Structural Plasticity of PAM Recognition by Engineered Variants of the RNA-Guided Endonuclease Cas9
Open accessAbstract
The RNA-guided endonuclease Cas9 from Streptococcus pyogenes (SpCas9) forms the core of a powerful genome editing technology. DNA cleavage by SpCas9 is dependent on the presence of a 5'-NGG-3' protospacer adjacent motif (PAM) in the target DNA, restricting the choice of targetable sequences. To address this limitation, artificial SpCas9 variants with altered PAM specificities have recently been developed. Here we report crystal structures of the VQR, EQR, and VRER SpCas9 variants bound to target DNAs containing their preferred PAM sequences. The structures reveal that the non-canonical PAMs are recognized by an induced fit mechanism. Besides mediating sequence-specific base recognition, the amino acid substitutions introduced in the SpCas9 variants facilitate conformational remodeling of the PAM region of the bound DNA. Guided by the structural data, we engineered a SpCas9 variant that specifically recognizes NAAG PAMs. Taken together, these studies inform further development of Cas9-based genome editing tools.
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Other variants such as VQR, VRER, VRQR, and QQR1 could identify different PAM sequences, allowing gene editing on a wider range of genetic loci.
“Besides, there are other variants that could identify different PAM sequences, such as VQR, VRER, VRQR, and QQR1, allowing gene editing on a wider range of genetic loci.”
From Machine learning-based prediction models to guide the selection of Cas9 variants for efficient gene editing (2024) · cites this paper as [26]
The Introduction (page not provided) says VQR and VRER recognize different PAM sequences, and the Results describe how they recognize those sequences. The paper discusses EQR as another variant, but the passages retrieved for VRQR and QQR1 do not support including those two in the list. (LLM, conf=0.88)
Experimental — accuracy not yet measuredSome non-NGG PAM Cas9 were generated to be applicable to a wider range of genomic sites.
“To be applicable to a wider range of genomic sites, some non-NGG PAM Cas9 were generated.”
From Machine learning-based prediction models to guide the selection of Cas9 variants for efficient gene editing (2024) · cites this paper as [26]
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References 41 · Cited by 201
- Authors
- Anders, C., Bargsten, K., & Jinek, M.
- Affiliations
- University of Zurich (CH)
- Venue
- Molecular Cell
- Publisher
- Elsevier BV
- Published
- 2016-03
- Volume
- 61
- Issue
- 6
- Pages
- 895-902
- ISSN
- 1097-2765 (ISSN-L)
- PMC
- PMC5065715
- OpenAlex
- W2302008724
- Cited by
- 201
- References
- 41
- Citations by year
- 2026: 6 · 2025: 13 · 2024: 18 · 2023: 16 · 2022: 28 · 2021: 22 · 2020: 20 · 2019: 27 · 2018: 20 · 2017: 17 · 2016: 12 · 2014: 1 · 2013: 1
- FWCI
- 13.34
- Type
- journal article
- Language
- en
- Open access
- bronze
- doi.org
- Licence
- Locations
- Molecular Cell (publishedVersion)
- PubMed (publishedVersion)
- Europe PMC (PubMed Central) (acceptedVersion)
- PubMed Central (acceptedVersion)
- DORA PSI (Paul Scherrer Institute) (submittedVersion)
- Funders
- Vallee Foundation
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA regulation and disease
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › DNA and Nucleic Acid Chemistry
- Keywords
- Cas9, protospacer adjacent motif, PAM recognition, induced fit, genome editing, crystal structure, Streptococcus pyogenes
- MeSH terms
- CRISPR-Associated Protein 9, RNA, Guide, CRISPR-Cas Systems, Bacterial Proteins, DNA, Endonucleases, Genetic Engineering, Mutation, Nucleic Acid Conformation, Substrate Specificity, Crystallography, X-Ray, Amino Acid Substitution, DNA, Intergenic, Nucleotide Motifs, CRISPR-Cas Systems
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