Precise allele-specific genome editing by spatiotemporal control of CRISPR-Cas9 via pronuclear transplantation

Yanhe Li, Yuteng Weng, Dandan Bai, Yanping Jia, Yingdong Liu, Yalin Zhang, Xiaochen Kou, Yanhong Zhao, Jingling Ruan, Jiayu Chen, Jiqing Yin, Hong Wang, Xiaoming Teng, Zuolin Wang, Wenqiang Liu () and Shaorong Gao ()
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Yanhe Li: Tongji University
Yuteng Weng: Tongji University
Dandan Bai: Tongji University
Yanping Jia: Tongji University
Yingdong Liu: Tongji University
Yalin Zhang: Tongji University
Xiaochen Kou: Tongji University
Yanhong Zhao: Tongji University
Jingling Ruan: Tongji University
Jiayu Chen: Tongji University
Jiqing Yin: Tongji University
Hong Wang: Tongji University
Xiaoming Teng: Tongji University
Zuolin Wang: Tongji University
Wenqiang Liu: Tongji University
Shaorong Gao: Tongji University

Nature Communications, 2020, vol. 11, issue 1, 1-12

Abstract: Abstract Gene-targeted animal models that are generated by injecting Cas9 and sgRNAs into zygotes are often accompanied by undesired double-strand break (DSB)-induced byproducts and random biallelic targeting due to uncontrollable Cas9 targeting activity. Here, we establish a parental allele-specific gene-targeting (Past-CRISPR) method, based on the detailed observation that pronuclear transfer-mediated cytoplasmic dilution can effectively terminate Cas9 activity. We apply this method in embryos to efficiently target the given parental alleles of a gene of interest and observed little genomic mosaicism because of the spatiotemporal control of Cas9 activity. This method allows us to rapidly explore the function of individual parent-of-origin effects and to construct animal models with a single genomic change. More importantly, Past-CRISPR could also be used for therapeutic applications or disease model construction.

Date: 2020
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DOI: 10.1038/s41467-020-18391-y

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