“Verifying the importation of gRNA rbcL into the chloroplast”
CRISPR technology is the latest genome editing craze because it edits DNA cheaply and efficiently. The Stern Lab focuses on utilizing the CRISPR/Cas9 system to edit the chloroplast genome. The two main required components of CRISPR are the Cas9 DNA endonuclease enzyme and the gRNA, which is a 20 base pair sequence designed to target a specific sequence of DNA. In our case, they both must be successfully imported into the chloroplast. My main goal was to verify the importation of a gRNA specific to the rbcL gene into the chloroplast. To do so, I needed to compare the RNA of the total cell to the RNA of the chloroplast to see if there was an enrichment of gRNA in the chloroplast. I first performed a total RNA extraction of chosen tobacco plants and amplified cDNA (RT-PCR) of the gRNA transcript and of transcripts for genes located in the chloroplast and nucleus. This allowed me to compare the expression of the gRNA to well known nuclear and chloroplastic genes. In a second step, I performed chloroplast purification of the same plants in order to specifically extract the chloroplast RNA and perform the same RT-PCR I just described. The comparison between the expression profile of the total RNA vs chloroplast RNA should allow me to decipher whether the gRNA is indeed imported into the chloroplast. This would be the first step toward the use of CRISPR genome editing in the chloroplast.
Throughout my internship at BTI, I discovered the complexity and intricacy of plant biological lab work mainly through the many mistakes and achievements I made during experimental procedures. I found it unsatisfying when things didn’t work out as planned. However, my missteps helped me to realize that scientific research is mostly about failing and learning from those mistakes. As a result, the successes that I had were extremely rewarding, as I gained valuable experiences and learned many techniques and information that I never would have known without interning at BTI. Making mistakes, identifying them, and then overcoming them has fueled an appreciation and passion for plant biology. My experience at BTI has been invaluable because it has given me a newfound confidence and fascination in plants, as I now have a slightly deeper understanding of the vast ocean that is plant biology.