The manipulation of gene expression in prokaryotes is usually achieved through the use of promoters whose activity can be modulated using small molecules (38
). This requires genetic engineering to place the gene of interest under the control of inducible promoters. Given the lack of RNAi (39
) in prokaryotes, methods that allow for simple and global regulation of gene expression are limited (40
). Here, we describe the use of an RNA-guided DNA-binding protein, dCas9, to either repress or activate genes in E. coli
and S. pneumoniae
. In this method, dCas9 can be directed to any region of the bacterial chromosome that is specified by the base-pair complementarity between the RNA guide and the cognate genomic sequence, i.e. without the need to modify the promoter sequence of the gene whose expression is manipulated.
Repression is achieved by directing dCas9 to either promoter or open-reading frame regions. Although binding of dCas9 to promoters prevents transcription initiation, binding to the open-reading frame prevents elongation, especially when the coding strand is targeted. While this article was in review, Qi and colleagues (41
) also showed that dCas9 can act as a transcription repressor by preventing initiation or elongation, both in E. coli
and human (HEK293) cells. As opposed to our system, which uses the natural CRISPR array and tracrRNA, Qi et al.
used a chimeric crRNA as a guide. Chimeric crRNAs combine critical crRNA and tracrRNA moieties into a single small RNA that can be loaded into dCas9 without the need for processing by RNAse III (8
). In this study, dCas9 was under the control of an inducible promoter, demonstrating that repression can be reversible if the inducer is withdrawn from the culture. By using several chimeric guide RNAs, Qi et al.
demonstrated that this methodology can be applied for repression of multiple loci at the same time. Multiplexing will also be possible with our system, as the CRISPR array can be engineered to contain multiple spacers encoding different crRNA guides.
Target specificity is an important aspect of all the recent Cas9-based technologies. Qi et al.
analyzed this by truncating the 5′ end of a chimeric guide RNA that prevents transcription elongation. They observed that repression is noticeable with at least 12 nt of homology between the chimeric RNA and the protospacer (41
). In addition, the authors performed RNAseq in the presence and absence of dCas9 targeting and determined that the only transcript with a significant change in abundance was the one specified by the RNA guide. We also determined the effects of the accumulation of mismatches at the 5′ end of crRNAs that prevent both transcription initiation and elongation. In accordance with the results of Qi et al.
, we found that a 12 nt match between crRNA and protospacer produces a weak but significant repression if the coding strand of an open-reading frame is targeted. In theory, as in addition to the crRNA:protospacer matches a perfect PAM is required for dCas9 repression, such off-target sequence occurs randomly about once every 414
bp, 268 Mbp, and thus is unlikely to be found in bacterial genomes [the largest size to date is 13 Mbp (42
)], but more likely to be present in larger eukaryotic genomes. Nonetheless, off target effects can happen, especially during studies that require the design of many crRNA guides. For example, during the course of this study, we were unable to clone one of the designed spacers on the pdCas9 vector. We later found that this spacer showed a 12 nt perfect match next to a good PAM in the essential murC
) (Supplementary Figure S3
). Such off-target effect could easily be avoided by a systematic blast of the engineered spacers. By analyzing the effect of mismatches between the crRNA and its target on dCas9 repression, we found that wild-type Cas9 can repress gene expression when directed by a crRNA with at least 4 nt of mismatch at the 5′end that prevents it from efficiently cleaving its target. This finding suggests that endogenous CRISPR systems could actively repress gene expression when an imperfect match exists between the crRNA and its targets. The inhibition of biofilm formation by the type I CRISPR system of Pseudomonas aeruginosa
, a phenomenon that requires mismatches between the crRNA and its target, could be an example of this side effect of CRISPR immunity on transcription (44
Finally, we demonstrated that dCas9 can be directed to promoter regions to activate gene expression. This requires the addition of an activation domain, in this study the ω subunit of RNAP, which was previously shown to provide effective recruitment of RNAP (29
). Activation levels depended on the distance between the dCas9-binding sequence and the −35 promoter element. A maximum of a 23-fold induction was achieved with a bottom strand target with its PAM positioned 59 nt upstream of the −35 element. This level of activation is low when compared with the activation achieved by a cI-ω fusion, which provides 70-fold induction (29
). We believe that activation can be further optimized by changing the protein linker between dCas9 and the activation domain and/or testing different activation domains.
Results presented here and in Qi et al.
) reveal a new powerful technology to regulate gene expression in prokaryotes with an exciting future. The use of larger CRISPR arrays generating multiple crRNA guides can provide the possibility of affecting many genes at the same time and will allow genetic network organization to be probed. dCas9 gene regulation can also be applied to the engineering of synthetic gene networks in living cells for biotechnological applications. There is also the possibility of tethering dCas9 fused to fluorescent proteins to specific loci to investigate the influence of gene function and expression on the subcellular positioning of chromosomal loci in bacteria (45
). We anticipate that dCas9-based technologies will contribute to the success of these applications.