Dead Clustered Regularly Interspaced Short Palindromic Repeats Associated Protein 9 (dCas9) is a catalytically dead Cas9 nuclease. It can be used in conjunction with a SunTag epitope array to recruit multiple copies of the transcriptional activator Viral Protein 64 (VP64) to any target gene, therefore increasing its expression. The system was initially developed in human cells and was shown to induce an up to 50-fold increase in gene expression. The aim of this study was to translate the technology into bread wheat, which is a vital global crop. SunTag constructs optimised for use in wheat were transiently and stably transformed into wheat protoplasts and plants respectively. The transcription, translation and interaction of these constructs were verified, although the results suggested they were weakly expressed or were deleterious in wheat. RNA sequencing (RNA-seq) analysis showed that SunTag could be used in protoplasts to increase expression of an exogenous target by 1.77-fold, however, no upregulation was detected when an endogenous gene was targeted. This study shows that the SunTag system can be implemented in wheat, however further development is necessary for it to become a useful tool. There are additional experiments which should be conducted to more comprehensively assess the power and specificity of SunTag in wheat.
|Date of Award||29 Sep 2020|
- The University of Bristol
|Supervisor||Keith J Edwards (Supervisor) & Gary L A Barker (Supervisor)|