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Crystal Structure of Cas9 in Complex with Guide RNA and Target DNA
Open accessAbstract
The CRISPR-associated endonuclease Cas9 can be targeted to specific genomic loci by single guide RNAs (sgRNAs). Here, we report the crystal structure of Streptococcus pyogenes Cas9 in complex with sgRNA and its target DNA at 2.5 Å resolution. The structure revealed a bilobed architecture composed of target recognition and nuclease lobes, accommodating the sgRNA:DNA heteroduplex in a positively charged groove at their interface. Whereas the recognition lobe is essential for binding sgRNA and DNA, the nuclease lobe contains the HNH and RuvC nuclease domains, which are properly positioned for cleavage of the complementary and noncomplementary strands of the target DNA, respectively. The nuclease lobe also contains a carboxyl-terminal domain responsible for the interaction with the protospacer adjacent motif (PAM). This high-resolution structure and accompanying functional analyses have revealed the molecular mechanism of RNA-guided DNA targeting by Cas9, thus paving the way for the rational design of new, versatile genome-editing technologies.
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“Upon binding of Streptococcus pyogenes Cas9 (SpCas9) to the target site, a stable strand separation is maintained through two kinds of interactions: the binding of gRNA to the target strand, and a positively-charged groove resulting from the unspecific interaction of both HNH and RuvC domains with the negatively-charged non-target strand [36].”
From CRISPR-Cas: Converting A Bacterial Defence Mechanism into A State-of-the-Art Genetic Manipulation Tool (2019) · cites this paper as [36]
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“tracrRNA is complementary to the repeat sequence and contains 3 stem-loop hairpin structures [35,36].”
From CRISPR-Cas: Converting A Bacterial Defence Mechanism into A State-of-the-Art Genetic Manipulation Tool (2019) · cites this paper as [36]
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“The structure that came out of the laboratory of Jennifer Doudna was of an apo-protein (PDBe ID 4cmp, PDBe DOI: 10.2210/pdb4cmp/pdb), while the research group of Osamu Nureki (University of Tokyo, Japan) succeeded in crystallising the protein in a complex with a “guide”-RNA and “target”-DNA (PDBe ID 4oo8, PDBe DOI: 10.2210/pdb4oo8/pdb).”
From CRISPR–Cas9: A History of Its Discovery and Ethical Considerations of Its Use in Genome Editing (2022) · cites this paper as [40]
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In the citing paper
“The key structures of the nuclease lobe of SpCas9 are 2 domains: HNH and RuvC, each of them cleaves one of the target DNA strands.”
From CRISPR–Cas9: A History of Its Discovery and Ethical Considerations of Its Use in Genome Editing (2022) · cites this paper as [40]
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“For example, SpCas9-NG shows reduced editing efficiency on 5′-NGC-3′ PAM targets and tends to increase off-target activity (Nishimasu et al., 2014).”
From Emerging applications of gene editing technologies for the development of climate-resilient crops (2025) · cites this paper as Nishimasu et al., 2014
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In the citing paper
“Crystal structures revealed that the Cas9 protein is spatially divided into 2 lobes: a target recognition lobe and a nuclease lobe, with the guide RNA and target DNA occupying the positively charged groove at their interface.”
From CRISPR–Cas9: A History of Its Discovery and Ethical Considerations of Its Use in Genome Editing (2022) · cites this paper as [40]
In the citing paper
“Attempts to obtain detailed information about the structure of this protein resulted in the first two crystallographic studies being published almost simultaneously: in February 2014 two crystal structures of Cas9 appeared in the database PDBe (“Protein Data Bank in Europe”), and the accompanying articles were published in the journals Nature and Cell [39, 40].”
From CRISPR–Cas9: A History of Its Discovery and Ethical Considerations of Its Use in Genome Editing (2022) · cites this paper as [40]
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“These, as well as many subsequent studies, used the Cas9 protein from S. pyogenes, SpCas9, which consists of 1368 amino acids and is a multidomain and multifunctional endonuclease.”
From CRISPR–Cas9: A History of Its Discovery and Ethical Considerations of Its Use in Genome Editing (2022) · cites this paper as [40]
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Metadata
- Authors
- Nishimasu, H., Ran, F. A., Hsu, P. D., Konermann, S., Shehata, S. I., Dohmae, N., Ishitani, R., Zhang, F., & Nureki, O.
- Affiliations
- Japan Science and Technology Agency (JP)
- Broad Institute (US)
- RIKEN (JP)
- The University of Tokyo (JP)
- Corresponding author
- Zhang, Feng
- Nureki, Osamu
- ORCID
- Venue
- Cell
- Publisher
- Elsevier BV
- Published
- 2014-02
- Volume
- 156
- Issue
- 5
- Pages
- 935-949
- ISSN
- 0092-8674 (ISSN-L)
- PMC
- PMC4139937
- OpenAlex
- W2107959600
- Cited by
- 2,306
- References
- 77
- Citations by year
- 2026: 130 · 2025: 193 · 2024: 221 · 2023: 177 · 2022: 229 · 2021: 218 · 2020: 215 · 2019: 212 · 2018: 191 · 2017: 193 · 2016: 149 · 2015: 93 · 2014: 80
- FWCI
- 70.34
- Type
- journal article
- Language
- en
- Open access
- bronze
- doi.org
- Licence
- Locations
- Cell (publishedVersion)
- PubMed (publishedVersion)
- Europe PMC (PubMed Central) (acceptedVersion)
- PubMed Central (acceptedVersion)
- DSpace@MIT (Massachusetts Institute of Technology) (submittedVersion, cc-by-nc-nd)
- eScholarship (California Digital Library) (submittedVersion)
- PMC
- Funders
- PRESTO from the Japan Science and Technology (JST) Agency
- Drug Discovery and Informatics
- Japan Society for the Promotion of Science (JSPS)
- Core Research for Evolutional Science and Technology (CREST)
- Japan Science and Technology Agency (JST)
- NIH (5DP1-MH100706)
- Keck
- McKnight
- Poitras
- Merkin
- Vallee
- Damon Runyon
- Searle Scholars
- Klingenstein
- Simons Foundations
- Bob Metcalfe
- Jane Pauley
- 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 › Structural Biology › Enzyme Structure and Function
- Keywords
- CRISPR/Cas12a, guide RNA, target DNA, RNA-guided DNA cleavage, protospacer adjacent motif, genome editing, crystal structure
- MeSH terms
- Amino Acid Sequence, Bacteria, DNA, Bacterial, Endonucleases, Models, Molecular, Molecular Sequence Data, RNA, Bacterial, Streptococcus pyogenes, Sequence Alignment, Protein Structure, Tertiary, Crystallography, X-Ray, RNA, Small Untranslated, CRISPR-Associated Proteins
About these checks
Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.
Statistics and arithmetic are recomputed, not judged.
Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.
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