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.

Fig. 1.. CRISPR- Cas9–, BE3-, or ABE7.10-mediated gene editing in one blastomere of two-cell embryos.

(A) Experimental design. 057, 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.