To generate him-8T64A, Q5 site-directed mutagenesis was used to insert a Cas9 targeting sequence for him-8
(ATTTACAGAATGAATTCGC) into pDD162 (Peft-3::Cas9 + empty sgRNA) (Dickinson et al., 2013) and to introduce silent mutations (ATTTACAGAACGAGTTTGC) into a repair template (pCFJ151 him-8T64A). N2 worms were injected with Cas9 + him-8
sgRNA construct (50 ng/ul), a repair template (50 ng/ul), pGH8 (10 ng/ul), pCFJ104 (5 ng/ul), and pCFJ90 (2.5 ng/ul). F1 progeny were lysed and screened for homologous recombination by PCR using a forward primer specific to the endogenous him-8
gene (TGAAGTTTAGTTTTCGCAGAATTCG) and a reverse primer specific to the mutated him-8
Cas9 targeting sequence on the repair template (CTGCCACCCGCAAACTCG). Successful introduction of the T64A mutation at the endogenous him-8
locus was verified by sequencing.