title: Nucleic Acid-Dependent Conformational Changes in CRISPR-Cas9 Revealed by Site-Directed Spin Labeling.
abstract: In a type II clustered regularly interspaced short palindromic repeats (CRISPR) system, RNAs that are encoded at the CRISPR locus complex with the CRISPR-associated (Cas) protein Cas9 to form an RNA-guided nuclease that cleaves double-stranded DNAs at specific sites. In recent years, the CRISPR-Cas9 system has been successfully adapted for genome engineering in a wide range of organisms. Studies have indicated that a series of conformational changes in Cas9, coordinated by the RNA and the target DNA, direct the protein into its active conformation, yet details on these conformational changes, as well as their roles in the mechanism of function of Cas9, remain to be elucidated. Here, nucleic acid-dependent conformational changes in Streptococcus pyogenes Cas9 (SpyCas9) were investigated using the method of site-directed spin labeling (SDSL). Single nitroxide spin labels were attached, one at a time, at one of the two native cysteine residues (Cys80 and Cys574) of SpyCas9, and the spin-labeled proteins were shown to maintain their function. X-band continuous-wave electron paramagnetic resonance spectra of the nitroxide attached at Cys80 revealed conformational changes of SpyCas9 that are consistent with a large-scale domain re-arrangement upon binding to its RNA partner. The results demonstrate the use of SDSL to monitor conformational changes in CRISPR-Cas9, which will provide key information for understanding the mechanism of CRISPR function.
authors.affiliation: Department of Chemistry, University of Southern California, Los Angeles, CA, 90089, USA.
authors.forename: Carolina
authors.initials: C
authors.lastname: Vazquez Reyes
authors.affiliation: Department of Chemistry, University of Southern California, Los Angeles, CA, 90089, USA.
authors.forename: Narin S
authors.initials: NS
authors.lastname: Tangprasertchai
authors.affiliation: Department of Chemistry and Biochemistry, University of Oklahoma, Norman, OK, 73019, USA.
authors.forename: S D
authors.initials: SD
authors.lastname: Yogesha
authors.affiliation: Department of Chemistry and Biochemistry, University of Oklahoma, Norman, OK, 73019, USA.
authors.forename: Richard H
authors.initials: RH
authors.lastname: Nguyen
authors.affiliation: Department of Chemistry, University of Southern California, Los Angeles, CA, 90089, USA.
authors.forename: Xiaojun
authors.initials: X
authors.lastname: Zhang
authors.affiliation: Department of Chemistry and Biochemistry, University of Oklahoma, Norman, OK, 73019, USA.
authors.forename: Rakhi
authors.initials: R
authors.lastname: Rajan
authors.affiliation: Department of Chemistry, University of Southern California, Los Angeles, CA, 90089, USA. pzq@usc.edu.
authors.forename: Peter Z
authors.initials: PZ
authors.lastname: Qin
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000076987:CRISPR-Associated Protein 9; D064113:CRISPR-Cas Systems; D003001:Cloning, Molecular; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D004247:DNA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D004720:Endonucleases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D008958:Models, Molecular; D009589:Nitrogen Oxides / Q000737:chemistry*; D011485:Protein Binding; D000072417:Protein Domains; D017433:Protein Structure, Secondary; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013113:Spin Labels; D013194:Staining and Labeling / Q000379:methods; D013297:Streptococcus pyogenes / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D009589:Nitrogen Oxides; D000094704:RNA, Guide, CRISPR-Cas Systems; D011994:Recombinant Proteins; D013113:Spin Labels; D004247:DNA; D000076987:CRISPR-Associated Protein 9; C000606107:Cas9 endonuclease Streptococcus pyogenes; D004720:Endonucleases; C039900:nitroxyl
keywords: CRISPR; Cas9; Conformational change; EPR; Spin labeling