Cas9 structure has once become a research hotspot for scientists owning to its powerful functions for genome editing and gene regulation in many eukaryotic organisms. Structures of Streptococcus pyogenes Cas9 alone or bound to single-guide RNA (sgRNA) and target DNA revealed a bilobed protein architecture. The architectures of Cas9 define nucleic acid binding clefts, and single-particle electron microscopy reconstructions show that the two structural lobes harboring these clefts undergo guide RNA–induced reorientation to form a central channel where DNA substrates are bound. The observation that extensive structural rearrangements occur before target DNA duplex binding implicates guide RNA loading as a key step in Cas9 activation.
Structures of Cas9 in the apo state have two distinct lobes, the alpha-helical recognition (REC) lobe and the nuclease (NUC) lobe containing the conserved HNH and the split RuvC nuclease domains as well as the more variable C-terminal domain (CTD). The two lobes are further connected through two linking segments, one formed by the arginine-rich bridge helix and the other by a disordered linker (residues 712–717).
The RuvC domain comprises three distinct motifs: motifs II and III are interrupted by the HNH domain, and motifs I and II are interrupted by the REC lobe, which is also composed of three alpha-helical domains (Hel-I, Hel-II, and Hel-III) and does not share structural similarity with other known proteins. The α-helical lobe forms a broad cleft that makes extensive contacts with the sgRNA and target DNA and undergoes a large rotation relative to the nuclease lobe upon guide RNA binding to create a central channel where target DNA is bound.
Fig 1. CAS9 secondary structure diagram in different bacteria
CTD is responsible for both the PAM recognition and the guide RNA repeat–antirepeat heteroduplex binding. It displays a Cas9-specific fold and contains PAM-interacting (PI) sites required for PAM interrogation. The PI domain forms an elongated structure comprising seven α-helices (α46–α52), a three-stranded antiparallel β-sheet (β18–β20), a five-stranded antiparallel β-sheet (β21–β23, β26 and β27), and a two-stranded antiparallel β-sheet (β24 and β25). Target recognition strictly requires the presence of a short PAM flanking the target site. PI domain is positioned to recognize the PAM sequence on the non-complementary DNA strand.
Here, we mainly introduce the crystal structure of Streptococcus pyogenes Cas9. Biochemical experiments show that PAM recognition occurs through a composite binding site that is largely disordered in the absence of guide RNA and substrate interactions, indicating that the apo-Cas9 enzyme is kept in an inactive configuration, and is unable to recognize the target DNA prior to binding to a guide RNA. This structural observation is in line with so-called DNA curtains assays showing that apo-Cas9 binds DNA nonspecifically and it can be rapidly detached from nonspecific sites in the presence of competitor RNA (guide RNA or heparin).
Structural superimposition of apo–Cas9 with sgRNA-bound and DNA-bound structures further demonstrates that the enzyme adopts a catalytically inactive conformation in the apo state, necessitating RNA-induced structural activation for DNA recognition and cleavage. This structural finding is consistent with the biochemical observation that Cas9 enzymes are inactive as nucleases in the absence of bound guide RNAs and further supports their function as RNA-guided endonucleases.
Fig 2. Crystal structure of SpyCas9. (a) Ribbon representation of the crystal structure of SpyCas9. (b) Ribbon diagram showing the apo structure of SpyCas9 aligned in the same orientation as Cas9–sgRNA pretargeting structure. (c) Ribbon representation of the SpyCas9 sgRNA pretargeting complex.
Guide RNA binding drives Cas9 to undergo a substantial structural rearrangement from an inactive conformation to a DNA recognition–competent conformation. The most prominent conformational change takes place in the REC lobe, in particular Hel-III, which moves ∼65 A˚ toward the HNH domain upon sgRNA binding. In contrast, Cas9 exhibits much smaller conformational changes upon binding to target DNA and PAM sequence, which indicates that the majority of the extensive structural rearrangements occur prior to target DNA binding, reinforcing the notion that guide RNA loading is a key regulator of Cas9 enzyme function.
1. Fuguo Jiang and Jennifer A. CRISPR–Cas9 Structures and Mechanisms. Annual Review of Biophysics. 2017 May 25. 46:505–29.
2. Addison V. Wright, Samuel H. Sternberg, et al. Rational design of a split-Cas9 enzyme complex. PNAS. March 10, 2015. vol. 112. no. 10:2984–2989.
3. Nishimasu et al. Crystal Structure of Cas9 in Complex with Guide RNA and Target DNA. Cell. 2014 February 27; 156(5): 935–949.
4. Jinek et al. Structures of Cas9 Endonucleases Reveal RNA-Mediated Conformational Activation. Science. 2014. 343(6176): 1247997–1247997.