Academic literature on the topic 'EDITING GENETICO'
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Journal articles on the topic "EDITING GENETICO"
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Sutton, Agneta. "Editing della linea germinale: quali sono i rischi sociali e morali? / Germ-line gene editing: What are the social and moral risks?" Medicina e Morale 65, no. 2 (September 21, 2016): 123–30. http://dx.doi.org/10.4081/mem.2016.430.
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Dovremmo accogliere tutti i possibili sviluppi dell’editing genetico? L’editing genetico delle cellule somatiche potrebbe essere considerato alla pari delle terapie convenzionali volte a trattare particolari patologie o ad alleviarne i sintomi. Tale intervento interesserebbe esclusivamente il singolo paziente trattato. Esso potrebbe quindi essere ben accolto come un nuovo tipo di trattamento per i tumori e le malattie del sangue, come ad esempio la beta-talassemia. Diversamente, l’editing della linea germinale avrebbe effetti ereditari. Ciò solleva preoccupazioni particolari riguardo al rischio medico. I rischi medici non sono, tuttavia, gli unici tipi di rischi che possono derivare dalla modificazione genetica della linea germinale. Nel contributo non vengono discussi i rischi medici, ma quelli sociali e morali correlati alla manipolazione genetica della linea-germinale. ---------- Should we welcome all developments in gene editing? Somatic cell gene editing would be on a par with conventional therapies aimed at treating particular conditions or alleviating symptoms. It would solely affect the individual patient treated. It could thus serve as a welcome new kind of treatment for cancers and blood diseases such as ß-thalassaemia. Germ-line gene editing, on the other hand, would have hereditary effects. This raises special concerns about medical mishaps. Medical risks are, however, not the only kinds of risks in the case of germline gene editing. Discussed here are not the medical risks, but the social and moral risks of germ-line-gene editing.
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Refolo, Pietro, Vincenzo L. Pascali, and Antonio G. Spagnolo. "Editing genetico: nuova questione bioetica? / Gene editing: a new issue for Bioethics?" Medicina e Morale 66, no. 3 (July 3, 2017): 291–304. http://dx.doi.org/10.4081/mem.2017.493.
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Modificazioni controllate nel genoma sono possibili, tramite svariate tecniche, sin dagli anni ’70. Nucleasi a dito di zinco, nucleasi TALE, ma soprattutto CRISPR-Cas9 sono tecniche di editing genetico che hanno reso più semplice effettuarle. Il sistema CRISPR-Cas9, in particolare, si sta dimostrando estremamente vantaggioso in termini di accessibilità, efficienza e versatilità. Gli obiettivi del presente contributo consistono nel: 1. ricostruire i “fatti” salienti che hanno determinato l’emergere del topic dell’“editing genetico”; 2. provare a dar risposta a un primo fondamentale interrogativo circa l’originalità dei dilemmi etici da esso sollevato con particolare riferimento al CRISPR-Cas9. La conclusione è che, allo stato attuale, l’impiego di queste nuove tecniche non solleva questioni etiche nuove. L’unica eccezione sembrerebbe essere data dal particolare tipo di mutazioni indotte da queste tecniche, indistinguibili a lungo termine da quelle prodotte dalla natura, fatto che sta già determinando qualche difficoltà nella classificazione degli OGM ottenuti tramite queste tecniche. ---------- Controlled genome mutations are made possible through several techniques since the ’70s. Zinc finger nucleases, TALE nucleases and above all CRISPR-Cas9 system are “gene editing” techniques which have made mutations easier. Particularly, CRISPR-Cas9 system seems to be extremely profitable in terms of accessibility, efficiency and versatility. The aims of the present article are: 1. to reconstruct the main “facts” about the birth of the topic on “gene editing”; to seek to answer a first question about the novelty of issues raised by this topic. Our conclusion is that, from an ethical point of view, using these techniques does not raise new ethical questions. Perhaps, the only exception refers to the specific mutations produced through these techniques which cannot be distinguished from natural mutations and makes GMO classification more difficult.
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Gentile, A., and S. La Malfa. "Il miglioramento genetico per la resistenza agli insetti: una sfida difficile ed attuale." Bullettin of the Gioenia Academy of Natural Sciences of Catania 52, no. 382/SFE (December 22, 2019): DECA10—DECA16. http://dx.doi.org/10.35352/gioenia.v52i382/sfe.80.
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Nello scenario di una nuova agricoltura, le tecniche di miglioramento genetico sono chiamate a svolgere un ruolo importante al fine di rendere i processi produttivi sostenibili anche sotto il profilo ambientale ed economico. Nei prossimi anni sarà necessario produrre di più in condizioni di risorse naturali decrescenti e al tempo stesso assicurare produzioni anche attraverso una riduzione degli input chimici. Negli ultimi cinquant’anni l’integrazione di diverse tecniche e lo sviluppo di metodi di rigenerazione in vitro e di strategie molecolari ha consentito la definizione di nuovi strumenti di miglioramento genetico. Le conoscenze genomiche degli ultimi decenni hanno, infatti, innovato gli strumenti per la realizzazione di programmi di miglioramento genetico, sia in termini di disponibilità di marcatori per selezione assistita o per selezione genomica, sia in termini di conoscenze sulla funzione dei geni responsabili di caratteri agronomici fondamentali, tra i quali la resistenza a stress biotici. I nuovi metodi molecolari, che consentono di ottenere piante cisgeniche, o con sequenze modificate mediante genome editing, si affiancano ai metodi tradizionali di miglioramento genetico superando i limiti dell’incrocio e della selezione, soprattutto con riferimento alla lunghezza dei tempi ed alla impossibilità di prevederne il risultato in termini di caratteristiche modificate. Nella nota vengono descritti i principali metodi di miglioramento genetico e le principali strategie utilizzabili per migliorare la resistenza delle piante agli insetti.
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Grion, Luca. "Post human e gene editing: riflessioni su perfezione e senso del limite." Medicina e Morale 68, no. 4 (December 20, 2019): 423–36. http://dx.doi.org/10.4081/mem.2019.597.
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A partire dalle opportunità terapeutiche e potenzianti dischiuse dalle recenti tecniche di editing genetico, il saggio si interroga sul senso umano della fragilità e sul desiderio di miglioramento che, da sempre, accompagna l’esperienza umana. Al centro della riflessione, che mette in dialogo innovazione tecnologica e riflessione antropologica, vi è la nozione di limite, da alcuni considerato come un male da rimuovere e, da altri, come una condizione essenziale da accogliere. Dopo aver ricostruito i termini essenziali del dibattito, nelle sue battute conclusive il saggio suggerisce la necessità di prendere posizione tra mito della perfezione e vocazione al compimento: ci vuole umiltà per accettarsi per ciò che si è, senza lasciarsi tentare dell’idea di un’umanità perfetta e invulnerabile; ci vuole coraggio per sfidare gli ostacoli che impediscono alla persona di fiorire in pienezza.
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Viglianisi Ferraro, Angelo, André Gonçalo Dias Pereira, and Antonio Casciano. "I NUOVI ORIZZONTI DELLA SPERIMENTAZIONE SUGLI ESSERI UMANI E SUGLI EMBRIONI ED I MOLTI INTERROGATIVI ETICO-GIURIDICI ANCORA DA SCIOGLIERE." Revista Direitos Fundamentais & Democracia 26, no. 1 (April 29, 2021): 135–60. http://dx.doi.org/10.25192/issn.1982-0496.rdfd.v26i12193.
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La possibilità di manipolare il genoma umano non è mai stata così vicina, come lo è oggi grazie alla recente evoluzione della tecnologia CRISPR. Le opportunità di superare i problemi di salute o migliorare gli esseri umani sono in esponenziale aumento. Pertanto, il dibattito sulla terapia genica nelle persone umane e negli embrioni umani non è solo un tema rilevante discusso nel mondo accademico, ma un imperativo di urgenza in tutta la società. L’articolo muove dal considerare dapprima il quadro normativo offerto dalle diverse convenzioni internazionali esistenti in materia – con un particolare focus dedicato alla legislazione portoghese ed italiana – al fine di far emergere i principi, per lo più di carattere negativo, che ispirano la disciplina in tema di editing genetico. La riflessione si sposta poi sulla considerazione della tematica relativa alla sperimentazione sugli embrioni umani, i cui limiti, nei diversi Stati, risentono della diversità delle visioni morali con le quali si affronta la questione della dignità dell’embrione. Ma, nonostante la varietà di tali posizioni di partenza, è parso possibile fissare taluni divieti accolti in tutti gli ordinamenti. Infine, il tema della difficile individuazione di confini chiari utili a distinguere tra l’utilizzazione delle terapie geniche e il diffondersi di una cultura eugenetica vera e propria, il cui rischio pare palesarsi non tanto a livello di scelte pubbliche, quanto piuttosto a livello di opzioni che le moderne biotecnologie in tema di procreazione medicalmente assistita mettono a disposizione dei privati cittadini.
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Hanrahan, Christopher J., Michael J. Palladino, Barry Ganetzky, and Robert A. Reenan. "RNA Editing of the Drosophila para Na+ Channel Transcript: Evolutionary Conservation and Developmental Regulation." Genetics 155, no. 3 (July 1, 2000): 1149–60. http://dx.doi.org/10.1093/genetics/155.3.1149.
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Abstract Post-transcriptional editing of pre-mRNAs through the action of dsRNA adenosine deaminases results in the modification of particular adenosine (A) residues to inosine (I), which can alter the coding potential of the modified transcripts. We describe here three sites in the para transcript, which encodes the major voltage-activated Na+ channel polypeptide in Drosophila, where RNA editing occurs. The occurrence of RNA editing at the three sites was found to be developmentally regulated. Editing at two of these sites was also conserved across species between the D. melanogaster and D. virilis. In each case, a highly conserved region was found in the intron downstream of the editing site and this region was shown to be complementary to the region of the exonic editing site. Thus, editing at these sites would appear to involve a mechanism whereby the edited exon forms a base-paired secondary structure with the distant conserved noncoding sequences located in adjacent downstream introns, similar to the mechanism shown for A-to-I RNA editing of mammalian glutamate receptor subunits (GluRs). For the third site, neither RNA editing nor the predicted RNA secondary structures were evolutionarily conserved. Transcripts from transgenic Drosophila expressing a minimal editing site construct for this site were shown to faithfully undergo RNA editing. These results demonstrate that Na+ channel diversity in Drosophila is increased by RNA editing via a mechanism analogous to that described for transcripts encoding mammalian GluRs.
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Guo, Shuyu, Ge Gao, Cuizhen Zhang, and Gang Peng. "Multiplexed Genome Editing for Efficient Phenotypic Screening in Zebrafish." Veterinary Sciences 9, no. 2 (February 19, 2022): 92. http://dx.doi.org/10.3390/vetsci9020092.
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Zebrafish are widely used to investigate candidate genes for human diseases. While the emergence of CRISPR-Cas9 technology has revolutionized gene editing, the use of individual guide RNAs limits the efficiency and application of this technology in functional genetics research. Multiplexed genome editing significantly enhances the efficiency and scope of gene editing. Herein, we describe an efficient multiplexed genome editing strategy to generate zebrafish mutants. Following behavioural tests and histological examination, we identified one new candidate gene (tmem183a) for hearing loss. This study provides a robust genetic platform to quickly obtain zebrafish mutants and to identify candidate genes by phenotypic readouts.
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Portela, Manuel. "'Nenhum Problema Tem Solução': Um Arquivo Digital do Livro do Desassossego." Matlit Revista do Programa de Doutoramento em Materialidades da Literatura 1, no. 1 (May 19, 2013): 9–33. http://dx.doi.org/10.14195/2182-8830_1-1_1.
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O projeto 'Nenhum Problema Tem Solução: Um Arquivo Digital do Livro do Desassossego' tem como objetivo a criação de um arquivo digital hipermédia dedicado à obra Livro do Desassossego [LdoD], de Bernardo Soares/Fernando Pessoa. O arquivo agregará fac-símiles digitais dos materiais documentais do LdoD, transcrições topográficas desses materiais, transcrições textuais das quatro edições críticas publicadas entre 1982 e 2010 (Coelho 1982 [2ª edição, 1997]; Cunha 1990-91 [2ª edição, 2008]; Zenith 1998 [10ª edição, 2012]; Pizarro 2010), e ainda ferramentas de pesquisa e análise textual. Esta agregação e codificação eletrónica dos fragmentos combina edição genética e edição social do LdoD, mostrando-o como rede potencial de intenções autorais e como construção conjetural dos seus sucessivos editores. O arquivo digital criará ainda um espaço de virtualização do LdoD que favoreça novas dinâmicas de leitura, edição, investigação e escrita. Este artigo apresenta o projeto nos seus aspetos teóricos, técnicos e metodológicos, enquadrando-os na investigação em curso neste domínio.
Abstract The research project 'No Problem Has Solution: A Digital Archive of the Book of Disquiet' aims to produce a digital hypermedia archive of the Book of Disquiet [LdoD], by Bernardo Soares/Fernando Pessoa. The archive will contain digital facsimiles of the documentary materials of LdoD, topographic transcriptions of those materials, textual transcriptions of the four critical editions published between 1982 and 2010 (Coelho 1982 [2nd edition, 1997]; Cunha 1990-91 [2nd edition, 2008]; Zenith 1998 [10th edition, 2012]; Pizarro 2010), and also tools for search and textual analysis. This aggregation and electronic encoding of textual fragments combines genetic and social editing of LdoD, showing it both as a network of potential authorial intentions and as a conjectural construction of its successive editors. This digital archive will also provide a space for virtualizing LdoD that encourages new dynamics of reading, editing, research, and writing. This article presents the project in its theoretical, technical, and methodological aspects, contextualizing them within ongoing research in the field.
DOI: http://dx.doi.org/10.14195/2182-8830_1-1_1
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Grauso, M., R. A. Reenan, E. Culetto, and D. B. Sattelle. "Novel Putative Nicotinic Acetylcholine Receptor Subunit Genes,Dα5,Dα6andDα7, inDrosophila melanogasterIdentify a New and Highly Conserved Target of Adenosine Deaminase Acting on RNA-Mediated A-to-I Pre-mRNA Editing." Genetics 160, no. 4 (April 1, 2002): 1519–33. http://dx.doi.org/10.1093/genetics/160.4.1519.
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AbstractGenome analysis of the fruit fly Drosophila melanogaster reveals three new ligand-gated ion channel subunits with the characteristic YXCC motif found only in α-type nicotinic acetylcholine receptor subunits. The subunits are designated Dα5, Dα6, and Dα7. Cloning of the Dα5 embryonic cDNAs reveals an atypically large N terminus, part of which is without identifiable sequence motifs and is specified by two polymorphic alleles. Embryonic clones from Dα6 contain multiple variant transcripts arising from alternative splicing as well as A-to-I pre-mRNA editing. Alternative splicing in Dα6 involves exons encoding nAChR functional domains. The Dα6 transcript is a target of the Drosophila adenosine deaminase acting on RNA (dADAR). This is the first case for any organism where a nAChR gene is the target of mRNA editing. Seven adenosines could be modified in the extracellular ligand-binding region of Dα6, four of which are also edited in the Dα6 ortholog in the tobacco budworm Heliothis virescens. The conservation of an editing site between the insect orders Diptera and Lepidoptera makes nAChR editing the most evolutionarily conserved invertebrate RNA editing site so far described. These findings add to our understanding of nAChR subunit diversity, which is increased and regulated by mechanisms acting at the genomic and mRNA levels.
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Ghanta, Krishna S., and Craig C. Mello. "Melting dsDNA Donor Molecules Greatly Improves Precision Genome Editing in Caenorhabditis elegans." Genetics 216, no. 3 (September 22, 2020): 643–50. http://dx.doi.org/10.1534/genetics.120.303564.
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CRISPR genome editing has revolutionized genetics in many organisms. In the nematode Caenorhabditis elegans, one injection into each of the two gonad arms of an adult hermaphrodite exposes hundreds of meiotic germ cells to editing mixtures, permitting the recovery of multiple indels or small precision edits from each successfully injected animal. Unfortunately, particularly for long insertions, editing efficiencies can vary widely, necessitating multiple injections, and often requiring coselection strategies. Here, we show that melting double-stranded DNA (dsDNA) donor molecules prior to injection increases the frequency of precise homology-directed repair (HDR) by several fold for longer edits. We describe troubleshooting strategies that enable consistently high editing efficiencies resulting, for example, in up to 100 independent GFP knock-ins from a single injected animal. These efficiencies make C. elegans by far the easiest metazoan to genome edit, removing barriers to the use and adoption of this facile system as a model for understanding animal biology.
Dissertations / Theses on the topic "EDITING GENETICO"
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BONOMELLI, SARA. "L'EDITING GENETICO GERMINALE UMANO, TRA PROBLEMI ETICI E QUESTIONI DI GOVERNANCE." Doctoral thesis, Università degli Studi di Milano, 2022. http://hdl.handle.net/2434/922688.
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The dissertation aims at identifying and analyzing the scientific, legal, and ethical issues raised by the perspective of intentional modification of human germline by the potential future use of gene editing techniques in the context of human reproduction.
Such a study makes it possible to formulate some critical considerations about human germline gene editing governance. The dissertation claims that the best option to regulate the use of this biotechnological innovation for reproductive purposes consists of a regulation on a state-by-state-basis, which should however be developed within an international governance framework. Several arguments are suggested to underpin this thesis, and some recent initiatives adhering to such governance pattern are examined.
The research is organized in three chapters.
The first chapter, which is introductive to the real research, focuses on the scientific and technical aspects of the thesis topic. More specifically, this chapter aims at laying the foundations for the subsequent discussion, by defining and explaining the notions of i) DNA, gene, chromosome; ii) genetic mutation and genetic disease; and iii) gene therapy and gene editing. Special attention is paid to this latter technology and especially to its potential use on the human germline. Such use is highly controversial, mainly – but not exclusively – since, unlike modifications made by somatic gene editing, those affecting germinal cells – namely, gametes and zygotes – are transmitted to descendants, and thus to next generations.
The second chapter is divided into two sections. The first section reconstructs and analyses the existing regulations in the field of human germline gene editing at international, supranational and national level, stressing their vagueness, fragmentation and lack of specificity. Given the impossibility of extensively examining all relevant domestic laws, guidelines and policies, those of four countries only – the USA, the UK, China and Italy – have been considered in detail. This choice is motivated by the geographical and cultural representativeness of their respective regulations, as well as by the fact that, except for Italy, those countries conducted nearly all the experiments carried out so far in the field of human germline gene editing. The second section of the chapter precisely focuses on these experiments – both for research and reproductive purposes. Jiankui He’s experiment – which resulted in the birth of the world’s first gene-edited babies in 2018 – and Denis Rebrikov’s germline gene editing clinical trial project are thoroughly described and analyzed.
The third and last chapter deals with the ethical issues raised by the perspective of the potential future implementation of germline gene editing interventions in the context of human reproduction. This chapter too is articulated into two sections. The first section provides the theoretical bases for the subsequent ethical analysis, by dividing the possible future uses of germline gene editing techniques into three categories: i) therapeutic interventions; ii) medical enhancement interventions; and iii) non-medical enhancement interventions. Such categorization is paramount, since the various ethical issues related to human germline gene editing do not always involve all three of these categories, and, even when they do, they tend to carry different connotations according to each category. This becomes clear in the second section of the chapter, which critically explores six main ethically problematic areas related to this biotechnological innovation and their numerous articulations.
Finally, the dissertation argues that the scientific, legal and ethical issues identified and examined throughout the research must be taken into account by proper germline gene editing governance mechanisms, which should be the result of parallel and complementary regulatory initiatives promoted both at national and international level.
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JACOB, AURELIEN MARC FLORENT. "IMPROVING TARGETED GENE EDITING IN HEMATOPOIETIC STEM CELLS FOR CLINICAL TRANSLATION." Doctoral thesis, Università degli Studi di Milano-Bicocca, 2021. http://hdl.handle.net/10281/304800.
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Negli ultimi anni, l’editing genetico nelle cellule staminali/progenitrici ematopoietiche umane (HSPC) per il trattamento di malattie genetiche del sangue è migliorato drasticamente trasformando inserzioni genetiche casuali in precise e mirate modificazioni del genoma. La modifica mirata dei geni mutati ereditati consente la correzione in situ e la ricostituzione funzionale con il mantenimento del controllo endogeno dell'espressione. Recentemente abbiamo dimostrato che sia le rotture del DNA a doppio filamento indotte dall’editing che il genoma stesso dell’Adeno-Associated Virus 6 (AAV) innescano una risposta dipendente da p53 nell'HSPC che risulta in un ritardo della proliferazione con conseguente diminuzione della ricostituzione ematopoietica dopo il trapianto delle cellule editate in animali immuno-compromessi. Per cui, abbiamo quindi dimostrato come la soppressione di questa risposta mediante l’espressione transitoria della forma negativa dominante di p53 preservi la ricostituzione del lineage ematopoietico. Tuttavia, la biologia sottostante è rimasta sconosciuta, così come l'impatto dell'editing genetico sulle dinamiche clonali dell'HSPC modificate con riparo diretto per omologia (Homology Directed Repair, HDR) al momento del trapianto. Inoltre, lo stato quiescente delle HSC primitive costituisce un limite per l’editing genetico mediato da HDR, riducendo le sue possibili applicazioni cliniche. In questo lavoro, abbiamo prima superato tale limite esprimendo transitoriamente la proteina dell'adenovirus 5 E4orf6/7, che regola il principale controllore del ciclo cellulare, E2F, insieme alla nucleasi. Mediante un'analisi dell'espressione genica globale e mirata, abbiamo dimostrato come E4orf6/7 spinga le cellule in fase S/G2 con concomitante sovra-regolazione di tutti i principali componenti del macchinario HDR, aumentando così l'efficienza dell'inserimento del transgene in cellule precedentemente quiescenti. Nel contesto dello xenotrapianto, l'espressione combinata di E4orf6/7 e l'inibizione di p53 hanno migliorato l'efficienza del HDR (>50%) all'interno dell'innesto umano totale, superando i livelli riportati fino ad ora in letteratura. Tale risultato è stato riprodotto in diversi donatori da diverse fonti di HSPC e sono stati modificati più loci genomici, dimostrando la maggior versatilità di questa piattaforma se paragonata ad altre strategie di editing. In parallelo, abbiamo ideato una nuova tecnologia (BAR-seq) che consente il monitoraggio clonale di singole HSC modificate con HDR. Questo approccio prevede l’introduzione di un codice a barre ereditabile univoco (BAR) nel templato AAV6 necessario al HDR. Il sequenziamento ad alta copertura di tali sequenze negli xenotrapianti ha mostrato come l’editing genetico risulti in un attecchimento di pochi cloni dominanti. Mentre l'inibizione transitoria di p53 durante l’editing ha consentito un aumento sostanziale della composizione clonale dell'innesto senza alterare la capacità ripopolante delle HSC. Inoltre, questi dati suggeriscono come la risposta mediata da p53 sia responsabile di un'ematopoiesi oligoclonale. È importante sottolineare che il BAR-seq ha fornito la prima prova diretta che le HSC umane modificate con HDR mantengono un potenziale multilineage e subiscono più cicli di divisioni simmetriche e asimmetriche nei trapianti primari e secondari. In conclusione, auspichiamo che i miglioramenti messi a punto nel nostro protocollo di editing possano ampliare le possibili applicazioni cliniche dell’editing genetico.The scope of genome engineering in hematopoietic stem/progenitor cells (HSPCs) has broadened from random to precise genome insertions for treating genetic diseases of the blood lineages. Targeted editing of inherited mutant genes allows in situ correction and functional reconstitution with preserved expression control. We recently showed that both the induced double-strand DNA breaks and the AAV6 genome trigger a p53-dependent DNA damage response in HSPC delaying proliferation and decreasing hematopoietic reconstitution after xenotransplantation. Suppression of this response by transient expression of a dominant negative p53 released cell-cycle block and rescued hematopoietic reconstitution. Yet, the underlying biology remained unknown as well as the impact of gene editing on clonal dynamics of HDR-edited HSPC upon transplantation. Moreover, it has long been contended that the quiescence of primitive HSC constrains HDR-mediated gene editing, thus limiting its perspective clinical applications in several diseases. Here, we first overcame such constraints by transiently expressing the adenovirus 5 protein E4orf6/7, which operates the major cell cycle controller E2F, together with the nuclease. By global and targeted gene expression analysis we showed engagement of targeted cells in S/G2 phases with concomitant upregulation of all major components of the HDR machinery, thus increasing the efficiency of targeted transgene insertion. Combined E4orf6/7 expression and p53 inhibition enhanced >50% HDR efficiency within human graft surpassing the levels reported until now in the literature. Such outcome was reproducible across several HSPC donors and sources, genomic loci and conceivably portable to most types of editing platforms. In parallel, we devised a novel technology (BAR-seq) which enables clonal tracking of individual HDR-edited HSC by introducing a unique heritable barcode in the AAV6 template. Deep sequencing of integrated BARs in human hematochimeric mice showed that only few (5-10) dominant clones of edited HSC robustly contributed to the hematopoietic graft long-term after transplant. Transient p53 inhibition during editing enabled substantial increase in polyclonal graft composition without altering individual HSC output, thus explaining the improved engraftment and highlighting the p53-mediated response as culprit of an otherwise oligoclonal hematopoiesis. Importantly, BAR-seq provided the first direct evidence that human HDR-edited HSC maintain multilineage potential and undergo multiple rounds of symmetric and asymmetric divisions in primary and secondary xenogeneic hosts. Altogether, we expect that the substantial gains obtained in HDR efficiency and polyclonal repopulation by our improved editing protocol should broaden applicability of HSC gene editing and pave its way to clinical translation.
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INSANGUINE, MINGARRO Ferdinando Achille. "MODIFICAZIONI GERMINALI DEL PATRIMONIO GENETICO E BIODIRITTO. I paradossi della de-differenziazione tra bioetica e biodiritto." Doctoral thesis, Università degli Studi di Palermo, 2021. http://hdl.handle.net/10447/507066.
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Il presente lavoro, incominciato nel novembre del 2017, è partito con l'ambizione di ricostruire la risposta che il sistema giuridico fornisce innanzi alle nuove tecniche di ingegneria genetica che, a fronte della loro applicabilità sugli esseri umani, hanno prodotto, negli ultimi anni, il sorgere di nuovi stakeholders e, ancor prima, di nuovi interessi meritevoli di tutela.
Se fino a qualche anno fa pareva impensabile modificare il genoma umano e, men che meno, farlo in maniera precisa, efficiente ed economica, oggi grazie al sistema di modificazione genetica CRISPR/Cas9 è possibile, intervenendo sulla linea germinale degli embrioni umani, prevenire la contrazione di odiose malattie genetiche e, addirittura, a medio termine sradicarle dalla nostra società. Le enormi potenzialità terapeutiche di questa tecnica hanno addirittura attirato l’attenzione dell’Accademia Reale Svedese delle Scienze che, proprio mentre si stanno scrivendo queste righe, ha attribuito alle sue inventrici, Jennifer Doudna ed Emmanuelle Charpentier, il Premio Nobel per la Chimica 2020, definendo CRISPR/Cas9 come “un rivoluzionario metodo di editing genetico che contribuisce allo sviluppo di nuove terapie contro il cancro e può realizzare il sogno di curare malattie ereditarie” (The Royal Swedish Academy of Sciences 2020a). Al fianco di queste prospettive, che dal 2017 ad oggi si sono fatte sempre più evidenti, si annidano però rischi e pericoli derivanti dall’uso delle tecniche d’ingegneria genetica che il diritto deve tenere in adeguata considerazione al momento della loro regolamentazione.
Nei primi mesi di lavoro, dedicati proprio alla ricostruzione delle fonti giuridiche applicabili, ci ha subito colpito che nonostante le tecniche in parola costituiscano, ancora oggi, un’assoluta novità in continuo cambiamento, le norme giuridiche, sia sovranazionali che nazionali, siano relativamente risalenti nel tempo: la legge 40 che, in Italia, si propone di regolare la procreazione medicalmente assistita e alla lett. b) del co. 3 del suo art. 13 si occupa delle manipolazioni genetiche è del 2004, mentre la norma più rilevante sul punto a livello internazionale, l’art. 13 della Convenzione di Oviedo, è addirittura datata aprile 1997. Insomma, in questo campo il diritto anziché presentarsi in fisiologico ritardo, ha enucleato delle regolamentazioni in sospetto anticipo. Questa constatazione, combinata con gli esiti della ricostruzione del dibattito dottrinale, dove anche autorevolissimi autori combinano continuamente argomentazioni etiche ed argomentazioni giuridiche, spesso senza neppure differenziarle, ci ha condotto ad appurare come prima di affrontare il tema della regolamentazione specifica del genome editing fosse necessario riflettere su come il diritto s’interfacci innanzi al bios come oggetto normativo e, soprattutto, in quale relazione si ponga con la bioetica nell’espletare siffatta funzione.
Pertanto, abbiamo deciso di dedicare la Parte Prima dell’opera proprio ad un’indagine sulla relazione tra la bioetica ed il biodiritto, che costituiscono la proiezione applicativa di etica e diritto al bios, finalizzata a dotare di un adeguato fondamento epistemologico l’intuizione della deriva di de-differenziazione tra essi. Per raggiungere tale obiettivo abbiamo ritenuto necessario partire, nel Capitolo I, da una breve genealogia della bioetica in cui ci siamo interrogati sulla nascita di questa disciplina e sulle sue successive svolte metodologiche. Il Capitolo II, invece, è stato dedicato alle origini di quello specifico ambito della comunicazione giuridica, comunemente identificato ormai come biodiritto, mettendo in evidenza i contributi interni che la scienza giuridica ha fornito per lo sviluppo dello stesso e riflettendo, in particolare, sul ruolo che ha giocato in tal senso l’istituzione giuridica dei diritti umani. Al contrario, il Capitolo III parte dai contributi esterni alla nascita del biodiritto e specificatamente quelli forniti dalla bioetica per proseguire, poi, con una riflessione sul rapporto tra questi. Mediante una ricostruzione delle posizioni dominanti in dottrina e soprattutto attraverso uno sguardo fisso alla prassi, si è posto in evidenza come, ad oggi, via sia un problema di de-differenziazione tra bioetica e biodiritto che ha portato quest’ultimo a trasformarsi in una scienza ancillare alla prima al punto da essere definito come “diritto della bioetica”. Lungi dal fermarci su posizioni unicamente critiche, abbiamo dotato l’ultima parte del Capitolo di una pars construens in cui abbiamo evidenziato i vantaggi di una relazione funzionalmente differenziata tra bioetica e biodiritto, senza però trascurare anche i problemi ad essa sottesi.
Con il chiaro intento di testare i nostri approdi teorici nell’esperienza empirica e, allo stesso tempo, per assolvere all’intento originario della nostra opera, abbiamo deciso di dedicare la Parte II interamente alle implicazioni etiche, sociologiche e giuridiche derivanti dalle tecniche di manipolazione genetica germinale. Per farlo si è reso necessario, anzi tutto, dedicare il Capitolo IV a comprendere, tecnicamente, cosa sia una modificazione genetica germinale e quali siano le posizioni rinvenibili all’interno della comunità scientifica. Il Capitolo V, invece, è stato dedicato ad affrontare i problemi, i rischi, le promesse e le speranze che si annidano intorno alla nostra tecnica: dal timore per una deriva eugenetica alla compatibilità delle modificazioni con l’autocomprensione e la dignità del genere umano, passando per le preoccupazioni delle comunità delle persone diversamente abili e dei genitori, che rischiano di restare schiacciati dalle pressioni sociali, giungendo a prendere in seria considerazione però anche le possibilità di sradicare odiose malattie genetiche una volta per tutte, liberando l’umanità di alcune atroci sofferenze. Con un quadro chiaro dei diversi valori che mette in gioco ed in potenziale conflitto tra loro la tecnica germinale, abbiamo finalmente affrontato il problema della regolamentazione delle nostre tecniche. Abbiamo cercato di farlo non con l’animo di produrre una mera attività compilativa sulle regolamentazioni esistenti e neanche con il solo intento di mostrare lacune e paradossi che in esse si annidano, ma con la finalità più ambiziosa di verificare se le nostre conclusioni teoriche della Parte Prima fossero fondate: se effettivamente il diritto si propone come un mero trasformatore permanente di principi bioetici in precetti coercitivi e se l’approccio regolativo vigente sia adeguato per cogliere i benefici che una tecnica premiata con il Nobel per la Chimica può dare alla società, senza rinunciare a tutelare i diritti fondamentali delle persone.
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INSANGUINE, MINGARRO Ferdinando Achille. "Modificazioni germinali del patrimonio genetico e biodiritto. I paradossi della de-differenziazione tra bioetica e biodiritto." Doctoral thesis, Università degli Studi di Palermo, 2021. http://hdl.handle.net/10447/499144.
Full textAbstract:
Il presente lavoro, incominciato nel novembre del 2017, è partito con l'ambizione di ricostruire la risposta che il sistema giuridico fornisce innanzi alle nuove tecniche di ingegneria genetica che, a fronte della loro applicabilità sugli esseri umani, hanno prodotto, negli ultimi anni, il sorgere di nuovi stakeholders e, ancor prima, di nuovi interessi meritevoli di tutela.
Se fino a qualche anno fa pareva impensabile modificare il genoma umano e, men che meno, farlo in maniera precisa, efficiente ed economica, oggi grazie al sistema di modificazione genetica CRISPR/Cas9 è possibile, intervenendo sulla linea germinale degli embrioni umani, prevenire la contrazione di odiose malattie genetiche e, addirittura, a medio termine sradicarle dalla nostra società. Le enormi potenzialità terapeutiche di questa tecnica hanno addirittura attirato l’attenzione dell’Accademia Reale Svedese delle Scienze che, proprio mentre si stanno scrivendo queste righe, ha attribuito alle sue inventrici, Jennifer Doudna ed Emmanuelle Charpentier, il Premio Nobel per la Chimica 2020, definendo CRISPR/Cas9 come “un rivoluzionario metodo di editing genetico che contribuisce allo sviluppo di nuove terapie contro il cancro e può realizzare il sogno di curare malattie ereditarie” (The Royal Swedish Academy of Sciences 2020a). Al fianco di queste prospettive, che dal 2017 ad oggi si sono fatte sempre più evidenti, si annidano però rischi e pericoli derivanti dall’uso delle tecniche d’ingegneria genetica che il diritto deve tenere in adeguata considerazione al momento della loro regolamentazione.
Nei primi mesi di lavoro, dedicati proprio alla ricostruzione delle fonti giuridiche applicabili, ci ha subito colpito che nonostante le tecniche in parola costituiscano, ancora oggi, un’assoluta novità in continuo cambiamento, le norme giuridiche, sia sovranazionali che nazionali, siano relativamente risalenti nel tempo: la legge 40 che, in Italia, si propone di regolare la procreazione medicalmente assistita e alla lett. b) del co. 3 del suo art. 13 si occupa delle manipolazioni genetiche è del 2004, mentre la norma più rilevante sul punto a livello internazionale, l’art. 13 della Convenzione di Oviedo, è addirittura datata aprile 1997. Insomma, in questo campo il diritto anziché presentarsi in fisiologico ritardo, ha enucleato delle regolamentazioni in sospetto anticipo. Questa constatazione, combinata con gli esiti della ricostruzione del dibattito dottrinale, dove anche autorevolissimi autori combinano continuamente argomentazioni etiche ed argomentazioni giuridiche, spesso senza neppure differenziarle, ci ha condotto ad appurare come prima di affrontare il tema della regolamentazione specifica del genome editing fosse necessario riflettere su come il diritto s’interfacci innanzi al bios come oggetto normativo e, soprattutto, in quale relazione si ponga con la bioetica nell’espletare siffatta funzione.
Pertanto, abbiamo deciso di dedicare la Parte Prima dell’opera proprio ad un’indagine sulla relazione tra la bioetica ed il biodiritto, che costituiscono la proiezione applicativa di etica e diritto al bios, finalizzata a dotare di un adeguato fondamento epistemologico l’intuizione della deriva di de-differenziazione tra essi. Per raggiungere tale obiettivo abbiamo ritenuto necessario partire, nel Capitolo I, da una breve genealogia della bioetica in cui ci siamo interrogati sulla nascita di questa disciplina e sulle sue successive svolte metodologiche. Il Capitolo II, invece, è stato dedicato alle origini di quello specifico ambito della comunicazione giuridica, comunemente identificato ormai come biodiritto, mettendo in evidenza i contributi interni che la scienza giuridica ha fornito per lo sviluppo dello stesso e riflettendo, in particolare, sul ruolo che ha giocato in tal senso l’istituzione giuridica dei diritti umani. Al contrario, il Capitolo III parte dai contributi esterni alla nascita del biodiritto e specificatamente quelli forniti dalla bioetica per proseguire, poi, con una riflessione sul rapporto tra questi. Mediante una ricostruzione delle posizioni dominanti in dottrina e soprattutto attraverso uno sguardo fisso alla prassi, si è posto in evidenza come, ad oggi, via sia un problema di de-differenziazione tra bioetica e biodiritto che ha portato quest’ultimo a trasformarsi in una scienza ancillare alla prima al punto da essere definito come “diritto della bioetica”. Lungi dal fermarci su posizioni unicamente critiche, abbiamo dotato l’ultima parte del Capitolo di una pars construens in cui abbiamo evidenziato i vantaggi di una relazione funzionalmente differenziata tra bioetica e biodiritto, senza però trascurare anche i problemi ad essa sottesi.
Con il chiaro intento di testare i nostri approdi teorici nell’esperienza empirica e, allo stesso tempo, per assolvere all’intento originario della nostra opera, abbiamo deciso di dedicare la Parte II interamente alle implicazioni etiche, sociologiche e giuridiche derivanti dalle tecniche di manipolazione genetica germinale. Per farlo si è reso necessario, anzi tutto, dedicare il Capitolo IV a comprendere, tecnicamente, cosa sia una modificazione genetica germinale e quali siano le posizioni rinvenibili all’interno della comunità scientifica. Il Capitolo V, invece, è stato dedicato ad affrontare i problemi, i rischi, le promesse e le speranze che si annidano intorno alla nostra tecnica: dal timore per una deriva eugenetica alla compatibilità delle modificazioni con l’autocomprensione e la dignità del genere umano, passando per le preoccupazioni delle comunità delle persone diversamente abili e dei genitori, che rischiano di restare schiacciati dalle pressioni sociali, giungendo a prendere in seria considerazione però anche le possibilità di sradicare odiose malattie genetiche una volta per tutte, liberando l’umanità di alcune atroci sofferenze. Con un quadro chiaro dei diversi valori che mette in gioco ed in potenziale conflitto tra loro la tecnica germinale, abbiamo finalmente affrontato il problema della regolamentazione delle nostre tecniche. Abbiamo cercato di farlo non con l’animo di produrre una mera attività compilativa sulle regolamentazioni esistenti e neanche con il solo intento di mostrare lacune e paradossi che in esse si annidano, ma con la finalità più ambiziosa di verificare se le nostre conclusioni teoriche della Parte Prima fossero fondate: se effettivamente il diritto si propone come un mero trasformatore permanente di principi bioetici in precetti coercitivi e se l’approccio regolativo vigente sia adeguato per cogliere i benefici che una tecnica premiata con il Nobel per la Chimica può dare alla società, senza rinunciare a tutelare i diritti fondamentali delle persone.
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Pérez, Álvarez Lucía. "Metabolic engineering and genome editing in rice." Doctoral thesis, Universitat de Lleida, 2018. http://hdl.handle.net/10803/665272.
Full textAbstract:
El meu programa d’investigació s’ha basat en la utilització de l’arròs com a model experimental per a estudiar mecanismes i colls d’ampolla que limiten la transició de l’enginyeria metabòlica a la biologia sintètica en les plantes. Em vaig concentrar en la caracterització a nivell molecular i bioquímica de plantes que vaig generar en dos conjunts de línies d’investigació diferents però interrelacionades. En el primer conjunt d’experiments vaig abordar la hipòtesi que en eliminar gens específics en una ruta metabòlica primària, concretament la biosíntesi de midó, les plantes mutants resultants podrien mostrar fenotips propicis per a aplicacions de biologia sintètica en termes de redireccionar el flux i limitar els precursors biosintètics a vies metabòliques secundàries específiques. En aquest context, vaig utilitzar CRISPR/Cas9 per crear dos mutants heterozigots, un amb una glucosa-1-fosfat adenil transferasa (AGPasa) citosòlica severament truncada i no funcional i l’altre amb una modificació estructural C-terminal causant una pèrdua parcial d’activitat. Inesperadament, vaig observar una reducció del midó en les fulles de tots dos mutants i un augment concomitant en el nivell de sucres solubles. Això es va reflectir en l’expressió no prevista d'OsAPL2 i OsAPS2b en les fulles, generant una AGPasa ectòpica completa en el citosol de la fulla, i una disminució en l’expressió de la subunitat petita plastidial OsAPS2a que es va complementar només parcialment amb un augment en l’expressió de OsAPS1
En un conjunt posterior d’experiments amb base similar, vaig investigar els efectes més amplis de les mutacions en un gen de la biosíntesi de midó, la midó sintasa unida a grànuls (GBBS, waxy). Vaig utilitzar CRISPR / Cas9 per introduir un rang de mutacions amb diferents efectes en aquest gen específic. Vaig trobar que les mutacions produïdes van reduir, però no van abolir totalment, l’activitat de GBSS en les llavors a causa d’una compensació parcial causada per la regulació ectòpica de GBSSII. L’activitat de GBSS en els mutants va ser de 61 a 71% dels nivells dels controls, però el contingut d'amilosa, va disminuir del 8 al 12% en llavors heterozigotes i va ser tan baix com nomes un 5% en llavors homozigotes, acompanyat per una organització cel•lular anormal en la capa d'aleurona i amb estructures del gra de midó amorfes. Gairebé tots els gens de la via del midó es van veure afectats a diferents nivells en les fulles i llavors. Aquests canvis en l’expressió gènica van donar com a resultat canvis en l’activitat de la AGPasa i de la sacarosa sintasa que coincidien amb els nivells corresponents de midó i sucres solubles.
La segona línia del meu programa d’investigació es va centrar en l’enginyeria d'una via ectòpica de MVA en plastidis d’arròs per investigar la hipòtesi que al reconstituir la via ectòpica, la regulació estricta de la via de MVA nativa podria relaxar-se en cert grau a mesura que augmentés el conjunt de precursors terpenoides essencials. Els resultats van indicar un augment molt elevat dels nivells d’àcids grassos, luteïna i tocoferol, i una reducció en els nivells d'esqualè i d’esterols.
Els meus resultats són el fonament per a futurs experiments encaminats a determinar si el germoplasma que he creat i caracteritzat pot servir de base per a intervencions d’enginyeria metabòlica i biologia sintètica més complexes.Mi programa de investigación se ha basado en la utilización del arroz como modelo experimental para estudiar mecanismos y cuellos de botella que limitan la transición de la ingeniería metabólica a la biología sintética en las plantas. Me concentré en la caracterización a nivel molecular y bioquímica de plantas que generé en dos conjuntos de líneas de investigación distintas pero interrelacionadas. En el primer conjunto de experimentos abordé la hipótesis de que al eliminar genes específicos en una ruta metabólica primaria, concretamente la biosíntesis de almidón, las plantas mutantes resultantes podrían mostrar fenotipos propicios para aplicaciones de biología sintética en términos de redireccionar el flujo y limitar los precursores biosintéticos a vías metabólicas secundarias específicas. En este contexto, utilicé CRISPR/Cas9 para crear dos mutantes heterocigotos, uno con una glucosa-1-fosfato adenil transferasa (AGPasa) citosólica severamente truncada y no funcional y el otro con una modificación estructural C-terminal causando una pérdida parcial de actividad. Inesperadamente, observamos una reducción del almidón en las hojas de ambos mutantes y un aumento concomitante en el nivel de azúcares solubles. Esto reflejó la expresión no prevista de OsAPL2 y OsAPS2b en las hojas, generando una AGPasa ectópica completa en el citosol de la hoja, y una disminución en la expresión de la subunidad pequeña plastidial OsAPS2a que se complementó solo parcialmente con un aumento en la expresión de OsAPS1 En un conjunto posterior de experimentos, con similar base, investigué los efectos más amplios de las mutaciones en un gen de la biosintésis de almidón, la almidon sintasa unida a gránulos (GBBS, waxy). Utilicé CRISPR/Cas9 para introducir un rango de mutaciones con diferentes efectos en este gen específico. Encontre que las mutaciones producidas redujeron, pero no abolieron la actividad de GBSS en las semillas, debido a una compensación parcial causada por la regulación ectópica de GBSSII. La actividad de GBSS en los mutantes fue de 61 a 71% de los niveles de los controles, pero el contenido de amilosa, sin embargo, disminuyó a 8 a 12% en semillas heterocigotas y fue tan bajo como 5% en semillas homocigotas, acompañado por una organización celular anormal en la capa de aleurona y con estructuras del grano de almidón amorfas. Casi todos los genes de la vía del almidón se vieron afectados a diferentes niveles en las hojas y semillas. Estos cambios en la expresión génica dieron como resultado cambios en la actividad de la AGPasa y de la sacarosa sintasa que coincidían con las alteraciones en los niveles de almidón y azúcares solubles. La segunda línea de mi programa de investigación se centró en la ingeniería de una vía ectópica de MVA en plastidios de arroz para investigar la hipótesis de que al reconstituir la vía ectópica, la regulación estricta de la vía de MVA nativa podría relajarse en cierto grado a medida que aumentase el conjunto de precursores terpenoides esenciales. Los resultados indicaron un aumento en los niveles de ácidos grasos, luteína y tocoferol, una reducción en los niveles de escualeno y niveles similares de esteroles. Mis resultados son el fundamento para futuros experimentos encaminados a determinar si el germoplasma que he creado y caracterizado puede servir de base para intervenciones de ingeniería metabólica y biología sintética más complejas.
My research program used rice as an experimental model to address fundamental bottlenecks and mechanisms limiting the transition from metabolic engineering to synthetic biology in plants. I concentrated on an in depth molecular and biochemical characterization of plants I generated in two distinct, yet interrelated sets of research lines. In the first set of experiments I addressed the hypothesis that by knocking out specific genes in a primary metabolic pathway, starch biosynthesis, resulting mutant plants might exhibit phenotypes conducive to synthetic biology applications in terms of redirecting flux and limiting biosynthetic precursors to specific secondary metabolic pathways. In this context I used CRISPR/Cas9 to create two heterozygous mutants, one with a severely truncated and non-functional cytosolic glucose-1-phosphate adenylyl transferase (AGPase) and the other with a C-terminal structural modification causing a partial loss of activity. Unexpectedly, both mutants exhibited depletion of starch in the leaves and a corresponding increase in the level of soluble sugars. This reflected the unanticipated expression of both OsAPL2 and OsAPS2b in the leaves, generating a complete ectopic AGPase in the leaf cytosol, and a corresponding decrease in the expression of the plastidial small subunit OsAPS2a that was only partially complemented by an increase in the expression of OsAPS1. In a subsequent set of experiments along similar lines I investigated the broader effects of mutations in an additional starch biosynthetic gene, granule bound starch synthase (GBBS, waxy). I used CRISPR/Cas9 to introduce a range of mutations with different effects in this specific gene. All mutations I recovered reduced but did not abolish GBSS activity in seeds due to partial compensation caused by the ectopic upregulation of GBSSII. The GBSS activity in the mutants was 61–71% of wild-type levels, but the amylose content nevertheless declined to 8–12% in heterozygous seeds and to as low as 5% in homozygous seeds, accompanied by abnormal cellular organization in the aleurone layer and amorphous starch grain structures. Almost every starch pathway gene was impacted at different degrees in leaves and seeds. These gene expression changes resulted in changes in AGPase and sucrose synthase activity that explained the corresponding levels of starch and soluble sugars. The second line of my program focused on the engineering of an ectopic MVA pathway in rice plastids in order to investigate the hypothesis that by reconstituting such an ectopic pathway the strict regulation of the native MVA pathway might be relieved to a certain degree in turns increasing the pool of essential terpenoid precursors. Results indicated a profound increase in the levels of fatty acids, lutein and tocopherol, a decrease in squalene levels and similar levels of sterols. My results set the stage for further experiments to ascertain whether germplasm I created and characterized, can serve as a basis for more complex metabolic engineering and synthetic biology interventions.
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McGurk, Leeane. "Drosophila lacking RNA editing." Thesis, University of Edinburgh, 2007. http://hdl.handle.net/1842/2695.
Full textAbstract:
ADAR is an adenosine deaminase that acts on dsRNA. Once bound to dsRNA, ADAR deaminates specific adenosines into inosines. If this occurs within the coding region of a transcript the inosine will be read as a guanosine. This can lead to a change in the amino acid at this position and increase protein diversity. In mammals there are three ADAR genes: ADAR1, ADAR2 and ADAR3. However, only ADAR1 and ADAR2 have been shown to be enzymatically active. ADAR1 is widely expressed and can edit both coding RNA and non-coding RNA. ADAR2 is restricted to the CNS and the key transcript that it edits encodes the GluR-B subunit of the glutamate-gated ion channel receptor. Editing of the Q/R site in the GluR-B transcript occurs with an efficiency of more than 99.9% and changes the genomically encoded glutamine into an arginine. This results in an ion channel that is impermeable to calcium. The ADAR2 knock-out mice are viable, but suffer from epileptic seizures and die by day 20. This phenotype can be rescued by expressing the edited R isoform of GluR-B, suggesting that this site is the most important target for ADAR2. Drosophila has only one Adar gene and its product has been reported to edit more than one hundred adenosines in different transcripts. Many of these transcripts encode subunits of ion channels, and it has been hypothesised that lack of ion channel editing causes the behavioural defects and age-related neurodegeneration observed in Adar deletion mutants. In this thesis I investigate the function of ADAR in an uncharacterised Adar mutant, Adar5G1. To characterise the Adar5G1mutant I not only used standard histology but a 3D imaging technique, optical projection tomography (OPT), that had not been reported to be used with Drosophila before this work. OPT allows the internal organs to be imaged without any manual sectioning or dissecting. I used OPT to identify neurodegenerative vacuoles from within the intact head and present the data both in 2D and in 3D. In addition to this, I demonstrate that this technique can be used to image global expression patterns in the Drosophila adult and I relate the TAU-β galactosidase expression pattern to the Drosophila anatomy. The neurodegeneration observed by OPT was confirmed by detailed analysis of stained wax sections. Complete loss of Adar, in the Adar5G1 mutant revealed age-dependent vacuolisation of the retina and mushroom body calyces. The vacuolisation observed in the Adar5G1 mutant was rescued by expression of Drosophila Adar and human ADAR1 p110, and ADAR2. However the cytoplasmic form of ADAR1, ADAR1 p150, did not rescue the vacuolisation of the Adar5G1 mutant. ADAR3, a catalytically inactive ADAR, rescued the vacuolisation phenotype of the Adar5G1 mutant, suggesting that ADAR may have an additional function independent of editing activity. The vacuolisation of the Adar5G1mutant was found not to be associated with type I programmed cell death. However, it was associated with swollen nerve fibres and degrading ommatidia containing multilamellar whorls. Neurodegeneration in various Drosophila mutant models and human neuropathies has been associated with similar cellular structures, suggesting that loss of ADAR results in neurodegeneration common to many of the known neuropathies. Finally, I found that expression of edited isoforms of the nicotinic receptor channel 34E subunit (Nic 34E) failed to rescue the locomotion phenotype of the Adar mutant. However, I found preliminary evidence that one of the lines generated for an edited isoform of Rdl, a subunit of the GABA receptor ion channel, gave a partial rescue of both locomotion and neurodegeneration of the Adar1F4 and Adar5G1 mutant.
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Chew, Wei Leong. "Postnatal Genome Editing With CRISPR." Thesis, Harvard University, 2016. http://nrs.harvard.edu/urn-3:HUL.InstRepos:33493352.
Full textAbstract:
Targeted genome editing holds tremendous promise for permanent correction of many genetic diseases. The recently developed CRISPR/Cas9 genome-editing tool exhibits facile programmability and robust gene-editing efficiency, and has been applied in cell cultures and animal tissues. However, multi-organ gene-editing in live mammals has not been examined or achieved. This study demonstrates genetic modification in multiple organs of postnatal mice by systemic delivery of CRISPR with adeno-associated viruses (AAVs). I resolved the AAV payload limitation by splitting Cas9 and reconstituting the native protein in vivo using scarless split-intein protein trans-splicing, which preserves full activity of Cas9. I determined that the delivery efficiency of AAV-CRISPR dictates gene-targeting rates in vivo, with the preferential gene-editing in liver and heart, and more modest editing efficiencies in skeletal muscle, brain and gonads, directly reflecting the infection profile of the virus serotype. To track CRISPR biodistribution, I established two reporter systems that apply in situ fluorescence activation to demarcate CRISPR-targeting events at single-cell resolution, identifying rare gene-edited cells that normally evade detection by sequencing. This exquisite detection sensitivity further allows evaluation of inter-generational transmission of gene-editing viruses. Finally, although Cas9 elicits host immune responses, these can be ameliorated by immunosuppression. I also identified a public Cas9-responsive T-cell clonotype and mapped the B-cell epitopes on Cas9 and AAV. Engineering tolerance to immunodominant epitopes may provide an avenue for avoiding immune rejection of AAV-CRISPR. The ability to create programmable genetic modifications in multiple organs of postnatal mammals provides a powerful tool for biological research, and foretells that the genomes of whole mammals may be rewritten at will.Medical Sciences
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Neadeau, Joseph Francis. "Comparing Genetic Modification and Genetic Editing Technolgies: Minimal Required Acreage." Thesis, North Dakota State University, 2018. https://hdl.handle.net/10365/29878.
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There are many technologies being developed for crop breeding. Two interesting technologies are genetic modification and genetic editing. Competitive pressures and changing consumer preferences are forcing organizations to invest heavily in these two technologies. Organizations must decide which traits they want to target and must commit significant time a money to the project. Traditionally, firms would decide which project to embark on if the project is net present value positive. Throughout the research and development process managers have flexibility to abandon the project once new information is received. That flexibility has value and real option analysis must be performed to value that flexibility. Once the value of a GM and GE project is determined, how might an organization decide which project to do? The concept of minimum required acreage (MRA) is developed in this study, allowing organizations to compare GM and GE technologies and decide which project to invest it.
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Robinson, Jason M. "Functional Significance of mtDNA Cytosine Modification Tested by Genome Editing." VCU Scholars Compass, 2016. http://scholarscompass.vcu.edu/etd/4561.
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The field of epigenetics is gaining popularity and speed, due in part to its capability to answer lingering questions about the root cause of certain diseases. Epigenetics plays a crucial role in regulation of the cell and cell survival, particularly by cytosine methylation. It remains controversial if DNMT’s which facilitate methylation are present in mammalian mitochondria and what the functional significance they may have on modification of mitochondrial DNA. CRISPR-Cas9 technology enabled genome editing to remove the MTS (mitochondrial targeting sequence) from DNMT1 of HCT116 cells, purposefully minimizing effects on nuclear cytosine methylation, while exclusively impacting mitochondrial modification. Removal of the DNMT1 MTS did not completely prevent the localization of this enzyme to the mitochondria according to immunoblot analysis. As well, deletion of the MTS in DNMT1 revealed only a small decline in transcription; not until removal of DNMT3B did we see a two-fold decrease in transcription from mitochondrial protein coding genes. No significant decline in transcription occurred when a DNMT3B knockout also lost the MTS of DNMT1; this study is evidencing that DNMT3B is possibly the more significant methyltransferase in the mitochondria. Our aim from this study and future research is to clearly characterize which enzymes in the mitochondria are controlling cytosine modifications and to understand the mechanistic complexities that accompany cause and consequence of epigenetic modifications.
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Rodríguez, José A. "Genetic editing with CRISPR/Cas9: A scientific, ethical, and pastoral approach." Thesis, Boston College, 2019. http://hdl.handle.net/2345/bc-ir:108890.
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Thesis advisor: Andrea ViciniThesis advisor: Colleen M. Griffith
Thesis (STL) — Boston College, 2019
Submitted to: Boston College. School of Theology and Ministry
Discipline: Sacred Theology
Books on the topic "EDITING GENETICO"
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Ulrich, Göringer H., ed. RNA editing. Heidelberg: Springer, 2008.
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Venables, Julian P. Alternative splicing in cancer. Trivandrum, Kerala, India: Transworld Research Network, 2006.
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J, Blencowe Benjamin, and Graveley Brenton R, eds. Alternative splicing in the postgenomic era. New York: Springer Science+Business Media, 2007.
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F, Gesteland Raymond, and SpringerLink (Online service), eds. Recoding: Expansion of Decoding Rules Enriches Gene Expression. New York, NY: Springer Science+Business Media, LLC, 2010.
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J, Hademenos George, ed. Genetics: Based on Schaum's outline of genetics, third edition. New York: McGraw-Hill, 2002.
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Russell, Peter J. iGenetics: A Molecular Approach (2nd Edition with CD-ROM) (The Genetics Place Series). 2nd ed. Benjamin Cummings, 2005.
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Genetic Analysis: An Integrated Approach, Global Edition. Pearson Education, Limited, 2015.
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Essentials of Genetics (4th Edition). Prentice Hall, 2002.
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Klug, William S., Charlotte A. Spencer, Michael A. Palladino, and Michael R. Cummings. Essentials of Genetics: International Edition. Pearson Education, Limited, 2012.
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Poatsy, Mary Anne, John L. Bowman, and Mark F. Sanders. Genetic Analysis: An Integrated Approach with MasteringGenetics, Global Edition. Pearson Education, Limited, 2015.
Find full textBook chapters on the topic "EDITING GENETICO"
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Wünschiers, Röbbe. "Editing Genetic Material." In Genes, Genomes and Society, 141–76. Berlin, Heidelberg: Springer Berlin Heidelberg, 2021. http://dx.doi.org/10.1007/978-3-662-64081-4_5.
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Endo, Masaki, Ayako Nishizawa-Yokoi, and Seiichi Toki. "Rice Genome Editing." In Rice Genomics, Genetics and Breeding, 523–39. Singapore: Springer Singapore, 2018. http://dx.doi.org/10.1007/978-981-10-7461-5_27.
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Wang, Wei, and Eduard Akhunov. "Application of CRISPR-Cas-Based Genome Editing for Precision Breeding in Wheat." In Wheat Improvement, 539–56. Cham: Springer International Publishing, 2022. http://dx.doi.org/10.1007/978-3-030-90673-3_29.
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AbstractWheat improvement relies on genetic diversity associated with variation in target traits. While traditionally the main sources of novel genetic diversity for breeding are wheat varieties or various wild relatives of wheat, advances in gene mapping and genome editing technologies provide an opportunity for engineering new variants of genes that could have beneficial effect on agronomic traits. Here, we provide the overview of the genome editing technologies and their application to creating targeted variation in genes that could enhance wheat productivity. We discuss the potential utility of the genome editing technologies and CRISPR-Cas-induced variation incorporated into the pre-breeding pipelines for wheat improvement.
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Rubio, Mary Anne T., and Juan D. Alfonzo. "tRNA Modification, Editing, and Import in Mitochondria." In Organelle Genetics, 359–80. Berlin, Heidelberg: Springer Berlin Heidelberg, 2011. http://dx.doi.org/10.1007/978-3-642-22380-8_14.
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Myers, Richard, and Edwin R. Hancock. "Genetic algorithms for structural editing." In Advances in Pattern Recognition, 159–68. Berlin, Heidelberg: Springer Berlin Heidelberg, 1998. http://dx.doi.org/10.1007/bfb0033234.
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Miglani, Gurbachan S., and Rajveer Singh. "Epigenome editing in crop improvement." In Quantitative genetics, genomics and plant breeding, 44–70. Wallingford: CABI, 2020. http://dx.doi.org/10.1079/9781789240214.0044.
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Zhang, Ying, and Hao Yin. "Genome Editing for Genetic Lung Diseases." In Pharmaceutical Inhalation Aerosol Technology, 389–402. Third edition. | Boca Raton, Florida : CRC Press, [2019] |: CRC Press, 2019. http://dx.doi.org/10.1201/9780429055201-16.
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Jin, Andrew, and Igor Linkov. "Synthetic Biology Brings New Challenges to Managing Biosecurity and Biosafety." In NATO Science for Peace and Security Series C: Environmental Security, 117–29. Dordrecht: Springer Netherlands, 2021. http://dx.doi.org/10.1007/978-94-024-2086-9_8.
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AbstractNovel biology technologies like gene editing and genetic engineering are creating a proliferation of breakthroughs in engineered biological systems that will change our world in areas ranging from medicine, to textiles, to energy. New developments in gene editing technologies, especially CRISPR-Cas9, have shown early signs of extraordinary potential in a variety of fields, including from basic research, applied biotechnology, and biomedical research. While the possibility of directly targeting and modifying genomic sequences in almost all eukaryotic cells could significantly improve standards of living, these technologies have the potential to pose serious biological hazards.
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Pillay, Michael. "Genome editing technologies for crop improvement." In Quantitative genetics, genomics and plant breeding, 33–43. Wallingford: CABI, 2020. http://dx.doi.org/10.1079/9781789240214.0033.
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Hundleby, Penny, and Wendy Harwood. "Regulatory Constraints and Differences of Genome-Edited Crops Around the Globe." In Genome Editing, 319–41. Cham: Springer International Publishing, 2022. http://dx.doi.org/10.1007/978-3-031-08072-2_17.
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AbstractPlant breeding for centuries has relied on the availability of genetic variation to introduce new desirable traits into crops. Biotechnology has already accelerated the ability to induce and utilize new genetic variation, through approaches such as mutation breeding and using technologies such as marker assisted breeding to rapidly identify the required variation. These technologies fall within the definition of “conventional and traditional” breeding and are lightly regulated. However, plant breeders are facing an urgent need for access to wider genetic variation to meet the needs of today’s farmers and consumers worldwide. New breeding technologies (NBTs), such as genome editing, are speeding up the breeding process and providing plant breeders with access to a far greater range of genetic variation. Coupled with a rapidly accelerating genomics era, genome editing is moving plant breeding into an exciting era of intelligent and precision-based plant breeding. The speed at which these new technologies are emerging has challenged the regulatory climate. Some countries consider genome edited crops to require the same regulatory oversight as genetically modified organisms (GMOs), while others have chosen to regulate with the same safety evaluations currently associated with bringing conventionally bred crops to market. Harmonization of the regulatory climate is urgently needed if there is to be equal access to this technology and to support international trade of these crops. The current chapter provides a global overview of the current regulatory status of genome-edited crops.
Conference papers on the topic "EDITING GENETICO"
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"Analysis and editing the maize gamete interactions and fusion genes." In Plant Genetics, Genomics, Bioinformatics, and Biotechnology. Novosibirsk ICG SB RAS 2021, 2021. http://dx.doi.org/10.18699/plantgen2021-137.
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"Genome editing in wheat: exploration of new challenges for crop improvement." In Plant Genetics, Genomics, Bioinformatics, and Biotechnology. Novosibirsk ICG SB RAS 2021, 2021. http://dx.doi.org/10.18699/plantgen2021-096.
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"CRISPR/Cas9 – mediated genome editing of bread wheat to modulate heading time." In Plant Genetics, Genomics, Bioinformatics, and Biotechnology. Novosibirsk ICG SB RAS 2021, 2021. http://dx.doi.org/10.18699/plantgen2021-135.
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"344. A simulator to evaluate gene editing assisted selection programs." In World Congress on Genetics Applied to Livestock Production. The Netherlands: Wageningen Academic Publishers, 2022. http://dx.doi.org/10.3920/978-90-8686-940-4_344.
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"Improvement of sorghum seed storage protein digestibility using RNA-interference and genome editing." In Plant Genetics, Genomics, Bioinformatics, and Biotechnology. Novosibirsk ICG SB RAS 2021, 2021. http://dx.doi.org/10.18699/plantgen2021-048.
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"StCDF1 gene editing strategy in potato wild species within de novo domestication concept." In Plant Genetics, Genomics, Bioinformatics, and Biotechnology. Novosibirsk ICG SB RAS 2021, 2021. http://dx.doi.org/10.18699/plantgen2021-055.
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Duclos, R., E. Delanoue, L. Journaux, D. Guéméné, M. Sourdioux, and J. P. Bidanel. "494. Perceptions of genome editing in farm animals by livestock stakeholders." In World Congress on Genetics Applied to Livestock Production. The Netherlands: Wageningen Academic Publishers, 2022. http://dx.doi.org/10.3920/978-90-8686-940-4_494.
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Huang, Yong Ping. "Genetic control of Lepidoptera using genome editing tools." In 2016 International Congress of Entomology. Entomological Society of America, 2016. http://dx.doi.org/10.1603/ice.2016.94052.
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Zhang, Xuecheng, Qiuxuan Wu, Lifeng Zhang, and Botao-Zhang. "Genetic Algorithm Inspired by Mimetic Octopus RNA Editing." In 2019 IEEE International Conference on Robotics and Biomimetics (ROBIO). IEEE, 2019. http://dx.doi.org/10.1109/robio49542.2019.8961399.
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Chu, Chi Nung, and Yu Ting Huang. "Assistive Technology for the Struggling Learner: Chinese PCS Editing Processor." In 2011 Fifth International Conference on Genetic and Evolutionary Computing (ICGEC). IEEE, 2011. http://dx.doi.org/10.1109/icgec.2011.44.
Full textReports on the topic "EDITING GENETICO"
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Wentworth, Jonathan, and David Rapley. Genome edited animals. Parliamentary Office of Science and Technology, November 2022. http://dx.doi.org/10.58248/pb50.
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Genome editing, also known as gene editing, encompasses a broad range of techniques that allows targeted changes in the DNA of animals (and plants). The Genetic Technology (Precision Breeding) Bill 2022 -2023, due for Second Reading in the House of Lords on 21 November 2022, intends to change the regulatory definition of certain genome-edited animals.
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Gothilf, Yoav, Roger Cone, Berta Levavi-Sivan, and Sheenan Harpaz. Genetic manipulations of MC4R for increased growth and feed efficiency in fish. United States Department of Agriculture, January 2016. http://dx.doi.org/10.32747/2016.7600043.bard.
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The hypothalamic melanocortin system plays a central role in the regulation of food consumption and energy homeostasis in mammals. Accordingly, our working hypothesis in this project was that genetic editing of the mc4r gene, encoding Melanocortin Receptor 4 (MC4R), will enhance food consumption, feed efficiency and growth in fish. To test this hypothesis and to assess the utility of mc4r editing for the enhancement of feed efficiency and growth in fish, the following objectives were set: Test the effect of the mc4r-null allele on feeding behavior, growth, metabolism and survival in zebrafish. Generate mc4r-null alleles in tilapia and examine the consequences for growth and survival, feed efficiency and body composition. Generate and examine the effect of naturally-occurring mc4r alleles found in swordfish on feeding behavior, growth and survival in zebrafish. Define the MC4R-mediated and MC4R-independent effects of AgRP by crossing mc4r- null strains with fish lacking AgRP neurons or the agrpgene. Our results in zebrafish did not support our hypothesis. While knockout of the agrpgene or genetic ablation of hypothalamic AgRP neurons led to reduced food intake in zebrafish larvae, knockout (KO) of the mc4r gene not only did not increase the rate of food intake but even reduced it. Since Melanocortin Receptor 3 (MC3R) has also been proposed to be involved in hypothalamic control of food intake, we also tested the effectofmc3r gene KO. Again, contrary to our hypothesis, the rate of food intake decreased. The next step was to generate a double mutant lucking both functional MC3R and MC4R. Again, the double KO exhibited reduced food intake. Thus, the only manipulation within the melanocortin system that affected food intake in consistent with the expected role of the system was seen in zebrafish larvae upon agrpKO. Interestingly, despite the apparent reduced food intake in the larval stage, these fish grow to be of the same size as wildtype fish at the adult stage. Altogether, it seems that there is a compensatory mechanism that overrides the effect of genetic manipulations of the melanocortin system in zebrafish. Under Aim 3, we introduced the Xna1, XnB1l, and XnB2A mutations from the Xiphophorus MC4R alleles into the zebrafish MC4R gene. We hypothesized that these MC4R mutations would act as dominant negative alleles to increase growth by suppressing endogenous MC4R activity. When we examined the activity of the three mutant alleles, we were unable to document any inhibition of a co-transfected wild type MC4R allele, hence we did not introduce these alleles into zebrafish. Since teleost fish possess two agrpgenes we also tested the effect of KO of the agrp2 gene and ablation of the AgRP2 cells. We found that the AgRP2 system does not affect food consumption but may rather be involved in modulating the stress response. To try to apply genetic editing in farmed fish species we turned to tilapia. Injection of exogenous AgRP in adult tilapia induced significant changes in the expression of pituitary hormones. Genetic editing in tilapia is far more complicated than in zebrafish. Nevertheless, we managed to generate one mutant fish carrying a mutation in mc4r. That individual died before reaching sexual maturity. Thus, our attempt to generate an mc4r-mutant tilapia line was almost successful and indicate out non-obvious capability to generate mutant tilapia.
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Jean Courret, Ezéchiel. For a Genetic and Diplomatic approach to Old Cadastres: Proposals and Advice for Editing Cadastral Sources. Edicions de la Universitat de Lleida, 2020. http://dx.doi.org/10.21001/itma.2020.14.01.
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Li, Li, Joseph Burger, Nurit Katzir, Yaakov Tadmor, Ari Schaffer, and Zhangjun Fei. Characterization of the Or regulatory network in melon for carotenoid biofortification in food crops. United States Department of Agriculture, April 2015. http://dx.doi.org/10.32747/2015.7594408.bard.
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The general goals of the BARD research grant US-4423-11 are to understand how Or regulates carotenoid accumulation and to reveal novel strategies for breeding agricultural crops with enhanced β-carotene level. The original objectives are: 1) to identify the genes and proteins in the Or regulatory network in melon; 2) to genetically and molecularly characterize the candidate genes; and 3) to define genetic and functional allelic variation of these genes in a representative germplasm collection of the C. melo species. Or was found by the US group to causes provitamin A accumulation in chromoplasts in cauliflower. Preliminary genetic study from the Israeli group revealed that the melon Or gene (CmOr) completely co-segregated with fruit flesh color in a segregating mapping population and in a wide melon germplasm collection, which set the stage for the funded research. Major conclusions and achievements include: 1). CmOris proved to be the gene that controls melon fruit flesh color and represents the previously described gflocus in melon. 2). Genetic and molecular analyses of CmOridentify and confirm a single SNP that is responsible for the orange and non-orange phenotypes in melon fruit. 3). Alteration of the evolutionarily conserved arginine in an OR protein to both histidine or alanine greatly enhances its ability to promote carotenoid accumulation. 4). OR promotes massive carotenoid accumulation due to its dual functions in regulating both chromoplast biogenesis and carotenoid biosynthesis. 5). A bulk segregant transcriptome (BSRseq) analysis identifies a list of genes associated with the CmOrregulatory network. 6). BSRseq is proved to be an effective approach for gene discovery. 7). Screening of an EMS mutation library identifies a low β mutant, which contains low level of carotenoids due to a mutation in CmOrto produce a truncated form of OR protein. 8). low β exhibits lower germination rate and slow growth under salt stress condition. 9). Postharvest storage of fruit enhances carotenoid accumulation, which is associated with chromoplast development. Our research uncovers the molecular mechanisms underlying the Or-regulated high level of carotenoid accumulation via regulating carotenoidbiosynthetic capacity and storage sink strength. The findings provide mechanistic insights into how carotenoid accumulation is controlled in plants. Our research also provides general and reliable molecular markers for melon-breeding programs to select orange varieties, and offers effective genetic tools for pro-vitamin A enrichment in other important crops via the rapidly developed genome editing technology. The newly discovered low β mutant could lead to a better understanding of the Or gene function and its association with stress response, which may explain the high conservation of the Or gene among various plant species.
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Paran, Ilan, and Allen Van Deynze. Regulation of pepper fruit color, chloroplasts development and their importance in fruit quality. United States Department of Agriculture, January 2014. http://dx.doi.org/10.32747/2014.7598173.bard.
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Pepper exhibits large natural variation in chlorophyll content in the immature fruit. To dissect the genetic and molecular basis of this variation, we conducted QTL mapping for chlorophyll content in a cross between light and dark green-fruited parents, PI 152225 and 1154. Two major QTLs, pc1 and pc10, that control chlorophyll content by modulation of chloroplast compartment size in a fruit-specific manner were detected in chromosomes 1 and 10, respectively. The pepper homolog of GOLDEN2- LIKE transcription factor (CaGLK2) was found as underlying pc10, similar to its effect on tomato fruit chloroplast development. A candidate gene for pc1was found as controlling chlorophyll content in pepper by the modulation of chloroplast size and number. Fine mapping of pc1 aided by bulked DNA and RNA-seq analyses enabled the identification of a zinc finger transcription factor LOL1 (LSD-One-Like 1) as a candidate gene underlying pc1. LOL1 is a positive regulator of oxidative stress- induced cell death in Arabidopsis. However, over expression of the rice ortholog resulted in an increase of chlorophyll content. Interestingly, CaAPRR2 that is linked to the QTL and was found to affect immature pepper fruit color in a previous study, did not have a significant effect on chlorophyll content in the present study. Verification of the candidate's function was done by generating CRISPR/Cas9 knockout mutants of the orthologues tomato gene, while its knockout experiment in pepper by genome editing is under progress. Phenotypic similarity as a consequence of disrupting the transcription factor in both pepper and tomato indicated its functional conservation in controlling chlorophyll content in the Solanaceae. A limited sequence diversity study indicated that null mutations in CaLOL1 and its putative interactorCaMIP1 are present in C. chinensebut not in C. annuum. Combinations of mutations in CaLOL1, CaMIP1, CaGLK2 and CaAPRR2 are required for the creation of the extreme variation in chlorophyll content in Capsicum.