Browse > Article
http://dx.doi.org/10.14348/molcells.2017.0262

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

Park, Dong-Seok (Department of Biomedical Sciences, University of Ulsan College of Medicine)
Yoon, Mijung (Department of Biomedical Sciences, University of Ulsan College of Medicine)
Kweon, Jiyeon (Department of Biomedical Sciences, University of Ulsan College of Medicine)
Jang, An-Hee (Department of Biomedical Sciences, University of Ulsan College of Medicine)
Kim, Yongsub (Department of Biomedical Sciences, University of Ulsan College of Medicine)
Choi, Sun-Cheol (Department of Biomedical Sciences, University of Ulsan College of Medicine)
Publication Information
Molecules and Cells / v.40, no.11, 2017 , pp. 823-827 More about this Journal
Abstract
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;
Citations & Related Records
연도 인용수 순위
  • Reference
1 Young, J.J., Cherone, J.M., Doyon, Y., Ankoudinova, I., Faraji, F.M., Lee, A.H., Ngo, C., Guschin, D.Y., Paschon, D.E., Miller, J.C., et al. (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.   DOI
2 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.   DOI
3 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.   DOI
4 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.   DOI
5 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.   DOI
6 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.   DOI
7 Harland, R.M., and Grainger, R.M. (2011). Xenopus research: metamorphosed by genetics and genomics. Trends Genet. 27, 507-515.   DOI
8 Kim, H., and Kim, J.S. (2014). A guide to genome engineering with programmable nucleases. Nat. Rev. Genet. 15, 321-334.   DOI
9 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.   DOI
10 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.   DOI
11 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.   DOI
12 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.   DOI
13 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.   DOI
14 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.   DOI
15 Liang, P., Sun, H., Sun, Y., Zhang, X., Xie, X., Zhang, J., Zhang, Z., Chen, Y., Ding, C., Xiong, Y., et al. (2017). Effective gene editing by high-fidelity base editor 2 in mouse zygotes. Protein Cell 8, 601-611.   DOI
16 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.   DOI
17 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.   DOI
18 Nishida, K., Arazoe, T., Yachie, N., Banno, S., Kakimoto, M., Tabata, M., Mochizuki, M., Miyabe, A., Araki, M., Hara, K.Y., et al. (2016). Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems. Science 353, pii: aaf8729.
19 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.   DOI
20 Shimatani, Z., Kashojiya, S., Takayama, M., Terada, R., Arazoe, T., Ishii, H., Teramura, H., Yamamoto, T., Komatsu, H., Miura, K., et al. (2017). Targeted base editing in rice and tomato using a CRISPR-Cas9 cytidine deaminase fusion. Nat. Biotechnol. 35, 441-443.   DOI