Researchers have developed a method to programmably repress and activate bacterial gene expression using an engineered CRISPR-Cas system. By modifying the Cas9 nuclease to lose its cutting activity (dCas9), they created a DNA-binding protein that can be directed to specific regions of the genome. When dCas9 binds to promoter regions, it represses transcription by preventing RNA polymerase (RNAP) from accessing the promoter. When directed to open reading frames, it blocks transcription elongation. Additionally, fusing dCas9 to the omega subunit of RNAP enables it to activate transcription by enhancing RNAP binding to promoters. The study demonstrates that dCas9 can be used to regulate gene expression in both E. coli and S. pneumoniae. By introducing mismatches in the crRNA guide, the level of repression can be modulated. The researchers also show that dCas9 can be used to activate gene expression when fused to the omega subunit of RNAP, which enhances transcription initiation. The effectiveness of this activation depends on the distance between the dCas9-binding site and the -35 promoter element, with the best activation achieved when the binding site is located on the non-coding strand. The study highlights the potential of dCas9-based technologies for regulating gene expression in prokaryotes, which could be useful for synthetic biology and biotechnology applications. The method allows for precise control of gene expression without the need to modify promoter sequences, making it a versatile tool for studying gene networks and engineering synthetic gene circuits. The findings suggest that dCas9 can be used to both repress and activate gene expression, offering a powerful tool for manipulating bacterial gene regulation.Researchers have developed a method to programmably repress and activate bacterial gene expression using an engineered CRISPR-Cas system. By modifying the Cas9 nuclease to lose its cutting activity (dCas9), they created a DNA-binding protein that can be directed to specific regions of the genome. When dCas9 binds to promoter regions, it represses transcription by preventing RNA polymerase (RNAP) from accessing the promoter. When directed to open reading frames, it blocks transcription elongation. Additionally, fusing dCas9 to the omega subunit of RNAP enables it to activate transcription by enhancing RNAP binding to promoters. The study demonstrates that dCas9 can be used to regulate gene expression in both E. coli and S. pneumoniae. By introducing mismatches in the crRNA guide, the level of repression can be modulated. The researchers also show that dCas9 can be used to activate gene expression when fused to the omega subunit of RNAP, which enhances transcription initiation. The effectiveness of this activation depends on the distance between the dCas9-binding site and the -35 promoter element, with the best activation achieved when the binding site is located on the non-coding strand. The study highlights the potential of dCas9-based technologies for regulating gene expression in prokaryotes, which could be useful for synthetic biology and biotechnology applications. The method allows for precise control of gene expression without the need to modify promoter sequences, making it a versatile tool for studying gene networks and engineering synthetic gene circuits. The findings suggest that dCas9 can be used to both repress and activate gene expression, offering a powerful tool for manipulating bacterial gene regulation.