Knockout of WRKY28 Transcription Factor Gene in Arabidopsis thaliana using a Two Pair sgRNA CRISPR-Cas9 Multiplex

<p style="font-weight: 400;">Throughout their evolutionary development, plants evolved mechanisms to combat the diverse array of microorganisms they encounter. Within the soil microbiome, they interact with beneficial and pathogenic microbes differently: defending against pathogenic bacteria while selectively allowing beneficial bacteria to colonize. These host-microbe interactions suggest that plants must possess the ability to differentiate between different bacterial strains. Commensal bacteria associated with plants influence a distinct and evolutionarily conserved sector of Arabidopsis MAMP-triggered immunity (MTI) during root colonization, which involves members of the WRKY gene family, including <em>WRKY28</em>. To better evaluate the function of the <em>WRKY28</em> gene, we generated a knockout mutant using CRISPR-Cas9 gene editing technology. We hypothesized that a two pair sgRNA CRISPR-Cas9 multiplex would generate multiple double-stranded breaks, allowing for the removal of a chromosomal segment. In this experiment, we designed and inserted a two pair CRISPR-Cas9 multiplex into <em>Arabidopsis thaliana</em>, generating a <em>WRKY28</em> knockout. This paves the way for investigating <em>WRKY28&rsquo;s</em> functional role in regulating immune responses and, more specifically, maintaining a host-microbe homeostasis state.