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Structural roles of guide RNAs in the nuclease activity of Cas9 endonuclease

Title
Structural roles of guide RNAs in the nuclease activity of Cas9 endonuclease
Author
배상수
Keywords
TARGET DNA RECOGNITION; SINGLE-MOLECULE FRET; COMPLEX; CRISPR-CAS9; SPECIFICITY; PROKARYOTES; RESISTANCE; CLEAVAGE; IMMUNITY; REPEATS
Issue Date
2016-11
Publisher
NATURE PUBLISHING GROUP
Citation
NATURE COMMUNICATIONS, v. 7, Page. 1-8, 13350
Abstract
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.
URI
https://www.nature.com/articles/ncomms13350https://repository.hanyang.ac.kr/handle/20.500.11754/101168
ISSN
2041-1723
DOI
10.1038/ncomms13350
Appears in Collections:
COLLEGE OF NATURAL SCIENCES[S](자연과학대학) > CHEMISTRY(화학과) > Articles
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