For him-3G280K, Q5 site-directed mutagenesis was used to insert a Cas9 targeting sequence for him-3
(CAAACCAATCGAAAAGGAA) into pDD162 (Peft-3::Cas9 + empty sgRNA) (Dickinson et al., 2013). Isothermal assembly with a synthetic gene block containing the G280K mutation and silent mutations in the Cas9 targeting sequence (CAAACCAAAGCAAGCGTAAA) was used to generate a repair template (pCFJ151 him-3G280K). N2 worms were injected with Cas9 + him-3
sg RNA construct (50 ng/ul), a repair template (50 ng/u), 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-3
gene (CCACCGAAATCCACAATTTCTCG) and a reverse primer specific to the mutated him-3
Cas9 targeting sequence on the repair template (GAAATTCGTCCTTTACGCTTGCT). The G280K mutation at the endogenous him-3
locus was verified by sequencing. Both him-8T64A and him-3G280K strains were outcrossed three times before analysis.