ScholarWorks@성균관대학교

초록

Proteins with multiple domains play pivotal roles in various biological processes, necessitating a thorough understanding of their structural stability and functional interplay. Here, a structure-guided protein engineering approach is proposed to develop thermostable Cas9 (CRISPR-associated protein 9) variant for CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) interference applications. By employing thermodynamic analysis, combining distance mapping and molecular dynamics simulations, deletable domains are identified to enhance stability while preserving the DNA recognition function of Cas9. The resulting engineered Cas9, termed small and dead form Cas9, exhibits improved thermostability and maintains target DNA recognition function. Cryo-electron microscopy analysis reveals structural integrity with reduced atomic density in the deleted domain. Fusion with functional elements enables intracellular delivery and nuclear localization, demonstrating efficient gene suppression in diverse cell types. Direct delivery in the mouse brain shows enhanced knockdown efficiency, highlighting the potential of structure-guided engineering to develop functional CRISPR systems tailored for specific applications. This study underscores the significance of integrating computational and experimental approaches for protein engineering, offering insights into designing tailored molecular tools for precise biological interventions. The thermostable Cas9 (CRISPR-associated protein 9) variant is developed using a structure-guided protein engineering approach. By thermodynamic analysis, deletable domains are identified to enhance stability while preserving function. Also, a 3D structure with novel conformation of nontarget-stranded DNA is confirmed through cryo-electron microscopy. Fusion with functional elements for CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) interference applications enables gene suppression efficiency, demonstrating the potential of tailored CRISPR systems. image

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