Sandbox Aryan 20221057 BI3323-Aug2025

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## '''Cas9-Nucleosome Complex (PDB: 8YNY)'''
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== Structure Overview ==
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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, with ~15 bp DNA peeled from the histone octamer, exposing H3 N-terminus.[1][2][8][11]
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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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## '''Key Interactions'''
 
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Cas9's PI domain (residues ~1100-1368) mediates contacts: electrostatic histone tail interactions (non-essential), PI edge lysine K1155 stabilizing the complex, and core DNA loops (H1264, R1298, K1300) causing inhibitory non-specific binding. Mutants disrupting PI-core DNA contacts enhance in vitro cleavage and rice genome editing.[2][11][1]
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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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## '''Biological Insights'''
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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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Nucleosomes inhibit Cas9 via DNA end inflexibility (access barrier) and PI domain trapping (motion restriction), with entry-exit asymmetry from sequence-dependent flexibility. This explains eukaryotic adaptation and guides chromatin-optimized Cas9 variants.[3][6][11]
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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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**Image 1: Overall complex (DNA unwrapping)**
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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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```
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fetch 8YNY; color chain H3 blue, H4 green, H2A yellow, H2B red;
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color chain A magenta, Cas9 orange; show cartoon, Cas9 sgRNA DNA;
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show surface, histones; distance hbond (Cas9.K1155), (DNA);
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orient; ray 1200,1000; png 8YNY_unwrap.png
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```
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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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**Image 2: PI domain-core DNA contacts**
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== Dual Inhibition Mechanism ==
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```
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Nucleosomes inhibit Cas9 via '''two sequential barriers''':
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select PI, resi 1100-1368; orient PI;
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show sticks, PI and resi 1155+1264+1298+1300; color red, those;
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distance hbond (PI.R1298), (DNA); surface DNA and core;
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ray 1200,1000; png 8YNY_PI_contacts.png
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```
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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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**Image 3: Key mutant sites**
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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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```
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show spheres, resi 1155+1264+1298+1300; color orange, those;
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hide everything; show cartoon; label those, "%s%r:%s";
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ray 1200,1000; png 8YNY_mutants.png
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```
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'''Aryan, BI3323-Aug2025'''
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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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[1](https://www.rcsb.org/structure/8YNY)
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[[Image:8YNYMutant.jpg|350px|thumb|3. Mutant sites (orange spheres): H1264/R1298/K1300]]
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[2](https://pdbj.org/mine/summary/8YNY)
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[3](https://pubmed.ncbi.nlm.nih.gov/39737984/)
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== Biological Context ==
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[4](https://rna.bgsu.edu/rna3dhub/pdb/8YNY)
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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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[5](https://swissmodel.expasy.org/repository/uniprot/Q99ZW2)
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[6](https://www.nature.com/articles/s41467-024-54768-z)
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== Experimental Validation ==
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[7](https://nakb.org/atlas=8YNY)
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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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[8](https://www.ebi.ac.uk/emdb/EMD-39431)
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* '''Native-PAGE''': Quantifies '''position-dependent cleavage''' (PAM1 >> PAM14/17)
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[9](https://www.emdataresource.org/EMD-39431)
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* '''Mutagenesis''': '''Triple PI mutant''' rescues inhibition '''in vitro/in vivo'''
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[10](https://pdbj.org/search/pdb-author?query=%22Kurumizaka%2C+H.%22)
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[11](https://ppl-ai-file-upload.s3.amazonaws.com/web/direct-files/attachments/53439927/6101bbe1-6927-494e-9e78-84ebfeb259b9/Structural_insights_into_how_Cas9_targets_nucleoso.pdf)
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[[8yny_scene2:1]] PI domain interactive 3D focus
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[12](https://pmc.ncbi.nlm.nih.gov/articles/PMC6842131/)
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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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