Structural basis for the altered PAM specificities of engineered CRISPR-cas9
Open accessAbstract
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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This could result in the development of wider-ranged VQR, EQR, and VRER SpCas9 variants, enabling the targeting of NGAN, NGNG, and NGCG PAMs, respectively.
“This could result in the development of wider-ranged VQR, EQR, and VRER SpCas9 variants, enabling the targeting of NGAN, NGNG, and NGCG PAMs, respectively (Kleinstiver et al., 2015; Hirano et al., 2016).”
From Emerging applications of gene editing technologies for the development of climate-resilient crops (2025) · cites this paper as Hirano et al., 2016
These modifications have led to the creation of SpCas9 variants VQR, EQR, and VRER, which can recognize broader PAM sequences like NGAN, NGNG, and NGCG, respectively.
“These modifications have led to the creation of SpCas9 variants such as VQR, EQR, and VRER, which can recognize broader PAM sequences like NGAN, NGNG, and NGCG, respectively (Kleinstiver et al., 2015; Hirano et al., 2016).”
From Emerging applications of gene editing technologies for the development of climate-resilient crops (2025) · cites this paper as Hirano et al., 2016
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- Authors
- Hirano, S., Nishimasu, H., Ishitani, R., & Nureki, O.
- Affiliations
- The University of Tokyo (JP)
- Japan Science and Technology Agency (JP)
- Corresponding author
- Nishimasu, Hiroshi
- Nureki, Osamu
- Venue
- Molecular Cell
- Publisher
- Elsevier BV
- Published
- 2016-03
- Volume
- 61
- Issue
- 6
- Pages
- 886-894
- ISSN
- 1097-2765 (ISSN-L)
- OpenAlex
- W2302341612
- Cited by
- 152
- References
- 32
- Citations by year
- 2026: 3 · 2025: 9 · 2024: 14 · 2023: 12 · 2022: 18 · 2021: 14 · 2020: 17 · 2019: 15 · 2018: 23 · 2017: 17 · 2016: 8 · 2014: 1 · 2013: 1
- FWCI
- 11.10
- Type
- journal article
- Language
- en
- Open access
- bronze
- doi.org
- Licence
- www.elsevier.com/tdm/userlicense/1.0/ (tdm)
- www.elsevier.com/legal/tdmrep-license (tdm)
- www.elsevier.com/open-access/userlicense/1.0/ (vor, 381 d)
- doi.org/10.15223/policy-017 (stm-asf)
- doi.org/10.15223/policy-037 (stm-asf)
- doi.org/10.15223/policy-012 (stm-asf)
- doi.org/10.15223/policy-029 (stm-asf)
- doi.org/10.15223/policy-004 (stm-asf)
- Locations
- Molecular Cell (publishedVersion)
- PubMed (publishedVersion)
- DORA PSI (Paul Scherrer Institute) (submittedVersion)
- Funders
- JST, PRESTO
- JSPS KAKENHI (26291010, 15H01463)
- Ministry of Education, Culture, Sports, Science and Technology
- Japan Agency for Medical Research and Development, AMED
- Council for Science, Technology and Innovation (CSTI)
- Bio-oriented Technology Research Advancement Institution, NARO
- 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
- CRISPR/Cas9, protospacer adjacent motif, SpCas9, single guide RNA, DNA duplex structure, crystal structure, genome editing
- MeSH terms
- CRISPR-Associated Protein 9, RNA, Guide, CRISPR-Cas Systems, Bacterial Proteins, DNA, Endonucleases, Genetic Engineering, Mutation, Substrate Specificity, RNA, Guide, Kinetoplastida, Crystallography, X-Ray, DNA, Intergenic, CRISPR-Cas Systems
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