Sandbox Aryan 20221057 BI3323-Aug2025

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Revision as of 17:49, 30 November 2025 (edit) Aryan ORCID 0009-0009-3985-5093 (Talk | contribs) ← Previous diff		Revision as of 17:56, 30 November 2025 (edit) (undo) Aryan ORCID 0009-0009-3985-5093 (Talk | contribs) Next diff →
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-	'''Key Interactions'''	+	=== Key Interactions ===
		+	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]
-	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.	+	=== Biological Insights ===
-	​	+	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]
-	​'''Biological Insights'''	+	[[Image:8YNYOverall.jpg|400px|thumb|Scene 1: Overall complex - DNA unwrapping]]
-		+	[[Image:8YNYPI.jpg|400px|thumb|Scene 2: PI domain contacts]]
-	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. These findings reveal Cas9's eukaryotic adaptation and guide chromatin-optimized variants for improved genome editing.	+	[[Image:8YNYMutant.jpg|400px|thumb|Scene 3: Mutant sites (orange)]]
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-		+
-	''Scene 1: Overall complex''	+
-	[[Image:8YNYOverall.jpg]]	+
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-	''Scene 2: PI domain contacts''	+
-	[[Image:8YNYPI.jpg]]	+
-	''	+
-	Scene 3: Mutant sites''	+
-	[[Image:8YNYMutant.jpg]]	+
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Revision as of 17:56, 30 November 2025

Cas9-Nucleosome Complex (PDB: 8YNY)

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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Key Interactions

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]

Biological Insights

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]

BI3323-Aug2025