Epsilon Archive for Student Projects

Abstract

This project aimed to develop a gene-editing system to knock out (KO) genes in Cluster 8 of Arabidopsis thaliana, which are involved in pollen development. Building on the findings of Reimegård et al. (2017) and Xie et al. (2015), the project generated a synthetic dicistronic sgRNA gene (SDRG) and CRISPR/Cas9 system to create targeted KOs of genes with high linkage disequilibrium. The Final Vector containing this system was successfully assembled and can be used for follow-up functional analyses. Phylogenetic analysis indicated significant genetic redundancy within the PME gene family, complicating functional studies. Removing gene redundancy is expected to reveal more pronounced phenotypes. Initial T1 generation screening showed promising results, with 15% of genotyped individuals scoring positive for the inserted transgene. The technology developed herein can help further elucidate plants’ reproductive development and holds the potential for developing alternative methods to induce male sterility, contributing to hybrid vigour in crops. Overall, this project underscored the complexity of multifactorial genetic experiments and provided a foundation for future investigations into the functional roles of Cluster 8 genes in pollen development.