Cytosine base editors induce off-target mutations and adverse phenotypic effects in transgenic mice

Base editors have been reported to induce off-target mutations in cultured cells, mouse embryos and rice, but their long-term effects in vivo remain unknown. Here, we develop a Systematic evaluation Approach For gene Editing tools by Transgenic mIce (SAFETI), and evaluate the off-target effects of BE3, high fidelity version of CBE (YE1-BE3-FNLS) and ABE (ABE7.10(F148A)) in similar to 400 transgenic mice over 15 months. Whole-genome sequence analysis reveals BE3 expression generated de novo mutations in the offspring of transgenic mice. RNA-seq analysis reveals both BE3 and YE1-BE3-FNLS induce transcriptome-wide SNVs, and the numbers of RNA SNVs are positively correlated with CBE expression levels across various tissues. By contrast, ABE7.10(F148A) shows no detectable off-target DNA or RNA SNVs. Notably, we observe abnormal phenotypes including obesity and developmental delay in mice with permanent genomic BE3 overexpression during long-time monitoring, elucidating a potentially overlooked aspect of side effects of BE3 in vivo.

Automated summary

We developed a systematic evaluation approach to assess the off-target effects of base editors (BE3, YE1-BE3-FNLS, and ABE7.10(F148A)) in transgenic mice. The study found that BE3 expression generated de novo mutations in the offspring of transgenic mice. Transcriptome analysis revealed that both BE3 and YE1-BE3-FNLS induced transcriptome-wide single nucleotide variants (SNVs), and the number of RNA SNVs was positively correlated with CBE expression levels. On the other hand, ABE7.10(F148A) showed no detectable off-target DNA or RNA SNVs. Importantly, long-term monitoring revealed abnormal phenotypes such as obesity and developmental delay in mice with permanent genomic BE3 overexpression, highlighting potential overlooked side effects of BE3 in vivo.