Using CRISPR/Cas9 to Investigate the Role of Egr1 and NAB2 in Neuronal Differentiation of PC12 Cells

Abstract

PC12 cells are a common model system used to study the molecular events underlying neuronal differentiation due to their ability to differentiate into neuronal cells following treatment with nerve growth factor (NGF). NGF induces neuronal differentiation through activation of the Ras/Raf/MEK/ERK signaling pathway, which in turn activates a transcriptional program leading to up‐regulation of neuronal genes, neurite outgrowth, and establish electrical excitability. Interestingly, treatment with epidermal growth factor (EGF) also activates Ras/Raf/MEK/ERK signaling, but induces proliferation rather than differentiation. The differential effects of NGF versus EGF are due to differences in ERK signal duration; EGF induces more transient ERK signaling that lasts 30–60 min, whereas NGF induces more sustained ERK signaling that lasts 4–6 h. Work in our lab and others indicate that sustained ERK signaling in response to NGF drives differentiation in part through sustained activity of the transcription factor Egr1 and, in turn, sustained expression of Egr1 target genes. Egr1 activity in response to EGF and NGF is regulated at two levels. First, Egr1 levels rapidly rise to peak levels by 1 h after treatment and then decline to baseline levels; similar to ERK signaling, Egr1 levels are sustained longer in response to NGF versus EGF. Second, one Egr1 target gene is its corepressor NAB2, which binds and represses Egr1 transactivation once expressed. This project was designed to further test our hypothesis that sustained Egr1 activity is a major driver of PC12 neuronal differentiation by evaluating whether knockout of NAB2 will lead to sustained Egr1 activity in response to EGF and, in turn, neuronal differentiation. To acquire PC12 cells that lack NAB2 protein, we used CRISPR/Cas9 technology to knockout the NAB2 gene. More specifically, a CRISPR NAB2‐KO plasmid was purchased from Santa Cruz Biotechnology, which encodes a green fluorescent protein (GFP) selectable marker, the Cas9 protein, and the guide RNA (gRNA). The plasmid had three variations for the gRNA which target different locations in the NAB2 gene. One day after transfection, GFP‐positive cells were sorted into 96‐well plates to isolate clones. Following clonal expansion, clones were first screened for NAB2 expression by Western blot, which identified eight putative knockout clones. Genomic DNA was then extracted from the eight clones, the NAB2 gene amplified by PCR, and the PCR project then subjected to Sanger sequencing to identify the INDELs responsible for NAB2 knockout. Ongoing work is examining the effects of NAB2 knockout on Egr1 levels and activity in response to EGF and NGF, as well as neuronal differentiation based on neurite outgrowth with the prediction that NAB2 knockout will result in sustained Egr1 activity and neuronal differentiation in response to both growth factors. Support or Funding Information Bridgewater State University Undergraduate Research Program