ArticleProceedings of the National Academy of Sciences of the United States of America2017
High-throughput biochemical profiling reveals sequence determinants of dCas9 off-target binding and unbinding.
Article in Proceedings of the National Academy of Sciences of the United States of America, 2017. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 117 papers.
What it found
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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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Trials whose registry record cites this paper, or whose number appears in the abstract. A trial that started after this paper was published is citing it as background, not reporting it.
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Who cites it
117 citing papers in PubMed.
- Active-site arginines differentially control Cas12a DNA cleavage and specificity.The Journal of biological chemistry · 2026Article
- Article
- Biophysical characterization ofSensors & diagnostics · 2026Article
- Automated high-throughput selection of DNA aptamers using a common optical next-generation sequencer.Nucleic acids research · 2026Article
- Mismatch tolerance of a gRNA for CRISPR-based gene activation confers broad activity critical for cell reprogramming.bioRxiv : the preprint server for biology · 2026Article
- Multiplexed single-molecule characterization at the library scale.Nature protocols · 2026Review
- Comprehensive profiling of CRISPR/dCas9 epigenome editors indicates a complex link between on and off target effects.Genome biology · 2026Article
- Article
- Bio-orthogonal chemistry-based strategy to Turn-OFF CRISPR-Cas9 activity in solution and live cells.NAR molecular medicine · 2026Article
- A guide RNA repeat checkpoint steers CRISPR-Cas9 catalysis.bioRxiv : the preprint server for biology · 2025Article
- Sensitive, direct detection of non-coding off-target base editor unwinding and editing in primary cells.bioRxiv : the preprint server for biology · 2025Article
- Off-target interactions in the CRISPR-Cas9 Machinery: mechanisms and outcomes.Biochemistry and biophysics reports · 2025Review
- CRISPR/Cas9 for achieving postintervention HIV control.Current opinion in HIV and AIDS · 2025Review
- CRISPR GENome and epigenome engineering improves loss-of-function genetic-screening approaches.Cell reports methods · 2025Article
- Modular and signal-responsive transcriptional regulation using CRISPRi-aided genetic switches in Escherichia coli.Journal of biological engineering · 2025Article
- Dynamic basis of supercoiling-dependent DNA interrogation by Cas12a via R-loop intermediates.Nature communications · 2025Article
- Modulation of CRISPR-Cas9 Cleavage with an Oligo-Ribonucleoprotein Design.Chembiochem : a European journal of chemical biology · 2025Article
- Multi-locus CRISPRi targeting with a single truncated guide RNA.Nature communications · 2025Article
- GLiDe: a web-based genome-scale CRISPRi sgRNA design tool for prokaryotes.BMC bioinformatics · 2025Article
- How to use CRISPR/Cas9 in plants: from target site selection to DNA repair.Journal of experimental botany · 2024Review
57 more citing papers are in PubMed but not listed here.
Corrections and comments
PubMed lists nothing against this paper. Absence here is not a guarantee, only a check that was made.
Authors and funding
8 authors.
Funding
Abstract
The bacterial adaptive immune system CRISPR-Cas9 has been appropriated as a versatile tool for editing genomes, controlling gene expression, and visualizing genetic loci. To analyze Cas9's ability to bind DNA rapidly and specifically, we generated multiple libraries of potential binding partners for measuring the kinetics of nuclease-dead Cas9 (dCas9) interactions. Using a massively parallel method to quantify protein-DNA interactions on a high-throughput sequencing flow cell, we comprehensively assess the effects of combinatorial mismatches between guide RNA (gRNA) and target nucleotides, both in the seed and in more distal nucleotides, plus disruption of the protospacer adjacent motif (PAM). We report two consequences of PAM-distal mismatches: reversal of dCas9 binding at long time scales, and synergistic changes in association kinetics when other gRNA-target mismatches are present. Together, these observations support a model for Cas9 specificity wherein gRNA-DNA mismatches at PAM-distal bases modulate different biophysical parameters that determine association and dissociation rates. The methods we present decouple aspects of kinetic and thermodynamic properties of the Cas9-DNA interaction and broaden the toolkit for investigating off-target binding behavior.
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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.