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TitleStructure-Guided Engineering of Thermodynamically Enhanced SaCas9 for Improved Gene Suppression.
Journal, issue, pagesAdv Mater, Page e2404680, Year 2024
Publish dateJun 22, 2024
AuthorsEun Sung Kang / Nam Hyeong Kim / Hyun-Kyoung Lim / Hyeyeon Jeon / Kayoung Han / Young Hyun No / Kyungtae Kim / Zinah Hilal Khaleel / Dongsun Shin / Kilho Eom / Jiyoung Nam / Bok-Soo Lee / Han-Joo Kim / Minah Suh / Jaecheol Lee / Trung Thanh Thach / Jaekyung Hyun / Yong Ho Kim /
PubMed AbstractProteins with multiple domains play pivotal roles in various biological processes, necessitating a thorough understanding of their structural stability and functional interplay. Here, a structure- ...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.
External linksAdv Mater / PubMed:38944889
MethodsEM (single particle)
Resolution2.97 Å
Structure data

39633

8ywh

EMDB-39633, PDB-8ywh:
Cryo-EM structure of small and dead form SaCas9-RNA-DNA ternary complex (sdCas9)
Method: EM (single particle) / Resolution: 2.97 Å

Source
  • staphylococcus aureus (bacteria)
  • synthetic construct (others)
KeywordsDNA BINDING PROTEIN / CRISPR/Cas9 / Thermostable protein engineering / Domain minimized Cas / engineered SaCas9

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