ArticlePLoS biology2019
Molecular basis for the PAM expansion and fidelity enhancement of an evolved Cas9 nuclease.
Article in PLoS biology, 2019. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 16 papers.
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Who cites it
16 citing papers in PubMed.
- Targeting Horizontal Gene Transfer to Combat Antimicrobial Resistance: A Review of Mechanisms, Drivers, and Multi-Omics Strategies.Infection and drug resistance · 2026Review
- Catalytic-state structure of Candidatus Hydrogenedentes Cas12b revealed by cryo-EM studies.Nucleic acids research · 2025Article
- In Vitro Correction of Point Mutations in theInternational journal of molecular sciences · 2025Article
- Article
- Flexibility in PAM Recognition Expands DNA Targeting in xCas9.bioRxiv : the preprint server for biology · 2025Article
- AlPaCas: allele-specific CRISPR gene editing through a protospacer-adjacent-motif (PAM) approach.Nucleic acids research · 2024Article
- Engineering Cas9: next generation of genomic editors.Applied microbiology and biotechnology · 2024Review
- CRISPR/Cas9 technology: applications in oocytes and early embryos.Journal of translational medicine · 2023Review
- Review
- Current genetic strategies to investigate gene functions in Trichoderma reesei.Microbial cell factories · 2023Review
- Exploring and engineering PAM-diverse Streptococci Cas9 for PAM-directed bifunctional and titratable gene control in bacteria.Metabolic engineering · 2023Article
- Structural biology of CRISPR-Cas immunity and genome editing enzymes.Nature reviews. Microbiology · 2022Review
- CRISPRactivation-SMS, a message for PAM sequence independent gene up-regulation in Escherichia coli.Nucleic acids research · 2022Article
- Engineering a PAM-flexible SpdCas9 variant as a universal gene repressor.Nature communications · 2021Article
- CRISPR/Cas9 for the treatment of haematological diseases: a journey from bacteria to the bedside.British journal of haematology · 2021Review
- Programmable adenine deamination in bacteria using a Cas9-adenine-deaminase fusion.Chemical science · 2020Article
Corrections and comments
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Authors and funding
6 authors.
Funding
No grant is acknowledged in the PubMed record.
Abstract
Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems have been harnessed as powerful genome editing tools in diverse organisms. However, the off-target effects and the protospacer adjacent motif (PAM) compatibility restrict the therapeutic applications of these systems. Recently, a Streptococcus pyogenes Cas9 (SpCas9) variant, xCas9, was evolved to possess both broad PAM compatibility and high DNA fidelity. Through determination of multiple xCas9 structures, which are all in complex with single-guide RNA (sgRNA) and double-stranded DNA containing different PAM sequences (TGG, CGG, TGA, and TGC), we decipher the molecular mechanisms of the PAM expansion and fidelity enhancement of xCas9. xCas9 follows a unique two-mode PAM recognition mechanism. For non-NGG PAM recognition, xCas9 triggers a notable structural rearrangement in the DNA recognition domains and a rotation in the key PAM-interacting residue R1335; such mechanism has not been observed in the wild-type (WT) SpCas9. For NGG PAM recognition, xCas9 applies a strategy similar to WT SpCas9. Moreover, biochemical and cell-based genome editing experiments pinpointed the critical roles of the E1219V mutation for PAM expansion and the R324L, S409I, and M694I mutations for fidelity enhancement. The molecular-level characterizations of the xCas9 nuclease provide critical insights into the mechanisms of the PAM expansion and fidelity enhancement of xCas9 and could further facilitate the engineering of SpCas9 and other Cas9 orthologs.
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