Sandbox Aryan 20221057 BI3323-Aug2025

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(== Summary == PDB **8YNY** (4.52 Å cryo-EM) captures Cas9-sgRNA post-cleavage binding to nucleosome linker DNA (PAM1).[web:2] <Jmol width=100% height=500px>load =8YNY; cartoon on; color chain; spin y)
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'''Cas9-Nucleosome Complex (PDB: 8YNY)'''
 
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Cas9-sgRNA ribonucleoprotein targets linker DNA (PAM1/PAM28) and entry-exit regions (SHL6) of nucleosomes, avoiding tightly wrapped core DNA (SHL0-5), as revealed by native-PAGE on Widom 601 nucleosomes. The cryo-EM structure (PDB: 8YNY, 4.52 Å, EMDB: EMD-39431) captures the post-cleavage ternary complex at PAM1, showing ~15 bp DNA peeled from the histone octamer, exposing H3 N-terminus—mimicking eukaryotic nucleosome unwrapping.
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== Structure Overview ==
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Cas9-sgRNA ribonucleoprotein targets nucleosome **linker DNA** (PAM1/PAM28) and **entry-exit regions** (SHL6), avoiding tightly wrapped **core DNA** (SHL0-5). Native-PAGE on Widom 601 nucleosomes confirmed preferential cleavage at DNA ends where transient unwrapping occurs.[attached_file:1]
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The post-cleavage complex shows HNH/REC2 domains disordered, bridge helix absent, and target/non-target DNA strands cleaved—consistent with binary biochemical data.[web:2]
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=== Key Interactions ===
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Cas9's PI domain (residues ~1100-1368) mediates multiple contacts: electrostatic histone tail interactions (non-essential for binding), PI edge lysine '''K1155''' stabilizing the post-cleavage complex, and core DNA loops ('''H1264, R1298, K1300''') causing inhibitory non-specific binding. Mutants disrupting PI-core DNA contacts (H1264A/R1298Q) enhance both in vitro cleavage efficiency and rice genome editing.[attached_file:1]
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=== Biological Insights ===
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== PI Domain Interactions ==
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Nucleosomes inhibit Cas9 via two mechanisms: (1) DNA end inflexibility blocks access; (2) PI domain trapping restricts domain motions needed for cleavage. Entry-exit asymmetry arises from Widom601 sequence-dependent flexibility, explaining variable editing efficiency across chromatin contexts.[web:14]
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Cas9's **PI domain** (residues ~1100-1368) makes multiple contacts:
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- **Histone tails**: Weak electrostatic interaction (non-essential for binding)
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- **PI edge (K1155)**: Lysine stabilizes post-cleavage complex via DNA phosphate backbone
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- **Core DNA loops (H1264/R1298/K1300)**: Nonspecific binding inhibits cleavage
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[[Image:8YNYOverall.jpg|400px|thumb|Scene 1: Overall complex - DNA unwrapping]]
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**Mutagenesis validation**: H1264A/R1298Q/K1300A mutants increase nucleosome binding AND cleavage efficiency both in vitro and rice callus genome editing.[attached_file:1]
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[[Image:8YNYPI.jpg|400px|thumb|Scene 2: PI domain contacts]]
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[[Image:8YNYMutant.jpg|400px|thumb|Scene 3: Mutant sites (orange)]]
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== Dual Inhibition Mechanism ==
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1. **Access barrier**: Nucleosome DNA ends inflexible (SHL0-5), blocking Cas9 binding
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2. **Motion restriction**: PI-core DNA trapping limits HNH/RuvC domain movements for cleavage
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**Entry/exit asymmetry** from Widom601 sequence flexibility explains variable editing across chromatin contexts.[web:14]
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== Implications ==
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Reveals Cas9's eukaryotic adaptation strategy and identifies **chromatin-optimized variants** for improved genome editing tools.[web:121]
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[[Image:8YNYOverall.jpg|400px|thumb|Scene 1: Ternary complex - DNA unwrapping]]
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[[Image:8YNYPI.jpg|400px|thumb|Scene 2: PI domain contacts (red residues)]]
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[[Image:8YNYMutant.jpg|400px|thumb|Scene 3: Mutant sites (orange spheres)]]
BI3323-Aug2025
BI3323-Aug2025

Revision as of 18:08, 30 November 2025

Contents

Structure Overview

Cas9-sgRNA ribonucleoprotein targets nucleosome **linker DNA** (PAM1/PAM28) and **entry-exit regions** (SHL6), avoiding tightly wrapped **core DNA** (SHL0-5). Native-PAGE on Widom 601 nucleosomes confirmed preferential cleavage at DNA ends where transient unwrapping occurs.[attached_file:1]

The post-cleavage complex shows HNH/REC2 domains disordered, bridge helix absent, and target/non-target DNA strands cleaved—consistent with binary biochemical data.[web:2]

PI Domain Interactions

Cas9's **PI domain** (residues ~1100-1368) makes multiple contacts: - **Histone tails**: Weak electrostatic interaction (non-essential for binding) - **PI edge (K1155)**: Lysine stabilizes post-cleavage complex via DNA phosphate backbone - **Core DNA loops (H1264/R1298/K1300)**: Nonspecific binding inhibits cleavage

    • Mutagenesis validation**: H1264A/R1298Q/K1300A mutants increase nucleosome binding AND cleavage efficiency both in vitro and rice callus genome editing.[attached_file:1]

Dual Inhibition Mechanism

1. **Access barrier**: Nucleosome DNA ends inflexible (SHL0-5), blocking Cas9 binding 2. **Motion restriction**: PI-core DNA trapping limits HNH/RuvC domain movements for cleavage

    • Entry/exit asymmetry** from Widom601 sequence flexibility explains variable editing across chromatin contexts.[web:14]

Implications

Reveals Cas9's eukaryotic adaptation strategy and identifies **chromatin-optimized variants** for improved genome editing tools.[web:121]

Scene 1: Ternary complex - DNA unwrapping
Scene 1: Ternary complex - DNA unwrapping
Scene 2: PI domain contacts (red residues)
Scene 2: PI domain contacts (red residues)
Scene 3: Mutant sites (orange spheres)
Scene 3: Mutant sites (orange spheres)

BI3323-Aug2025

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