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

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

Teresa Börding et al. Front Physiol.

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.

Keywords: CRISPR/Cas9; U-2 OS; circadian; cryptochrome; duplex.

Figures

FIGURE 1
FIGURE 1
Workflow to create and analyze circadian clock gene knockout cells using duplex CRISPR/Cas9 technology.
FIGURE 2
FIGURE 2
CRISPR/Cas9-mediated generation of CRY1 and CRY2 knockout cells (A) Schematic overview of CRISPR/Cas9-mediated exon deletion strategy. SgRNAs were designed to introduce a double strand break in intron regions upstream and downstream of target exons (scissors). Deletion of target exons induces a frameshift resulting in a premature STOP codon. For each CRY gene three (out of six) different guide RNA combinations are exemplarily shown in different colors. E.g., c1e2-4 refers to a sgRNA combination that aimed at the deletion of exons 2 to 4 of CRY1. E: exon. (B) Relative genomic abundance of targeted exons in single cell clones. Cell populations as indicated in Supplementary Figure 1 were sub-cloned, and genomic DNA of individual clones was analyzed for exon deletions using qPCR. As control C, abundance of 3′-UTR regions of each gene were quantified. Single cell clones with undetectable levels of targeted exons were maintained for further analysis (marked in the corresponding color), single cell clones with minimal reduction in all screened genomic loci were maintained as controls (marked in gray). (C) Generation of CRY1/CRY2 double knockout cells. The CRY2 knockout cell clone G5 [see (A)], was transduced with Cas9 and sgRNA expression vector targeting exons 2–4 of CRY1. Single cell clones were analyzed for genomic deletion of target exons as described above. Shown are results for six clones, four of which were selected for further downstream analysis.
FIGURE 3
FIGURE 3
Sequences of single cell clone genomic regions confirm deletion of targeted exons. Out-out-PCR products for each KO clone (see marked bands in Supplementary Figure 2B) were gel-purified, sequenced and aligned to corresponding wild-type sequence. SgRNA recognition sequences are indicated in red, PAM sequence in green and expected Cas9 cutting site by an arrow. Sequence ambiguities suggest that genome editing was not identical for the two alleles.
FIGURE 4
FIGURE 4
CRY protein levels in CRISPR/Cas9-generated knockout cell clones. Whole cell lysates from indicated single cell clones were analyzed using SDS-PAGE and western blotting. The blots were stained for CRY1 or CRY2 as well as for GAPDH as a loading control. CRY1/CRY2 double knockout clone D5 (blue) was not analyzed for CRY2 expression, since for its “parental” clone, the CRY2 knockout clone G5 (dark green), we did not detect CRY2 protein. Colored headers indicate the identity of the analyzed clones and their potential genomic deletions. Note, for “gray clones,” we did not detect reduction in relative genomic abundance of target exons (see Figure 2B). marks an unspecific band with slightly lower electrophoretic mobility.
FIGURE 5
FIGURE 5
Deletion of CRY proteins alters circadian dynamics. (A) Indicated single clones containing a Bmal1-luciferase reporter were synchronized with dexamethasone, and bioluminescence rhythms were analyzed for several days. Shown are representative detrended time series. (B) Quantification of circadian period for indicated single cell clones (DKO = double knockout). Shown are means of two independent experiments with twelve technical replicas each (clone D5 was measured once with eight technical replicas). Dots indicate the two mean values of the two experiments.

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