ArticleNucleic acids research2021
Guide-target mismatch effects on dCas9-sgRNA binding activity in living bacterial cells.
Article in Nucleic acids research, 2021. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 22 papers.
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Who cites it
22 citing papers in PubMed.
- Mesoscopic Calculation of Single Mismatches in RNA/DNA Hybrids: Strong Hydrogen Bonds of dTrG Affect CRISPR Off-Target Binding.The journal of physical chemistry. B · 2026Article
- Biophysical characterization ofSensors & diagnostics · 2026Article
- ARROW: Allele-Specific Recombined gRNA Design for Reduced Off-Target with Enhanced Specificity.Bioengineering (Basel, Switzerland) · 2025Article
- Off-target interactions in the CRISPR-Cas9 Machinery: mechanisms and outcomes.Biochemistry and biophysics reports · 2025Review
- Population-level amplification of gene regulation by programmable gene transfer.Nature chemical biology · 2025Article
- Generation and validation of a versatile inducible multiplex CRISPRi system to examine bacterial regulation in the Euprymna-Vibrio fischeri symbiosis.Archives of microbiology · 2025Article
- Construction of multi-targeted CRISPR libraries in tomato to overcome functional redundancy at genome-scale level.Nature communications · 2025Article
- GLiDe: a web-based genome-scale CRISPRi sgRNA design tool for prokaryotes.BMC bioinformatics · 2025Article
- CRISPR/Cas9 gene editing: a novel strategy for fighting drug resistance in respiratory disorders.Cell communication and signaling : CCS · 2024Review
- Advances and applications of CRISPR/Cas-mediated interference inEngineering microbiology · 2024Review
- Genome-scale CRISPRi screening: A powerful tool in engineering microbiology.Engineering microbiology · 2023Review
- Vector enabled CRISPR gene editing - A revolutionary strategy for targeting the diversity of brain pathologies.Coordination chemistry reviews · 2023Article
- CRISPR-Cas effector specificity and cleavage site determine phage escape outcomes.PLoS biology · 2023Article
- Computational design of CRISPR guide RNAs to enable strain-specific control of microbial consortia.Proceedings of the National Academy of Sciences of the United States of America · 2023Article
- Exploring and engineering PAM-diverse Streptococci Cas9 for PAM-directed bifunctional and titratable gene control in bacteria.Metabolic engineering · 2023Article
- Deep learning in CRISPR-Cas systems: a review of recent studies.Frontiers in bioengineering and biotechnology · 2023Review
- Biosensor-assisted titratable CRISPRi high-throughput (BATCH) screening for over-production phenotypes.Metabolic engineering · 2023Article
- Efficient Single-Nucleotide Microbial Genome Editing Achieved Using CRISPR/Cpf1 with Maximally 3'-End-Truncated crRNAs.ACS synthetic biology · 2022Article
- From DNA-protein interactions to the genetic circuit design using CRISPR-dCas systems.Frontiers in molecular biosciences · 2022Review
- HUGE pipeline to measure temporal genetic variation inFrontiers in insect science · 2022Article
Corrections and comments
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Authors and funding
5 authors.
Funding
No grant is acknowledged in the PubMed record.
Abstract
As an effective programmable DNA targeting tool, CRISPR-Cas9 system has been adopted in varieties of biotechnological applications. However, the off-target effects, derived from the tolerance towards guide-target mismatches, are regarded as the major problems in engineering CRISPR systems. To understand this, we constructed two sgRNA libraries carrying saturated single- and double-nucleotide mismatches in living bacteria cells, and profiled the comprehensive landscape of in vivo binding affinity of dCas9 toward DNA target guided by each individual sgRNA with particular mismatches. We observed a synergistic effect in seed, where combinatorial double mutations caused more severe activity loss compared with the two corresponding single mutations. Moreover, we found that a particular mismatch type, dDrG (D = A, T, G), only showed moderate impairment on binding. To quantitatively understand the causal relationship between mismatch and binding behaviour of dCas9, we further established a biophysical model, and found that the thermodynamic properties of base-pairing coupled with strand invasion process, to a large extent, can account for the observed mismatch-activity landscape. Finally, we repurposed this model, together with a convolutional neural network constructed based on the same mechanism, as a predictive tool to guide the rational design of sgRNA in bacterial CRISPR interference.
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