Promoting Cas9 degradation reduces mosaic mutations in non-human primate embryos

• Authors: Zhuchi Tu, Chinese Academy of Sciences; Weili Yang, Chinese Academy of Sciences; Sen Yan, Jinan University; An Yin, Chinese Academy of Sciences; Jinquan Gao, Chinese Academy of Sciences; Xudong Liu, Chinese Academy of Sciences; Yinghui Zheng, Chinese Academy of Sciences; Jiezhao Zheng, Chinese Academy of Sciences; Zhujun Li, Yuanxi Biotech Inc.; Su Yang, Emory University; Shihua Li, Emory University; Xiangyu Guo, Jinan University; Xiao-Jiang Li, Emory University
• Journal Title: Scientific Reports
• Volume: Volume 7
• Publisher: Nature Publishing Group: Open Access Journals - Option C | 2017-02-03, Pages 42081-42081
• Type of Work: Article | Final Publisher PDF
• Publisher DOI: http://doi.org/10.1038/srep42081
• Subjects: Biology, Molecular; Biology, Genetics
• Keywords: Development of the nervous system; Functional genomics

Abstract

CRISPR-Cas9 is a powerful new tool for genome editing, but this technique creates mosaic mutations that affect the efficiency and precision of its ability to edit the genome. Reducing mosaic mutations is particularly important for gene therapy and precision genome editing. Although the mechanisms underlying the CRSIPR/Cas9-mediated mosaic mutations remain elusive, the prolonged expression and activity of Cas9 in embryos could contribute to mosaicism in DNA mutations. Here we report that tagging Cas9 with ubiquitin-proteasomal degradation signals can facilitate the degradation of Cas9 in non-human primate embryos. Using embryo-splitting approach, we found that shortening the half-life of Cas9 in fertilized zygotes reduces mosaic mutations and increases its ability to modify genomes in non-human primate embryos. Also, injection of modified Cas9 in one-cell embryos leads to live monkeys with the targeted gene modifications. Our findings suggest that modifying Cas9 activity can be an effective strategy to enhance precision genome editing.