User talk:Swasti Pradhan

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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 and crystallographic studies reveal three major conformational states in the catalytic cycle:

1. In State I (checkpoint), HNH is positioned >30 Å from the scissile phosphate and REC2 blocks access. 2. In State II (postcatalytic), HNH undergoes a ~34 Å swing to dock onto the target-strand cleavage site while REC2 becomes disordered. 3. In State III (product-bound), HNH becomes disordered, REC2 returns to its checkpoint position, and REC3/RuvC stabilize 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. Cas9 binds a single-guide RNA (sgRNA) to form a stable Cas9–RNA complex capable of scanning DNA for complementarity.

DNA interrogation requires recognition of an NGG PAM sequence, after which the sgRNA forms an RNA–DNA hybrid (R-loop), displacing the non-target strand.

Cas9 contains two nuclease domains:

• HNH – cleaves the target DNA strand
• RuvC – cleaves the non-target strand

This architecture allows programmable, sequence-specific editing.

1.2 Why structure matters

Structural studies reveal:

• an arginine-rich PAM clamp
• stabilization of the RNA–DNA hybrid
• routing of the displaced NTS toward RuvC
• large domain motions that activate catalysis

These define fidelity checkpoints which prevent cleavage unless the RNA–DNA pairing is correct.

Structural Features

Cas9 uses multiple structural elements to ensure accurate recognition and cleavage:

• An arginine-rich PAM clamp explains NGG specificity.
• Structures reveal the complete RNA–DNA hybrid and the path of the displaced NTS.
• The REC3 domain contains mismatch-sensing loops that prevent HNH docking when mismatches occur.
• Structures on chromatin show Cas9 preferentially binds DNA near nucleosome entry–exit sites.

Cryo-EM snapshots further support three Mg²⁺-dependent conformations:

State I – HNH far from scissile bond; REC2 blocks access

State II – HNH swings ~34 Å to dock

State III – HNH disordered; REC2 reforms its checkpoint role

Mechanism

Cas9 activation involves:

• PAM recognition by the arginine clamp
• stabilization of DNA in the central channel
• R-loop formation
• routing of NTS toward RuvC
• HNH docking (TS cleavage)
• RuvC cleavage of the NTS
• HNH disordering to promote product release

Integration of Structural Findings

The collected structures outline a coherent mechanical cycle for Cas9 action. Cas9 begins with PAM scanning and R-loop formation. In the checkpoint state, REC2 blocks catalysis until proper guide–target pairing is confirmed. HNH then performs a large conformational swing to reach the scissile phosphate, enabling coordinated cleavage with RuvC. After cleavage, HNH becomes disordered and REC2 reforms the checkpoint configuration, enabling product release while maintaining high specificity.

Implications for Genome Editing

This structural framework guides protein engineering. REC3 modifications can enhance mismatch discrimination for high-fidelity variants. Targeted alterations in or around HNH can tune catalytic efficiency. Surface-charge engineering may improve activity on chromatin. RuvC channel redesign can influence non-target strand cleavage. Understanding Cas9’s conformational cycle enables rational design of safer, more precise genome-editing tools.

About

Proteopedia assignment by Swasti Pradhan for BI3323-Aug2025 (Structural Biology)

References

Liu et al. (2019), Structures of Cas9 in Catalytically Active States