Developing circadian clock reporter cell lines using a CRISPR gene editing knockin approach

January 3 – Febuary 28, 2021

Meeting Abstract

P30-7  Sat Jan 2  Developing circadian clock reporter cell lines using a CRISPR gene editing knockin approach Stanton, DS*; He, H; Liu, AC; University of Florida, Gainesville FL; University of Florida , Gainesville FL; University of Florida , Gainesville FL

Circadian rhythms are important in the regulation of physiology and behavior of animals. The molecular clock underlying these rhythms is based on a transcriptional/translational negative feedback mechanism, in which transcriptional activators BMAL1 and CLOCK regulate the expression of their own repressors PER1, PER2, CRY1 and CRY2. Nucleocytoplasmic translocation of the negative regulators play key roles in establishing circadian oscillations. However, the precise spatiotemporal dynamics and regulatory mechanisms of PER and CRY are not well understood. Many studies use molecular methods to gain a snapshot perspective of the translocation process, however this prohibits the visualization circadian protein movement in time and space within a single cell. In our study, we leverage our understanding of previously developed cellular clock models including human U2OS osteosarcoma cells and mouse MMH-D3 hepatocytes that display cell-autonomous robust circadian rhythms, permitting mechanistic studies in single cells with spatial-temporal resolution. Here, we present the development of circadian reporter cell lines using CRISPR-Cas9 gene editing technology to knockin fluorescent reporter genes in the N or C terminus of PER and CRY proteins. Studies using these developed cell lines will improve our understanding of circadian negative regulator translocation dynamics in response to pharmaceuticals, nutrition, and disease pertubations with spatiotemporal resolution in a single cell.

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