Generation of transgenic pluripotent stem cells to overexpress neuronal survival genes against stroke using CRISPR-activation technology
In: Student Reports, Summer 2023, The Jackson Laboratory
Dan Cortés, M.D., Ph.D., and Martin Pera, Ph.D.
Despite being a leading cause of death, suitable treatment for stroke is lacking. There is a need for further understanding of the genetic mechanisms that affect stroke resilience. Stem cell research has opened new possibilities in the study of stroke. Using a genetically diverse panel of mouse and human stem cells, the lab previously found transcriptional factors and other genes associated with neuronal survival. The goal of this research was to validate those findings using CRISPRa to activate those (IRF7, TFAP2C, VDR, MGARP, and PTN) endogenous genes on neurons and brain organoids. Previous attempts to generate the transgenic cells failed due to silencing effects. Therefore, iPSCs were differentiated into NPCs and then transfected with the various plasmids that targeted the endogenous genes. Once transfected, the NPCs were reprogrammed back into iPSCs to generate stable cell lines. NPCs showed incorporation of the dCAS9-VPR system via transfection and were successfully reprogrammed into iPSCs. iPSC- derived NPCs can incorporate the dCAS9-VPR plasmid, indicating integration of the first part of the overexpression system, but more work is needed to determine how to incorporate the second part (gRNA plasmids) without causing massive cell death. As a second approach, iPSCs were transfected with a dCAS9-VPH plasmid into a safe harbor locus. Nonetheless, efficiency is extremely low and we are still selecting positive cells after transfection. In parallel, we sought to validated this system on H293 cells since these cells are more accessible to genetic manipulation. We found successful transfection of two CRISPRa systems (dCAS9-VPR and dCAS9-VPH), but through PCR it was determined that the endogenous genes were not activated when transfected with gRNAs.
Saavedra-Weis, Pilar, "Generation of transgenic pluripotent stem cells to overexpress neuronal survival genes against stroke using CRISPR-activation technology" (2023). Summer and Academic Year Student Reports. 2765.