Comparison of effectiveness in gene silencing of phytoene desaturase (PDS) gene through RNAi and CRISPR techniques
Targeted genome editing approach- CRISPR/Cas (Clustered Regularly Interspaced short palindromic repeats/CRISPR-associated) is relatively new, and reported to be highly specific and effective in genome modification in a variety of living organisms. These systems are a bacterial defense against invading foreign nucleic acids.They use an array of small CRISPR RNAs (crRNAs) consisting of repetitive sequences flanking unique spacers to recognize their targets, and conserved Cas proteins to mediate target degradation. This project examined the differences in effectiveness between CRISPR and RNA interference (RNAi) in silencing Phytoene Desaturase (PDS) gene in tomato leaves. Constructs for both CRISPR and RNAi with PDS sequence were designed and infiltrated into the tomato leaves via agrobacterium infiltration method. Tissues from yellow patches, which appeared yellow because of the CRISPR and RNAi effect, were collected and subjected to further analyses. DNA was extracted from yellow tissue from leaves transfected with CRISPR constructs and subjected to DNA sequencing. Transcript levels were examined and found reduced in case of RNAi when compared to wild type tomato. In comparison, yellow patches were more prominent in case of CRISPR than in plants infiltrated with RNAi constructs. Together, these findings indicate that CRISPR/Cas method is more promising compared to RNAi.
Participating in the PGRP Internship program at BTI has taught me so many great things about plant genetic research. My mentor, Sarika Gupta, as well as other people in Dr. Van Eck’s lab truly guided me and provided me with so much knowledge and exposure to plant gene silencing techniques as well as plant tissue culture techniques, both of which I didn’t have much experience before. The weekly science seminars and many professional development activities at BTI also enhanced my knowledge about plant science, future career and graduate school pathways. This internship definitely has helped me to determine what I want to do after graduating from college. I am so glad to have this opportunity to meet so many other summer interns and people at BTI who are passionate about plant biology research. Outside of work, I enjoyed hiking around the Cornell Campus, exploring around the finger lake area, and seeing so many wonderful waterfalls around Ithaca!