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

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.