Structural roles of guide RNAs in the nuclease activity of Cas9 endonuclease.
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.
Department of Biophysics and Chemical Biology, Seoul National University, Seoul 151-747, Republic of Korea.
Youngbin
Y
Lim
Department of Chemistry, Seoul National University, Seoul 151-747, Republic of Korea.
So Young
SY
Bak
Department of Chemistry, Seoul National University, Seoul 151-747, Republic of Korea.
Keewon
K
Sung
Department of Chemistry, Seoul National University, Seoul 151-747, Republic of Korea.
Euihwan
E
Jeong
Center for Genome Engineering, Institute for Basic Science, Seoul 151-747, Republic of Korea.
Seung Hwan
SH
Lee
Department of Chemistry, Seoul National University, Seoul 151-747, Republic of Korea.|Center for Genome Engineering, Institute for Basic Science, Seoul 151-747, Republic of Korea.
Jin-Soo
JS
Kim
Department of Chemistry, Seoul National University, Seoul 151-747, Republic of Korea.|Center for Genome Engineering, Institute for Basic Science, Seoul 151-747, Republic of Korea.
Sangsu
S
Bae
Department of Biophysics and Chemical Biology, Seoul National University, Seoul 151-747, Republic of Korea.|Department of Chemistry, Seoul National University, Seoul 151-747, Republic of Korea.
Seong Keun
SK
Kim
D064130:CRISPR-Associated Proteins / Q000378:metabolism*; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004720:Endonucleases / Q000737:chemistry / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D008954:Models, Biological; D009690:Nucleic Acid Conformation; D009692:Nucleic Acid Heteroduplexes / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000737:chemistry* / Q000378:metabolism*
D064130:CRISPR-Associated Proteins; D009692:Nucleic Acid Heteroduplexes; D000094704:RNA, Guide, CRISPR-Cas Systems; D004247:DNA; D004720:Endonucleases