Abstract

The  CRISPR-associated endonuclease Cas9 binds to a guide RNA and cleaves  double-stranded DNA with a sequence complementary to the RNA guide. The  Cas9–RNA system has been harnessed for numerous applications, such as  genome editing. Here we use high-speed atomic force microscopy (HS-AFM)  to visualize the real-space and real-time dynamics of CRISPR-Cas9 in  action. HS-AFM movies indicate that, whereas apo-Cas9 adopts unexpected  flexible conformations, Cas9–RNA forms a stable bilobed structure and  interrogates target sites on the DNA by three-dimensional diffusion.  These movies also provide real-time visualization of the Cas9-mediated  DNA cleavage process. Notably, the Cas9 HNH nuclease domain fluctuates  upon DNA binding, and subsequently adopts an active conformation, where  the HNH active site is docked at the cleavage site in the target DNA.  Collectively, our HS-AFM data extend our understanding of the action  mechanism of CRISPR-Cas9.

Introduction

In  the microbial CRISPR-Cas (clustered regularly interspaced short  palindromic repeats-CRISPR-associated proteins) adaptive immune system^1,2,3, the RNA-guided DNA endonuclease Cas9 associates with a CRISPR RNA (crRNA) and a trans-activating crRNA (tracrRNA), and cleaves the double-stranded (ds) DNA complementary to the crRNA guide^4,5,6,7 (Supplementary Fig. 1a). Cas9 can function with a single-guide RNA (sgRNA), in which the crRNA and the tracrRNA are fused with an artificial linker⁷. The two-component system, consisting of Cas9 from Streptococcus pyogenes and the sgRNA, has been harnessed for genome-engineering technologies in a variety of cell types and organisms^8,9,10. Cas9–RNA first recognizes a short nucleotide sequence (NGG for S. pyogenes  Cas9; N represents any nucleotide) next to a target sequence in the  dsDNA, called a protospacer adjacent motif (PAM). Cas9–RNA then  initiates DNA unwinding at the PAM-proximal region, which is followed by  the directional formation of an R-loop, consisting of the RNA–DNA  hybrid and the displaced non-target DNA strand¹¹.  After the R-loop formation, the HNH and RuvC nuclease domains of Cas9  cleave the target DNA strand (complementary to the RNA guide) and the  non-target DNA strand, respectively^6,7.

Previous structural studies showed that apo-Cas9 adopts a closed auto-inhibited conformation¹²,  whereas the Cas9–RNA binary complex adopts a bilobed architecture  comprising an α-helical recognition (REC) lobe and a nuclease (NUC) lobe¹³ (Fig. 1a, b).  A comparison between these structures suggests that Cas9 undergoes a  closed-to-open structural rearrangement upon binding the guide RNA.  Moreover, the crystal structures of Cas9–RNA bound to the target DNA  provided mechanistic insights into RNA-guided DNA targeting by Cas9^14,15 (Fig. 1b).  Furthermore, the Cas9 R-loop complex structure revealed drastic  conformational changes in the linker regions between the RuvC and HNH  nuclease domains, thereby translocating the HNH domain closer to the  target DNA strand¹⁶ (Fig. 1b, Supplementary Fig. 1b).  Consistently, bulk and single-molecule Förster resonance energy  transfer (FRET) studies indicated that the HNH domain undergoes a  structural transition during DNA cleavage and adopts three major  conformations: RNA-bound (R), intermediate (I) and active docked (D)  states^17,18. The R and I conformations predominantly correspond to the crystal structures of Cas9–RNA¹³ and Cas9–RNA–DNA^14,15, respectively. A structure of the D conformation was predicted by modeling^17,18, but it has not been determined, although the D conformation approximates the crystal structure of the Cas9 R-loop complex¹⁶.  These structural and imaging studies provided mechanistic insights into  the Cas9-mediated DNA recognition and cleavage, but its action  mechanism has not been fully clarified. It is unknown how apo-Cas9 in  the closed conformation assembles with the guide RNA to form an effector  complex. In addition, Cas9 in the catalytically-active D conformation  has not been visualized.

HS-AFM observations of apo-Cas9 and Cas9–RNA. a Domain structure of S. pyogenes Cas9. BH, Bridge helix. b Crystal structures of apo-Cas9 (PDB: 4CMP)¹², Cas9–RNA (PDB: 4ZT0)¹³, Cas9–RNA bound to its single-stranded DNA target (PDB: 4OO8)¹⁵, Cas9–RNA bound to a partial DNA duplex (PDB: 4UN3)¹⁴ and Cas9–RNA bound to its dsDNA target (a Cas9 R-loop complex) (PDB: 5F9R)¹⁶.  The guide RNA and the target DNA are colored red and yellow,  respectively. The PAM is colored purple. The 98-nt guide RNA (PDB: 4OO8)  was used for HS-AFM observations. c, d Sequential HS-AFM images of apo-Cas9 (c) and Cas9–RNA (d) on the AP-mica surface. The color (from black to white) corresponds to the height. The scale bars are 10 nm. e Time courses of correlation coefficients between the sequential HS-AFM images of apo-Cas9 and Cas9–RNA

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High-speed atomic force microscopy (HS-AFM) is a  powerful technique that enables real-space and real-time observations  of macromolecules, which are not feasible by other techniques¹⁹.  HS-AFM imaging has elucidated the dynamics of various proteins with  unprecedented details; for example, the photo-induced conformational  change of bacteriorhodopsin²⁰, myosin V walking on an actin filament²¹, cellulose degradation by a cellulase enzyme²², rotary catalysis of F₁-ATPase²³, lipid membrane remodeling by ESCRT-III polymerization²⁴ and lipid membrane stabilization by annexin V²⁵.

In  this study, we employed HS-AFM to visualize the real-space and  real-time dynamics of CRISPR-Cas9 in action, at nanometer resolution.  HS-AFM movies revealed that apo-Cas9 adopts flexible conformations,  whereas Cas9–RNA forms a stable bilobed architecture. Furthermore, the  HS-AFM movies directly visualized the Cas9-mediated DNA cleavage  reaction accompanied by the drastic structural transition of the HNH  nuclease domain. Overall, the HS-AFM movies provided distinct scenes of  Cas9 in action, comprising the complex assembly, target search, target  cleavage and product release, thus substantially improving our  mechanistic understanding of CRISPR-Cas9.