ArticleNature chemical biology2020
Bridge helix arginines play a critical role in Cas9 sensitivity to mismatches.
Article in Nature chemical biology, 2020. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 40 papers.
What it found
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Who cites it
40 citing papers in PubMed, 75 citations in OpenAlex.
- Helicity of the bridge helix of Cas12a regulates on-target DNA cleavage efficiency and off-target cleavage propensity.Biochemical and biophysical research communications · 2026Article
- Bridge helix of Cas12a is an allosteric regulator of R-loop formation and RuvC activation.Nature communications · 2026Article
- Screening strategy to identify Cas9 variants with higher HDR activity based on diphtheria toxin.Journal of biomedical science · 2025Article
- RNA-DNA hybrid binding domain broadens the editing window of base editors.Molecular therapy : the journal of the American Society of Gene Therapy · 2025Article
- Genome Editing in Gynecological Oncology: The Emerging Role of CRISPR/Cas9 in Precision Cancer Therapy.Therapeutic innovation & regulatory science · 2025Review
- The virulence regulator CovR boosts CRISPR-Cas9 immunity in Group B Streptococcus.Nature communications · 2025Article
- Structural integrity and side-chain interaction at the loop region of the bridge helix modulate Cas9 substrate discrimination.Nucleic acids research · 2025Article
- A rational engineering strategy for structural dynamics modulation enables target specificity enhancement of the Cas9 nuclease.Nucleic acids research · 2025Article
- A universal and wide-range cytosine base editor via domain-inlaid and fidelity-optimized CRISPR-FrCas9.Nature communications · 2025Article
- Quantifying Protein-Nucleic Acid Interactions for Engineering Useful CRISPR-Cas9 Genome-Editing Variants.Methods in molecular biology (Clifton, N.J.) · 2025Article
- Overcoming CRISPR-Cas9 off-target prediction hurdles: A novel approach with ESB rebalancing strategy and CRISPR-MCA model.PLoS computational biology · 2024Article
- Role of CRISPR-Cas systems and anti-CRISPR proteins in bacterial antibiotic resistance.Heliyon · 2024Review
- Mutational rescue of the activity of high-fidelity Cas9 enzymes.Cell reports methods · 2024Article
- SpCas9-HF1 enhances accuracy of cell cycle-dependent genome editing by increasing HDR efficiency, and by reducing off-target effects and indel rates.Molecular therapy. Nucleic acids · 2024Article
- Engineering Cas9: next generation of genomic editors.Applied microbiology and biotechnology · 2024Review
- CRISPR-Cas Systems in the Fight Against Antimicrobial Resistance: Current Status, Potentials, and Future Directions.Infection and drug resistance · 2024Review
- Genome-wide CRISPR off-target prediction and optimization using RNA-DNA interaction fingerprints.Nature communications · 2023Article
- A cleavage rule for selection of increased-fidelity SpCas9 variants with high efficiency and no detectable off-targets.Nature communications · 2023Article
- Sniper2L is a high-fidelity Cas9 variant with high activity.Nature chemical biology · 2023Article
- A roadmap to establish a comprehensive platform for sustainable manufacturing of natural products in yeast.Nature communications · 2023Review
Corrections and comments
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Authors and funding
9 authors at 6 institutions in 3 countries.
Funding
No grant is acknowledged in the PubMed record.
Abstract
The RNA-programmable DNA-endonuclease Cas9 is widely used for genome engineering, where a high degree of specificity is required. To investigate which features of Cas9 determine the sensitivity to mismatches along the target DNA, we performed in vitro biochemical assays and bacterial survival assays in Escherichia coli. We demonstrate that arginines in the Cas9 bridge helix influence guide RNA, and target DNA binding and cleavage. They cluster in two groups that either increase or decrease the Cas9 sensitivity to mismatches. We show that the bridge helix is essential for R-loop formation and that R63 and R66 reduce Cas9 specificity by stabilizing the R-loop in the presence of mismatches. Additionally, we identify Q768 that reduces sensitivity of Cas9 to protospacer adjacent motif-distal mismatches. The Cas9_R63A/Q768A variant showed increased specificity in human cells. Our results provide a firm basis for function- and structure-guided mutagenesis to increase Cas9 specificity for genome engineering.
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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.