Dissecting the Role of Hes1 Human-Specific Enhancers

Abstract

Human brain development is a complex process shaped by regulatory elements such as enhancers. Hes1 is an important gene controlling the proliferation of Neural Stem Cells (NSCs) and Neural Progenitor Cells (NPCs) and is believed to be regulated by multiple enhancers. The study of human-specific enhancers allows us to further comprehend species-specific differences between humans and chimpanzees. In this work, I investigate the role of silencing Hes1 human-specific enhancers. I have used human neural stem cells (hNSCs) as a cell model and performed silencing through CRISPR interference (CRISPRi). The targets for CRISPRi were candidate enhancers that we designed computationally. I transfected the CRISPRi (dCas9-KRAB-MeCP2) and guide RNA construct into the hNSCs through nucleofection and performed gene expression analysis through qPCR. During my project work, I encountered plenty of difficulties with the cells, including low vitality and differentiation problems. Nonetheless, I managed to establish common principles for better cell growth and to improve transfection experiments in the future.