Structural insights into how Cas9 targets nucleosomes
Abstract The CRISPR-associated endonuclease Cas9 derived from prokaryotes is used as a genome editing, which targets specific genomic loci by single guide RNAs (sgRNAs). The eukaryotes, the target of genome editing, store their genome DNA in chromatin, in which the nucleosome is a basic unit. Despit...
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Nature Portfolio
2024-12-01
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| Series: | Nature Communications |
| Online Access: | https://doi.org/10.1038/s41467-024-54768-z |
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| author | Reina Nagamura Tomoya Kujirai Junko Kato Yutaro Shuto Tsukasa Kusakizako Hisato Hirano Masaki Endo Seiichi Toki Hiroaki Saika Hitoshi Kurumizaka Osamu Nureki |
| author_facet | Reina Nagamura Tomoya Kujirai Junko Kato Yutaro Shuto Tsukasa Kusakizako Hisato Hirano Masaki Endo Seiichi Toki Hiroaki Saika Hitoshi Kurumizaka Osamu Nureki |
| author_sort | Reina Nagamura |
| collection | DOAJ |
| description | Abstract The CRISPR-associated endonuclease Cas9 derived from prokaryotes is used as a genome editing, which targets specific genomic loci by single guide RNAs (sgRNAs). The eukaryotes, the target of genome editing, store their genome DNA in chromatin, in which the nucleosome is a basic unit. Despite previous structural analyses focusing on Cas9 cleaving free DNA, structural insights into Cas9 targeting of DNA within nucleosomes are limited, leading to uncertainties in understanding how Cas9 operates in the eukaryotic genome. In the present study, we perform native-polyacrylamide gel electrophoresis (PAGE) analyses and find that Cas9 targets the linker DNA and the entry-exit DNA region of the nucleosome but not the DNA tightly wrapped around the histone octamer. We further determine cryo-electron microscopy (cryo-EM) structure of the Cas9-sgRNA-nucleosome ternary complex that targets linker DNA in nucleosomes. The structure suggests interactions between Cas9 and nucleosomes at multiple sites. Mutants that reduce the interaction between nucleosomal DNA and Cas9 improve nucleosomal DNA cleavage activity in vitro, although inhibition by the interaction between Cas9 and nucleosomes is limited in vivo. These findings will contribute to the development of novel genome editing tools in chromatin. |
| format | Article |
| id | doaj-art-67b1bc5a32d9478991f62ef587db2674 |
| institution | Kabale University |
| issn | 2041-1723 |
| language | English |
| publishDate | 2024-12-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Nature Communications |
| spelling | doaj-art-67b1bc5a32d9478991f62ef587db26742025-01-05T12:36:12ZengNature PortfolioNature Communications2041-17232024-12-0115111010.1038/s41467-024-54768-zStructural insights into how Cas9 targets nucleosomesReina Nagamura0Tomoya Kujirai1Junko Kato2Yutaro Shuto3Tsukasa Kusakizako4Hisato Hirano5Masaki Endo6Seiichi Toki7Hiroaki Saika8Hitoshi Kurumizaka9Osamu Nureki10Department of Biological Sciences, Graduate School of Science, The University of TokyoInstitute for Quantitative Biosciences, Department of Biological Sciences, Graduate School of Science, The University of TokyoInstitute for Quantitative Biosciences, Department of Biological Sciences, Graduate School of Science, The University of TokyoDepartment of Biological Sciences, Graduate School of Science, The University of TokyoDepartment of Biological Sciences, Graduate School of Science, The University of TokyoDepartment of Biological Sciences, Graduate School of Science, The University of TokyoDivision of Crop Genome Editing Research, Institute of Agrobiological Sciences, National Agriculture and Food Research OrganizationDivision of Crop Genome Editing Research, Institute of Agrobiological Sciences, National Agriculture and Food Research OrganizationDivision of Crop Genome Editing Research, Institute of Agrobiological Sciences, National Agriculture and Food Research OrganizationInstitute for Quantitative Biosciences, Department of Biological Sciences, Graduate School of Science, The University of TokyoDepartment of Biological Sciences, Graduate School of Science, The University of TokyoAbstract The CRISPR-associated endonuclease Cas9 derived from prokaryotes is used as a genome editing, which targets specific genomic loci by single guide RNAs (sgRNAs). The eukaryotes, the target of genome editing, store their genome DNA in chromatin, in which the nucleosome is a basic unit. Despite previous structural analyses focusing on Cas9 cleaving free DNA, structural insights into Cas9 targeting of DNA within nucleosomes are limited, leading to uncertainties in understanding how Cas9 operates in the eukaryotic genome. In the present study, we perform native-polyacrylamide gel electrophoresis (PAGE) analyses and find that Cas9 targets the linker DNA and the entry-exit DNA region of the nucleosome but not the DNA tightly wrapped around the histone octamer. We further determine cryo-electron microscopy (cryo-EM) structure of the Cas9-sgRNA-nucleosome ternary complex that targets linker DNA in nucleosomes. The structure suggests interactions between Cas9 and nucleosomes at multiple sites. Mutants that reduce the interaction between nucleosomal DNA and Cas9 improve nucleosomal DNA cleavage activity in vitro, although inhibition by the interaction between Cas9 and nucleosomes is limited in vivo. These findings will contribute to the development of novel genome editing tools in chromatin.https://doi.org/10.1038/s41467-024-54768-z |
| spellingShingle | Reina Nagamura Tomoya Kujirai Junko Kato Yutaro Shuto Tsukasa Kusakizako Hisato Hirano Masaki Endo Seiichi Toki Hiroaki Saika Hitoshi Kurumizaka Osamu Nureki Structural insights into how Cas9 targets nucleosomes Nature Communications |
| title | Structural insights into how Cas9 targets nucleosomes |
| title_full | Structural insights into how Cas9 targets nucleosomes |
| title_fullStr | Structural insights into how Cas9 targets nucleosomes |
| title_full_unstemmed | Structural insights into how Cas9 targets nucleosomes |
| title_short | Structural insights into how Cas9 targets nucleosomes |
| title_sort | structural insights into how cas9 targets nucleosomes |
| url | https://doi.org/10.1038/s41467-024-54768-z |
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