Human 8-cell embryos enable efficient induction of disease-preventive mutations without off-target effect by cytosine base editor.

Yinghui Wei; Meiling Zhang; Jing HU; Yingsi Zhou; Mingxing Xue; Jianhang Yin; Yuanhua Liu; Hu Feng; Ling Zhou; Zhifang LI; Dongshuang Wang; Zhiguo Zhang; Yin Zhou; Hongbin Liu; Ning Yao; Erwei Zuo; Jiazhi HU; Yanzhi DU; Wen LI; Chunlong XU; Hui Yang

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Human 8-cell embryos enable efficient induction of disease-preventive mutations without off-target effect by cytosine base editor.

Author: Yinghui WEI ¹ ; Meiling ZHANG ¹ ; Jing HU ² ; Yingsi ZHOU ² ; Mingxing XUE ² ; Jianhang YIN ³ ; Yuanhua LIU ² ; Hu FENG ⁴ ; Ling ZHOU ⁴ ; Zhifang LI ⁴ ; Dongshuang WANG ⁵ ; Zhiguo ZHANG ⁶ ; Yin ZHOU ⁵ ; Hongbin LIU ⁷ ; Ning YAO ⁵ ; Erwei ZUO ⁴ ; Jiazhi HU ³ ; Yanzhi DU ⁵ ; Wen LI ¹ ; Chunlong XU ⁸ ; Hui YANG ²
Author Information

1. Center for Reproductive Medicine, International Peace Maternity and Child Health Hospital, Innovative Research Team of High-level Local Universities in Shanghai, School of Medicine, Shanghai Jiao Tong University, Shanghai 200030, China.
2. Institute of Neuroscience, State Key Laboratory of Neuroscience, Key Laboratory of Primate Neurobiology, CAS Center for Excellence in Brain Science and Intelligence Technology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China.
3. The MOE Key Laboratory of Cell Proliferation and Differentiation, School of Life Sciences, Genome Editing Research Center, Peking University, Beijing 100871, China.
4. Shenzhen Branch, Guangdong Laboratory for Lingnan Modern Agriculture, Genome Analysis Laboratory of the Ministry of Agriculture, Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences, Shenzhen 518000, China.
5. Center for Reproductive Medicine, Ren Ji Hospital, School of Medicine, Shanghai Key Laboratory for Assisted Reproduction and Reproductive Genetics, Shanghai Jiao Tong University, Shanghai 200127, China.
6. Reproductive Medicine Center, Department of Obstetrics and Gynecology, the First Affiliated Hospital of Anhui Medical University, Hefei 230022, China.
7. Center for Reproductive Medicine, Shandong University, Jinan 250012, China.
8. Lingang Laboratory, Shanghai Research Center for Brain Science and Brain-Inspired Intelligence Technology, Shanghai 200031, China.
Publication Type:Research Support, Non-U.S. Gov't
Keywords: APOE4; base editor; disease-preventive mutations; human embryo
MeSH: Humans; Apolipoprotein E4/genetics*; Cytosine; Mutation; Blastocyst; Heterozygote; Gene Editing; CRISPR-Cas Systems
From: Protein & Cell 2023;14(6):416-432
CountryChina
Language:English
Abstract: Approximately 140 million people worldwide are homozygous carriers of APOE4 (ε4), a strong genetic risk factor for late onset familial and sporadic Alzheimer's disease (AD), 91% of whom will develop AD at earlier age than heterozygous carriers and noncarriers. Susceptibility to AD could be reduced by targeted editing of APOE4, but a technical basis for controlling the off-target effects of base editors is necessary to develop low-risk personalized gene therapies. Here, we first screened eight cytosine base editor variants at four injection stages (from 1- to 8-cell stage), and found that FNLS-YE1 variant in 8-cell embryos achieved the comparable base conversion rate (up to 100%) with the lowest bystander effects. In particular, 80% of AD-susceptible ε4 allele copies were converted to the AD-neutral ε3 allele in human ε4-carrying embryos. Stringent control measures combined with targeted deep sequencing, whole genome sequencing, and RNA sequencing showed no DNA or RNA off-target events in FNLS-YE1-treated human embryos or their derived stem cells. Furthermore, base editing with FNLS-YE1 showed no effects on embryo development to the blastocyst stage. Finally, we also demonstrated FNLS-YE1 could introduce known protective variants in human embryos to potentially reduce human susceptivity to systemic lupus erythematosus and familial hypercholesterolemia. Our study therefore suggests that base editing with FNLS-YE1 can efficiently and safely introduce known preventive variants in 8-cell human embryos, a potential approach for reducing human susceptibility to AD or other genetic diseases.