Advancing the toolkit for understanding and manipulating RNA movement:
A mobile CRISPR system in Solanum lycopersicum

Mobile signaling is the process by which molecules travel in response to stimuli. Messenger RNAs (mRNAs) are part of this signaling system. There is a limited understanding of how specific mRNAs are trafficked to their intended destinations and which tissues these RNAs are capable of becoming functional signals in (i.e., translated proteins). I established a system to test the mobilization of CRISPR-Cas9 using dexamethasone in Solanum lycopersicum. The TLS motif that is appended to the Cas9 and gRNA has been established to induce RNA mobility in traditionally non-mobile RNAs. This system targets a phytoene desaturase (PDS), providing a visual marker upon editing, indicating functional delivery. I validated 50 T0 plants transformed to contain my constructs of interest. I also crossed over 50 plants containing the independent constructs to develop a fully functional two-component system (i.e., driver and experimental effector containing TLS motif or driver and control effector lacking TLS motif). I then tested the functionality of this system by exposing leaves to dexamethasone, which will allow the driver to induce transcription of Cas9 in the effector. This system provides a method to validate when and where mobile signals may be functional. It also provides a basis for the future to test factors that may influence mobile transcripts by changing the mobility tag associated with the Cas9 and gRNA. Thus, providing evidence for or against the postal code hypothesis, a hypothesis stating that specific sequences direct mobile signals to particular tissues within the plant.

I am very grateful to have had the opportunity to spend my summer in the Lab of Dr. Margaret Frank under the guidance of Michelle Heeney as part of the BTI and CROPPS REU program. This area of research was new to me, as all of my previous experience has been with bacterial plant pathogens. I have enjoyed this new challenge, and this experience has renewed my interest in developmental biology and plant response to external stimuli. I developed expertise in molecular biology by genotyping over 50 T0 transformed plants using DNA extractions and PCR. I also spent numerous hours caring for my greenhouse and conducting cross-pollinations. If you’re interested in learning how to cross yourself, search for my video tutorial explaining the protocol. I have gained experience in plant tissue culture and sterile technique by culturing my DEX inducible system. This experience has also solidified my plan to apply to PhD programs this fall. I have become a more thoughtful and passionate scientist and researcher because of this experience, and will forever be grateful to everyone who made this experience possible and so memorable. This community was incredible, and I have made lifelong friends that I look forward to catching up with at conferences for the rest of our careers.