Genomic target sequences for generating <i>tsp-15</i> knockin and <i>csnk-1</i> knockout strains using the CRISPR/Cas9 method.

ND: not determined. For tsp-15 repair template, the letter in red was for introducing the missense mutation and letters in blue were for introducing silent mutations. (TIFF)</p

Representative morphologies of double homozygous mutants between <i>csnk-1(lf)</i> and <i>bli-3(lf)</i> or <i>doxa-1(lf)</i>.

bli-3(lf) csnk-1(lf) double homozygous mutants were derived from bli-3(lf) csnk-1(lf)/hT2 heterozygous mutants. csnk-1(lf); doxa-1(lf) double homozygous mutants were derived from csnk-1(lf)/hT2; doxa-1(lf)/hT2 heterozygous mutants. Arrows point to typical blisters. All images are of the same scale. (TIFF)</p

Depletion of wildtype <i>csnk-1</i> transcripts in L4 <i>csnk-1(lf)</i> mutants.

(A) Positions and sequences of PCR primers for detecting all or wildtype-only csnk-1 transcripts. Partial wildtype and mutant csnk-1 sequences are aligned to show the specificity of the primers for wildtype-only transcripts. (B) Relative total csnk-1 transcript levels. (C, D) Relative wildtype-only csnk-1 transcript levels in wildtype, csnk-1(mac494lf) or csnk-1(mac495lf) animals. tba-1 was the loading control. Statistics: two-tailed unpaired Student’s t-test. *: p p p (TIFF)</p

Effects of <i>csnk-1</i> mutations or variable feeding RNAis on the Sisi phenotype.

Mammalian orthologs or homologs are indicated in the parentheses. (TIFF)</p

Effects of <i>csnk-1</i> transgenes on the Sisi phenotype of <i>csnk-1(lf)</i> mutants in 5 mM NaI.

Effects of csnk-1 transgenes on the Sisi phenotype of csnk-1(lf) mutants in 5 mM NaI.</p

Raw data for Figures and Tables.

Oxidative stress response is a fundamental biological process mediated by conserved mechanisms. The identities and functions of some key regulators remain unknown. Here, we report a novel role of C. elegans casein kinase 1 gamma CSNK-1 (also known as CK1γ or CSNK1G) in regulating oxidative stress response and ROS levels. csnk-1 interacted with the bli-3/tsp-15/doxa-1 NADPH dual oxidase genes via genetic nonallelic noncomplementation to affect C. elegans survival in oxidative stress. The genetic interaction was supported by specific biochemical interactions between DOXA-1 and CSNK-1 and potentially between their human orthologs DUOXA2 and CSNK1G2. Consistently, CSNK-1 was required for normal ROS levels in C. elegans. CSNK1G2 and DUOXA2 each can promote ROS levels in human cells, effects that were suppressed by a small molecule casein kinase 1 inhibitor. We also detected genetic interactions between csnk-1 and skn-1 Nrf2 in oxidative stress response. Together, we propose that CSNK-1 CSNK1G defines a novel conserved regulatory mechanism for ROS homeostasis.</div

<i>csnk-1(lf)</i> and <i>skn-1(gf)</i> mutations exhibit synergy on the Sisi phenotype in 50 mM NaI.

csnk-1(lf) and skn-1(gf) mutations exhibit synergy on the Sisi phenotype in 50 mM NaI.</p

Characterization of <i>csnk-1</i>.

(A) csnk-1 gene structure (based on wormbase.org). The positions of mac397, mac494 and mac495 mutations are indicated. The position of the sgRNA used for CRISPR/Cas9-based mutagenesis is shown as a red bar. (B) Percentage of L1 larva that grew into adults on plates with 5 mM NaI. Results were based on two biological replicates. 100 L1 larva were analyzed in each replicate. Statistics: two-tailed unpaired Student’s t-test. *: p csnk-1p::GFP transgene in adults. (C) Fluorescent picture of a transgenic adult. The head, tail and vulva are indicated. (D-H) Higher-resolution pictures of transgenic adults. Cells with obvious GFP expression are indicated.</p

Representative morphologies of <i>csnk-1(lf)</i>, <i>bli-3(lf)</i> and <i>doxa-1(lf)</i> single mutants.

Representative morphologies of csnk-1(lf), bli-3(lf) and doxa-1(lf) single mutants.</p

CSNK-1::mCherry does not colocalize with GFP and DOXA-1::GFP does not colocalize with mCherry.

(A, B, C) A transgenic L3 larvae co-expressing GFP and CSNK-1::mCherry in epithelial cells. (D, E, F) A transgenic 3-fold embryo co-expressing DOXA-1::GFP and mCherry in epithelial cells. For unclear reason, DOXA-1::GFP was strongly expressed in embryos but was not visible at larval stages in these transgenic lines. We therefore observed whether DOXA-1::GFP colocalizes with mCherry in embryos. (TIFF)</p