RT Journal Article
SR Electronic
T1 Conformational dynamics of Cas9 governing DNA cleavage revealed by single molecule FRET
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 167627
DO 10.1101/167627
A1 Mengyi Yang
A1 Sijia Peng
A1 Ruirui Sun
A1 Jingdi Lin
A1 Nan Wang
A1 Chunlai Chen
YR 2017
UL http://biorxiv.org/content/early/2017/07/24/167627.abstract
AB Off-target binding and cleavage by Cas9 pose as major challenges in its applications. How conformational dynamics of Cas9 governs its nuclease activity under on- and off-target conditions remains largely unknown. Here, using intra-molecular single molecule fluorescence resonance energy transfer measurements, we revealed that Cas9 in apo, sgRNA-bound, and dsDNA/sgRNA-bound forms all spontaneously transits between three major conformational states, mainly reflecting significant conformational mobility of the catalytic HNH domain. We furthermore uncovered a surprising long-range allosteric communication between the HNH domain and RNA/DNA heteroduplex at the PAM-distal end to ensure correct positioning of the catalytic site, which demonstrated a unique proofreading mechanism served as the last checkpoint before DNA cleavage. Several Cas9 residues were likely to mediate the allosteric communication and proofreading step. Modulating interactions between Cas9 and heteroduplex at the distal end by introducing mutations on these sites provides an alternative route to improve and optimize the CRISPR/Cas9 toolbox.