CRISPR-Cas9 Engineered C. elegans Mutations

The brc-1 mutations (km88 deletion and S266A point mutation) and the dgk-3 mutations (km89 insertion and km90 deletion) were obtained using the CRISPR–Cas9 system as described previously (Dokshin et al., 2018 (link)). The CRISPR RNAs [5′-UGGAAACAUGUGGACAGAAU-3′ for brc-1(km88), 5′-UUGCGAGUUCUCAAGAUCUU-3′ for brc-1(S266A), and 5′-UAUCACCGGAGCAAUUCUCG-3′ for dgk-3(km89, km90)] and the single-stranded donor template DNA [5′-ATCAGAGAAACCAGCGAATCGAAGAGTAgccTTTGCGAGTTCTCAAGATCTTGAAAACA\TAAAAATTATG-3′ for brc-1(S266A)] were synthesized (Integrated DNA Technologies; IDT), co-injected with the trans-activating CRISPR RNA (IDT), Streptococcus pyogenes Cas9 3NLS (IDT) protein, and the pRF4(rol-6d) plasmid into the KU501 [for brc-1(km88) and brc-1(S266A)] and KU1448 [for dgk-3(km89, km90)] strains. Each of the F1 animals carrying the transgene was transferred onto a new dish and used for single-worm PCR, followed by DNA sequencing to detect the mutations. The brc-1(km88) mutation is a 2-bp deletion in the brc-1 gene, causing a frameshift and premature stop codon in exon 2. The dgk-3(km89) mutation is a 20-bp insertion that contains an in-frame stop codon, thus terminating translation in the middle of exon 1. The dgk-3(km90) mutation is a 5-bp deletion, causing a frameshift and premature stop codon in exon 1.