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

Youngbin Lim, So Young Bak, Keewon Sung, Euihwan Jeong, Seung Hwan Lee, Jin-Soo Kim, Sangsu Bae () and Seong Keun Kim ()
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Youngbin Lim: Seoul National University
So Young Bak: Seoul National University
Keewon Sung: Seoul National University
Euihwan Jeong: Seoul National University
Seung Hwan Lee: Center for Genome Engineering, Institute for Basic Science
Jin-Soo Kim: Seoul National University
Sangsu Bae: Seoul National University
Seong Keun Kim: Seoul National University

Nature Communications, 2016, vol. 7, issue 1, 1-8

Abstract: 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.

Date: 2016
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DOI: 10.1038/ncomms13350

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