Genome editing in bread wheat using CRISPR/Cas9

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dc.contributor.advisorBurger, Johanen_ZA
dc.contributor.advisorCampa, Manuelaen_ZA
dc.contributor.advisorBotes, Willemen_ZA
dc.contributor.authorDijkerman, Alexanderen_ZA
dc.contributor.otherStellenbosch University. Faculty of AgriSciences. Dept. of Genetics. Institute for Plant Biotechnology.en_ZA
dc.date.accessioned2022-03-09T09:11:36Z
dc.date.accessioned2022-04-29T09:44:33Z
dc.date.available2022-03-09T09:11:36Z
dc.date.available2022-04-29T09:44:33Z
dc.date.issued2022-04
dc.descriptionThesis (MScAgric)--Stellenbosch University, 2022.en_ZA
dc.description.abstractENGLISH ABSTRACT: Bread wheat (Triticum aestivum) is one of the most important food crops consumed by humans, providing approximately 20% of the world’s total caloric intake. However, wheat yields must be increased to supply the growing demand of an increasing global population. Traditional breeding techniques will not be sufficient to confront this challenge and improved genetic engineering and molecular-based techniques will be a necessity. The CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR associated) technology has emerged as a promising genetic engineering tool for the purposes of plant breeding. The CRISPR/Cas9 system has recently been developed as a DNA-free genome editing technique allowing for a precise and efficient method to genetically improve bread wheat while mitigating regulatory concerns. This study, therefore, aimed to establish CRISPR/Cas9 system in bread wheat and investigate the feasibility of this system in a DNA-free format. To achieve this, ribonucleoproteins (RNPs) were assembled by complexing single guide RNA (sgRNA) sequences targeting regions of a gene involved in the carotenoid pathway to the Cas9 protein. The RNPs were subsequently introduced to immature embryos through biolistic bombardment. Immature embryos were assessed to confirm successful editing of the target genes. No editing was detected in the experimental target. Evidence is provided for successful editing in embryos bombarded with RNPs mediated by a previously validated sgRNA. To establish an efficient method of achieving CRISPR/Cas9 edited wheat plants, a multiplex CRISPR/Cas9 DNA construct was assembled and introduced into wheat tissues through Agrobacterium-mediated transformation and particle bombardment. No edits were detected in plantlets that were regenerated on selective media from embryos transformed with Agrobacterium carrying the CRISPR/Cas9 construct. However, indels were detected in pre-initiated calli bombarded with the multiplex CRISPR/Cas9 DNA construct when analysed with the ICE v2 online software. Furthermore, various wheat transformation and regeneration protocols are assessed. Overall, the results provide insights into methods to deliver CRISPR/Cas9 components into bread wheat explant tissue for genome editing. A rapid and accessible method of screening for edits in pooled samples through PCR/RE assays followed by ICE v2 software analyses is demonstrated. Further, a vector delivery method that could circumvent challenging tissue culture procedures through the bombardment of mature embryos is explored. Possible optimisations of CRISPR/Cas delivery systems and experimental design are highlighted for future studies.en_ZA
dc.description.abstractAFRIKAANSE OPSOMMING: Broodkoring (Triticum aestivum) is een van die belangrikste voedselgewasse wat deur mense verbruik word, wat ongeveer 20% van die wêreld se totale kalorie-inname verskaf. Koringopbrengste moet egter verhoog word om in die groeiende aanvraag van 'n toenemende wêreldbevolking te voorsien. Tradisionele teeltegnieke sal nie voldoende wees om hierdie uitdaging die hoof te bied nie en verbeterde genetiese ingenieurswese en molekulêr-gebaseerde tegnieke sal 'n noodsaaklikheid wees. Die CRISPR/Cas-tegnologie het na vore gekom as 'n belowende genetiese ingenieurswerktuig vir die doeleindes van plantteling. Die CRISPR/Cas9-stelsel is onlangs ontwikkel as 'n DNS-vrye genoomredigeringstegniek wat voorsiening maak vir 'n presiese en doeltreffende metode om broodkoring geneties te verbeter terwyl regulatoriese bekommernisse versag word. Hierdie studie het dus ten doel gehad om CRISPR/Cas9-stelsel in broodkoring te vestig en die uitvoerbaarheid van hierdie stelsel in 'n DNS-vrye formaat te ondersoek. Om dit te bereik, is ribonukleoproteïene (RNP's) saamgestel deur enkelgids-RNA (sgRNA)-volgordes te ontwerp wat streke van 'n geen in die karotenoïedweg, teiken, aan die Cas9-proteïen te bind. Die RNP's is daarna in onvolwasse embrio's ingedra deur biolistiese bombardement. Onvolwasse embrios is geassesseer om suksesvolle redigering van die teikengene te bevestig. Geen redigering is in die eksperimentele teiken bespeur nie. Bewyse word egter verskaf vir suksesvolle redigering in embrios wat gebombardeer is met RNP's wat vooraf-bevestigde sgRNA bevat het. Om 'n doeltreffende metode te vestig om CRISPR/Cas9 geredigeerde koringplante te genereer, is 'n multipleks CRISPR/Cas9 DNA-vektor saamgestel en in koringweefsels ingevoeg deur Agrobacterium- gemedieerde transformasie en partikelbombardement. Geen redigering is opgemerk in plantjies wat op selektiewe media geregenereer is vanaf embrios wat getransformeer is met Agrobacterium met die CRISPR/Cas9-konstruk nie. Indels is egter opgespoor in vooraf-geïnisieerde kallusse wat gebombardeer is met die multipleks CRISPR/Cas9 DNA-konstruksie wanneer dit met die ICE v2 aanlyn sagteware ontleed is. Verder word verskeie koringtransformasie- en regenerasieprotokolle geassesseer. Oor die algemeen verskaf die resultate insig in metodes om CRISPR/Cas9- komponente in broodkoring ex-plantweefsel vir genoomredigering te lewer. 'n Vinnige en toeganklike metode van sifting vir redigering in saamgevoegde monsters deur PCR/RE-toetse, gevolg deur ICE v2-sagteware-ontledings word gedemonstreer. Verder word 'n vektor-afleweringsmetode ondersoek wat uitdagende weefselkultuurprosedures kan omseil deur die bombardement van volwasse embrios. Moontlike optimalisering van CRISPR/Cas-afleweringstelsels en eksperimentele ontwerp word uitgelig vir toekomstige studies.af_ZA
dc.description.versionMastersen_ZA
dc.format.extentx, 90 pages : illustrations (some color)en_ZA
dc.identifier.urihttp://hdl.handle.net/10019.1/124977
dc.language.isoen_ZAen_ZA
dc.publisherStellenbosch : Stellenbosch Universityen_ZA
dc.rights.holderStellenbosch Universityen_ZA
dc.subjectGenome editingen_ZA
dc.subjectRibonucleoproteinsen_ZA
dc.subjectWheat genetic transformationen_ZA
dc.subjectDNA -- Analysisen_ZA
dc.subjectWheat (Triticum eastivum) -- Yieldsen_ZA
dc.subjectNucleoproteinsen_ZA
dc.subjectCRISPR-associated protein 9.en_ZA
dc.subjectGenetic engineeringen_ZA
dc.subjectUCTDen_ZA
dc.titleGenome editing in bread wheat using CRISPR/Cas9en_ZA
dc.typeThesisen_ZA
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