Figure 1

Yaowu Zheng

and 12 more

Most human genetic diseases arise from point mutations with G:C>A:T or T:A>C:G base changes and they represent nearly half of the pathogenic single-nucleotide polymorphisms (SNPs). Animal models for human genetic diseases are important in dissecting pathogenic mechanism, drug screening, and drug efficacy testing. Mouse models are mostly generated by traditional gene knockout that is costly and time-consuming. CRISPR/Cas9 is a recently developed system that is efficient and cost-effective in generating genetic deletion, insertion and point mutation. It has been widely used to generate mouse models with deletions and point mutations. But, size and location of deletions by CRISPR/Cas9 is unpredictable. Mouse models of point mutation are still the best human disease models which can precisely mimic human pathology. Cytidine base editor BE4 is a newly developed version of cytidine base editing system. It has a cytidine deaminase and two uracil glycosylase inhibitors fused to C terminus of Cas9n, A cas9 mutant with D10A amino acid change. BE4 enables direct conversion of cytidine (C) to uridine (U) in targeted bases of DNA sequence. But this system has never been tested in vivo. In this study, we have confirmed that BE4 system can introduce site-specific and single-base substitution with high precision and efficiency in mouse. The designed nonsense mutation has a high efficiency up to 56.25%. Results confirm BE4 system has great potentials in modeling human genetic diseases and pharmaceutical screenings.