CRISPR/Cas9 mediated gene editing

Knock-in strains were generated using a dpy-10 Co-CRISPR strategy (Arribere et al., 2014; Paix et al., 2015; Paix et al., 2017). All crRNA and repair template sequences are in Table 2. An injection mix containing 0.12 µl dpy-10 crRNA (0.6 mM) (Horizon Discovery/Dharmacon), 0.3 µl target gene crRNA (0.6 mM) for one locus editing or 0.21 µl of each crRNA (0.6 mM) for multi-loci editing and 1.46 µl (one locus) or 1.88 µl (two loci) universal tracrRNA (0.17 mM) (Horizon Discovery/Dharmacon) precomplexed with purified 7.6 µl of 40 µM Cas9 protein (UC Berkeley QB3) and 0.29 µl of the dpy-10 single-strand DNA oligonucleotide (ssODN) (500 ng/µl) repair templates and 0.21 µl ssODN repair template (25 µM) for the target gene editing or up to 500 ng double-strand DNA were injected to the germline of the hermaphrodite young adults. For anc-1(yc52[∆25KASH]) I, anc-1(yc69[∆29KASH]) I, anc-1(yc62[ΔF1]) I, anc-1(yc61[∆6RPS]) I, anc-1(yc80[∆F2]) I, anc-1(yc78[∆5RPS]) I, anc-1(yc91[gfp::anc-1b::Δ5rps]) I, anc-1(yc70[gfp::anc-1b::Δ6rps]) I, anc-1(yc92[gfp::anc-1b::Δf1]) I, anc-1(yc93[anc-1AI972,973**::gfp]) I, anc-1(yc106[gfp::anc-1b::Δkash]) I, anc-1(yc94[gfp::anc-1b::Δtk]), I single-strand DNA (SSD) (synthesized by Integrated DNA Technologies, IDT) was used as repair template. For GFP knock-in strains, double-strand DNA repair templates were amplified with PCR from the plasmids pSL779 for gfp using Phusion polymerase and the primers listed in Table 2 (New England Biolabs) (Bone et al., 2016; Cain et al., 2018).

*all nucleotide sequences are displayed as single strand in the 5’ to 3’ orientation.

†In many cases a ssDNA oligonucleotide was used. For larger inserts, a PCR product was used.

§An imprecise NHEJ event led to an in-frame deletion without using the repair template.

¶Underlined sequences introduce silent mutations so the repair template is not cut by Cas9.

‡Underline indicates the silent mutation in the repair template.

Strains anc-1[yc41(anc-1::gfp3Xflg::kash,Δch)]I and anc-1[yc36(anc-1::gfp3Xflag::kash)]I were generated by Dickinson Self-Excising Drug Selection Cassette (SEC) method (Dickinson et al., 2015). In anc-1[yc41(anc-1::gfp3Xflg::kash,Δch)]I, both CH domains were deleted (starting with 23KAQK26 and ending with 322QFVR325) and replaced with GFP flanked with 9-residue long linkers (GASGASGAS).