Transgenic knockout (KO) strains were constructed using the CRISPR-sdm approach (49). In brief, an injection mix containing the standard CRISPR components [single-guide RNA (sgRNAs), donor homology (ODN), Cas9 protein, and a co-CRISPR selection tool (dpy-10 sgRNA and dpy-10 ODN)] were microinjected into wild-type animals. The F1 generation animals were screened for the presence of the co-CRISPR phenotype (this phenotype will not be present in the final strain). Deletion of the selected genomic region was confirmed using PCR. Animals positive for the KO were homozygosed, and the homozygous KO was further confirmed by PCR and sequencing. Strain JRS37 carrying KO in col-80 was generated by precise deletion of 1171 bp spanning exons 1 and 2 and insertion of a three-frame stop codon. Strain JRS38 carrying KO in col-98 was generated by precise deletion of 1203 bp spanning exons 1, 2, and 3 and insertion of a three-frame stop codon. More strain details are available upon request.

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