Efficient base editing by RNA-guided cytidine base editors (CBEs) in pigs
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.
KeywordsBase editing Cytidine base editors (CBEs) Pigs BE4-Gam AncBE4max
The authors thank Zhuang Shao, Chuang Gao, and Kang Yang for assistance at the Embryo Engineering Center for the critical technical assistance.
Conceived and designed the experiments: HO and DP. Performed the experiments: HY, TY, LW, LY, YZ, HL, ML, XT, ZL, ZL, CL, and XC. Wrote the manuscript: HO and DP. All authors reviewed the manuscript.
This work was supported by Special Funds for Cultivation and Breeding of New Transgenic Organisms (No. 2016ZX08006001), the Program for Changjiang Scholars and Innovative Research Team in University (PCSIRT, No. IRT_16R32), the Program for JLU Science and Technology Innovative Research Team (2017TD-28), and the Fundamental Research Funds for the Central Universities.
Compliance with ethical standards
Conflict of interest
The authors declare no competing interest.
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