Promoting Cas9 degradation reduces mosaic mutations in non-human primate embryos.
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.
State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, 100101 China.|Guangdong-Hongkong-Macau Institute of CNS Regeneration, Ministry of Education CNS Regeneration Collaborative Joint Laboratory, Jinan University, Guangzhou, 510632, China.
Zhuchi
Z
Tu
State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, 100101 China.|Guangdong-Hongkong-Macau Institute of CNS Regeneration, Ministry of Education CNS Regeneration Collaborative Joint Laboratory, Jinan University, Guangzhou, 510632, China.
Weili
W
Yang
Guangdong-Hongkong-Macau Institute of CNS Regeneration, Ministry of Education CNS Regeneration Collaborative Joint Laboratory, Jinan University, Guangzhou, 510632, China.
Sen
S
Yan
State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, 100101 China.
An
A
Yin
State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, 100101 China.
Jinquan
J
Gao
State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, 100101 China.
Xudong
X
Liu
State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, 100101 China.
Yinghui
Y
Zheng
Yuanxi Biotech Inc., Guangzhou, 510663 China.
Jiezhao
J
Zheng
Yuanxi Biotech Inc., Guangzhou, 510663 China.
Zhujun
Z
Li
Department of Human Genetics, Emory University School of Medicine, Atlanta, GA 30322, USA.
Su
S
Yang
Department of Human Genetics, Emory University School of Medicine, Atlanta, GA 30322, USA.
Shihua
S
Li
Guangdong-Hongkong-Macau Institute of CNS Regeneration, Ministry of Education CNS Regeneration Collaborative Joint Laboratory, Jinan University, Guangzhou, 510632, China.
Xiangyu
X
Guo
State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, 100101 China.|Guangdong-Hongkong-Macau Institute of CNS Regeneration, Ministry of Education CNS Regeneration Collaborative Joint Laboratory, Jinan University, Guangzhou, 510632, China.|Department of Human Genetics, Emory University School of Medicine, Atlanta, GA 30322, USA.
Xiao-Jiang
XJ
Li
D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D000076987:CRISPR-Associated Protein 9; D004720:Endonucleases / Q000235:genetics / Q000378:metabolism*; D000072669:Gene Editing / Q000379:methods*; D005786:Gene Expression Regulation; D008967:Molecular Biology / Q000379:methods*; D009030:Mosaicism; D009154:Mutation; D011323:Primates / Q000196:embryology*; D059748:Proteolysis; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D025801:Ubiquitin / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; D025801:Ubiquitin; D000076987:CRISPR-Associated Protein 9; C000606107:Cas9 endonuclease Streptococcus pyogenes; D004720:Endonucleases