User talk:Swasti Pradhan

From Proteopedia

Revision as of 15:35, 30 November 2025

ABSTRACT

Cas9 from Streptococcus pyogenes is a programmable RNA-guided endonuclease that mediates targeted double-stranded DNA cleavage. Structural studies have shown how Cas9 recognizes a protospacer adjacent motif (PAM), forms an RNA–DNA R-loop, and aligns its two nuclease domains, HNH and RuvC, for strand-specific catalysis.

Cryo-EM structures of wild-type Cas9 bound to sgRNA and a 40-bp DNA substrate in the presence of Mg²⁺ reveal three distinct conformational states capturing key transitions in the catalytic cycle.

1. In State I (checkpoint), HNH is positioned over 30 Å away from the scissile phosphate while REC2 blocks access.

2. In State II (postcatalytic), HNH undergoes a dramatic ~34 Å swing to dock onto the target-strand cleavage site while REC2 becomes disordered and REC3 forms new PAM-distal contacts.

3. In State III (product-bound), HNH becomes highly disordered, REC2 returns to its checkpoint position, and REC3 together with RuvC stabilizes the cleaved DNA.

These structural snapshots reveal the dynamic energy landscape governing Cas9 specificity and catalysis and form the basis for engineering high-fidelity Cas9 variants.

1. INTRODUCTION

1.1 Background: What is Cas9?

Cas9 from Streptococcus pyogenes is an RNA-guided endonuclease within Type II CRISPR adaptive immune systems. It binds a single-guide RNA (sgRNA) to form a Cas9–RNA complex that searches DNA for complementarity.

DNA binding begins with recognition of a PAM (NGG). PAM engagement enables sgRNA–DNA pairing and formation of an R-loop that displaces the non-target DNA strand. Cas9 contains two nuclease domains: HNH, which cleaves the target strand, and RuvC, which cleaves the non-target strand. These features make Cas9 a programmable sequence-specific nuclease.

1.2 Why structure matters

High-resolution structures show how Cas9 recognizes PAM sites, stabilizes the RNA–DNA hybrid, positions the non-target strand, and undergoes major domain rearrangements required for cleavage. These structures also reveal fidelity checkpoints, mismatch detection mechanisms, and activation steps underlying accurate DNA targeting.

1.3 Purpose of this review

This page summarizes structural insights from major studies—including those in Nature, Science, Cell, and Nature Structural & Molecular Biology—illustrating how conformational changes in Cas9 govern target binding, activation, cleavage, and specificity.

Structural Features

Cas9 contains several structural elements that ensure accurate DNA recognition. The arginine-rich PAM clamp (R1333 and R1335) explains the strict requirement for NGG PAM. Cryo-EM structures revealed the complete RNA–DNA hybrid and the trajectory of the displaced non-target strand leading toward the RuvC domain. The REC3 domain contains mismatch-sensing loops that prevent HNH docking when RNA–DNA complementarity is insufficient.

Studies using nucleosomal substrates demonstrate that Cas9 preferentially interacts with partially accessible DNA at nucleosome entry–exit sites, explaining reduced cleavage efficiency on chromatin.

Cryo-EM snapshots revealed three Mg²⁺-dependent states:

State I – HNH far from the scissile phosphate; REC2 blocks access State II – HNH swings ~34 Å and rotates to dock at the cleavage site State III – HNH becomes disordered, and REC2 returns to a checkpoint-like position

Mechanism

Cas9 initiates target interrogation through PAM binding, mediated by arginine residues that clamp the PAM duplex. PAM recognition stabilizes the DNA in the central channel and triggers R-loop formation. The REC lobe supports the RNA–DNA hybrid, while the displaced non-target strand is guided toward the RuvC active site.

Disordering of REC2 correlates with catalytic activation (State II), allowing HNH to swing into position and cleave the target strand. RuvC cleaves the non-target strand. After cleavage, disordering of the HNH domain and repositioning of REC2 support formation of a stable product-bound state before final release.

Integration of Structural Findings

Cas9 follows a coordinated mechanical cycle beginning with PAM scanning and progressing through R-loop formation, checkpoint verification, activation through REC2 displacement, and the characteristic HNH swing that enables target-strand cleavage. Dual-strand cleavage is followed by stabilization of the product complex through REC3 and RuvC interactions. These structural transitions explain how Cas9 achieves high specificity and catalytic efficiency.

Implications for Genome Editing

Structural understanding directly informs Cas9 engineering strategies. Modifying residues within the REC3 domain can enhance mismatch discrimination, producing high-fidelity variants with reduced off-target cleavage. Alterations within or adjacent to the HNH domain can tune catalytic rates, generating hyperactive or attenuated Cas9 variants. Changes to Cas9’s interaction surfaces with nucleosomal DNA can improve cleavage efficiency within chromatin environments, addressing limitations posed by DNA compaction.

ABOUT

Proteopedia assignment Swasti Pradhan for BI3323 – Aug2025 Structural Biology.

Source – Cryo-EM structures reveal coordinated domain motions that govern DNA cleavage by Cas9, by Xing Zhu et al. ^[1]

Interactive Scenes

Overall_Cas9_6O0X

State_I_6O0Z

State_II_6O0Y

PAM_Pocket_6O0Z

Cleaved_DNA_Ends_6O0X

REC3_Mismatch_Sensing

HNH_Swing_Animation

References

1. ↑ Zhu X, Clarke R, Puppala AK, Chittori S, Merk A, Merrill BJ, Simonovic M, Subramaniam S. Cryo-EM structures reveal coordinated domain motions that govern DNA cleavage by Cas9. Nat Struct Mol Biol. 2019 Jul 8. pii: 10.1038/s41594-019-0258-2. doi:, 10.1038/s41594-019-0258-2. PMID:31285607 doi:http://dx.doi.org/10.1038/s41594-019-0258-2