Worm handling and strains

C. elegans strains were maintained on nematode growth medium (NGM) plates at 20°C and fed a diet of OP50 E. coli bacteria, unless noted otherwise [81]. Experiments were carried out at room temperature (~22°C).

Strains generated for this study are indicated below. All strains used and details on strain construction information are listed in S1 Table. GC1459 naSi2 II; unc-119(ed3) III?; daf-18(ok480) IV, GC1527 glh-1(sam24[glh-1::gfp::3xFLAG]) I; daf-18(ok480) IV, GC1537 vbh-1(na110[GFP::vbh-1]) I; naSi2 II, GC1576 vbh-1(na110[GFP::vbh-1]) I; naSi2 II; daf-18(ok480) IV, GC1456 naSi2 II; unc-119(ed3) III?; daf-18(ok480) IV; naEx261 (daf-18P::daf-18::daf-18 3’UTR::SL2::GFP::H2B::unc-54 3’UTR), GC1460 naSi2 II; unc-119(ed3) III?; daf-18(ok480) IV; naEx264 (rab-3P::daf-18::daf-18 3’UTR::SL2::GFP::H2B::unc-54 3’UTR), GC1464 naSi2 II; unc-119(ed3) III?; daf-18(ok480) IV; naEx268 (nlp-40P::daf-18::daf-18 3’UTR::SL2::GFP::H2B::unc-54 3’UTR), GC1465 naSi2 II; unc-119(ed3) III?; daf-18(ok480) IV; naEx269 (dpy-7P::daf-18::daf-18 3’UTR::SL2::GFP::H2B::unc-54 3’UTR), GC1485 naSi2 II; unc-119(ed3) III?; daf-18(ok480) IV; naEx275 (elt-2P::daf-18::daf-18 3’UTR::SL2::GFP::H2B::unc-54 3’UTR), GC1484 naSi2 II; unc-119(ed3) III?; daf-18(ok480) IV; naEx274 (ehn-3P::daf-18::daf-18 3’UTR::SL2::GFP::H2B::unc-54 3’UTR), GC1479 naSi22 (mex-5P::daf-18::nos-2 3’UTR::SL2::GFPo::PH::tbb-2 3’UTR + unc-119(+)) II; unc-119(ed3) III?; daf-18(ok480) IV, GC1483 glh-1(sam24[glh-1::gfp::3xFLAG]) I; naSi22 (mex-5P::daf-18::nos-2 3’UTR::SL2::GFPo::PH::tbb-2 3’UTR) II; unc-119(ed3) III?; daf-18(ok480) IV, GC1627 glh-1(sam24[glh-1::gfp::3xFLAG]) I; daf-18; naEx261 (daf-18P::daf-18::daf-18 3’UTR::SL2::GFP::H2B::unc-54 3’UTR), GC1621 ppw-1 (pk2505) I; naSi2 II, GC1390 rrf-1 (pk1417) I; naSi2 II, GC1638 naSi2 II; unc-119(ed3) III?; otIs45 (unc-119::GFP) V, GC1639 naSi2 II; unc-119(ed3) III?; daf-18(ok480) IV; otIs45 (unc-119::GFP) V.

Transgenic strains were generated by three different methods, as indicated below (see S1 Table):

Microinjection (generating extrachromosomal arrays) [82] directly into GC1459 naSi2 II; daf-18(ok480) IV. All arrays used pJB134 (flp-17P::dsred) as a co-injection marker [83].

Cas9-triggered long-range HDR using plasmid repair template, and plasmid Cas9 and single guide RNA [43]. Although the Phsp::peel-1 was included in the injection mix we found that the heat shock step was not necessary (Used to generate germline::daf-18 (naSi22)).

CRISPR-based "hybrid" GFP donor method [84], with minor modifications from J. Nance that used dpy-10 co-CRISPR instead of rol-6(su1006d). Injection was done into GC1171 naSi2 II to generate GFP::vbh-1 (na110). crRNA targeting sequence for vbh-1 and dpy-10 (co-CRISPR) were generated by IDT. https://www.idtdna.com/site/order/designtool/index/CRISPR_CUSTOM. Cas9 and tracrRNA were purchased from IDT.