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

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.

SUBMITTER: Wei Y

PROVIDER: S-EPMC10246723 | biostudies-literature | 2023 Jun

REPOSITORIES: biostudies-literature

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

Wei Yinghui Y Zhang Meiling M Hu Jing J Zhou Yingsi Y Xue Mingxing M Yin Jianhang J Liu Yuanhua Y Feng Hu H Zhou Ling L Li Zhifang Z Wang Dongshuang D Zhang Zhiguo Z Zhou Yin Y Liu Hongbin H Yao Ning N Zuo Erwei E Hu Jiazhi J Du Yanzhi Y Li Wen W Xu Chunlong C Yang Hui H Yang Hui H

Protein & cell 20230601 6

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 cy ...[more]

PMID: 37285261

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Project description:Base editing is an efficient and precise gene-editing technique, by which a single base can be changed without introducing double-strand breaks, and it is currently widely used in studies of various species. In this study, we used hA3A-BE3-Y130F to simultaneously introduce premature stop codons (TAG, TGA, and TAA) into three tumor suppressor genes, TP53, PTEN, and APC, in large white porcine fetal fibroblasts (PFFs). Among the isolated 290 single-cell colonies, 232 (80%) had premature stop codons in all the three genes. C-to-T conversion was found in 98.6%, 92.8%, and 87.2% of these cell colonies for TP53, PTEN, and APC, respectively. High frequencies of bystander C-to-T edits were observed within the editing window (positions 3-8), and there were nine (3.01%) clones with the designed simultaneous three-gene C-to-T conversion without bystander conversion. C-to-T conversion outside the editing window was found in 9.0%, 14.1%, and 26.2% of the 290 cell colonies for TP53, PTEN, and APC, respectively. Low-frequency C-to-G or C-to-A transversion occurred in APC. The mRNA levels of the three genes showed significant declines in triple-gene-mutant (Tri-Mut) cells as expected. No PTEN and a significantly lower (p < 0.05) APC protein expression were detected in Tri-Mut cells. Interestingly, the premature stop codon introduced into the TP53 gene did not eliminate the expression of its full-length protein in the Tri-Mut cells, suggesting that stop codon read-through occurred. Tri-Mut cells showed a significantly higher (p < 0.05) proliferation rate than WT cells. Furthermore, we identified 1418 differentially expressed genes (DEGs) between the Tri-Mut and WT groups, which were mainly involved in functions such as tumor progression, cell cycle, and DNA repair. This study indicates that hA3A-BE3-Y130F can be a powerful tool to create diverse knockout cell models without double-strand breaks (DSBs), with further possibilities to produce porcine models with various purposes.

Dataset Information

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

Publications

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

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