CRISPR/Cas9 Genome Editing in C. elegans

CRISPR/Cas9 genome editing was performed in C. elegans as previously described with some modifications [47 (link)]. In brief, a 20μl CRISPR injection mixture containing 4.2μM tracrRNA, 2.5μM target sgRNA, 1.7μM rol-6 sgRNA, 1.56μM recombinant Alt-RspCas9 protein (~5μg), and 2.5μM of each repair template for rol-6 and the target gene was prepared. All reagents were purchased from Integrated DNA Technologies, Inc. The tracrRNA, sgRNAs, and Cas9 protein were first incubated at 37°C for 10 minutes in duplex buffer (Integrated DNA Technologies, Inc.) for reconstitution, followed by mixing with the repair templates. Prior to injection, the mixture was filtered through a 0.22μm cellulose filter (Corning Life Sciences). Twenty to thirty young adult worms were injected and maintained individually on plates. The F1 rollers were further maintained and their genotypes were checked. The sequences for the sgRNA and repair templates are listed in (S6 Table).