Mol. Cells

Targeted Base Editing via RNA-Guided Cytidine Deaminases in Xenopus laevis Embryos

Dong-Seok Park, Mijung Yoon, Jiyeon Kweon, An-Hee Jang, Yongsub Kim, and Sun-Cheol Choi

Additional article information


Genome editing using programmable nucleases such as CRISPR/Cas9 or Cpf1 has emerged as powerful tools for gene knock-out or knock-in in various organisms. While most genetic diseases are caused by point mutations, these genome-editing approaches are inefficient in inducing single-nucleotide substitutions. Recently, Cas9-linked cytidine deaminases, named base editors (BEs), have been shown to convert cytidine to uridine efficiently, leading to targeted single-base pair substitutions in human cells and organisms. Here, we first report on the generation of Xenopus laevis mutants with targeted single-base pair substitutions using this RNA-guided programmable deaminase. Injection of base editor 3 (BE3) ribonucleoprotein targeting the tyrosinase (tyr) gene in early embryos can induce site-specific base conversions with the rates of up to 20.5%, resulting in oculocutaneous albinism phenotypes without off-target mutations. We further test this base-editing system by targeting the tp53 gene with the result that the expected single-base pair substitutions are observed at the target site. Collectively, these data establish that the programmable deaminases are efficient tools for creating targeted point mutations for human disease modeling in Xenopus.

Keywords: base editing, CRISPR/Cas9, genome engineering, Xenopus laevis

Supplementary data

Article information

Mol. Cells.Nov 30, 2017; 40(11): 823-827.
Published online 2017-11-20. doi:  10.14348/molcells.2017.0262
1Department of Biomedical Sciences, University of Ulsan College of Medicine, Seoul 05505, Korea
*Correspondence: (YK); (SCC)
Received October 17, 2017; Accepted November 6, 2017.
Articles from Mol. Cells are provided here courtesy of Mol. Cells


  • Aslan, Y., Tadjuidje, E., Zorn, A.M., and Cha, S.W. (2017). High-efficiency non-mosaic CRISPR-mediated knock-in and indel mutation in F0 Xenopus. Development. 144, 2852-2858.
  • Bae, S., Park, J., and Kim, J.S. (2014). Cas-OFFinder: a fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases. Bioinformatics. 30, 1473-1475.
  • Blitz, I.L., Biesinger, J., Xie, X., and Cho, K.W. (2013). Biallelic genome modification in F(0) Xenopus tropicalis embryos using the CRISPR/Cas system. Genesis. 51, 827-834.
  • Harland, R.M., and Grainger, R.M. (2011). Xenopus research: metamorphosed by genetics and genomics. Trends Genet. 27, 507-515.
  • Kim, H., and Kim, J.S. (2014). A guide to genome engineering with programmable nucleases. Nat Rev Genet. 15, 321-334.
  • Kim, Y., Cheong, S.A., Lee, J.G., Lee, S.W., Lee, M.S., Baek, I.J., and Sung, Y.H. (2016). Generation of knockout mice by Cpf1-mediated gene targeting. Nat Biotechnol. 34, 808-810.
  • Kim, K., Ryu, S.M., Kim, S.T., Baek, G., Kim, D., Lim, K., Chung, E., Kim, S., and Kim, J.S. (2017a). Highly efficient RNA-guided base editing in mouse embryos. Nat Biotechnol. 35, 435-437.
  • Kim, Y.B., Komor, A.C., Levy, J.M., Packer, M.S., Zhao, K.T., and Liu, D.R. (2017b). Increasing the genome-targeting scope and precision of base editing with engineered Cas9-cytidine deaminase fusions. Nat Biotechnol. 35, 371-376.
  • Komor, A.C., Kim, Y.B., Packer, M.S., Zuris, J.A., and Liu, D.R. (2016). Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature. 533, 420-424.
  • Lei, Y., Guo, X., Liu, Y., Cao, Y., Deng, Y., Chen, X., Cheng, C.H., Dawid, I.B., Chen, Y., and Zhao, H. (2012). Efficient targeted gene disruption in Xenopus embryos using engineered transcription activator-like effector nucleases (TALENs). Proc Natl Acad Sci USA. 109, 17484-17489.
  • Liang, P., Sun, H., Sun, Y., Zhang, X., Xie, X., Zhang, J., Zhang, Z., Chen, Y., Ding, C., and Xiong, Y. (2017). Effective gene editing by high-fidelity base editor 2 in mouse zygotes. Protein Cell. 8, 601-611.
  • Ma, Y., Zhang, J., Yin, W., Zhang, Z., Song, Y., and Chang, X. (2016). Targeted AID-mediated mutagenesis (TAM) enables efficient genomic diversification in mammalian cells. Nat Methods. 13, 1029-1035.
  • Nakayama, T., Fish, M.B., Fisher, M., Oomen-Hajagos, J., Thomsen, G.H., and Grainger, R.M. (2013). Simple and efficient CRISPR/Cas9-mediated targeted mutagenesis in Xenopus tropicalis. Genesis. 51, 835-843.
  • Nishida, K., Arazoe, T., Yachie, N., Banno, S., Kakimoto, M., Tabata, M., Mochizuki, M., Miyabe, A., Araki, M., and Hara, K.Y. (2016). Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems. Science. 353, pii: aaf8729.
  • Rees, H.A., Komor, A.C., Yeh, W.H., Caetano-Lopes, J., Warman, M., Edge, A.S.B., and Liu, D.R. (2017). Improving the DNA specificity and applicability of base editing through protein engineering and protein delivery. Nat Commun. 8, 15790.
  • Sakane, Y., Sakuma, T., Kashiwagi, K., Kashiwagi, A., Yamamoto, T., and Suzuki, K.T. (2014). Targeted mutagenesis of multiple and paralogous genes in Xenopus laevis using two pairs of transcription activator-like effector nucleases. Dev Growth Differ. 56, 108-114.
  • Shimatani, Z., Kashojiya, S., Takayama, M., Terada, R., Arazoe, T., Ishii, H., Teramura, H., Yamamoto, T., Komatsu, H., and Miura, K. (2017). Targeted base editing in rice and tomato using a CRISPR-Cas9 cytidine deaminase fusion. Nat Biotechnol. 35, 441-443.
  • Young, J.J., Cherone, J.M., Doyon, Y., Ankoudinova, I., Faraji, F.M., Lee, A.H., Ngo, C., Guschin, D.Y., Paschon, D.E., and Miller, J.C. (2011). Efficient targeted gene disruption in the soma and germ line of the frog Xenopus tropicalis using engineered zinc-finger nucleases. Proc Natl Acad Sci USA. 108, 7052-7057.
  • Zhang, Y., Qin, W., Lu, X., Xu, J., Huang, H., Bai, H., Li, S., and Lin, S. (2017). Programmable base editing of zebrafish genome using a modified CRISPR-Cas9 system. Nat Commun. 8, 118.
  • Zong, Y., Wang, Y., Li, C., Zhang, R., Chen, K., Ran, Y., Qiu, J.L., Wang, D., and Gao, C. (2017). Precise base editing in rice, wheat and maize with a Cas9-cytidine deaminase fusion. Nat Biotechnol. 35, 438-440.

Figure 1

Figure 2