Efficient base editing by RNA-guided cytidine base editors (CBEs) in pigs

  • Hongming Yuan
  • Tingting Yu
  • Lingyu Wang
  • Lin Yang
  • Yuanzhu Zhang
  • Huan Liu
  • Mengjing Li
  • Xiaochun Tang
  • Zhiquan Liu
  • Zhanjun Li
  • Chao Lu
  • Xue Chen
  • Daxin PangEmail author
  • Hongsheng OuyangEmail author
Original Article


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.


Base 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.

Author contributions

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.

Supplementary material

18_2019_3205_MOESM1_ESM.tif (10.4 mb)
Supplementary Figure 1 Targeted base conversion in porcine blastocysts with BE4-Gam by injecting single sgRNA. A-C The DNA fragments of GGTA1 (A), B4galNT2 (B) and CMAH (C) from the porcine blastocysts were sub-cloned into pGM-T vectors and then sequenced, respectively. The number of clones for each sequence pattern is indicated. The targeted sequence was underlined, the PAM sequence was shown with green and substituted nucleotide was shown with red. (TIFF 10622 kb)
18_2019_3205_MOESM2_ESM.tif (15.5 mb)
Supplementary Figure 2 Targeted base conversion in porcine blastocysts with BE4-Gam by injecting three gene sgRNAs. A-C DNA fragments of GGTA1 (A), B4galNT2 (B) and CMAH (C) from the porcine blastocysts were sub-cloned into pGM-T vectors and sequenced, respectively. The number of clones for each sequence pattern is indicated. The targeted sequence was underlined, the PAM sequence was shown in green and substituted nucleotide was shown in red. (TIFF 15896 kb)
18_2019_3205_MOESM3_ESM.tif (9 mb)
Supplementary Figure 3 The results of deep sequencing from six IVF derived pigs. A-C The results of deep sequencing of GGTA1 (A), B4galNT2 (B) and CMAH (C)genes from all IVF derived F0 pigs, respectively. At each position, 1170407-1562083 sequencing reads were used. (TIFF 9229 kb)
18_2019_3205_MOESM4_ESM.tif (2.3 mb)
Supplementary Figure 4 Off-target detection in the pig # 420. Chromatogram sequence analysis of potential off-target sites (POTS) for sgRNA in the pig #420 genome of GGTA1 (A), B4galNT2 (B), CMAH (C), respectively. 20 bp of the POTS and the PAM are represented in shadow. (TIFF 2333 kb)
18_2019_3205_MOESM5_ESM.tif (10.6 mb)
Supplementary Figure 5 Off-target detection in the pig # 502. Chromatogram sequence analysis of potential off-target sites (POTS) for sgRNA in the pig #502 genome of GGTA1 (A), B4galNT2 (B), CMAH (C), respectively. 20 bp of the POTS and the PAM are represented in shadow. (TIFF 10862 kb)
18_2019_3205_MOESM6_ESM.tif (20.1 mb)
Supplementary Figure 6 Efficiently multiple genes base editing in porcine parthenogenesis embryo with AncBE4max. A-F The genotypes of GGTA1, B4galNT2 and CMAH mutant blastocysts which injected AncBE4max and three gene sgRNAs, shown in A, C and E, respectively. The genotypes of GGTA1, B4galNT2 and CMAH mutant blastocysts which injected BE4-Gam and three gene sgRNAs, shown in B, D, F respectively. The number of clones for each sequence pattern is indicated. Target sequence (underlined), PAM region (green), and substituted nucleotide (red). (TIFF 20544 kb)
18_2019_3205_MOESM7_ESM.tif (13.9 mb)
Supplementary Figure 7 Off-target detection in the Anc-18 PFFs. A Representative sequencing chromatograms at the GGTA1, B4galNT2, and CMAH targets of WT and Bama miniature Anc-18 PFFs after transfected three gene sgRNAs and Anc-BE4max express plasmid. Target amino acid are indicated by red box. B-D Chromatogram sequence analysis of potential off-target sites (POTS) for sgRNA in the pig #502 genome of GGTA1 (B), B4galNT2 (C), CMAH (D), respectively. 20 bp of the POTS and the PAM are represented in shadow. (TIFF 14256 kb)
18_2019_3205_MOESM8_ESM.docx (16 kb)
Supplementary material 8 (DOCX 15 kb)
18_2019_3205_MOESM9_ESM.xlsx (14 kb)
Supplementary material 9 (XLSX 13 kb)


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Copyright information

© Springer Nature Switzerland AG 2019

Authors and Affiliations

  1. 1.Key Lab for Zoonoses Research, Ministry of Education, Jilin Provincial Key Laboratory of Animal Embryo Engineering, College of Animal SciencesJilin UniversityChangchunPeople’s Republic of China

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