最先端研究セミナー
講演者： 濡木 理 （東京大学 大学院理学系研究科 生物科学専攻 教授）
演題： Molecular mechanism of CRISPR and structure-based development of genome editing tool towards medical applications
日時： 2月5日（月） 14：00-15：00
会場： 発生医学研究所1階 カンファレンス室
Abstract
The CRISPR-associated endonuclease Cas9 can be targeted to specific genomic loci by single guide RNAs (sgRNAs). We first solved the crystal structure of Streptococcus pyrogenes Cas9 (SpCas9) in complex with sgRNA and its target DNA at 2.5 A resolution. The structure revealed a bilobed architecture consisting of target recognition and nuclease lobes (Rec and Nuc lobes, respectively), accommodating the sgRNA:DNA heteroduplex in a positively-charged groove at their interface. While Rec lobe is essential for binding sgRNA and DNA, Nuc lobe contains the HNH and RuvC nuclease domains, which are properly located for cleavage of the complementary and noncomplemantary strands of the target DNA, respectively. Nuc lobe also contains a C-terminal domain responsible for the recognition of the protospacer adjacent motif (PAM). Recently, our high-speed atomic force microscopy (HS-AFM) analysis of SpCas9 visualized real-space and real-time dynamics of Cas9. We further solved the crystal structure of more compact Staphyrococcus aureus Cas9 (SaCas9), minimal Campylobacter jejuni Cas9 (CjCas9), and larger Francisella novicida Cas9 (FnCas9), complexed with their guide RNAs and double-stranded target DNAs at 2.6, 2.4 and 1.8 A resolutions, respectively. These high-resolution structures combined with functional analyses revealed the generality and diversity of molecular mechanism of RNA-guided DNA targeting by Cas9, and uncovered the distinct mechanisms of PAM recognition. On the basis of the structures, we succeeded in changing the specificity of PAM recognition, which paves the way for rational design of new, versatile genome-editing technologies. Recently, we solved the crystal structure of type-V CRISPR, Cpf1 in complex with crRNA and target dsDNA. The structure explains striking similarity and major differences between Cas9 and Cpf1.
References
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- “Crystal structure of Staphylococcus aureus Cas9” H. Nishimasu, L. Cong, W. X. Yan, F. A. Ran, B. Zetsche, Y. Li, A. Kurabayashi, R. Ishitani, F. Zhang and O. Nureki
Cell 162, 1113-1126 (2015). doi: 10.1016/j.cell.2015.08.007.
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Cell 164, 950-961 (2016) doi: 10.1016/j.cell.
- “Structural basis for the altered Pam specificities of engineered CRISPR-Cas9” S. Hirano, H. Nishimasu, R. Ishitani and O. Nureki.
Mol. Cell 61, 886-894 (2016) doi 10.1016/j.molcel.2016.02.018.
- “Crystal structure of Cpf1 in complex with guide RNA and target DNA” T. Yamano, H. Nishimasu, B. Zetsche, H. Hirano, I. M. Slaymaker, Y Li, I. Fedorova, T. Nakane, K. S. Makarova, E. V. Koonin, R. Ishitani, F. Zhang and O. Nureki.
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- “Crystal Structure of the Minimal Cas9 from Campylobacter jejuni Reveals the Molecular Diversity in the CRISPR-Cas9 Systems” M. Yamada, Y. Watanabe, J. S. Gootenberg, H. Hirano, F. A. Ran, T. Nakane, R. Ishitani, F. Zhang, H. Nishimasu and O. Nureki. Mol. Cell. 65, 1109-1121 (2017).
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担当分野： 機能分子構造解析学 山縣ゆり子（内線：4638）
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