ArticleNucleic acids research2021
Efficient DNA interrogation of SpCas9 governed by its electrostatic interaction with DNA beyond the PAM and protospacer.
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 13 papers.
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Who cites it
13 citing papers in PubMed.
- Engineered Un1Cas12f1 with boosted gene-editing activity and expanded genomic coverage.Proceedings of the National Academy of Sciences of the United States of America · 2025Article
- PAM-interacting domain turn-helix 51 motifs can improve Cas9-SpRY activity.Nucleic acids research · 2025Article
- Spuriously transcribed RNAs from CRISPR-sgRNA expression plasmids scaffold biomolecular condensate formation and hamper accurate genomic imaging.Nucleic acids research · 2025Article
- Single-molecule perspectives of CRISPR/Cas systems: target search, recognition, and cleavage.BMB reports · 2025Review
- CRISPR-AsCas12f1 couples out-of-protospacer DNA unwinding with exonuclease activity in the sequential target cleavage.Nucleic acids research · 2024Article
- A platform to deliver single and bi-specific Cas9/guide RNA to perturb genes in vitro and in vivo.Molecular therapy : the journal of the American Society of Gene Therapy · 2024Article
- RNA compaction and iterative scanning for small RNA targets by the Hfq chaperone.Nature communications · 2024Article
- Engineering Cas9: next generation of genomic editors.Applied microbiology and biotechnology · 2024Review
- Enlarged DNA unwinding by Nme2Cas9 permits a broadened base editing window beyond the protospacer.Science China. Life sciences · 2024Article
- A generalizable Cas9/sgRNA prediction model using machine transfer learning with small high-quality datasets.Nature communications · 2023Article
- Full-Length Model of SaCas9-sgRNA-DNA Complex in Cleavage State.International journal of molecular sciences · 2023Article
- Discrete RNA-DNA hybrid cleavage by the EXD2 exonuclease pinpoints two rate-limiting steps.The EMBO journal · 2023Article
- Molecular mechanisms ofBiophysics reports · 2021Article
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Authors and funding
12 authors.
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No grant is acknowledged in the PubMed record.
Abstract
Streptococcus pyogenes Cas9 (SpCas9), a programmable RNA-guided DNA endonuclease, has been widely repurposed for biological and medical applications. Critical interactions between SpCas9 and DNA confer the high specificity of the enzyme in genome engineering. Here, we unveil that an essential SpCas9-DNA interaction located beyond the protospacer adjacent motif (PAM) is realized through electrostatic forces between four positively charged lysines among SpCas9 residues 1151-1156 and the negatively charged DNA backbone. Modulating this interaction by substituting lysines with amino acids that have distinct charges revealed a strong dependence of DNA target binding and cleavage activities of SpCas9 on the charge. Moreover, the SpCas9 mutants show markedly distinguishable DNA interaction sites beyond the PAM compared with wild-type SpCas9. Functionally, this interaction governs DNA sampling and participates in protospacer DNA unwinding during DNA interrogation. Overall, a mechanistic and functional understanding of this vital interaction explains how SpCas9 carries out efficient DNA interrogation.
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