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

Abstract

In a type II Clustered-Regularly-Interspersed-Short-Palindromic-Repeats (CRISPR) system, RNAs derived from 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 its 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.

Mapping out the Degree of Freedom of Hosted Enzymes in Confined Spatial Environments

“…[22][23][24][25][26][27][28][29][30][31][32][33][34][35][36] Moreover, advances in techniques are noteworthy as site-directed spin labeling (SDSL), in combination with electron paramagnetic resonance (EPR) spectroscopy, has proven to be unique for characterizing protein topography, local and global structure, and dynamics regardless of complexities caused by enzyme-matrix signals and/or enzyme-matrix interactions. [37][38][39][40][41][42][43][44][45][46] Accordingly, the application of SDSL-EPR for mapping the host-guest interactions of the enzymes within various functional COF pores may be able to answer key questions about the impact of a confined spatial environment on the degree of freedom of the infiltrated enzyme and thereby the accompanying reactivity. These results are also anticipated to offer unique insight into the chemistry of the bespoke host environments in the COFs.…”

Striking Plasticity of CRISPR-Cas9 and Key Role of Non-target DNA, as Revealed by Molecular Simulations

“…Overall, structural analysis and biochemical experiments have suggested that conformational plasticity of the protein underlies the processes of nucleic acid association and subsequent cleavage. 7,12,13 However, the conformational changes triggering the binding of the nucleic acids remain speculative. It is unclear, in fact, how the conformational plasticity of Cas9 would allow the transition from an “open” apo state to a “closed” conformation in which the protein binds its guide RNA and DNA target.…”

“…This structure is thought to depict a precatalytic state of the system (Cas9:pre-cat), given that a distance of ∼18 Å separates the catalytic H840 from the cleavage site on the t -DNA, suggesting that a further conformational transition is required for the formation of a catalytically competent Cas9. Overall, structural analysis and biochemical experiments have suggested that conformational plasticity of the protein underlies the processes of nucleic acid association and subsequent cleavage. ,, However, the conformational changes triggering the binding of the nucleic acids remain speculative. It is unclear, in fact, how the conformational plasticity of Cas9 would allow the transition from an “open” apo state to a “closed” conformation in which the protein binds its guide RNA and DNA target.…”

Spin‐labeling of polymeric micelles and application in probing micelle swelling using EPR spectroscopy

“…EPR in combination with spin trapping has been applied to detect the radicals formed during the initiation, propagation, and termination of polymerization and polymer degradation . EPR has also been applied to probe the structure and dynamics of macromolecules with arbitrary size, heterogeneity, and complexity, as demonstrated in polymers, proteins, and nucleic acids . Typical information from EPR measurements include long‐range (a few nm) intra‐macromolecular distance distributions and site‐specific conformational dynamics (ns to ms) .…”