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Nucleic Acid-Dependent Conformational Changes in CRISPR–Cas9 Revealed by Site-Directed Spin Labeling

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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.

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Acknowledgments

This work has been supported, in part, by the National Science Foundation (P.Z.Q., CHE-1213673) and the National Institutes of Health (P.Z.Q., RR028992). Research in RR’s lab was supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under the Grant Number P20GM103640 and partly by a grant from the Research Council of the University of Oklahoma Norman Campus.

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    Authors and Affiliations

    1. Department of Chemistry, University of Southern California, Los Angeles, CA, 90089, USA

      Carolina Vazquez Reyes, Narin S. Tangprasertchai, Xiaojun Zhang & Peter Z. Qin

    2. Department of Chemistry and Biochemistry, University of Oklahoma, Norman, OK, 73019, USA

      S. D. Yogesha, Richard H. Nguyen & Rakhi Rajan

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    1. Carolina Vazquez Reyes
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    2. Narin S. Tangprasertchai
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    Correspondence to Peter Z. Qin.

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    Carolina Vazquez Reyes and Narin S. Tangprasertchai have contributed equally.

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    Vazquez Reyes, C., Tangprasertchai, N.S., Yogesha, S.D. et al. Nucleic Acid-Dependent Conformational Changes in CRISPR–Cas9 Revealed by Site-Directed Spin Labeling. Cell Biochem Biophys 75, 203–210 (2017). https://doi.org/10.1007/s12013-016-0738-5

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    • DOI: https://doi.org/10.1007/s12013-016-0738-5

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