Abstract
Cytidine base editors (CBEs) have been demonstrated to be useful for precisely inducing C:G-to-T:A base mutations in various organisms. In this study, we showed that the BE4-Gam system induced the targeted C-to-T base conversion in porcine blastocysts at an efficiency of 66.7-71.4% via the injection of a single sgRNA targeting a xeno-antigen-related gene and BE4-Gam mRNA. Furthermore, the efficiency of simultaneous three gene base conversion via the injection of three targeting sgRNAs and BE4-Gam mRNA into porcine parthenogenetic embryos was 18.1%. We also obtained beta-1,4-N-acetyl-galactosaminyl transferase 2, alpha-1,3-galactosyltransferase, and cytidine monophosphate-N-acetylneuraminic acid hydroxylase deficient pig by somatic cell nuclear transfer, which exhibited significantly decreased activity. In addition, a new CBE version (termed AncBE4max) was used to edit genes in blastocysts and porcine fibroblasts (PFFs) for the first time. While this new version demonstrated a three genes base-editing rate of 71.4% at the porcine GGTA1, B4galNT2, and CMAH loci, it increased the frequency of bystander edits, which ranged from 17.8 to 71.4%. In this study, we efficiently and precisely mutated bases in porcine blastocysts and PFFs using CBEs and successfully generated C-to-T and C-to-G mutations in pigs. These results suggest that CBEs provide a more simple and efficient method for improving economic traits, reducing the breeding cycle, and increasing disease tolerance in pigs, thus aiding in the development of human disease models.