Structural roles of guide RNAs in the nuclease activity of Cas9 endonuclease.
2016; 7: 13350
Recent advancement of light-based single-molecule approaches for studying biomolecules
WILEY INTERDISCIPLINARY REVIEWS-SYSTEMS BIOLOGY AND MEDICINE
2019; 11 (4)
Incorporation of STED technique into single-molecule spectroscopy to break the concentration limit of diffusing molecules in single-molecule detection
2018; 54 (69): 9667–70
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.
View details for DOI 10.1038/ncomms13350
View details for PubMedID 27804953
Noncanonical DNA-binding mode of repressor and its disassembly by antirepressor
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2016; 113 (18): E2480-E2488
By incorporating STED (stimulated emission depletion) nanoscopy into single-molecule spectroscopy, we demonstrate that the concentration limit imposed by optical diffraction can be overcome in diffusion-based single-molecule measurement. We showed that single-molecule detection is feasible at a concentration of 5 nM, which is 100-times higher than the limit of conventional single-molecule measurements.
View details for DOI 10.1039/c8cc05726e
View details for Web of Science ID 000442605100026
View details for PubMedID 30101240
New sub-diffraction-limit microscopy technique: Dual-point illumination AND-gate microscopy on nanodiamonds (DIAMOND)
2012; 20 (12): 13347-13356
DNA-binding repressors are involved in transcriptional repression in many organisms. Disabling a repressor is a crucial step in activating expression of desired genes. Thus, several mechanisms have been identified for the removal of a stably bound repressor (Rep) from the operator. Here, we describe an uncharacterized mechanism of noncanonical DNA binding and induction by a Rep from the temperate Salmonella phage SPC32H; this mechanism was revealed using the crystal structures of homotetrameric Rep (92-198) and a hetero-octameric complex between the Rep and its antirepressor (Ant). The canonical method of inactivating a repressor is through the competitive binding of the antirepressor to the operator-binding site of the repressor; however, these studies revealed several noncanonical features. First, Ant does not compete for the DNA-binding region of Rep. Instead, the tetrameric Ant binds to the C-terminal domains of two asymmetric Rep dimers. Simultaneously, Ant facilitates the binding of the Rep N-terminal domains to Ant, resulting in the release of two Rep dimers from the bound DNA. Second, the dimer pairs of the N-terminal DNA-binding domains originate from different dimers of a Rep tetramer (trans model). This situation is different from that of other canonical Reps, in which two N-terminal DNA-binding domains from the same dimeric unit form a dimer upon DNA binding (cis model). On the basis of these observations, we propose a noncanonical model for the reversible inactivation of a Rep by an Ant.
View details for DOI 10.1073/pnas.1602618113
View details for Web of Science ID 000375395700005
View details for PubMedID 27099293
We introduce a new, easily implementable sub-diffraction-limit microscopy technique utilizing the optical AND-gate property of fluorescent nanodiamond (FND). We demonstrate that when FND is illuminated by two spatially-offset lights of different wavelengths, emission comes only from the region of their overlap, which is used to reduce the effective point spread function from ~300 nm to ~130 nm in lateral plane, well below the diffraction limit.
View details for DOI 10.1364/OE.20.013347
View details for Web of Science ID 000305463600072
View details for PubMedID 22714363