28 Feb 2019

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 vs. non-edited blastomeres at E14.5 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. In contrast, cytosine base editing induced SNVs with over 20-fold higher frequencies, requiring a solution to address its fidelity.

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Fig. 3

Characteristics of BE3 induced off-target SNVs.

(A) Off-target SNVs are enriched in the transcribed regions of the genome compared to random permutation.

(B) Genes containing off-target SNVs were significantly higher expressed than random simulated genes in 4-cell embryos.

(C) SNVs identified from each embryo were non-overlapping.

(D) Overlap among SNVs detected by GOTI with predicted off-targets by Cas-OFFinder and CRISPOR. P values were calculated by two-sided Wilcoxon rank sum test in (A) and (B).