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

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Title
Structural roles of guide RNAs in the nuclease activity of Cas9 endonuclease
Author(s)
Toungbin Lim; So Young Bak; Keewon Sung; Euihwan Jeong; Seung Hwan Lee; Jin-Soo Kim; Sangsu Bae; Seong Keun Kim
Publication Date
2016-11
Journal
NATURE COMMUNICATIONS, v.7, no., pp.13350 -
Publisher
NATURE PUBLISHING GROUP
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. © The Author(s) 2016
URI
https://pr.ibs.re.kr/handle/8788114/3100
ISSN
2041-1723
Appears in Collections:
Center for Genome Engineering(유전체 교정 연구단) > Journal Papers (저널논문)
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