ts allele rescue oligonucleotide contained the nucleotide changes of interest mutant Thymine 712 to WT Cytosine and an additional 50 nucleotides of perfect homology on both sides flanking nucleotide 716 and the PAM site (120 ng/l) (Paix et al. 2014; Zhao et al. 2014). Rescue of the Cytosine 716 to Thymine mutation (codon 239) in cdc-25.2
ts) was performed by editing the single nucleotide 716 from T to a wild-type C using the CRISPR/Cas9 technology. An additional silent nucleotide change was made to remove the PAM Cas9 target site to prevent possible recutting of the rescue oligo or the target site (Kim et al. 2014). This additional change (C681G silent mutation) created an EaeI recognition site, which was used to distinguish the rescued line from N2. cdc-25.2
was targeted for Cas9 cleavage using the guide sequence (ATGTCTCTCAATGTTTCGG) in pDD162 (Dickinson et al. 2013), targeting the noncoding strand.