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Cytosine base editor generates substantial off-target single-nucleotide variants in mouse embryos.

ABSTRACT: Genome editing holds promise for correcting pathogenic mutations. However, it is difficult to determine off-target effects of editing due to single-nucleotide polymorphism in individuals. Here we developed a method named GOTI (genome-wide off-target analysis by two-cell embryo injection) to detect off-target mutations by editing one blastomere of two-cell mouse embryos using either CRISPR-Cas9 or base editors. Comparison of the whole-genome sequences of progeny cells of edited and nonedited blastomeres at embryonic day 14.5 showed that off-target single-nucleotide variants (SNVs) were rare in embryos edited by CRISPR-Cas9 or adenine base editor, with a frequency close to the spontaneous mutation rate. By contrast, cytosine base editing induced SNVs at more than 20-fold higher frequencies, requiring a solution to address its fidelity.

SUBMITTER: Zuo E

PROVIDER: S-EPMC7301308 | biostudies-literature | 2019 Apr

REPOSITORIES: biostudies-literature

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Cytosine base editor generates substantial off-target single-nucleotide variants in mouse embryos.

Zuo Erwei E Zuo Erwei E Sun Yidi Y Wei Wu W Yuan Tanglong T Ying Wenqin W Sun Hao H Yuan Liyun L Steinmetz Lars M LM Li Yixue Y Yang Hui H Yang Hui H

Science (New York, N.Y.) 20190228 6437

Genome editing holds promise for correcting pathogenic mutations. However, it is difficult to determine off-target effects of editing due to single-nucleotide polymorphism in individuals. Here we developed a method named GOTI (genome-wide off-target analysis by two-cell embryo injection) to detect off-target mutations by editing one blastomere of two-cell mouse embryos using either CRISPR-Cas9 or base editors. Comparison of the whole-genome sequences of progeny cells of edited and nonedited blas ...[more]

PMID: 30819928

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Cytosine base editor 4 but not adenine base editor generates off-target mutations in mouse embryos.

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Cytosine base editor generates substantial off-target single nucleotide variants in mouse embryos

Project description:Genome editing holds promise for correcting pathogenic mutations. However, it is difficult to determine off-target effects of editing due to single-nucleotide polymorphism in individuals. Here we developed a method named GOTI (genome-wide off-target analysis by two-cell embryo injection) to detect off-target mutations by editing one blastomere of two-cell mouse embryos using either CRISPR-Cas9 or base editors. Comparison of the whole-genome sequences of progeny cells of edited and nonedited blastomeres at embryonic day 14.5 showed that off-target single-nucleotide variants (SNVs) were rare in embryos edited by CRISPR-Cas9 or adenine base editor, with a frequency close to the spontaneous mutation rate. By contrast, cytosine base editing induced SNVs at more than 20-fold higher frequencies, requiring a solution to address its fidelity.

| OEX001547 | NODE

Cytosine base editor generates substantial off-target single nucleotide variants in mouse embryos

| OEX001548 | NODE

Targeting fidelity of adenine and cytosine base editors in mouse embryos.

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Project description:Base Editor (BE) and Target-AID (activation-induced cytidine deaminase) are engineered genome-editing proteins composed of Cas9 and cytidine deaminases. These base-editing tools convert C:G base pairs to T:A at target sites. Here, we inject either BE or Target-AID mRNA together with identical single-guide RNAs (sgRNAs) into mouse zygotes, and compare the base-editing efficiencies of the two distinct tools in vivo. BE consistently show higher base-editing efficiency (10.0-62.8%) compared to that of Target-AID (3.4-29.8%). However, unexpected base substitutions and insertion/deletion formations are also more frequently observed in BE-injected mice or zygotes. We are able to generate multiple mouse lines harboring point mutations in the mouse presenilin 1 (Psen1) gene by injection of BE or Target-AID. These results demonstrate that BE and Target-AID are highly useful tools to generate mice harboring pathogenic point mutations and to analyze the functional consequences of the mutations in vivo.

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Cytosine Base Editor (hA3A-BE3-NG)-Mediated Multiple Gene Editing for Pyramid Breeding in Pigs.

Project description:Pig is an important agricultural economic animal, providing large amount of meat products. With the development of functional genomics and bioinformatics, lots of genes and functional single nucleotide polymorphisms (SNPs) related to disease resistance and (or) economic traits in pigs have been identified, which provides the targets for genetic improvement by genome editing. Base editors (BEs), combining Cas9 nickase and cytidine or adenine deaminase, achieve all four possible transition mutations (C-to-T, A-to-G, T-to-C, and G-to-A) efficiently and accurately without double strand breaks (DSBs) under the protospacer adjacent motif (PAM) sequence of NGG. However, the NGG PAM in canonical CRISPR-Cas9 can only cover approximately 8.27% in the whole genome which limits its broad application. In the current study, hA3A-BE3-NG system was constructed with the fusion of SpCas9-NG variant and hA3A-BE3 to create C-to-T conversion at NGN PAM sites efficiently. The editing efficiency and scope of hA3A-BE3-NG were confirmed in HEK293T cells and porcine fetal fibroblast (PFF) cells. Results showed that the efficiency of hA3A-BE3-NG was much higher than that of hA3A-BE3 on NGH (H = A, C, or T) PAM sites (21.27 vs. 2.81% at average). Further, nonsense and missense mutations were introduced efficiently and precisely via hA3A-BE3-NG in multiple pig economic trait-related genes (CD163, APN, MSTN, and MC4R) in PFF cells by one transfection. The current work indicates the potential applications of hA3A-BE3-NG for pyramid breeding studies in livestock.

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