Pluripotent stem cells can differentiate into any cell in the body and show tremendous promise for regenerative medicine applications. We hypothesize that we can control stem cell fate on demand by activating key developmentally regulated genes in an inducible and reversible manner with light. To test this hypothesis, we constructed a modular, two-component system based on the light-mediated heterodimerization of two exogenous proteins, Cry2 and CibN. We tethered CibN to an enzymatically dead version of Cas9 (dCas9) to facilitate recruitment of this fusion protein to target genes via CRISPR/Cas9 sgRNAs. We also constructed plasmids encoding Cry2 and a fluorescent marker or antibiotic resistance to aid in visualization and downstream selection. In parallel, we have designed a light box to effectively stimulate the interaction of Cry2 and CibN, accounting for wavelength, LED intensity, and pulsing parameters. Our long-term goal is to use our cloned plasmids and light box to activate the critical pluripotency genes Sox2 and Klf4. These results will enable researchers to understand the role of gene expression in healthy development and disease progression.
Receiving the Class of 1971 Robert J Holtz Research Award has augmented my educational and professional career because the funding enabled me to do lab work full-time. This summer of full-time lab work has enhanced my chances of getting into graduate programs by giving me the opportunity to be a critical member of a team working on high-level, impactful research. Additionally, I have been able to further develop my understanding of and ability to do important technical skills in molecular biology techniques and electronics design. Finally, full-time work in the Cremins lab has helped me develop the intangible skills necessary for a career in research, like communicating my results and progress to research advisors, troubleshooting experiments, and designing follow-up studies. I am extremely grateful for the support from CURF in allowing me to dive into a cutting-edge project that has the potential to positively impact the lives of many patients.
Pluripotent stem cells can differentiate into any cell in the body and show tremendous promise for regenerative medicine applications. We hypothesize that we can control stem cell fate on demand by activating key developmentally regulated genes in an inducible and reversible manner with light. To test this hypothesis, we constructed a modular, two-component system based on the light-mediated heterodimerization of two exogenous proteins, Cry2 and CibN. We tethered CibN to an enzymatically dead version of Cas9 (dCas9) to facilitate recruitment of this fusion protein to target genes via CRISPR/Cas9 sgRNAs. We also constructed plasmids encoding Cry2 and a fluorescent marker or antibiotic resistance to aid in visualization and downstream selection. In parallel, we have designed a light box to effectively stimulate the interaction of Cry2 and CibN, accounting for wavelength, LED intensity, and pulsing parameters. Our long-term goal is to use our cloned plasmids and light box to activate the critical pluripotency genes Sox2 and Klf4. These results will enable researchers to understand the role of gene expression in healthy development and disease progression.
Receiving the Class of 1971 Robert J Holtz Research Award has augmented my educational and professional career because the funding enabled me to do lab work full-time. This summer of full-time lab work has enhanced my chances of getting into graduate programs by giving me the opportunity to be a critical member of a team working on high-level, impactful research. Additionally, I have been able to further develop my understanding of and ability to do important technical skills in molecular biology techniques and electronics design. Finally, full-time work in the Cremins lab has helped me develop the intangible skills necessary for a career in research, like communicating my results and progress to research advisors, troubleshooting experiments, and designing follow-up studies. I am extremely grateful for the support from CURF in allowing me to dive into a cutting-edge project that has the potential to positively impact the lives of many patients.