Extending CRISPR design software features for tomato protein kinase silencing

Project Summary

The discovery of the CRISPR/Cas9 method in 2012 represents a revolutionary advance in genome editing. The system relies on two main functional groups: A Cas9 protein that cuts the targeted DNA strand and an sgRNA (single guide RNA) that guides the protein to specific locations in the genome. One of the primary challenges in implementing CRISPR systems is designing optimal guide RNAs with minimum off-target matches. Currently, several algorithms have been developed to score guide RNAs effectiveness by a variety of experimentally determined factors. My research has extended the scope of currently published CRISPR tools, such as CCTop and CRISPR-P. With added functionality, researchers will have greater specificity when selecting sgRNAs. Researchers will be able to analyze guide RNA positions within a gene, rewarding preference for RNAs near the 5’ end, and view whether they target specific gene domains for increased desirability. Additionally, researchers will have the option to design sequences intended to target multiple genes within a family. By extending guide RNAs to target several genes, scientists studying multiple related genes or genes from the same family will more easily be able to experiment with simultaneously silencing several genes. Utilizing these extended techniques, I have designed multiple guide sequences for the Receptor Like Cytoplasmic Kinase (RLCK) gene family, a family that plays an important role in plant immune responses. Overall, extending the functionality of current CRISPR tools allows for more specialized guide sequences to be obtained, enabling scientists a greater range of experiments when researching genes through genetic manipulation.

My Experience

Working as an intern at the Boyce Thompson Institute (BTI) has been a truly invaluable experience. Although I started the internship with a minimal background in bioinformatics and plant biology, through the amazing guidance of my mentor Dr. Noe Fernandez, I have gained an immense amount of knowledge about these areas. Every day presented an array of different challenges, teaching me the rigor and dedication needed to conduct research. My experience over the last six weeks has shown me the excitement of being in the pursuit of discovery, and has opened my eyes to the possibility of continuing to conduct research in the future. The opportunity to be imaginative, creative, and to think critically about a problem strongly appeals to my interest as an individual. I would like to extend my sincere gratitude to Tiffany Fleming, Nicole Waters Fisher, my mentor Dr. Noe Fernandez, Professor Lukas Mueller, and the entire Mueller lab for making my summer internship at BTI such an amazing and memorable experience.