Date of this Version
Campbell, D. (2020). Using CRISPR-Cas9 to construct knockout mutants in DNA repair genes in Arabidopsis thaliana. Undergraduate Honors Thesis. University of Nebraska - Lincoln
The mitochondria are known as the powerhouse of the cell, and just like a real powerhouse, it can be a dangerous place to store sensitive information. Energy generation and redox reactions in the mitochondria can cause damage to the DNA stored there, resulting in a higher mutation rate. Compared to their animal counterparts, however, plant mitochondria exhibit a lower mutation rate and a higher recombination rate. It is hypothesized that the unique DNA repair methods present in plant mitochondria are responsible for the phenomena observed there. To study the mechanics of DNA-repair in this organelle, however, researchers must be able to generate knockout mutants of the nuclear genes encoding mitochondrial repair proteins. This study addresses the need for efficient mutant generation in plant mitochondrial repair proteins using the CRISPR-Cas9 system to generate knockouts. The gRNA sequences targeting exons in mitochondrial genome maintenance proteins were ligated into the pEN entry vector and then transferred to the pMR333 destination vector containing the Cas9-protein sequence. The pMR333-gRNA plasmid was introduced to A. tumefaciens using electroporation and A. thaliana plants were transformed using the floral dip method. Transformed plants were identified using the BlpR gene present in the plasmid which confers resistance to the herbicide BASTA. PCR and sequencing demonstrated the successful knockout mutation of a mitochondrial DNA-repair protein, MSH1. Future research will utilize CRISPR-generated mutants and double-mutants to investigate the plant mitochondrial genome’s reliance on Double-Strand Break Repair methods.