Sandbox Aryan 20221057 BI3323-Aug2025

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Contents

Structure Overview

Template:StructureSection

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: Structure of Cas9-sgRNA ribonucleoprotein bound to nucleosome
  2. ↑ 2.0 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 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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