MDM2 antagonists promote CRISPR/Cas9-mediated precise genome editing in sheep primary cells

Yan Li; Di Lian; Jiahao Wang; Yue Zhao; Yao Li; Guoshi Liu; Sen Wu; Shoulong Deng; Xuguang Du; Zhengxing Lian

Molecular Therapy: Nucleic Acids (Mar 2023)

MDM2 antagonists promote CRISPR/Cas9-mediated precise genome editing in sheep primary cells

Yan Li,
Di Lian,
Jiahao Wang,
Yue Zhao,
Yao Li,
Guoshi Liu,
Sen Wu,
Shoulong Deng,
Xuguang Du,
Zhengxing Lian

Affiliations

Yan Li: Beijing Key Laboratory for Animal Genetic Improvement, National Engineering Laboratory for Animal Breeding, College of Animal Science and Technology, China Agricultural University, Beijing 100193, China; State Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing 100193, China; Laboratory Animal Center of the Academy of Military Medical Sciences, Beijing 100071, China; These authors contributed equally
Di Lian: State Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing 100193, China; These authors contributed equally
Jiahao Wang: Beijing Key Laboratory for Animal Genetic Improvement, National Engineering Laboratory for Animal Breeding, College of Animal Science and Technology, China Agricultural University, Beijing 100193, China; Department of Biomedical Engineering, College of Future Technology, Peking University, Beijing 100871, China; These authors contributed equally
Yue Zhao: Beijing Key Laboratory for Animal Genetic Improvement, National Engineering Laboratory for Animal Breeding, College of Animal Science and Technology, China Agricultural University, Beijing 100193, China
Yao Li: Beijing Key Laboratory for Animal Genetic Improvement, National Engineering Laboratory for Animal Breeding, College of Animal Science and Technology, China Agricultural University, Beijing 100193, China
Guoshi Liu: Beijing Key Laboratory for Animal Genetic Improvement, National Engineering Laboratory for Animal Breeding, College of Animal Science and Technology, China Agricultural University, Beijing 100193, China
Sen Wu: State Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing 100193, China
Shoulong Deng: NHC Key Laboratory of Human Disease Comparative Medicine, Institute of Laboratory Animal Sciences, Chinese Academy of Medical Sciences and Comparative Medicine Center, Peking Union Medical College, Beijing 100021, China; Corresponding author: Shoulong Deng, NHC Key Laboratory of Human Disease Comparative Medicine, Institute of Laboratory Animal Sciences, Chinese Academy of Medical Sciences and Comparative Medicine Center, Peking Union Medical College, 5 Panjiayuannanli, Chaoyang District, Beijing 100021, China.
Xuguang Du: State Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing 100193, China; Corresponding author: Xuguang Du, State Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing 100193, China.
Zhengxing Lian: Beijing Key Laboratory for Animal Genetic Improvement, National Engineering Laboratory for Animal Breeding, College of Animal Science and Technology, China Agricultural University, Beijing 100193, China; Corresponding author: Zhengxing Lian, Beijing Key Laboratory for Animal Genetic Improvement, National Engineering Laboratory for Animal Breeding, College of Animal Science and Technology, China Agricultural University, 2 Mingyuanxilu, Haidian District, Beijing 100193, China. .

Journal volume & issue: Vol. 31
pp. 309 – 323

Abstract

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CRISPR-Cas9-mediated genome editing in sheep is of great use in both agricultural and biomedical applications. While targeted gene knockout by CRISPR-Cas9 through non-homologous end joining (NHEJ) has worked efficiently, the knockin efficiency via homology-directed repair (HDR) remains lower, which severely hampers the application of precise genome editing in sheep. Here, in sheep fetal fibroblasts (SFFs), we optimized several key parameters that affect HDR, including homology arm (HA) length and the amount of double-stranded DNA (dsDNA) repair template; we also observed synchronization of SFFs in G2/M phase could increase HDR efficiency. Besides, we identified three potent small molecules, RITA, Nutlin3, and CTX1, inhibitors of p53-MDM2 interaction, that caused activation of the p53 pathway, resulting in distinct G2/M cell-cycle arrest in response to DNA damage and improved CRISPR-Cas9-mediated HDR efficiency by 1.43- to 4.28-fold in SFFs. Furthermore, we demonstrated that genetic knockout of p53 could inhibit HDR in SFFs by suppressing the expression of several key factors involved in the HDR pathway, such as BRCA1 and RAD51. Overall, this study offers an optimized strategy for the usage of dsDNA repair template, more importantly, the application of MDM2 antagonists provides a simple and efficient strategy to promote CRISPR/Cas9-mediated precise genome editing in sheep primary cells.

Published in Molecular Therapy: Nucleic Acids

ISSN: 2162-2531 (Online)
Publisher: Elsevier
Country of publisher: United States
LCC subjects: Medicine: Therapeutics. Pharmacology
Website: https://www.cell.com/molecular-therapy-family/nucleic-acids/latest-content

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