Single-copy knock-in loci for defined gene expression in Caenorhabditis elegans

Parts used

Key findings

• A SKI LODGE cassette has the architecture tissue-specific promoter::3xFLAG::dpy-10 protospacer and PAM (30 bases)::3′UTR and is inserted as a single copy at MosSCI safe-harbor intergenic loci on different chromosomes of Caenorhabditis elegans. Because the transplanted dpy-10 site matches the endogenous dpy-10 gene, one dpy-10 crRNA with Cas9 cuts both the cassette and the endogenous dpy-10 locus, so the same guide drives knock-in of any PCR template carrying about 35-bp homology arms and acts as the Co-CRISPR marker, selected as dumpy or roller animals 3-4 days after injection.
• Four SKI LODGE cassettes drive tissue-specific single-copy expression in Caenorhabditis elegans from defined loci: the eft-3 promoter (ubiquitous, chromosome V), the rab-3 promoter (neurons, chromosome IV), the myo-3 promoter (muscle, chromosome I) and the pie-1 promoter (germline, chromosome III). Knock-in of wrmScarlet into the 3xFLAG-dpy-10 site gives the expected ubiquitous, neuronal and muscle patterns from the eft-3, rab-3 and myo-3 cassettes, and knock-in of GFP gives germline expression from the pie-1 cassette.
• A wrmScarlet coding sequence, which lacks introns, is not expressed in the germline when knocked into the pie-1 promoter SKI LODGE cassette in Caenorhabditis elegans, whereas GFP carrying artificial introns knocked into the same cassette is expressed in the germline. This is consistent with introns enhancing germline transgene expression.
• A rab-3 promoter SKI LODGE cassette carrying the unc-54 3′UTR shows ectopic intestinal expression of knocked-in wrmScarlet in Caenorhabditis elegans, and replacing the unc-54 3′UTR with the rab-3 3′UTR removes identifiable intestinal expression at days 1 and 6 of adulthood. The rab-3 promoter with the rab-3 3′UTR therefore drives cleaner neuronal expression, and the unc-54 3′UTR (unc-54 lies upstream of the intestinally expressed aex-5) may explain non-neuronal leaky expression of pan-neuronal promoters.
• SKI LODGE strains for the eft-3, rab-3, myo-3 and pie-1 promoters, outcrossed six times, are indistinguishable from wild-type Caenorhabditis elegans in fertility, embryonic lethality, developmental timing and lifespan. Sequencing of the three genes closest to each insertion site, and of the R12E2.15 gene for dpy-10 off-target events, finds no mutations, so the lines are phenotypically wild type and easier to microinject than unc-119 mutant backgrounds used for other single-copy methods.
• PCR-template knock-in efficiency into SKI LODGE lines decreases with template length in Caenorhabditis elegans: a 774-bp wrmScarlet template inserts in up to 13.04% of F1 animals, a 1872-bp template in 0.44% and a 2319-bp template in 2.08%, using blunt-ended PCR products with at least 35-bp homology arms. Templates above about 3000 bp are better inserted with plasmid templates carrying long homology arms.
• Two PCR templates with overlapping sequences can be knocked into a SKI LODGE line in Caenorhabditis elegans in a single injection, giving insertion in 13.39% of F1 animals for a 1534-bp plus 778-bp pair and in 9.56% for an 829-bp plus 778-bp pair, using a single dpy-10 crRNA.
• PCR products with single-strand ends, made as hybrid asymmetric molecules from donors with 120-bp homology arms and no homology arms, insert wrmScarlet into SKI LODGE lines in Caenorhabditis elegans at the same or higher frequency than blunt-ended PCR products. Insertion into the myo-3 SKI LODGE line is 13.33% with single-strand ends versus 13.04% with blunt ends, and insertion into the eft-3 line rises from 8.57% with blunt ends to 26% with single-strand ends.
• SKI LODGE lines for the eft-3 (ubiquitous), rab-3 (neuronal) and ges-1 (intestinal) promoters carry the gpd-2 SL2 trans-splicing sequence between 3xFLAG and wrmScarlet in Caenorhabditis elegans, so a gene of interest can be co-expressed with wrmScarlet from the same transcript without making a gene::wrmScarlet translational fusion.
• CRISPR knock-in efficiency in SKI LODGE lines of Caenorhabditis elegans depends on template length, template sequence complexity, template concentration, homology-arm size and microinjection proficiency. Short sequences such as the 3xFLAG tag insert easily, repetitive sequences such as those in promoters reduce efficiency, and larger homology arms increase insertion frequency.

Abstract

We have generated a single-copy knock-in loci for defined gene expression (SKI LODGE) system to insert any DNA by CRISPR/Cas9 at defined safe harbors in the Caenorhabditis elegans genome. Utilizing a single crRNA guide, which also acts as a Co-CRISPR enrichment marker, any DNA sequence can be introduced as a single copy, regulated by different tissue-specific promoters. The SKI LODGE system provides a fast, economical, and effective approach for generating single-copy ectopic transgenes in C. elegans.