“Determining successful targeting of the Cas9 protein to chloroplast”
Chloroplasts are crucial components of plant cells in that they serve as the site for photosynthesis, among other important metabolic processes, and contain their own genome encoding few but necessary proteins involved in plant metabolism. Modifying the chloroplast genome is important for advancing plant research, developing drought tolerant crops, and producing biofuels. At this point, however, the tools available for chloroplast genome editing are limited and not universally successful across plant species, creating a demand for new and better genome editing tools.
The Stern lab is working to repurpose the CRISPR/Cas9 gene editing system, originally found in bacteria as an immune defense against viruses, as a tool for modifying the chloroplast genome. As part of this process, the lab has successfully transformed the nucleus of Nicotiana tabacum to encode the Cas9 protein with an N-terminal chloroplast transit peptide to direct the Cas9 protein to the chloroplast. We expect that the chloroplast in our transformed plant lines will contain the Cas9 protein.
To confirm this, extracted proteins from plant tissue and from isolated chloroplasts was analyzed by immunoblots and a Cas9 antibody. We expect to see an enrichment in Cas9 protein within the chloroplast compared to the total plant protein. To confirm that our chloroplast extraction was successful, we are doing additional immunoblots using antibodies that target cytosolic or chloroplast proteins. Confirming the presence of the Cas9 protein in chloroplast is the first step in being able to use the CRISPR/Cas9 system to modify the chloroplast genome.
I really enjoyed my time working as an intern in the Stern Lab. I loved the dynamics of the other researchers and everyone was so friendly. I knew I wanted to go into research in the future but this summer has reassured me that plant genetics is absolutely the field I am interested.
This entire program has served to make me more aware of the types of research available to a plant scientist, which is a topic I had very little knowledge in. I am truly grateful to have had this opportunity to actually “test run” my career in plant science. Along with learning several new lab techniques, I also learned that I want to focus my research on more molecular based genetic work.