Generation of Human CRY1 and CRY2 Knockout Cells Using Duplex CRISPR/Cas9 Technology
Citations Over TimeTop 17% of 2019 papers
Abstract
Circadian clocks are endogenous oscillators essential for orchestrating daily rhythms in physiology, metabolism and behavior. While mouse models have been instrumental to elucidate the molecular mechanism of circadian rhythm generation, our knowledge about the molecular makeup of circadian oscillators in humans is still limited. Here, we used duplex CRISPR/Cas9 technology to generate three cellular models for studying human circadian clocks: CRY1 knockout cells, CRY2 knockout cells as well as CRY1/CRY2 double knockout cells. Duplex CRISPR/Cas9 technology efficiently removed whole exons of CRY genes by using two guide RNAs targeting exon-flanking intron regions of human osteosarcoma cells (U-2 OS). Resulting cell clones did not express CRY proteins and showed short period, low-amplitude rhythms (for CRY1 knockout), long period rhythms (for CRY2 knockout) or were arrhythmic (for CRY1/CRY2 double knockout) similar to circadian phenotypes of cells derived from classical knockout mouse models.
Related Papers
- → Cryptochromes Are Required for Phytochrome Signaling to the Circadian Clock but Not for Rhythmicity(2000)334 cited
- → Cryptochrome Mediates Light-Dependent Magnetosensitivity of Drosophila's Circadian Clock(2009)226 cited
- → Arabidopsis cryptochrome 2 forms photobodies with TCP22 under blue light and regulates the circadian clock(2022)45 cited
- → The circadian clock controls the expression pattern of the circadian input photoreceptor, phytochrome B(1999)134 cited
- → Chlamydomonas reinhardtii strain CC-124 is highly sensitive to blue light in addition to green and red light in resetting its circadian clock, with the blue-light photoreceptor plant cryptochrome likely acting as negative modulator(2013)29 cited