Establishing the CRISPR/Cas13a genome editing system in Nicotiana benthamiana for RNA targeting applications

Date
2021-12
Journal Title
Journal ISSN
Volume Title
Publisher
Stellenbosch : Stellenbosch University
Abstract
ENGLISH ABSTRACT: Globally, grapevine (Vitis vinifera) is a widely cultivated fruit crop and makes a vital contribution to the South African agricultural sector. Highly susceptible to a plethora of virus species, grapevine faces severe constraints to overall crop productivity and durable antiviral strategies are necessary to control the spread of viral diseases. The CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR associated) technology has emerged as a valuable genetic engineering tool for plant breeding purposes. Recently, genome editing by the CRISPR/Cas system has been expanded beyond DNA targeting. A novel class 2 Cas effector, Cas13a, has been revealed as a programmable RNA- targeting nuclease. Using CRISPR/Cas13a, this study therefore aimed to investigate the potential of this system to mediate targeted RNA cleavage and viral RNA interference in Nicotiana benthamiana. The choice of this model plant allowed for an immediate test of the functionality and efficacy of this system. For this, binary Cas13a vectors targeting regions of an annotated mRNA transcript from the carotenoid pathway were assembled and transgenic N. benthamiana lines were established. A CRISPR/Cas13a-based down-regulation of gene expression was not observed in these lines. However, after improving the cellular localisation of the Cas13a/crRNA constructs, a transgenic line expressing a cytoplasmic-localised Cas13a/crRNA vector showed a significant two-fold reduction in target gene expression, further correlated with a lowered concentration of total carotenoid content from a preliminary measurement. To demonstrate virus inhibition, a modified tobacco mosaic virus (TMV) system expressing a green fluorescent protein (TRBO-GFP), was used to visually and molecularly measure CRISPR/Cas13a-mediated interference activity in N. benthamiana. A LwaCas13a/crRNA vector targeting a conserved region of the reporter mRNA was assembled and after performing a series of transient assays, phenotypic quantifications of GFP signal intensity confirmed a significant attenuation (~50%) in virus accumulation. However, RT-qPCR analyses showed that GFP mRNA abundance was not directly proportional to that of the observed GFP signal intensity, suggesting a possible limitation in the method of molecular quantification of the GFP mRNA transcript levels. Overall, the results provide insight into the functionality of the CRISPR/Cas13a system for RNA targeting of both an endogenous transcript and a viral genome in plants. Further optimisation of crRNA design features and the method of CRISPR component delivery are highlighted for future studies, especially for an application in grapevine.
AFRIKAANSE OPSOMMING: Wingerd (Vitis vinifera) is wêreldwyd 'n algemeen-verboude vrugtegewas en maak 'n belangrike bydrae tot die Suid-Afrikaanse landbousektor. Aangesien wingerd hoogs vatbaar is vir 'n groot aantal virusspesies, staar die bedryf ernstige beperkings in die gesig ten opsigte van produktiwiteit, en word duur virus-beheerstrategieë benodig om die verspreiding van virussiektes te beheer. Die CRISPR/Cas (gegroepeerde eweredig-gespasieërde kort palindromiese herhalings/CRISPR-geassosieerde) tegnologie het as 'n genetiese manipulasie toepassing vir planttelingsdoeleindes na vore gekom. Onlangs is genoom-redigering deur middel van CRISPR/Cas verder as net DNS as teiken uitgebrei. Die nuwe klas-2 Cas effektor, Cas13a, het as ‘n programmeerbare RNS-teiken nuklease te voorskyn gekom. Die doel van hierdie studie was om die potensiaal van CRISPR/Cas13a op geteikende RNS kliewing en virus-RNS inhibisie in Nicotiana benthamiana, te ondersoek. Die keuse van hierdie modelplant het die vinnige toets van beide funksionaliteit en doeltreffendheid van hierdie stelsel moontlik gemaak. Hiervoor is binêre Cas13a vektore ontwikkel wat areas van ‘n geannoteerde bRNS transkrip van die karotenoïed-weg teiken, en transgeniese N. benthamiana lyne is daargestel. 'n CRISPR/Cas13a- gebaseerde afregulering van geenuitdrukking was nie in hierdie lyne waargeneem nie. Na die verbetering van die sellulêre lokalisering van die Cas13a/crRNS konstrukte egter, is 'n transgeniese lyn, wat 'n sitoplasmies-gelokaliseerde Cas13a/crRNA vektor uitdruk, ontwikkel wat 'n beduidende tweevoudige vermindering in teiken geenuitdrukking getoon het, en wat ook goed gekorreleer het met 'n verlaagde konsentrasie van totale karotenoïed-inhoud. Om virus-inhibisie te demonstreer, is 'n gemodifiseerde tabak mosaïek virus (TMV) stelsel, wat 'n groen-fluoresserende proteïen uitdruk (TRBO-GFP), gebruik om die CRISPR/Cas13a-gemedieërde inhibisie-aktiwiteit in N. benthamiana, beide visueel en molekulêr, te meet. 'n Cas13a/crRNS vektor wat 'n gekonserveerde streek van die merker-geen mRNS teiken, was ontwikkel wat in ‘n reeks tydelike uitdrukkingseksperimente ‘n beduidende verswakking (~50%) in virus-akkumulasie bevestig het. Kwantitatiewe molekulêre ontledings van GFP bRNS uitdrukking kon egter nie ‘n direkte korrelasie met die waargenome GFP sein-intensiteit aantoon nie, wat moontlik dui op 'n beperking van die molekulêre kwantifiseringsmetode. Saamgevat, gee die resultate ‘n insig in die funksionaliteit van die CRISPR/Cas13a stelsel vir RNS teikening van beide 'n endogene transkrip asook 'n virusgenoom in plante. Verdere optimisering van crRNS ontwerp en die metode van CRISPR-komponent toediening word benodig vir toekomstige studies, veral vir toepassings in wingerd.
Description
Thesis (MScAgric)--Stellenbosch University, 2021.
Keywords
Grapevine genome editing, RNA editing, Plants -- Virus resistance, Grapevines (Vitis vinifera) -- South Africa, Nicotiana benthamiana, UCTD
Citation