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

By David Bikard, Wenyan Jiang, Poulami Samai, A. Hochschild, Feng Zhang, L. Marraffini. Nucleic Acids Research, 2013

1,113 citations recorded by the source corpus.

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.

Subfields: Genetics, Molecular Biology

View Paper: https://www.semanticscholar.org/paper/ec83203ad33da37ffe3dc16921d7385b35679aa9

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Citations in the corpus

Diverse evolutionary roots and mechanistic variations of the CRISPR-Cas systems (2016, methodology): “The dCas9 has been used for transcriptional activation and repression [134-137], for localizing fluorescent protein labels [138], and for recruiting histone modifying enzymes [139, 140].”
CRISPR/Cas9-Induced Mutagenesis of Semi-Rolled Leaf1,2 Confers Curled Leaf Phenotype and Drought Tolerance by Influencing Protein Expression Patterns and ROS Scavenging in Rice (Oryza sativa L.) (2019, methodology): “The CRISPR/Cas9 system has also been employed for the simultaneous editing of multiple genes [22], as well as target gene repression and activation [23].”
CRISPR/Cas9 in Genome Editing and Beyond. (2016, methodology): “In addition to CRISPRi, CRISPR activation (CRISPRa) has been created by fusing dCas9 to transcriptional activators, such as VP64 and p65AD in mammalian cells (Figure 2b) (37, 76, 175, 177) and the ω subunit of RNA polymerase in bacteria (176).”
Development of sequence-specific antimicrobials based on programmable CRISPR-Cas nucleases (2014, positive): “6).. Altogether, these results demonstrate that delivery of the sequence-specific Cas9 nuclease dramatically reduces the plasmid content in a bacterial population without killing the host, can be used to immunize non-pathogenic strains against the transfer of virulence and/or antibiotic-resistant pl”
CRISPR RNA-guided activation of endogenous human genes (2013, background): “RNA-mediated recruitment of a catalytically inactive mutant form of Cas9 (referred to as dCas9) can induce repression of endogenous genes in bacterial and human cells 8,9 .”
The new frontier of genome engineering with CRISPR-Cas9 (2014, positive): “CRISPRi has been used to repress multiple target genes simultaneously, and its effects are reversible ( 127 – 130 ).”
CRISPR-Cas systems: prokaryotes upgrade to adaptive immunity (2014, background): “Their biological role remained elusive until 2005, when three groups reported that the spacers were homologous to foreign genetic elements, including viruses and plasmids (Bolotin et al., 2005; Mojica et al., 2005; Pourcel et al., 2005).”
Identifying and visualizing functional PAM diversity across CRISPR-Cas systems (2016, methodology): “We used the catalytically dead Cas9 (dCas9) containing point mutations to the RuvC and HNH endonuclease domains (D10A, H840A), which was shown to bind target DNA and block transcription in E. coli (Bikard et al., 2013; Jinek et al., 2012; Qi et al., 2013).”