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Modeling SHANK3-associated autism spectrum disorder in Beagle dogs via CRISPR/Cas9 gene editing

Molecular Psychiatry volume 28, pages 3739–3750 (2023)Cite this article

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Abstract

Despite intensive studies in modeling neuropsychiatric disorders especially autism spectrum disorder (ASD) in animals, many challenges remain. Genetic mutant mice have contributed substantially to the current understanding of the molecular and neural circuit mechanisms underlying ASD. However, the translational value of ASD mouse models in preclinical studies is limited to certain aspects of the disease due to the apparent differences in brain and behavior between rodents and humans. Non-human primates have been used to model ASD in recent years. However, a low reproduction rate due to a long reproductive cycle and a single birth per pregnancy, and an extremely high cost prohibit a wide use of them in preclinical studies. Canine model is an appealing alternative because of its complex and effective dog–human social interactions. In contrast to non-human primates, dog has comparable drug metabolism as humans and a high reproduction rate. In this study, we aimed to model ASD in experimental dogs by manipulating the Shank3 gene as SHANK3 mutations are one of most replicated genetic defects identified from ASD patients. Using CRISPR/Cas9 gene editing, we successfully generated and characterized multiple lines of Beagle Shank3 (bShank3) mutants that have been propagated for a few generations. We developed and validated a battery of behavioral assays that can be used in controlled experimental setting for mutant dogs. bShank3 mutants exhibited distinct and robust social behavior deficits including social withdrawal and reduced social interactions with humans, and heightened anxiety in different experimental settings (n = 27 for wild-type controls and n = 44 for mutants). We demonstrate the feasibility of producing a large number of mutant animals in a reasonable time frame. The robust and unique behavioral findings support the validity and value of a canine model to investigate the pathophysiology and develop treatments for ASD and potentially other psychiatric disorders.

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Fig. 1: CRISPR/Cas9-mediated bShank3 mutations in dogs.
Fig. 2: Reduced neuronal markers and GluN2B in bShank3 mutant brains.
Fig. 3: bShank3 mutants show impaired social interaction and increased social withdrawal in three-chamber test.
Fig. 4: Deficits of social interaction with human in bShank3 mutants.
Fig. 5: Reduced tail wagging accompanied by increased blood cortisol in bShank3 mutants.
Fig. 6: Delayed attention-seeking behavior in bShank3 mutants.

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Data availability

The raw whole-genome sequence data from founder mutants and WT parents are deposited in the Genome Sequence Archive in National Genomics Data Center, China National Center for Bioinformation, Chinese Academy of Sciences, under accession number CRA004090 and publicly accessible at https://bigd.big.ac.cn/gsa.

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Acknowledgements

We thank Prof. X. Yu, X. Li, X. Xu, K. Guo, R. Zhang and A. Andics for discussion. This work was supported in part by the Ministry of Science and Technology of China (2019YFA0707100 and 2021ZD0203901), the National Natural Science Foundation of China (31830036, 31730039, and 31921002), the National Major Scientific Instruments and Equipment Development Project (ZDYZ2015-2), the Chinese Academy of Sciences Strategic Priority Research Program B grants (XDBS1020100, and XDB32010300), and Spring City Plan: the High-level Talent Promotion and Training Project of Kunming (2022SCP001).

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Author notes

  1. Yong Q. Zhang

    Present address: School of Life Sciences, Hubei University, Wuhan, China

  2. These authors contributed equally: Rui Tian, Yuan Li, Hui Zhao.

  3. These authors jointly supervised this work: Jidong Mi, Yong-hui Jiang, Yong Q. Zhang.

Authors and Affiliations

  1. State Key Laboratory of Molecular Developmental Biology, and CAS Center for Excellence in Brain Science and Intelligence Technology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, 100101, China

    Rui Tian, Hui Zhao, Wen Lyu, Huijuan Xu, Wei Ren, Qing-quan Tan, Qi Shi & Yong Q. Zhang

  2. Beijing Sinogene Biotechnology Co. Ltd, Beijing, China

    Yuan Li, Jianping Zhao & Jidong Mi

  3. Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530, China

    Hui Zhao, Xiaomin Wang & Liangxue Lai

  4. Department of Life Science and Medicine, University of Science and Technology of China, Hefei, 230022, China

    Heng Lu

  5. State Key Laboratory of Genetic Resources and Evolution, and Center for Excellence in Animal Evolution and Genetics, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, 650201, China

    Heng Lu, Guo-dong Wang & Ya-ping Zhang

  6. Department of Genetics and Neuroscience, Yale University School of Medicine, New Haven, CT, 06510, USA

    Yong-hui Jiang

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Contributions

YHJ and YQZ conceptualized the project, supervised data collection and analysis. XW and LL performed gene targeting. HL, YZ, and GW analyzed sequencing data. HX and HZ performed Western analysis. RT, HZ, YL, and QS designed and performed behavioral experiments. JZ, ZS, JM, WL, and WR assisted data collection and analysis. RT, HZ, LY and YQZ wrote the manuscript, and YHJ and YQZ finalized the manuscript.

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Correspondence to Yong-hui Jiang or Yong Q. Zhang.

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Tian, R., Li, Y., Zhao, H. et al. Modeling SHANK3-associated autism spectrum disorder in Beagle dogs via CRISPR/Cas9 gene editing. Mol Psychiatry 28, 3739–3750 (2023). https://doi.org/10.1038/s41380-023-02276-9

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