AUTHOR=Börding Teresa , Abdo Ashraf N. , Maier Bert , Gabriel Christian , Kramer Achim 

TITLE=Generation of Human CRY1 and CRY2 Knockout Cells Using Duplex CRISPR/Cas9 Technology

JOURNAL=Frontiers in Physiology

VOLUME=10

YEAR=2019

URL=https://www.frontiersin.org/journals/physiology/articles/10.3389/fphys.2019.00577

DOI=10.3389/fphys.2019.00577

ISSN=1664-042X

ABSTRACT=<p>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: <italic>CRY1</italic> knockout cells, <italic>CRY2</italic> knockout cells as well as <italic>CRY1</italic>/<italic>CRY2</italic> double knockout cells. Duplex CRISPR/Cas9 technology efficiently removed whole exons of <italic>CRY</italic> 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 <italic>CRY1</italic> knockout), long period rhythms (for <italic>CRY2</italic> knockout) or were arrhythmic (for <italic>CRY1</italic>/<italic>CRY2</italic> double knockout) similar to circadian phenotypes of cells derived from classical knockout mouse models.</p>