CRISPR interference (CRISPRi) is a recently developed tool used to study single guide RNA (gRNA)-mediated sequence-specific repression of transcription in both prokaryotic and eukaryotic cells1,2,3,4,5. Transcription initiation and elongation of a gene can be interfered by the presence of gRNA:DNA hetero-duplex/dCas9 (a catalytically inactive form of Cas9) complex in its promoter and exons. If Krüppel associated box (KRAB) domain is fused to dCas9, repression of target gene is more efficient. Likewise, fusion of a transcription activator such as VP16 (CRISPR-on) can increase target gene expression through three to four gene-specific gRNAs (up to seven) that recognize the proximal promoter of a target gene in cultured cells and in vivo2. We applied both CRISPRi and CRISPR-on tools in worm and zebrafish, and demonstrated here that our dCas9 fusion systems modify gene expression at or near their endogenous expression location(s) through target-specific gRNAs (ts-gRNAs).

Decrease in dpy-5 expression levels produces short body and dumpy phenotype, known as Dpy. Dbl-1 is a TGF-β family member expressed primarily in neurons and regulates lon-1 to adjust body length. When dbl-1 is overexpressed, worm body length is elongated (Lon phenotype). We targeted dpy-5 by selecting seven ts-gRNAs that recognize its non-template DNA strand of coding region spanning about 400 bp downstream of the transcription start site of dpy-5 (Figure 1D). Transgenic worms carrying Pdpy-5::dCas9 or Pdpy-5::dCas9-KRAB and one of seven dpy-5ts-gRNAs did not exhibit obvious changes in dpy-5 expression and body length (Figure 1F and Supplementary information, Table S1a). When all seven dpy-5ts-gRNAs(1-7) were included, suppression of dpy-5 expression and Dpy phenotype occurred (Figure 1F and Supplementary information, Figure S1aA–S1aC and S1aE). It appeared that Pdpy-5::dCas9-KRAB and dpy-5ts-gRNAs(1-7) combination showed slightly stronger effect on dpy-5 expression than that of Pdpy-5::dCas9 and dpy-5ts-gRNAs(1-7) combination, although both combinations resulted in the Dpy phenotype (Figure 1F and Supplementary information, Figure S1aA–S1aC, S1aE and Table S1a). We also found that combined with dCas9-KRAB, at least five ts-gRNAs were required to knockdown dpy-5 (Supplementary information, Figure S1bA and S1bC). These results suggest that recruiting multiple dCas9/dpy-5ts-gRNA complexes to exonic region of dpy-5 may account for a synergistic suppression. To directly visualize tissue-specific gene suppression, we generated Pdpy-30::NLSSV40-GFP-NLSegl-13; Pdpy-30::NLSSV40-mCherry-NLSegl-13 transgenic worms expressing GFP and mCherry in all cells (Supplementary information, Figure S1cA–S1cD). When dCas9-KRAB driven by an intestine-specific promoter Pges-1 and five gfpts-gRNAs driven by PU6 promoter were introduced into double-transgenic animals, GFP was largely diminished in intestine nuclei while mCherry signal remained in most animals examined (92.7%, n = 83; Supplementary information, Figure S1cE–S1cH), suggesting that tissue- and gene-specific knockdown is achievable with our dCas9 fusion system. Using Pdpy-7::GFP worms that only express GFP in the hypodermis, we found that only dCas9-KRAB driven by the hypodermis-specific dpy-7 promoter, not that by the intestine-specific Pges-1, reduced GFP expression in hypodermal cells (Supplementary information, Figure S1d), indicating that dCas9 functions in a cell-autonomous manner.

Log in or create a free account to read this content

Gain free access to this article, as well as selected content from this journal and more on nature.com

Access through your institution

or

Sign in or create an account
Continue with Google
Continue with ORCiD