CRISPR editing and generation of extrachromosomal array lines in C. elegans

To create a deletion of lea-1 and to insert mNG and mYPET fluorescent tags into the endogenous gene locus, CRISPR methods were employed [34, 35]. Using the self-excising cassette (SEC) system, sgRNA sequences were added to the sgRNA-Cas9 plasmid pDD162 using the NEB Q5 site-directed mutagenesis kit. Homology arms of ~ 500–700 bp were added to plasmids carrying the SEC and repair templates. To insert a myo-2p::GFP::myo-2 3′UTR reporter to track the null deletion of lea-1, homology arms were cloned into plasmid pDD317. To insert mNG or mPYET tags, homology arms were cloned into pUA77 and pDD283 respectively.

To replace the endogenous lea-1 sequence with LEA motifs, sequence was first codon-optimized for C. elegans and synthesized by Integrated DNA Technologies (IDT). These stretches were cloned into pUA77 with the same upstream homology arm for insertion of mNG to the endogenous locus and the same downstream homology arm for insertion of mYPET into the endogenous locus. The same sgRNAs for each of the initial mNG and mYPET insertions were used in combination to excise the lea-1 locus.

Worms were injected with 50 ng/μL of plasmid containing the sgRNA and Cas9, and 10–20 ng/μL plasmid containing the repair template, along with a co-injection mix [35]. Two to three days after injection, worms were treated with Hygromycin and selected for transgene-carrying rollers lacking red co-injection mix extra-chromosomal arrays. L1 worms were heat shocked at 32 °C for 5 h to excise the SEC. Genomic edits were confirmed by visualization of fluorescent reporters and with PCR genotyping.

Construction of most TIR1-expressing strains is described in [52]. The col-10p::TIR1 line was generated by cloning to combine the col-10 promoter with the TIR1 construct of pDD356 (NEB Hifi Assembly Master Mix). Plasmid pAP082 was used to express Cas9 and a sgRNA to target the chromosome I insertion site. Worms were injected with 50 ng/μL of pAP082 and 20 ng/μL plasmid containing the repair template.

To establish extrachromosomal array lines expressing lea-1a or motifs specifically in body wall muscle, the myo-3 promoter and an mNG fluorescent tag was cloned onto each of lea-1 isoform A, a 44-mer motif sequence, and a 97-mer motif sequence. DNA was injected into the gonads of LP852 daf-2(e1370);lea-1Δ(cp423[myo-2p::GFP::myo-2 3′UTR]) animals at a concentration of 50 ng/μL. Progeny reliably expressing and transmitting the mNG labeled construct in body wall muscle were selected and established as transgenic array lines.