ArticleNucleic acids research2021
Systematic in vitro specificity profiling reveals nicking defects in natural and engineered CRISPR-Cas9 variants.
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 17 papers.
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Who cites it
17 citing papers in PubMed, 22 citations in OpenAlex.
- Harnessing Deep Learning Models for Guide RNA Optimization and Off-Target Prediction in CRISPR Systems.Biotechnology journal · 2026Review
- Gene Editing Strategies for Duchenne Muscular Dystrophy: From Molecular Mechanisms to Clinical Translation.Cells · 2026Review
- Article
- Cooperative Mismatch Discrimination by PNA and DNAzyme Enables High-Fidelity Cleavage of Plasmid DNA.Biochemistry · 2025Article
- Rapid CRISPR-Cas9 target-strand nicking can provide phage resistance by reducing DNA abundance.Nucleic acids research · 2025Article
- Off-target interactions in the CRISPR-Cas9 Machinery: mechanisms and outcomes.Biochemistry and biophysics reports · 2025Review
- Development and validation of optimized lentivirus-like particles for gene editing tool delivery with Gag-Only strategy.European journal of medical research · 2025Article
- Analysis of metal-dependent DNA nicking activities by Cas endonucleases.Methods in enzymology · 2025Article
- Gene Editing for Duchenne Muscular Dystrophy: From Experimental Models to Emerging Therapies.Degenerative neurological and neuromuscular disease · 2025Review
- CRISPR-Cas12a exhibits metal-dependent specificity switching.Nucleic acids research · 2024Article
- CRISPR-Cas12a exhibits metal-dependent specificity switching.bioRxiv : the preprint server for biology · 2024Article
- CRISPR-Cas effector specificity and cleavage site determine phage escape outcomes.PLoS biology · 2023Article
- The PROTECTOR strategy employs dCas orthologs to sterically shield off-target sites from CRISPR/Cas activity.Scientific reports · 2023Article
- Systematically attenuating DNA targeting enables CRISPR-driven editing in bacteria.Nature communications · 2023Article
- Review
- A detailed protocol for expression, purification, and activity determination of recombinant SaCas9.STAR protocols · 2022Article
- CRISPR-Cas9 library screening approach for anti-cancer drug discovery: overview and perspectives.Theranostics · 2022Review
Corrections and comments
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Authors and funding
5 authors at 1 institution in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
Cas9 is an RNA-guided endonuclease in the bacterial CRISPR-Cas immune system and a popular tool for genome editing. The commonly used Streptococcus pyogenes Cas9 (SpCas9) is relatively non-specific and prone to off-target genome editing. Other Cas9 orthologs and engineered variants of SpCas9 have been reported to be more specific. However, previous studies have focused on specificity of double-strand break (DSB) or indel formation, potentially overlooking alternative cleavage activities of these Cas9 variants. In this study, we employed in vitro cleavage assays of target libraries coupled with high-throughput sequencing to systematically compare cleavage activities and specificities of two natural Cas9 variants (SpCas9 and Staphylococcus aureus Cas9) and three engineered SpCas9 variants (SpCas9 HF1, HypaCas9 and HiFi Cas9). We observed that all Cas9s tested could cleave target sequences with up to five mismatches. However, the rate of cleavage of both on-target and off-target sequences varied based on target sequence and Cas9 variant. In addition, SaCas9 and engineered SpCas9 variants nick targets with multiple mismatches but have a defect in generating a DSB, while SpCas9 creates DSBs at these targets. Overall, these differences in cleavage rates and DSB formation may contribute to varied specificities observed in genome editing studies.
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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 Socratic graph.