Abstract
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.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 51 print issues and online access
$199.00 per year
only $3.90 per issue
Buy this article
Purchase on Springer Link
Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816–821 (2012)
Gasiunas, G., Barrangou, R., Horvath, P. & Siksnys, V. Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proc. Natl Acad. Sci. USA 109, 2579–2586 (2012)
Mali, P., Esvelt, K. M. & Church, G. M. Cas9 as a versatile tool for engineering biology. Nature Methods 10, 957–963 (2013)
Garneau, J. E. et al. The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA. Nature 468, 67–71 (2010)
Sapranauskas, R. et al. The Streptococcus thermophilus CRISPR/Cas system provides immunity in Escherichia coli . Nucleic Acids Res. 39, 9275–9282 (2011)
Sternberg, S. H., Redding, S., Jinek, M., Greene, E. C. & Doudna, J. A. DNA interrogation by the CRISPR RNA-guided endonuclease Cas9. Nature 507, 62–67 (2014)
Deltcheva, E. et al. CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III. Nature 471, 602–607 (2011)
Mali, P. et al. RNA-guided human genome engineering via Cas9. Science 339, 823–826 (2013)
Cong, L. et al. Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819–823 (2013)
Jinek, M. et al. RNA-programmed genome editing in human cells. elife 2, e00471 (2013)
Hwang, W. Y. et al. Efficient genome editing in zebrafish using a CRISPR-Cas system. Nature Biotechnol. 31, 227–229 (2013)
Wang, H. et al. One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell 153, 910–918 (2013)
Bassett, A. R., Tibbit, C., Ponting, C. P. & Liu, J.-L. Highly efficient targeted mutagenesis of Drosophila with the CRISPR/Cas9 system. Cell Rep. 4, 220–228 (2013)
Gratz, S. J. et al. Genome engineering of Drosophila with the CRISPR RNA-guided Cas9 nuclease. Genetics 194, 1029–1035 (2013)
Friedland, A. E. et al. Heritable genome editing in C. elegans via a CRISPR-Cas9 system. Nature Methods 10, 741–743 (2013)
Qi, L. S. et al. Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell 152, 1173–1183 (2013)
Bikard, D. et al. Programmable repression and activation of bacterial gene expression using an engineered CRISPR-Cas system. Nucleic Acids Res. 41, 7429–7437 (2013)
Gilbert, L. A. et al. CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell 154, 442–451 (2013)
Mali, P. et al. CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nature Biotechnol. 31,. 833–838 (2013)
Jinek, M. et al. Structures of Cas9 endonucleases reveal RNA-mediated conformational activation. Science 343, 1215 (2014)
Nishimasu, H. et al. Crystal structure of Cas9 in complex with guide RNA and target DNA. Cell 156, 935–949 (2014)
Fonfara, I. et al. Phylogeny of Cas9 determines functional exchangeability of dual-RNA and Cas9 among orthologous type II CRISPR-Cas systems. Nucleic Acids Res. 42, 2577–2590 (2013)
Esvelt, K. M. et al. Orthogonal Cas9 proteins for RNA-guided gene regulation and editing. Nature Methods 10, 1116–1121 (2013)
Luscombe, N. M., Laskowski, R. A. & Thornton, J. M. Amino acid-base interactions: a three-dimensional analysis of protein-DNA interactions at an atomic level. Nucleic Acids Res. 29, 2860–2874 (2001)
Briner, A. E. & Barrangou, R. Lactobacillus buchneri genotyping on the basis of clustered regularly interspaced short palindromic repeat (CRISPR) locus diversity. Appl. Environ. Microbiol. 80, 994–1001 (2014)
Redondo, P. et al. Molecular basis of xeroderma pigmentosum group C DNA recognition by engineered meganucleases. Nature 456, 107–111 (2008)
Ashworth, J. et al. Computational reprogramming of homing endonuclease specificity at multiple adjacent base pairs. Nucleic Acids Res. 38, 5601–5608 (2010)
Jiang, W., Bikard, D., Cox, D., Zhang, F. & Marraffini, L. A. RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nature Biotechnol. 31, 233–239 (2013)
Hsu, P. D. et al. DNA targeting specificity of RNA-guided Cas9 nucleases. Nature Biotechnol. 31, 827–832 (2013)
Kabsch, W. XDS. Acta Crystallogr. D 66, 125–132 (2010)
Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D 66, 213–221 (2010)
Vonrhein, C., Blanc, E., Roversi, P. & Bricogne, G. Automated structure solution with autoSHARP. Methods Mol. Biol. 364, 215–230 (2007)
Vagin, A. & Teplyakov, A. Molecular replacement with MOLREP. Acta Crystallogr. D 66, 22–25 (2010)
Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D 60, 2126–2132 (2004)
Afonine, P. V. et al. Towards automated crystallographic structure refinement with phenix.refine. Acta Crystallogr. D 68, 352–367 (2012)
Katoh, K. & Standley, D. M. MAFFT: iterative refinement and additional methods. Methods Mol. Biol. 1079, 131–146 (2014)
Acknowledgements
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.
Author information
Authors and Affiliations
Contributions
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.
Corresponding author
Ethics declarations
Competing interests
M.J. is a co-founder of Caribou Biosciences, Inc. The authors have filed a related patent application.
Extended data figures and tables
Extended Data Figure 1 The PAM duplex binds in a positively charged cleft on the C-terminal PAM-interacting domain.
a, Enlarged view of the PAM binding site in Cas9. Nucleic acids are shown in stick representation, coloured according to the scheme in Fig. 1 and overlaid with experimentally phased, solvent-flattened electron density map (grey mesh, contoured at 1σ). b, PAM binding site in Cas9, shown in the same orientation as in panel a. The molecular surface of Cas9 is coloured according to electrostatic potential.
Extended Data Figure 2 The PAM binding site is pre-ordered in the Cas9–RNA complex.
a, Comparison of the structures of Cas9–sgRNA bound to a PAM-containing target DNA duplex (left) and single-stranded DNA target21 (right). The target DNA strands of the complexes were superimposed using a least-squares algorithm in Coot34 and the complexes are shown in identical orientations. Bound nucleic acids are shown in stick format and coloured according to the scheme in Fig. 1. b, Superimposed Cas9 molecules from the PAM-containing and ssDNA-bound complexes. The colour scheme is the same as in panel a. In both complexes, the HNH domain is in an inactive conformation, with the active site located approximately 40 Å away from the scissile phosphate in the target DNA strand, suggesting that the domain undergoes a further conformational rearrangement upon target-strand cleavage. c, Superimposed nucleic acid ligands. sgRNA and target DNA from the single-stranded target complex are coloured grey. d, Detailed view of the PAM binding site in the superimposed complexes, indicating a slight tightening of the PAM binding cleft.
Extended Data Figure 3 Endonuclease activities of Cas9 proteins containing mutations in the PAM binding motif.
a, Endonuclease activity assay of wild-type and mutant Cas9 proteins using supercoiled circular (SC) plasmid DNA containing a target sequence fully complementary to the sgRNA in Fig. 1a. Nucleotide sequences of target sites are provided in Extended Data Table 2. b, Endonuclease activity assay of wild-type and mutant Cas9 proteins using an oligonucleotide duplex containing a target sequence fully complementary to the sgRNA in Fig. 1a.
Extended Data Figure 4 PAM binding motifs in Cas9 orthologues.
a, Cas9 orthologues with known PAM sequences4,19,22,23. The PAM of Lactobacillus buchneri Cas9 has been inferred from known protospacer sequences, but has not been experimentally validated25. b, Alignment of the amino acid sequences of the major groove interacting regions of Cas9 orthologues. Primary sequences of type II-A Cas9 proteins from S. pyogenes (GI 15675041), Listeria innocua Clip 11262 (GI 16801805), S. mutans UA159 (GI 24379809), S. thermophilus LMD-9 (S. thermophilus A, GI 11662823; S. thermophilus B, GI 116627542), Lactobacillus buchneri NRRL B-30929 (GI 331702228), Treponema denticola ATCC 35405 (GI 42525843), type II-B Cas9 from Francisella novicida U112 (GI 118497352), and type II-C Cas9 proteins from Campylobacter jejuni subsp. jejuni NCTC 11168 (GI 218563121), Pasteurella multocida subsp. multocida str. Pm70 (GI 218767588) and Neisseria meningitidis Zs491 (GI 15602992) were aligned using MAFFT36. Amino acids are coloured in shades of blue according to their degree of conservation. The red boxes denote amino acid residues inferred to be involved in PAM recognition in type II-A and type II-B Cas9 proteins based on the sequence alignment and the crystal structure of the Cas9–sgRNA–DNA complex elucidated in this study.
Extended Data Figure 5 Glutamine substitution of Arg 1333 and Arg 1335 in S. pyogenes Cas9.
a, Endonuclease activity assay of wild-type and mutant Cas9 proteins using a linearized plasmid containing a target sequence fully complementary to sgRNA-2 and a 5′-TGG-3′ PAM (Extended Data Table 2). Bands at 2,014 and 598 base pairs (bp) correspond to Cas9 cleavage products. b, Endonuclease activity assay as in panel a using linearized plasmid DNA containing a 5′-TAA-3′ PAM. c, Endonuclease activity assay as in panel a using linearized plasmid DNA containing an extended 5′-TAAAA-3′ PAM.
Extended Data Figure 6 PAM-dependent interaction of the +1 phosphate with the phosphate lock loop.
a, Comparison of the bound target DNA (left) and the modelled B-form DNA (right). Docking of the ideal B-form duplex yields a steric clash with the phosphate lock loop. The arrow indicates the rotation of the +1 phosphate group (+1P) needed for interaction with the phosphate lock loop. b, Comparison of the phosphate lock loop and the +1 phosphate positions in the Cas9–sgRNA–DNA complex containing a PAM (left) and the Cas9–sgRNA–ssDNA target complex21 (right). Molecule A from the crystallographic asymmetric unit of the Cas9–sgRNA–ssDNA complex is shown. In molecule B, the nucleotides upstream of the +1 phosphate are structurally ordered due to crystal packing interactions, and the +1 phosphate is positioned within hydrogen-bonding distance as a result. Numbers indicate interatomic distances in Å. c, Superposition of the two structures shown in panel b.
Extended Data Figure 7 Endonuclease activity of phosphate lock loop Cas9 mutants against mismatch- and bubble-containing DNA substrates.
a, Endonuclease activity assay of wild-type and mutant Cas9 proteins using double-stranded oligonucleotide DNA containing a target sequence fully complementary to the sgRNA shown in Fig. 1a. Samples were taken after 15 s, 30 s, 1 min, 2 min, 5 min, 15 min, 1 h and 2 h. b, Endonuclease activity assay using an oligonucleotide duplex containing mismatches to the sgRNA at positions 1–2. c, Endonuclease activity assay using a bubble-containing oligonucleotide duplex in which the target strand is mismatched to the sgRNA at positions 1–2 and the target and non-target strands are themselves mismatched at positions 1–2. d, Quantification of cleavage defects observed with mismatch- and bubble-containing substrates from a–c. For each protein, the amount of cleaved product obtained after 2 h was normalized to the amount of product obtained from a perfectly complementary DNA substrate. Experiments were performed in triplicate. Error bars report standard error of the mean (s.e.m.).
Supplementary information
Supplementary Information
This file contains Supplementary Figure 1. (PDF 1674 kb)
Rights and permissions
About this article
Cite this article
Anders, C., Niewoehner, O., Duerst, A. et al. Structural basis of PAM-dependent target DNA recognition by the Cas9 endonuclease. Nature 513, 569–573 (2014). https://doi.org/10.1038/nature13579
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/nature13579
This article is cited by
-
CRISPR/Cas-mediated germplasm improvement and new strategies for crop protection
Crop Health (2024)
-
CRISPR/Cas9 assisted stem cell therapy in Parkinson's disease
Biomaterials Research (2023)
-
CRISPR-Cas9 mediated phage therapy as an alternative to antibiotics
Animal Diseases (2023)
-
Group B Streptococcus Cas9 variants provide insight into programmable gene repression and CRISPR-Cas transcriptional effects
Communications Biology (2023)
-
A generalizable Cas9/sgRNA prediction model using machine transfer learning with small high-quality datasets
Nature Communications (2023)
Comments
By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.