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

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Science  19 Apr 2019:
Vol. 364, Issue 6437, pp. 289-292
DOI: 10.1126/science.aav9973
  • Fig. 1 CRISPR-Cas9–, BE3-, or ABE7.10-mediated gene editing in one blastomere of two-cell embryos.

    (A) Experimental design. C57, an inbred strain of mice. (B) FACS analysis in indicated embryos. (C) Percentage of on-target efficiency for tdTomato+ and tdTomato cells on the basis of WGS. On-target efficiencies of Cas9, BE3, and ABE7.10 in tdTomato+ cells were 66 ± 12% SEM indels (n = 5), 83 ± 10% SEM nucleotide substitutions (n = 4), and 47 ± 18% SEM nucleotide substitutions (n = 2), respectively.

  • Fig. 2 Substantial off-target SNVs generated in BE3-treated mouse embryos.

    (A) Comparison of the total number of detected off-target SNVs. The number of SNVs for Cre-, Cas9-, BE3-, and ABE7.10-treated embryos were 14 ± 12 SEM (n = 2), 12 ± 4 SEM (n = 11), 283 ± 32 SEM (n = 6), and 10 ± 5 SEM (n = 4) SNVs, respectively. (B) Distribution of mutation types. The number in each cell indicates the proportion of a certain type of mutation among all mutations. (C) Proportion of G·C to A·T mutations for Cre, Cas9, BE3, and ABE7.10 groups. (D) Proportion of A·T to G·C mutations for Cre, Cas9, BE3, and ABE7.10 groups. Two Cre, 11 Cas9, 6 BE3, and 4 ABE7.10 samples were analyzed. In (A), (C), and (D), the P values shown above the horizontal bars were calculated by two-sided Wilcoxon rank sum test, and error bars indicate SEM.

  • Fig. 3

    Characteristics of BE3-induced off-target SNVs. (A) Off-target SNVs are enriched in the transcribed regions of the genome compared with random permutation. (B) Genes containing off-target SNVs were significantly more highly expressed than random simulated genes in four-cell embryos. RSEM, RNA sequencing by expectation maximization. (C) SNVs identified from each embryo were nonoverlapping. (D) Overlap among SNVs detected by GOTI with predicted off-targets by Cas-OFFinder and CRISPOR. In (A) and (B), P values were calculated by two-sided Wilcoxon rank sum test.

Supplementary Materials

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

    Erwei Zuo, Yidi Sun, Wu Wei, Tanglong Yuan, Wenqin Ying, Hao Sun, Liyun Yuan, Lars M. Steinmetz, Yixue Li, Hui Yang

    Materials/Methods, Supplementary Text, Tables, Figures, and/or References

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    • Materials and Methods
    • Figs. S1 to S15
    • Tables S1 to S5 and S8 to S12
    • Captions for Tables S6 and S7
    • References
    Table S6
    SNVs detected in Cre-, CRISPR-Cas9- and ABE7.10-treated samples.
    Table S7
    SNVs detected in all BE3-treated samples.

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