Single-molecule Fluorescence Study on Structural Roles of Guide RNAs in Activation of Cas9 Endonuclease

Single-molecule spectroscopy;Total internal reflection fluorescence microscopy;Fluorescence resonance energy transfer;CRISPR/Cas9;Structural dynamics;Enzymatic mechanism;540

자연과학대학 화학부 ;

University:서울대학교 대학원

关键词: Single-molecule spectroscopy;    Total internal reflection fluorescence microscopy;    Fluorescence resonance energy transfer;    CRISPR/Cas9;    Structural dynamics;    Enzymatic mechanism;    540;   

Others  :  http://s-space.snu.ac.kr/bitstream/10371/134949/1/000000141057.pdf

美国|英语

来源: Seoul National University Open Repository

【 摘 要 】

Clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated (Cas) system has recently been discovered as a prokaryotic adaptive immune mechanism against invading viruses and plasmids. Among various types of the CRISPR/Cas systems, type-II consists of Cas9 endonuclease and two guide RNAs (gRNA), CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA), whose ribonucleoprotein complex recognizes and cleaves target DNA. Although extensive structural studies of Cas9-gRNA complex has been carried out since the CRISPR/Cas9 system was successfully employed to genome editing, the detailed mechanism and dynamics of gRNAs in the structural activation of the Cas9 endonuclease are still elusive.Herein, we review our studies on the structural roles of the two guide RNAs in the activation of Cas9 endonuclease using pseudo-ensemble and single-molecule fluorescence spectroscopic assays, thereby providing mechanistic details which are not available from other static and/or ensemble-averaged measurements. Firstly, tracrRNA was found to be critical in the formation of functional Cas9-gRNA complex (Cas9:gRNA). In the absence of tracrRNA, the Cas9 protein became inactivated so that it failed to form Cas9:gRNA, whereas tracrRNA-Cas9 interaction prevented the inactivation pathway by leading Cas9 toward a complexation pathway for Cas9:gRNA. Secondly, crRNA was found to regulate the nuclease activation of Cas9 after Cas9:gRNA bound to the target DNA with forming Cas9-gRNA-DNA ternary complex (Cas9:gRNA:DNA). R-loop structure between crRNA and the target DNA in Cas9:gRNA:DNA exhibited intrinsically repetitive transitions between two distinct sub-conformations termed ;;open’ and ;;zipped’ conformations. This crRNA-related conformational dynamics was proved to be a crucial factor for the nuclease activation by the observation that the bound DNA was cleaved only when the zipped conformation formed. Overall, these results show mechanistic insights into the gRNA-associated structural activation of the Cas9 endonuclease.