonka todettiin olevan lähtöisin Pohjois-Karjalan alueelta ja siten osa suomalaista tautiperimää. Taudin nimi on Jokela tyypin spinaalinen lihasatrofia (Spinal Muscular Atrophy of Jokela Type = SMAJ) ja sitä sairastaa yhteensä arviolta 200–400 potilasta. SMAJ on autosomaalisesti vallitseva, alempien liikehermosolujen rappeumasairaus, jonka aiheuttaa pistemutaatio c.197G>T geenissä CHCHD10 ja yhden aminohapon vaihtuminen p.G66V vastaavassa proteiinissa. CHCHD10 on saman nimisestä geenistä tuotettu proteiini, jota esiintyy mitokondrioiden ulko- ja sisäkalvon välissä. Sen tarkkaa toimintaa ei tunneta, eikä miten mutaatio siihen vaikuttaa. Taudin aiheuttavan mekanismin selvittäminen on kuitenkin olennaisen tärkeää, jotta mahdollisia hoitoja ja diagnostisia testejä on mahdollista kehittää. Tutkielman tavoite on korjata tämä mutaatio heterotsygootin SMAJ-potilaan myoblasteissa (lihassolun esiaste) käyttämällä CRISPR-Cas9 geenieditointi teknologiaa. Korjaamalla mutaatio, voidaan luoda solulinja, joka on muuten täysin identtinen potilaan solujen kanssa, mutta mutaatio geenissä CHCHD10 on korjattu vastaamaan normaalia. Vertailemalla tätä solulinjaa potilaan soluihin, voidaan selvittää ainoastaan mutaatiosta johtuvat erot soluissa. Tutkielmassa pyritään selvittämään taudin aiheuttavan mutaation vaikutuksia ihmisen lihassoluissa. Myoblasti-solujen CRISPR-Cas9 geenieditointi ei ole yleistä, sillä usein vastaavan kaltaisia tutkimuksia tehdään indusoiduilla kantasoluilla, joista kohdekudoksen soluja on helppo erilaistaa. Nyt käytettävissä oli kuitenkin potilaan tutkimuskäyttöön luovuttamia lihaksen kantasoluja ja oli mielenkiintoista tutkia mitokondriaalisen CHCHD10 proteiinin aiheuttamaa tautia mitokondriorikkaissa lihassoluissa. CRISPR-Cas9 ribonukleproteiinikompleksia (RNP) ja sitä vastaavaa korjaustemplaattia käytettiin mutaation korjaamisessa. RNP-kompleksi transfektoitiin soluihin elektroporaation avulla, jota ennen elektroporaatio-olosuhteet optimoitiin lihassoluille edullisiksi. Elektroporoitujen solulinjojen geenieditoinnin onnistuminen arvioitiin sekä restriktioentsyymianalyysin, että Synthego ICE CRISPR internettyökalun avulla. Klonaaliset solulinjat luotiin fluoresenssiavusteisella solun lajittelu teknologialla (FACS) ja manuaalisesti poimimalla kolonioita solu maljoilta. Kloonien genotyypit selvitettiin Sanger sekvensoimalla ja arvioitiin off-target geenieditoinnin varalta. Yksi korjattu solulinja saatiin valmistettua ja geenieditointiprosessin optimisaatio myoblasti-soluille onnistui. Tuotetun isogeenisen solulinjan avulla voidaan tulevaisuudessa tutkia CHCHD10:n proteiini- ja mRNA-tasojen eroja verrattuna potilassolulinjaan, ja näin saada arvokasta informaatiota sairauden vaikutuksista lihassoluissa. Tulevaisuuden tehtäviin lukeutuu myös mutaation vaikutuksien hermolihasliitokseen tutkiminen indusoiduista kantasoluista erikoistettujen hermosolujen avulla. Yhteissolukulttuurit mahdollistavat editoitujen myoblastien, sekä hermosolujen kasvattamisen ja hermolihasliitoksen tutkimisen in vitro. Hermolihasliitos on tärkeä yhdistäjä alempien liikehermosolujen ja luustolihasten välissä ja mutaation vaikutuksen tutkiminen voisi valaista SMAJ-tautimekanismia entisestään.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

Ocean Energy in Belgium - 2019

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The Future of Biotechnology Crime: A Parallel Delphi Study with Non-Traditional Experts

BACKGROUND: The way science is practiced is changing and forecasting biotechnology crime trends remains a challenge as future misuses become more sophisticated. METHODS: A parallel Delphi study was conducted to elicit future biotechnology scenarios from two groups of experts. Traditional experts, such as professionals in national security/intelligence, were interviewed. They were asked to forecast emerging crime trends facilitated by biotechnology and what should be done to safeguard against them. Non-traditional experts, such as “biohackers” who experiment with biotechnology in unexpected ways, were also interviewed. The study entailed three rounds to obtain consensus on (i) biotechnology misuse anticipated and (ii) potential prevention strategies expected. RESULTS: Traditional and non-traditional experts strongly agreed that misuse is anticipated within the cyber-infrastructure of, for example, medical devices and hospitals, through breaches and corporate espionage. Preventative steps that both groups strongly advocated involved increasing public biosecurity literacy, and funding towards addressing biotechnology security. Both groups agreed that the responsibility for mitigation includes government bodies. Non-traditional experts generated more scenarios and had a greater diversity of views. DISCUSSION: A systematic, anonymous and independent interaction with a diverse panel of experts provided meaningful insights for anticipating emerging trends in biotechnology crime. A multi-sector intervention strategy is proposed