A cryptic binding pocket regulates the metal-dependent activity of Cas9
Abstract
Cas9 is a metal-dependent nuclease used for genome editing across diverse cells and organisms exhibiting distinct ionic environments, yet how metal ions regulate its catalytic function, and consequently its editing efficiency remains unclear. Here, molecular simulations, Markov state models, and mixed quantum–classical approaches reveal that divalent metals promote activation of the catalytic HNH domain through formation of a divalent metal-binding pocket (DBP) at the HNH–RuvC interface. This cryptic pocket emerges dynamically during HNH activation and is supported by NMR measurements revealing metal-dependent structural perturbations within the HNH–L2 region. Mutations targeting the DBP disrupt HNH activation and impair the coupled catalytic activity of both nucleases, identifying the pocket as a key regulator of Cas9’s metal-dependent activity. Together with quantum–classical simulations revealing distinct catalytic behaviours among divalent metals, these findings uncover an ion-dependent regulatory mechanism in Cas9 with insights for genome editing across diverse biological environments.
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BibTeX
@article{ahsan2026cryptic,
title = {{A cryptic binding pocket regulates the metal-dependent activity of Cas9}},
author = {Mohd Ahsan and Alexa L. Knight and Aakash Saha and Delisa Ramos and Isabel Strohkendl and Erin Skeens and Madeline S. West and Jinping Luo and Charlotte S. Dresser and David W. Taylor and George P. Lisi and Giulia Palermo},
journal = {Nature Communications},
year = {2026},
month = oct,
doi = {10.1038/s41467-026-78458-0},
url = {https://www.nature.com/articles/s41467-026-78458-0},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - A cryptic binding pocket regulates the metal-dependent activity of Cas9
AU - Mohd Ahsan
AU - Alexa L. Knight
AU - Aakash Saha
AU - Delisa Ramos
AU - Isabel Strohkendl
AU - Erin Skeens
AU - Madeline S. West
AU - Jinping Luo
AU - Charlotte S. Dresser
AU - David W. Taylor
AU - George P. Lisi
AU - Giulia Palermo
PY - 2026
DA - 2026/10/09
JO - Nature Communications
DO - 10.1038/s41467-026-78458-0
UR - https://www.nature.com/articles/s41467-026-78458-0
AB - Cas9 is a metal-dependent nuclease used for genome editing across diverse cells and organisms exhibiting distinct ionic environments, yet how metal ions regulate its catalytic function, and consequently its editing efficiency remains unclear. Here, molecular simulations, Markov state models, and mixed quantum–classical approaches reveal that divalent metals promote activation of the catalytic HNH domain through formation of a divalent metal-binding pocket (DBP) at the HNH–RuvC interface. This cryptic pocket emerges dynamically during HNH activation and is supported by NMR measurements revealing metal-dependent structural perturbations within the HNH–L2 region. Mutations targeting the DBP disrupt HNH activation and impair the coupled catalytic activity of both nucleases, identifying the pocket as a key regulator of Cas9’s metal-dependent activity. Together with quantum–classical simulations revealing distinct catalytic behaviours among divalent metals, these findings uncover an ion-dependent regulatory mechanism in Cas9 with insights for genome editing across diverse biological environments.
SN - 2041-1723
ER -