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: | , , , , , , , , |
|---|---|
| Other Authors: | |
| Format: | Article |
| Published: |
Elsevier
2017
|
| Online Access: | http://hdl.handle.net/1721.1/112727 https://orcid.org/0000-0001-7915-1685 https://orcid.org/0000-0003-2782-2509 |
| _version_ | 1826206948138156032 |
|---|---|
| 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 | 2024-09-23T13:41:05Z |
| format | Article |
| id | mit-1721.1/112727 |
| institution | Massachusetts Institute of Technology |
| last_indexed | 2024-09-23T13:41:05Z |
| publishDate | 2017 |
| publisher | Elsevier |
| record_format | dspace |
| spelling | mit-1721.1/1127272022-09-28T15:31:53Z 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 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. National Institutes of Health (U.S.) (Grant 5DP1-MH100706) 2017-12-13T15:02:26Z 2017-12-13T15:02:26Z 2014-02 2014-02 2017-12-13T14:04:54Z Article http://purl.org/eprint/type/JournalArticle 0092-8674 1097-4172 http://hdl.handle.net/1721.1/112727 Nishimasu, Hiroshi et al. “Crystal Structure of Cas9 in Complex with Guide RNA and Target DNA.” Cell 156, 5 (February 2014): 935–949 © 2014 Elsevier Inc https://orcid.org/0000-0001-7915-1685 https://orcid.org/0000-0003-2782-2509 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 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/1721.1/112727 https://orcid.org/0000-0001-7915-1685 https://orcid.org/0000-0003-2782-2509 |
| work_keys_str_mv | AT nishimasuhiroshi crystalstructureofcas9incomplexwithguidernaandtargetdna AT dohmaenaoshi crystalstructureofcas9incomplexwithguidernaandtargetdna AT ishitaniryuichiro crystalstructureofcas9incomplexwithguidernaandtargetdna AT nurekiosamu crystalstructureofcas9incomplexwithguidernaandtargetdna AT ranfei crystalstructureofcas9incomplexwithguidernaandtargetdna AT hsupatrick crystalstructureofcas9incomplexwithguidernaandtargetdna AT konermannsilvanam crystalstructureofcas9incomplexwithguidernaandtargetdna AT shehatasorayai crystalstructureofcas9incomplexwithguidernaandtargetdna AT zhangfeng crystalstructureofcas9incomplexwithguidernaandtargetdna |