Youngbin Lim1, So Young Bak2, Keewon Sung2, Euihwan Jeong2, Seung Hwan Lee3, Jin-Soo Kim2,3, Sangsu Bae2,3,† & Seong Keun Kim1,2
1 Department of Biophysics and Chemical Biology, Seoul National University, Seoul 151-747, Republic of Korea. 2 Department of Chemistry, Seoul National University, Seoul 151-747, Republic of Korea. 3 Center for Genome Engineering, Institute for Basic Science, Seoul 151-747, Republic of Korea.† Present address: Department of Chemistry, Hanyang University, Seoul 133-791, Republic of Korea.
Correspondence to Sangsu Bae or Seong Keun Kim.
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.