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Sci Rep. 2017 Feb 3;7:42081. doi: 10.1038/srep42081.

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

Tu Z1,2, Yang W1,2, Yan S2, Yin A1, Gao J1, Liu X1, Zheng Y1, Zheng J3, Li Z3, Yang S4, Li S4, Guo X2, Li XJ1,2,4.

Author information

1
State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, 100101 China.
2
Guangdong-Hongkong-Macau Institute of CNS Regeneration, Ministry of Education CNS Regeneration Collaborative Joint Laboratory, Jinan University, Guangzhou, 510632, China.
3
Yuanxi Biotech Inc., Guangzhou, 510663 China.
4
Department of Human Genetics, Emory University School of Medicine, Atlanta, GA 30322, USA.

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.

PMID:
28155910
PMCID:
PMC5290468
DOI:
10.1038/srep42081
[Indexed for MEDLINE]
Free PMC Article

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