Document Type
Poster
Publication Date
Summer 2025
Abstract
The moss P. patens utilizes seasonal cues, such as daylength and temperature, to regulate the timing of its sexual reproduction. We are using a variety of forward and reverse genetic approaches to identify candidate genes that are involved in this regulatory pathway. To determine if a gene is necessary for seasonal reproduction in P. patens, we use CRISPRCas9 technology to induce deleterious mutations. In the past, we have created knockouts of candidate genes with a CRISPR-Cas9 system by transforming multiple bacterial plasmids into protoplasts, including at least three separate plasmids for expressing the endonuclease Cas9, a single guide RNA (sgRNA), and an antibiotic resistance gene. We’ve simplified this process by utilizing a destination vector containing Cas9 and selectable markers to then load an sgRNA onto the plasmid via Golden Gate Cloning (Mallett et al. 2019, Prigge and Estelle). This method possesses numerous advantages, including cheaper costs and greater simplicity for protoplast transformation protocols.
Recommended Citation
Lee, Joseph; Suhm, Gabriel; and Hicks, Karen, "Generating CRISPR-Cas9 plasmids for targeted gene knockouts in Physcomitrium patens via Golden Gate cloning" (2025). Kenyon Summer Science Scholars Program. Paper 772.
https://digital.kenyon.edu/summerscienceprogram/772
Rights Statement
