Crystal Structure of Cas9 in Complex with Guide RNA and Target DNA
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...
| Main Authors: | , , , , , , , , |
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| Format: | Article |
| Language: | en_US |
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Elsevier B.V.
2017
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| Online Access: | http://hdl.handle.net/1721.1/110860 https://orcid.org/0000-0001-7915-1685 https://orcid.org/0000-0003-2782-2509 |
| _version_ | 1826213723515125760 |
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| author | Nishimasu, Hiroshi Dohmae, Naoshi Ishitani, Ryuichiro Nureki, Osamu Ran, Fei Hsu, Patrick Konermann, Silvana M Shehata, Soraya I. Zhang, Feng |
| author2 | Massachusetts Institute of Technology. Department of Biological Engineering |
| author_facet | Massachusetts Institute of Technology. Department of Biological Engineering Nishimasu, Hiroshi Dohmae, Naoshi Ishitani, Ryuichiro Nureki, Osamu Ran, Fei Hsu, Patrick Konermann, Silvana M Shehata, Soraya I. Zhang, Feng |
| author_sort | Nishimasu, Hiroshi |
| collection | MIT |
| description | 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. |
| first_indexed | 2024-09-23T15:53:46Z |
| format | Article |
| id | mit-1721.1/110860 |
| institution | Massachusetts Institute of Technology |
| language | en_US |
| last_indexed | 2024-09-23T15:53:46Z |
| publishDate | 2017 |
| publisher | Elsevier B.V. |
| record_format | dspace |
| spelling | mit-1721.1/1108602022-09-29T16:54:18Z Crystal Structure of Cas9 in Complex with Guide RNA and Target DNA Nishimasu, Hiroshi Dohmae, Naoshi Ishitani, Ryuichiro Nureki, Osamu Ran, Fei Hsu, Patrick Konermann, Silvana M Shehata, Soraya I. Zhang, Feng Massachusetts Institute of Technology. Department of Biological Engineering Massachusetts Institute of Technology. Department of Biology Massachusetts Institute of Technology. Department of Brain and Cognitive Sciences McGovern Institute for Brain Research at MIT Ran, Fei Hsu, Patrick Konermann, Silvana M Shehata, Soraya I. Zhang, Feng 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. Japan. Science and Technology Agency (PRESTO) Japan Society for the Promotion of Science National Institutes of Health (U.S.) (NIH Director’s Pioneer Award (5DP1-MH100706)) 2017-07-26T18:59:19Z 2017-07-26T18:59:19Z 2014-02 Article http://purl.org/eprint/type/JournalArticle 00928674 http://hdl.handle.net/1721.1/110860 Nishimasu, Hiroshi, F. Ann Ran, Patrick D. Hsu, Silvana Konermann, Soraya I. Shehata, Naoshi Dohmae, Ryuichiro Ishitani, Feng Zhang, and Osamu Nureki. “Crystal Structure of Cas9 in Complex with Guide RNA and Target DNA.” Cell 156, no. 5 (February 2014): 935–949. https://orcid.org/0000-0001-7915-1685 https://orcid.org/0000-0003-2782-2509 en_US http://dx.doi.org/10.1016/j.cell.2014.02.001 Cell Creative Commons Attribution-NonCommercial-NoDerivs License http://creativecommons.org/licenses/by-nc-nd/4.0/ application/pdf Elsevier B.V. PMC |
| spellingShingle | Nishimasu, Hiroshi Dohmae, Naoshi Ishitani, Ryuichiro Nureki, Osamu Ran, Fei Hsu, Patrick Konermann, Silvana M Shehata, Soraya I. Zhang, Feng Crystal Structure of Cas9 in Complex with Guide RNA and Target DNA |
| title | Crystal Structure of Cas9 in Complex with Guide RNA and Target DNA |
| title_full | Crystal Structure of Cas9 in Complex with Guide RNA and Target DNA |
| title_fullStr | Crystal Structure of Cas9 in Complex with Guide RNA and Target DNA |
| title_full_unstemmed | Crystal Structure of Cas9 in Complex with Guide RNA and Target DNA |
| title_short | Crystal Structure of Cas9 in Complex with Guide RNA and Target DNA |
| title_sort | crystal structure of cas9 in complex with guide rna and target dna |
| url | http://hdl.handle.net/1721.1/110860 https://orcid.org/0000-0001-7915-1685 https://orcid.org/0000-0003-2782-2509 |
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