Gene Repression in Bacteria

The CRISPR-dCas9-based repression has been shown to efficiently down-regulate genes in eukaryotes and it was also used efficiently in bacteria. The first demonstration of CRISPR-dCas9 CRISPRi tool was in bacteria, where it was shown that dCas9 is able to abrogate the transcription of targeted genes either by disrupting transcription factor binding or by interfering with transcriptional elongation. CRISPR repression (CRISPR interference or CRISPRi) has been extensively characterized in the model bacteria Escherichia coli and Bacillus subtilis. 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. Now several CRISPR-Cas systems can be used for gene repression.

Mechanism of Gene Repression in Bacteria

CRISPR interference (CRISPRi) employs the CRISPR-Cas9 system for repress gene expression. To purpose Cas9 for transcriptional repression, Cas9 nuclease activity is inactivated by point mutations within its two nuclease domains, called dCas9. As bacterial cells lack the machinery for RNAi, the dCas9-based repression allows for research into gene function that was not previously possible in microbes. As CRISPR-dCas9 interferes with the transcription of the targeted genes by sterically hindering the elongation of RNA polymerase (RNAP) or inhibiting the initial binding of RNAP to the promoter, this approach is termed CRISPRi. 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.

CRISPR-dCas9-mediated gene repression in E. coli

Fig 1. CRISPR-dCas9-mediated gene repression in E. coli.

In CRISPRi, a dCas9 is coupled with designed sgRNAs complementary to a desired DNA target. The dCas9-sgRNA complex acts to sterically hinder transcription of the targeted DNA, causing gene repression. Because repression by CRISPRi depends on base pairing between a short segment of the sgRNA and targeted DNA, new DNA targets can be specified simply by altering the sgRNA sequence. Furthermore, repression by CRISPR is titratable and reversible through use of inducible promoters for both Cas9 protein and sgRNA expression. When targeting the promoter, sgRNAs specific for either the template or non-template strand can be used, whereas an elongation block is most effective when targeting the non-template strand, producing up to 300-fold repression. CRISPRi is highly specific with minimal off-target effects in bacterial cells and allows for tunable regulation of individual genes and multiplexable control of many genes using multiple sgRNAs.

CRISPRi using only dCas9 and an sgRNA is highly efficient and can silence gene expression by up to 99.9% in prokaryotes. While, some applications require a precise tuning of gene expression rather than its complete repression. Scientists have done a lot of reaserch to achieve intermediate repression levels through the introduction of mismatches that will weaken the cr RNA/target interactions. And some researches have demonstrated that the introduction of mismatches in the crRNA guide allows for the modulation of dCas9 repression. However, the number of the mismatches required to achieve this modulation depends on the mode of dCas9 repression, i.e. blocking transcription initiation or elongation.

Gene Repression in Bacteria using Other CRISPR-Cas Systems

Another CRISPR-Cas system (type III-B) has been developed to target and degrade mRNAs and thus is applicable for targeted mRNA degradation. For this application the Cas protein complex from type III is required, thus it is most convenient to use in cells having an endogenous CRISPR-Cas type III system. In Escherichia coli a CRISPR-Cas type I system was developed into a CRISPRi tool. Here, the endogenous CRISPR-Cas type I-E system was harnessed and modified to use for transcription regulation. In the CRISPR-Cas type I-E system the Cas protein complex (called Cascade for Cas protein complex for antiviral defence) binds via the crRNA to the target DNA and recruits the Cas3 protein to degrade the target DNA. To prevent target DNA degradation, a cas3 gene deletion strain was generated, resulting in a strain that had a functional Cascade complex binding to specific DNA sequences but unable to cleave those sequences.

Gene repression reaction of CRISPR-Cas type I in E. coli
Gene repression reaction of CRISPR-Cas type I in E. coli

Fig 2. Gene repression reaction of CRISPR-Cas type I in E. coli.

In CRISPRi, a dCas9 is coupled with designed sgRNAs complementary to a desired DNA target. The dCas9-sgRNA complex acts to sterically hinder transcription of the targeted DNA, causing gene repression. Because repression by CRISPRi depends on base pairing between a short segment of the sgRNA and targeted DNA, new DNA targets can be specified simply by altering the sgRNA sequence. Furthermore, repression by CRISPR is titratable and reversible through use of inducible promoters for both Cas9 protein and sgRNA expression. When targeting the promoter, sgRNAs specific for either the template or non-template strand can be used, whereas an elongation block is most effective when targeting the non-template strand, producing up to 300-fold repression. CRISPRi is highly specific with minimal off-target effects in bacterial cells and allows for tunable regulation of individual genes and multiplexable control of many genes using multiple sgRNAs.

CRISPRi using only dCas9 and an sgRNA is highly efficient and can silence gene expression by up to 99.9% in prokaryotes. While, some applications require a precise tuning of gene expression rather than its complete repression. Scientists have done a lot of reaserch to achieve intermediate repression levels through the introduction of mismatches that will weaken the cr RNA/target interactions. And some researches have demonstrated that the introduction of mismatches in the crRNA guide allows for the modulation of dCas9 repression. However, the number of the mismatches required to achieve this modulation depends on the mode of dCas9 repression, i.e. blocking transcription initiation or elongation.

Gene Repression in Archaea using CRISPR-Cas I-B System

Since the discovery of CRISPR-Cas system, it has been developed into numerous applications like gene editing and regulation of gene expression in eukaryotes and bacteria. But, for archaea, no tools for transcriptional repression is previously available. Because more archaeal organisms are studied over the years, and molecular biology analyses in archaea also become more and more widespread such a tool is vital for investigating the biological function of essential genes in archaea. The prevalent subtypes of the CRISPR-Cas I system found in archaea are subtypes I-A, I-B, and I-D. Because many archaea have the type I-B system it is possible to harness the endogenous system for the CRISPR mediated gene repression and to convert it into a transcriptional regulator.

In this system crRNAs targeting the template strand in the promoter region have the highest effect. This is in contrast to the CRISPR-Cas9 CRISPRi tool used in eukarya and bacteria and the type I-E CRISPRi system in bacteria, where the targeting efficiency in the promoter region is independent of the nature of the strand. In these systems the nature of the strand only makes a difference upon targeting of the coding region, here targeting of the coding strand is more efficient than targeting of the template strand. Differences between transcriptional regulatory processes have been reported in archaea, bacteria, and eukarya. However, to finally determine the cause for the differences more data about the systems are required.

Gene Repression in Bacteria Related Information

Gene Repression in Bacteria Related References

1. Hawkins et al. Targeted Transcriptional Repression in Bacteria Using CRISPR Interference (CRISPRi). Methods Mol Biol. 2015; 1311:349–362.
2. Lo A and Qi L. Genetic and epigenetic control of gene expression by CRISPR-Cas systems. F1000Research 2017, 6(F1000 Faculty Rev):747.
3. Aris-Edda Stachler and Anita Marchfelder. Gene Repression in Haloarchaea Using the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas I-B System. THE JOURNAL OF BIOLOGICAL CHEMISTRY. July 15, 2016. 291(29):pp. 15226-15242.
4. Bikard et al. Programmable repression and activation of bacterial gene expression using an engineered CRISPR-Cas system. Nucleic Acids Research. 2013;41(15):7429-7437.