ArticleNucleic acids research2023
Cas9 off-target binding to the promoter of bacterial genes leads to silencing and toxicity.
Article in Nucleic acids research, 2023. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 53 papers.
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The abstract states no effect estimate the extractor could read, or names no intervention and outcome on the map, so this paper lights no cell and moves no belief. It is still indexed, cited and linked below.
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Who cites it
53 citing papers in PubMed, 84 citations in OpenAlex.
- One Plasmid Is All You Need: Genome Editing in Escherichia coli Using Endogenous TnpB and Endogenous Recombination System.Biotechnology and bioengineering · 2026Article
- An inducer-independent, single-plasmid CRISPR-Cas9 system for genome editing in Bacillus species.Applied microbiology and biotechnology · 2026Article
- Markerless fliC Knockout inBiology · 2026Article
- Engineering the Gut Microbiome: Emerging Genome-Editing Strategies and Therapeutic Applications.Microorganisms · 2026Review
- Minute-scale control of ubiquitin-mediated degradation reveals dynamics of bacterial secreted effector-functions.Nature communications · 2026Article
- Tailoring Corynebacterium glutamicum for Sustainable Biomanufacturing: From Traditional to Cutting-Edge Technologies.Molecular biotechnology · 2026Review
- Enhanced editing ofApplied and environmental microbiology · 2026Article
- A platform for CRISPRi-seq inmBio · 2026Article
- Antibody-Antibiotic Conjugates: Mechanisms, Clinical Progress, and Next-Generation Strategies Against Multidrug-Resistant Bacterial Infections.MicrobiologyOpen · 2026Review
- Functional Analysis of CRISPR-Cas9-Mediated Gene Deletion inMicroorganisms · 2026Article
- Multilayered regulation by RNA thermometers enables precise control of Cas9 expression in E. coli.Nucleic acids research · 2026Article
- CRISPR-Cas-Mediated Reprogramming Strategies to Overcome Antimicrobial Resistance.Pharmaceutics · 2026Review
- Design and validation of an inducible and curable EvolvR system for directed evolution inFrontiers in bioengineering and biotechnology · 2026Article
- Evaluating the scientific and regulatory case for the deployment of the radioresistant genusFrontiers in microbiology · 2026Review
- CRISPR-Cas9: Transforming Functional Genomics, Precision Medicine, and Drug Development - Opportunities, Challenges, and Future Directions.Current gene therapy · 2026Review
- Automation and machine learning drive rapid optimization of isoprenol production in Pseudomonas putida.Nature communications · 2025Article
- The stress of carrying CRISPR-Cas.Virulence · 2025Article
- Multigenerational proteolytic inactivation of restriction upon subtle genomic hypomethylation in Pseudomonas aeruginosa.Nature microbiology · 2025Article
- A programmable, selection-free CRISPR interference system iniScience · 2025Article
- EASY-edit: a toolbox for high-throughput single-step custom genetic editing in bacteria.Nucleic acids research · 2025Article
Corrections and comments
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Authors and funding
7 authors at 1 institution in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
Genetic tools derived from the Cas9 RNA-guided nuclease are providing essential capabilities to study and engineer bacteria. While the importance of off-target effects was noted early in Cas9's application to mammalian cells, off-target cleavage by Cas9 in bacterial genomes is easily avoided due to their smaller size. Despite this, several studies have reported experimental setups in which Cas9 expression was toxic, even when using the catalytic dead variant of Cas9 (dCas9). Specifically, dCas9 was shown to be toxic when in complex with guide RNAs sharing specific PAM (protospacer adjacent motif)-proximal sequence motifs. Here, we demonstrate that this toxicity is caused by off-target binding of Cas9 to the promoter of essential genes, with silencing of off-target genes occurring with as little as 4 nt of identity in the PAM-proximal sequence. Screens performed in various strains of Escherichia coli and other enterobacteria show that the nature of toxic guide RNAs changes together with the evolution of sequences at off-target positions. These results highlight the potential for Cas9 to bind to hundreds of off-target positions in bacterial genomes, leading to undesired effects. This phenomenon must be considered in the design and interpretation of CRISPR-Cas experiments in bacteria.
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Registered trials
Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.