Programmable repression and activation of bacterial gene expression using an engineered CRISPR-Cas system

Abstract

The ability to artificially control transcription is essential both to the study of gene function and to the construction of synthetic gene networks with desired properties. Cas9 is an RNA-guided double-stranded DNA nuclease that participates in the CRISPR-Cas immune defense against prokaryotic viruses. We describe the use of a Cas9 nuclease mutant that retains DNA-binding activity and can be engineered as a programmable transcription repressor by preventing the binding of the RNA polymerase (RNAP) to promoter sequences or as a transcription terminator by blocking the running RNAP. In addition, a fusion between the omega subunit of the RNAP and a Cas9 nuclease mutant directed to bind upstream promoter regions can achieve programmable transcription activation. The simple and efficient modulation of gene expression achieved by this technology is a useful asset for the study of gene networks and for the development of synthetic biology and biotechnological applications.

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

“…When all five fgf8ats-gRNAs were co-injected with dCas9-KRAB mRNA into one-cell stage wild-type fertilized eggs, the resulted embryos showed reduced fgf8a expression at 11 hpf ( Figure 1J and Supplementary information, Figure S1hC) and smaller otic vesicle at 32 hpf (Supplementary information, Figure S1iA-S1iB). Similarly, we observed that foxi1 expression and otic vesicle size were reduced when dCas9-KRAB mRNA and all five foxi1ts-gRNAs (1)(2)(3)(4)(5) were injected ( Figure 1K, Supplementary information, Figure S1hD, S1iA and S1iC). If dCas9-VP160 mRNA and fgf8ats-gRNAs (6-10) or foxi1ts-gRNAs (6-10) were co-injected, an elevated expression of fgf8a or foxi1 was observed ( Figure 1L and 1M and Supplementary information, Figure S1hE and S1hF), leading to enlarged otic vesicles (Supplementary information, Figure S1iD-S1iF), and enhanced fgf8a or foxi1 expression appeared to be near their endogenous expression locations (Supplementary information, Figure S1j).…”

“…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.…”

“…In addition, using transgenic worms carrying a copy of integrated pud-2.2-promoter-driven GFP (Ppud-2.2::GFP) that expresses GFP strongly and uniformly in the intestine and weakly in the hypodermis, we were able to directly monitor changes of GFP in these two tissues. Ppud-2.2::GFP worms carrying Pges-1::dCas9 or Pges-1::dCas9-KRAB and all five gfpts-gRNAs (1)(2)(3)(4)(5) showed suppressed gfp expression (Supplementary information, Figure S1e). Ppud-2.2::GFP worms carrying Pges-1::dCas9-VP160 and all five pud-2.2ts-gRNAs (6-10) that recognize pud-2.2 promoter showed significantly enhanced GFP expression mainly in the intestine (Supplementary information, Figure S1e and S1fC-S1fH).…”

“…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)(2)(3)(4)(5)(6)(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 dpy5ts-gRNAs (1)(2)(3)(4)(5)(6)(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).…”

CRISPR-based screening of genomic island excision events in bacteria

“…Interference results from a corresponding transcript that is processed selectively at each repeat sequence, forming CRISPR RNAs (crRNAs) that guide Cas proteins for sequence-specific recognition and cleavage of target DNA complementary to the spacer (7). CRISPR-Cas technology has applications in strain typing and detection (8)(9)(10), exploitation of natural/engineered immunity against mobile genetic elements (11), programmable genome editing in diverse backgrounds (12), transcriptional control (13,14), and manipulation of microbial populations in defined consortia (15).…”