期刊
NATURE COMMUNICATIONS
卷 7, 期 -, 页码 -出版社
NATURE PUBLISHING GROUP
DOI: 10.1038/ncomms13350
关键词
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资金
- National Research Foundation of Korea through the Global Frontier Program for Bioconvergence [NRF-2014M3A6A4075063]
- Fostering Core Leaders of the Future Basic Science Program [NRF-2011-0002378]
- Institute for Basic Science [IBS-R021-D1]
- Plant Molecular Breeding Center of Next Generation BioGreen 21 Program [PJ01119201]
- SNU Brain Fusion grant
The type II CRISPR-associated protein Cas9 recognizes and cleaves target DNA with the help of two guide RNAs (gRNAs; tracrRNA and crRNA). However, the detailed mechanisms and kinetics of these gRNAs in the Cas9 nuclease activity are unclear. Here, we investigate the structural roles of gRNAs in the CRISPR-Cas9 system by single-molecule spectroscopy and reveal a new conformation of inactive Cas9 that is thermodynamically more preferable than active apo-Cas9. We find that tracrRNA prevents Cas9 from changing into the inactive form and leads to the Cas9: gRNA complex. For the Cas9: gRNA complex, we identify sub-conformations of the RNA-DNA heteroduplex during R-loop expansion. Our single-molecule study indicates that the kinetics of the sub-conformations is controlled by the complementarity between crRNA and target DNA. We conclude that both tracrRNA and crRNA regulate the conformations and kinetics of the Cas9 complex, which are crucial in the DNA cleavage activity of the CRISPR-Cas9 system.
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