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.

Figure 1.

dCas9-mediated repression in E. coli. (A) Plasmid pdCas9 encodes a cas9 mutant containing D10A and H840A substitutions (red asterisks) that abrogate nuclease activity. dCas9 binds to a tracrRNA:precursor crRNA and recruits RNase III to process the precursor and liberate the crRNA. The crRNA directs binding of dCas9 to promoter or open reading frame regions to prevent RNAP binding or elongation, respectively. (B) GFP fluorescence of cells expressing dCas9 guided to different regions of the gfp-mut2 gene, relative to the fluorescence of cells expressing a non-targeting dCas9, as a function of the position of the target sequence within the gene (+1, transcription start). Squares indicate the PAM position, lines the extension of complementarity between the crRNA guide and the reporter gene. Red and blue lines indicate crRNAs sequences identical to top or bottom DNA strand, respectively. Error bars show one standard deviation from the mean of three relative fluorescence values. The gfp-mut2 gene (green), its promoter, including the −35 and −10 elements (gray shade) and the ribosome binding site (rbs) are shown as reference for the localization of the dCas9 binding sites. (C) Nothern blot with probes annealing either upstream or downstream of the T10 and B10 target sites using RNA extracted from cells expressing T5-, T10-, B10-guided dCas9 or a control strain without a target. Detection of 5S RNA serves as control.

Figure 2.

Effect of mismatches between crRNA guide and target sequence. (A) Protospacer and crRNA-guide sequences for the B10 target site. Mutations in the 5′ region of the crRNA are shown in lower case; Watson–Crick complementary bases were introduced. (B) Effect of an increasing number of mutations in the 5′ end of the B1, T5 and B10 crRNAs on gfp-mut2 repression mediated by dCas9. Repression by wild-type Cas9 guided by mutant versions of the B10 crRNA is also shown. Fluorescence values are normalized to the fluorescence of a strain expressing gfp-mut2 and dCas9, but no crRNA guide (Ø). Error bars show one standard deviation from the mean of three relative fluorescence values. (C) Effect of Cas9 targeting using a B10 crRNA guide with increasing numbers of mutations at the 5′ end on the transformation of the GFP reporter plasmid, pDB127, or an empty vector control, pZS*24. The mean of three independent enumerations of the total number of colony forming units (CFU) per transformation is shown; error bars indicate one standard deviation. (D) Agarose gel electrophoresis of SacI-digested purified plasmids from cells obtained after transformation of the pDB127-target plasmid into cells expressing wild-type Cas9 and different 5′ mutant versions of the B10 crRNA. Individual plasmids are shown to indicate the electrophoretic mobility of each plasmid.

Figure 3.

Activation of gene expression in E. coli using dCas9 fused to the ω subunit of RNAP. (A) dCas9 is directed to the promoter region and is fused to the ω subunit of RNAP, which recruits the polymerase by interacting with the β′ subunit. A host with a deletion of rpoZ, encoding ω, is used. (B) Either N- or C-terminal fusions of the ω subunit to dCas9 were directed to four regions of the top strand upstream of the −35 element of the lacZ gene. (C) lacZ gene expression levels in the different strains were measured as β-galactosidase activity (Miller units). Activation is reported as the relative Miller units normalized against the units obtained with cells expressing a C-terminal dCas9-ω fusion but no crRNA guide (Ø). The average of three independent experiments is indicated; error bars indicate one standard deviation. Asterisks indicate the P-values associated with each measurement, compared with the no crRNA guide control (Ø). *P ≤ 0.05; **P ≤ 0.005; ***P ≤ 0.001.

Figure 4.

Activation of gene expression using a dCas9-ω fusion. (A) The positions of the different crRNA guides tested (W101–W110), relative to the ribosome binding site (rbs) and the −35 and −10 promoter elements of the gfp-mut2 gene are shown. (B) GFP fluorescence levels, relative to the fluorescence produced by a control strain expressing a non-targeting dCas9-ω, as a function of the position of the target sequence within the gfp-mut2 upstream region (+1, transcription start). Squares indicate the PAM position, lines the extension of complementarity between the crRNA guide and the reporter gene; red lines, top strand targets; blue, bottom strand. Error bars show one standard deviation from the mean of three relative fluorescence values. (C) Activation of three variants gfp-mut2 containing promoters of different strengths (J23117, J23116 and J23110) by W103- or W108-guided dCas9-ω. The relative induction, compared with the fluorescence of cells expressing a non-targeting dCas9-ω, is shown. The average of three independent experiments is indicated; error bars indicate one standard deviation. Asterisks indicate the P-values associated with each measurement, compared with the no spacer control. *P ≤ 0.05; ***P ≤ 0.001.