User talk:Swasti Pradhan

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== ABSTRACT ==
== 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.
+
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.
+
Cryo-EM and crystallographic studies reveal **three major conformational states** in the catalytic cycle:
-
1. In '''State I (checkpoint)''', HNH is positioned over 30 Å away from the scissile phosphate while REC2 blocks access.
+
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.
-
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 disordered, REC2 returns to its checkpoint position, and REC3/RuvC stabilize the cleaved DNA.
-
 
+
-
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.
These structural snapshots reveal the dynamic energy landscape governing Cas9 specificity and catalysis and form the basis for engineering high-fidelity Cas9 variants.
-
<Structure load='6O0X' size='350' align='right' caption='Cas9 State III (PDB 6O0X)' scene='Overall_Cas9_6O0X' />
+
<Structure load='6O0Q' size='350' align='right' caption='Cas9 Product-Bound State (PDB 6O0Q)' scene='Overall_Cas9_6O0Q' />
== 1. INTRODUCTION ==
== 1. INTRODUCTION ==
=== 1.1 Background: What is Cas9? ===
=== 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.
+
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 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.
+
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 ===
=== 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.
+
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.
=== 1.3 Purpose of this review ===
=== 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.
+
This Proteopedia page summarizes major structural insights explaining Cas9 targeting, activation, cleavage, and specificity.
== Structural Features ==
== 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.
+
Cas9 uses multiple structural elements to ensure accurate recognition and cleavage:
-
Studies using nucleosomal substrates demonstrate that Cas9 preferentially interacts with partially accessible DNA at nucleosome entry–exit sites, explaining reduced cleavage efficiency on chromatin.
+
* 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 revealed three Mg²⁺-dependent states:
+
Cryo-EM snapshots further support three Mg²⁺-dependent conformations:
-
'''State I''' – HNH far from the scissile phosphate; REC2 blocks access
+
'''State I''' – HNH far from scissile bond; REC2 blocks access
-
'''State II''' – HNH swings ~34 Å and rotates to dock at the cleavage site
+
'''State II''' – HNH swings ~34 Å to dock
-
'''State III''' – HNH becomes disordered, and REC2 returns to a checkpoint-like position
+
'''State III''' – HNH disordered; REC2 reforms its checkpoint role
-
<Structure load='6O0Z' size='350' align='left' caption='State I: Checkpoint (PDB 6O0Z)' scene='State_I_6O0Z' />
+
<Structure load='5F9R' size='350' align='left' caption='State I: Checkpoint (PDB 5F9R)' scene='State_I_5F9R' />
== Mechanism ==
== 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.
+
Cas9 activation involves:
-
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.
+
* 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
-
<Structure load='6O0Y' size='350' align='right' caption='State II: HNH Docked (PDB 6O0Y)' scene='State_II_6O0Y' />
+
<Structure load='5Y36' size='350' align='right' caption='State II: HNH Docked (PDB 5Y36)' scene='State_II_5Y36' />
== Integration of Structural Findings ==
== 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.
+
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 ==
== 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.
+
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 '''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.'' <ref>DOI: 10.1038/s41594-019-0258-2</ref>
+
 +
== About ==
 +
Proteopedia assignment by Swasti Pradhan for BI3323-Aug2025 (Structural Biology)
== Interactive Scenes ==
== Interactive Scenes ==
-
=== Overall_Cas9_6O0X ===
+
=== Overall_Cas9_6O0Q ===
<scene>
<scene>
-
load 6O0X;
+
load 6O0Q;
select all; cartoon on; color chain;
select all; cartoon on; color chain;
select rna; color green;
select rna; color green;
Line 75: Line 91:
</scene>
</scene>
-
=== State_I_6O0Z ===
+
=== State_I_5F9R ===
<scene>
<scene>
-
load 6O0Z;
+
load 5F9R;
select all; cartoon on; color grey;
select all; cartoon on; color grey;
-
select (resid 775-905); color yellow;
+
select (residue=775-905); color yellow; # HNH domain region
zoom 150; background white;
zoom 150; background white;
</scene>
</scene>
-
=== State_II_6O0Y ===
+
=== State_II_5Y36 ===
<scene>
<scene>
-
load 6O0Y;
+
load 5Y36;
select all; cartoon on; color grey;
select all; cartoon on; color grey;
-
select (resid 775-905); color yellow;
+
select (residue=775-905); color yellow; # HNH in docked position
select rna; color green;
select rna; color green;
select dna; color red;
select dna; color red;
Line 93: Line 109:
</scene>
</scene>
-
=== PAM_Pocket_6O0Z ===
+
=== PAM_Pocket_5F9R ===
<scene>
<scene>
-
load 6O0Z;
+
load 5F9R;
select all; cartoon on; color grey;
select all; cartoon on; color grey;
-
select (dna and within(4.0, resno=1:3)); color red;
+
select (dna and within(4.0, resno=1:3)); color red; # PAM region
-
select (resno=1333,1335); color blue; spacefill 0.6;
+
select (resno=1333,1335); color blue; spacefill 0.6; # arginine clamp
zoom 150; background white;
zoom 150; background white;
</scene>
</scene>
-
=== Cleaved_DNA_Ends_6O0X ===
+
=== Cleaved_DNA_Ends_6O0Q ===
<scene>
<scene>
-
load 6O0X;
+
load 6O0Q;
select all; cartoon on; color grey;
select all; cartoon on; color grey;
select dna; color red;
select dna; color red;
Line 113: Line 129:
=== REC3_Mismatch_Sensing ===
=== REC3_Mismatch_Sensing ===
<scene>
<scene>
-
load 6O0Z;
+
load 5F9R;
select all; cartoon on; color grey;
select all; cartoon on; color grey;
-
select (resid 530-537,574-588,686-689); color magenta; spacefill 0.7;
+
select (residue=530-537,574-588,686-689); color magenta; spacefill 0.7;
select rna; color green;
select rna; color green;
select dna; color red;
select dna; color red;
Line 123: Line 139:
=== HNH_Swing_Animation ===
=== HNH_Swing_Animation ===
<scene>
<scene>
-
load 6O0Z;
+
load 5F9R;
-
load append 6O0Y;
+
load append 5Y36;
-
load append 6O0X;
+
load append 6O0Q;
select all; cartoon on; color chain;
select all; cartoon on; color chain;
animation mode palindrome;
animation mode palindrome;

Revision as of 15:48, 30 November 2025

Contents

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.

Cas9 Product-Bound State (PDB 6O0Q)

Drag the structure with the mouse to rotate

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.

1.3 Purpose of this review

This Proteopedia page summarizes major structural insights explaining Cas9 targeting, activation, cleavage, and specificity.

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

State I: Checkpoint (PDB 5F9R)

Drag the structure with the mouse to rotate

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

State II: HNH Docked (PDB 5Y36)

Drag the structure with the mouse to rotate

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)

Interactive Scenes

Overall_Cas9_6O0Q

State_I_5F9R

State_II_5Y36

PAM_Pocket_5F9R

Cleaved_DNA_Ends_6O0Q

REC3_Mismatch_Sensing

HNH_Swing_Animation

References

Retrieved from "http://52.214.119.220/wiki/index.php/User_talk:Swasti_Pradhan"
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