현재경 (성균관대학교 약학대학)

한빛사논문

조회165

성균관대학교 약학대학

CV File 62 kb

Adv. Mater., Jun 22 2024, | https://doi.org/10.1002/adma.202404680 Structure-Guided Engineering of Thermodynamically Enhanced SaCas9 for Improved Gene Suppression

Eun Sung Kang^1,2,3, Nam Hyeong Kim ^1,2,3, Hyun-Kyoung Lim ⁴, Hyeyeon Jeon ^1,3,5, Kayoung Han ⁴, Young Hyun No ^1,3, Kyungtae Kim ⁶, Zinah Hilal Khaleel ^1,3,7, Dongsun Shin ^1,3,5, Kilho Eom ⁸, Jiyoung Nam ^1,2,3, Bok-Soo Lee^1,2,3, Han-Joo Kim ², Minah Suh^{2,4,7,9,10,11}, Jaecheol Lee ^2,4,6,12,13, Trung Thanh Thach ², Jaekyung Hyun ⁶, Yong Ho Kim ^{1,2,3,4,5,7,9}

¹SKKU Advanced Institute of Nanotechnology (SAINT), Sungkyunkwan University, Suwon, 16419, Republic of Korea.

²IMNEWRUN INC., Suwon, 16419, Republic of Korea.

³Department of Nano Science and Technology, Sungkyunkwan University, Suwon, 16419, Republic of Korea.

⁴Biomedical Institute for Convergence at SKKU (BICS), Sungkyunkwan University, Suwon, 16419, Republic of Korea.

⁵Department of Nano Engineering, Sungkyunkwan University, Suwon, 16419, Republic of Korea.

⁶School of Pharmacy, Sungkyunkwan University, Suwon, 16419, Republic of Korea.

⁷Center for Neuroscience Imaging Research (CNIR), Institute for Basic Science (IBS), Suwon, 16419, Republic of Korea.

⁸Biomechanics Laboratory, College of Sport Science, Sungkyunkwan University (SKKU), Suwon, 16419, Republic of Korea.

⁹Department of Biomedical Engineering, Sungkyunkwan University, Suwon, 16419, Republic of Korea.

¹⁰Department of Intelligent Precision Healthcare Convergence (IPHC), Sungkyunkwan University, Suwon, 16419, Republic of Korea.

¹¹KIST-SKKU Brain Research Center, Sungkyunkwan University, Suwon, 16419, Republic of Korea.

¹²Department of Biopharmaceutical Convergence, Sungkyunkwan University, Suwon, 16419, Republic of Korea.

¹³Department of Biohealth Regulatory Science, Sungkyunkwan University, Suwon, 16419, Republic of Korea.

E.S.K. and N.H.K. contributed equally to this work.

CORRESPONDING AUTHORS: Trung Thanh Thach, Jaekyung Hyun, Yong Ho Kim

Abstract

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.

논문정보

형식Research article
게재일2024년 06월 (BRIC 등록일 2024-07-08)
연구진국내 연구진
분야 생명과학 > 구조생물 및 생물물리학

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