Cytosine base editors induce off-target mutations and adverse phenotypic effects in transgenic mice
Nana Yan, Feng Hu, Yongsen Sun, Ying Xin, Haihang Zhang, Hongjiang Lu, Jitan Zheng, Chenfei He, Zhenrui Zuo, Tanglong Yuan, Nana Li, Long Xie, Wei Wu, Yidi Sun, Erwei Zuo, Yidi Sun, Erwei Zuo
Abstract
Abstract 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 ~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.