Sandbox Aryan 20221057 BI3323-Aug2025

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#REDIRECT [[Monkeypox DNA Polymerase]]
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== Structure Overview ==
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PDB '''8YNY''' (4.52 Å '''cryo-EM''', EMDB: '''EMD-39431''') captures '''Cas9-sgRNA ribonucleoprotein''' in '''post-cleavage ternary complex''' with '''Widom 601 nucleosome'''.<ref name="pdb8yny">RCSB PDB - 8YNY</ref><ref name="nature">Nagamura R, et al. Structural insights into how Cas9 targets nucleosomes. Nat Commun. 2024</ref>
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=== Components ===
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- '''Cas9''' (Chain A): '''HNH/REC2 domains disordered''', '''bridge helix absent'''
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- '''sgRNA''' (Chain B): Guides PAM1 targeting
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- '''Nucleosome''': '''Histone octamer''' (H2A/H2B/H3/H4) + '''147 bp Widom601 DNA'''
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- '''DNA state''': '''~15 bp unwrapped''' from histone octamer at '''PAM1 site'''
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<Structure load='8YNY' size='350' frame='true' align='right' caption='Insert caption here' scene='Insert optional scene name here' />
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== Summary ==
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PDB '''8YNY''' (4.52 Å cryo-EM, EMDB: EMD-39431) captures '''Cas9-sgRNA ribonucleoprotein''' in '''post-cleavage complex''' with Widom 601 nucleosome.<ref name="pdb8yny"/> Cas9 targets '''linker DNA''' (PAM1/PAM28 sites) and '''entry-exit regions''' (SHL6), unwrapping ~15 bp DNA from histone octamer while avoiding tightly wrapped '''core DNA''' (SHL0-5).<ref name="nature">Nagamura R, et al. Structural insights into how Cas9 targets nucleosomes. Nat Commun. 2024</ref>
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== Structure Overview ==
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'''Native-PAGE analysis''' confirms Cas9 preferentially cleaves '''nucleosome DNA ends''' where transient '''DNA breathing''' occurs, exploiting spontaneous unwrapping from histone octamer.<ref name="nature"/> Post-cleavage state reveals '''HNH/REC2 domains disordered''', '''bridge helix absent''', and both '''target/non-target DNA strands cleaved'''—consistent with binary biochemical assays.<ref name="nature"/>
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[[8yny_scene1:1]] Interactive 3D overview (community scene + student analysis)
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== PI Domain Interactions ==
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Cas9 '''PI domain''' (residues ~1100-1368) establishes '''multiple contacts''' with nucleosome:
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- '''Histone tails''' (H2A/H3): Weak '''electrostatic interactions''' (acidic PI loop + basic tails) — '''non-essential''' for binding<ref name="nature"/>
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- '''K1155''' (PI edge): '''Stabilizes post-cleavage complex''' via DNA phosphate backbone
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- '''Core DNA loops''' ('''H1264/R1298/K1300'''): '''Inhibitory nonspecific binding''' to SHL0-5 DNA
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**Mutagenesis validation**: '''H1264A/R1298Q/K1300A triple mutant''' ↑ nucleosome binding '''2-fold''' + cleavage efficiency '''3-5 fold''' (in vitro assays + rice callus genome editing).<ref name="nature"/>
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[[Image:8YNYOverall.jpg|350px|thumb|1. Overall ternary complex - ~15 bp DNA unwrapping]]
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[[Image:8YNYPI.jpg|350px|thumb|2. PI domain contacts - key residues highlighted]]
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== Dual Inhibition Mechanism ==
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Nucleosomes inhibit Cas9 via '''two sequential barriers''':
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# '''Access barrier''' (Stage 1): '''DNA end inflexibility''' at SHL0-5 prevents Cas9 binding to embedded PAM sites. '''Nucleosome breathing''' (transient unwrapping) + '''chromatin remodelers''' (SNF2h/RSC) enable access.<ref name="isaac">Isaac RS, et al. Nucleosome breathing and remodeling constrain CRISPR-Cas9 function. eLife. 2016</ref>
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# '''Motion restriction''' (Stage 2): '''PI-core DNA trapping''' (H1264/R1298/K1300) physically '''stabilizes post-cleavage complex''', preventing '''HNH/RuvC domain rearrangements''' required for product release.<ref name="nature"/>
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'''Entry/exit asymmetry''' from '''Widom601 sequence flexibility''' explains variable editing efficiency across chromatin contexts.<ref name="nature"/>
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[[Image:8YNYMutant.jpg|350px|thumb|3. Mutant sites (orange spheres): H1264/R1298/K1300]]
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== Biological Context ==
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**Prokaryotic Cas9 in eukaryotic chromatin**: Reveals '''natural adaptation limitations''' + identifies '''chromatin-optimized variants''' (PI mutants) for therapeutic genome editing. '''Sequence-dependent nucleosome positioning''' + '''PTMs''' modulate endogenous Cas9 efficiency.<ref name="nature"/><ref name="isaac"/>
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== Experimental Validation ==
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* '''Cryo-EM''': 4.52 Å resolution, captures '''DNA-attached state''' with fluctuating Cas9-nucleosome proximity<ref name="nature"/>
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* '''Native-PAGE''': Quantifies '''position-dependent cleavage''' (PAM1 >> PAM14/17)
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* '''Mutagenesis''': '''Triple PI mutant''' rescues inhibition '''in vitro/in vivo'''
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[[8yny_scene2:1]] PI domain interactive 3D focus
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== References ==
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<references />
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BI3323-Aug2025
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*Sandbox: proteopedia.org/wiki/Sandbox_BI3323_8YNY
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*3D scenes: proteopedia.org/wiki/8yny (community resource)
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*PNGs + 850-word analysis: Original PyMOL renders + primary literature synthesis
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[[Category:CRISPR]] [[Category:Nucleosomes]] [[Category:Genome Editing]]

Current revision

Contents

Structure Overview

PDB 8YNY (4.52 Å cryo-EM, EMDB: EMD-39431) captures Cas9-sgRNA ribonucleoprotein in post-cleavage ternary complex with Widom 601 nucleosome.[1][2]


Components

- Cas9 (Chain A): HNH/REC2 domains disordered, bridge helix absent - sgRNA (Chain B): Guides PAM1 targeting - Nucleosome: Histone octamer (H2A/H2B/H3/H4) + 147 bp Widom601 DNA - DNA state: ~15 bp unwrapped from histone octamer at PAM1 site

Insert caption here

Drag the structure with the mouse to rotate

Summary

PDB 8YNY (4.52 Å cryo-EM, EMDB: EMD-39431) captures Cas9-sgRNA ribonucleoprotein in post-cleavage complex with Widom 601 nucleosome.[1] Cas9 targets linker DNA (PAM1/PAM28 sites) and entry-exit regions (SHL6), unwrapping ~15 bp DNA from histone octamer while avoiding tightly wrapped core DNA (SHL0-5).[2]

Structure Overview

Native-PAGE analysis confirms Cas9 preferentially cleaves nucleosome DNA ends where transient DNA breathing occurs, exploiting spontaneous unwrapping from histone octamer.[2] Post-cleavage state reveals HNH/REC2 domains disordered, bridge helix absent, and both target/non-target DNA strands cleaved—consistent with binary biochemical assays.[2]

8yny_scene1:1 Interactive 3D overview (community scene + student analysis)

PI Domain Interactions

Cas9 PI domain (residues ~1100-1368) establishes multiple contacts with nucleosome: - Histone tails (H2A/H3): Weak electrostatic interactions (acidic PI loop + basic tails) — non-essential for binding[2] - K1155 (PI edge): Stabilizes post-cleavage complex via DNA phosphate backbone - Core DNA loops (H1264/R1298/K1300): Inhibitory nonspecific binding to SHL0-5 DNA

    • Mutagenesis validation**: H1264A/R1298Q/K1300A triple mutant ↑ nucleosome binding 2-fold + cleavage efficiency 3-5 fold (in vitro assays + rice callus genome editing).[2]
1. Overall ternary complex - ~15 bp DNA unwrapping
1. Overall ternary complex - ~15 bp DNA unwrapping
2. PI domain contacts - key residues highlighted
2. PI domain contacts - key residues highlighted

Dual Inhibition Mechanism

Nucleosomes inhibit Cas9 via two sequential barriers:

  1. Access barrier (Stage 1): DNA end inflexibility at SHL0-5 prevents Cas9 binding to embedded PAM sites. Nucleosome breathing (transient unwrapping) + chromatin remodelers (SNF2h/RSC) enable access.[3]
  1. Motion restriction (Stage 2): PI-core DNA trapping (H1264/R1298/K1300) physically stabilizes post-cleavage complex, preventing HNH/RuvC domain rearrangements required for product release.[2]

Entry/exit asymmetry from Widom601 sequence flexibility explains variable editing efficiency across chromatin contexts.[2]

3. Mutant sites (orange spheres): H1264/R1298/K1300
3. Mutant sites (orange spheres): H1264/R1298/K1300

Biological Context

    • Prokaryotic Cas9 in eukaryotic chromatin**: Reveals natural adaptation limitations + identifies chromatin-optimized variants (PI mutants) for therapeutic genome editing. Sequence-dependent nucleosome positioning + PTMs modulate endogenous Cas9 efficiency.[2][3]

Experimental Validation

  • Cryo-EM: 4.52 Å resolution, captures DNA-attached state with fluctuating Cas9-nucleosome proximity[2]
  • Native-PAGE: Quantifies position-dependent cleavage (PAM1 >> PAM14/17)
  • Mutagenesis: Triple PI mutant rescues inhibition in vitro/in vivo

8yny_scene2:1 PI domain interactive 3D focus

References

  1. ↑ 1.0 1.1 RCSB PDB - 8YNY
  2. ↑ 2.0 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9 Nagamura R, et al. Structural insights into how Cas9 targets nucleosomes. Nat Commun. 2024
  3. ↑ 3.0 3.1 Isaac RS, et al. Nucleosome breathing and remodeling constrain CRISPR-Cas9 function. eLife. 2016

BI3323-Aug2025

  • Sandbox: proteopedia.org/wiki/Sandbox_BI3323_8YNY
  • 3D scenes: proteopedia.org/wiki/8yny (community resource)
  • PNGs + 850-word analysis: Original PyMOL renders + primary literature synthesis
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