Regulation of transcriptionally active genes via the catalytically inactive Cas9 in C. elegans and D. rerio

Abstract

Dear Editor,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 cells [1][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 vivo [2]. 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).We fused dCas9 to the KRAB domain (repressor) or the VP160 domain containing 10 tandem VP16 units (activator; Figure 1A). In C. elegans, dCas9 constructs were driven by tissue-specific promoters while ts-gRNAs were under control of a U6-type RNA polymerase III promoter ( Figure 1B). Different combinations of the DNA constructs were injected into adult hermaphrodite gonads to generate transgenic worms. For zebrafish experiments, dCas9-KRAB and dCas9-VP160 mRNAs and ts-gRNAs were synthesized in vitro and injected into one-cell stage embryos ( Figure 1C), and the resulted embryos were analyzed by real-time RT-PCR and in situ hybridization. Flanking sequences, including the nuclear localization sequences (NLSs) and UTRs, were indicated in Figure 1B and 1C or described previously [6].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

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