ArticleMolecular cell2016
Structural Basis for the Altered PAM Specificities of Engineered CRISPR-Cas9.
Article in Molecular cell, 2016. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 79 papers.
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Who cites it
79 citing papers in PubMed.
- Cas9-Embedding Hyperactive TadA8e Confers Efficient and Highly Specific A-To-G Base Editing in Rice.Plant biotechnology journal · 2026Article
- Customizing CRISPR-Cas PAM specificity with protein language models.Nature biotechnology · 2026Article
- Conformational changes induced by K949A mutation in the CRISPR-Cas12a complex drives an effective target-binding mechanism.Current research in structural biology · 2025Article
- DNA targeting by compact Cas9d and its resurrected ancestor.Nature communications · 2025Article
- Engineering of CRISPR-Cas PAM recognition using deep learning of vast evolutionary data.bioRxiv : the preprint server for biology · 2025Article
- Emerging applications of gene editing technologies for the development of climate-resilient crops.Frontiers in genome editing · 2025Review
- Graph theory approaches for molecular dynamics simulations.Quarterly reviews of biophysics · 2024Article
- AlPaCas: allele-specific CRISPR gene editing through a protospacer-adjacent-motif (PAM) approach.Nucleic acids research · 2024Article
- PAM-flexible Engineered FnCas9 variants for robust and ultra-precise genome editing and diagnostics.Nature communications · 2024Article
- Precision in Action: The Role of Clustered Regularly Interspaced Short Palindromic Repeats/Cas in Gene Therapies.Vaccines · 2024Review
- CRISPR technologies for genome, epigenome and transcriptome editing.Nature reviews. Molecular cell biology · 2024Review
- Unraveling the mechanisms of PAMless DNA interrogation by SpRY-Cas9.Nature communications · 2024Article
- High-Throughput Screening of PAM-Flexible Cas9 Variants for Expanded Genome Editing in the Silkworm (Insects · 2024Article
- Recent Updates of the CRISPR/Cas9 Genome Editing System: Novel Approaches to Regulate Its Spatiotemporal Control by Genetic and Physicochemical Strategies.International journal of nanomedicine · 2024Review
- Discovery of Diverse CRISPR-Cas Systems and Expansion of the Genome Engineering Toolbox.Biochemistry · 2023Review
- Substrate-independent activation pathways of the CRISPR-Cas9 HNH nuclease.Biophysical journal · 2023Article
- Shifted PAMs generate DNA overhangs and enhance SpCas9 post-catalytic complex dissociation.Nature structural & molecular biology · 2023Article
- CRISPR applications in cancer diagnosis and treatment.Cellular & molecular biology letters · 2023Review
- Vision rescue via unconstrained in vivo prime editing in degenerating neural retinas.The Journal of experimental medicine · 2023Article
- Strategies and Methods for Improving the Efficiency of CRISPR/Cas9 Gene Editing in Plant Molecular Breeding.Plants (Basel, Switzerland) · 2023Review
19 more citing papers are in PubMed but not listed here.
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Authors and funding
4 authors.
Funding
No grant is acknowledged in the PubMed record.
Abstract
The RNA-guided endonuclease Cas9 cleaves double-stranded DNA targets bearing a PAM (protospacer adjacent motif) and complementarity to the guide RNA. A recent study showed that, whereas wild-type Streptococcus pyogenes Cas9 (SpCas9) recognizes the 5'-NGG-3' PAM, the engineered VQR, EQR, and VRER SpCas9 variants recognize the 5'-NGA-3', 5'-NGAG-3', and 5'-NGCG-3' PAMs, respectively, thus expanding the targetable sequences in Cas9-mediated genome editing applications. Here, we present the high-resolution crystal structures of the three SpCas9 variants in complexes with a single-guide RNA and its altered PAM-containing, partially double-stranded DNA targets. A structural comparison of the three SpCas9 variants with wild-type SpCas9 revealed that the multiple mutations synergistically induce an unexpected displacement in the phosphodiester backbone of the PAM duplex, thereby allowing the SpCas9 variants to directly recognize the altered PAM nucleotides. Our findings explain the altered PAM specificities of the SpCas9 variants and establish a framework for further rational engineering of CRISPR-Cas9.
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Registered trials
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