Letter | Published:

Structural basis of PAM-dependent target DNA recognition by the Cas9 endonuclease

Nature volume 513, pages 569573 (25 September 2014) | Download Citation


The CRISPR-associated protein Cas9 is an RNA-guided endonuclease that cleaves double-stranded DNA bearing sequences complementary to a 20-nucleotide segment in the guide RNA1,2. Cas9 has emerged as a versatile molecular tool for genome editing and gene expression control3. RNA-guided DNA recognition and cleavage strictly require the presence of a protospacer adjacent motif (PAM) in the target DNA1,4,5,6. Here we report a crystal structure of Streptococcus pyogenes Cas9 in complex with a single-molecule guide RNA and a target DNA containing a canonical 5′-NGG-3′ PAM. The structure reveals that the PAM motif resides in a base-paired DNA duplex. The non-complementary strand GG dinucleotide is read out via major-groove interactions with conserved arginine residues from the carboxy-terminal domain of Cas9. Interactions with the minor groove of the PAM duplex and the phosphodiester group at the +1 position in the target DNA strand contribute to local strand separation immediately upstream of the PAM. These observations suggest a mechanism for PAM-dependent target DNA melting and RNA–DNA hybrid formation. Furthermore, this study establishes a framework for the rational engineering of Cas9 enzymes with novel PAM specificities.

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Data deposits

Atomic coordinates and structure factors have been deposited in the Protein Data Bank under accession numbers 4un3, 4un4 and 4un5.


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We are grateful to J. Doudna for agreement on research directions, helpful discussions and encouragement throughout the project. We thank B. Blattmann and C. Stutz-Ducommun for crystallization screening, N. Ban and M. Leibundgut for the gift of iridium hexamine, and R. Dutzler for sharing synchrotron beam time and crystallographic advice. We thank E. Charpentier, I. Fonfara, S. Sternberg, P. Sledz, A. May and S. Kassube for critical reading of the manuscript. Part of this work was performed at the Swiss Light Source at the Paul Scherrer Institute, Villigen, Switzerland. We thank T. Tomizaki, V. Olieric and M. Wang for assistance with X-ray data collection. This work was supported by the European Research Council Starting Grant no. 337284 ANTIVIRNA and by start-up funds from the University of Zurich.

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  1. Department of Biochemistry, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland

    • Carolin Anders
    • , Ole Niewoehner
    • , Alessia Duerst
    •  & Martin Jinek


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C.A. designed experiments, performed site-directed mutagenesis, prepared guide RNAs, purified and crystallized the Cas9–sgRNA–target-DNA complex, determined its structure together with M.J., and performed plasmid cleavage assays. O.N. purified Cas9 mutants, performed EMSA assays and assisted with cleavage assays. A.D. performed site-directed mutagenesis, prepared guide RNAs and assisted with cleavage assays. M.J. designed experiments and supervised the study. C.A. and M.J. wrote the manuscript.

Competing interests

M.J. is a co-founder of Caribou Biosciences, Inc. The authors have filed a related patent application.

Corresponding author

Correspondence to Martin Jinek.

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