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Highly efficient RNA-guided base editing in mouse embryos

Nature Biotechnology volume 35, pages 435–437 (2017)Cite this article

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Abstract

Base editors (BEs) composed of a cytidine deaminase fused to CRISPR–Cas9 convert cytidine to uridine, leading to single-base-pair substitutions in eukaryotic cells. We delivered BE mRNA or ribonucleoproteins targeting the Dmd or Tyr gene via electroporation or microinjection into mouse zygotes. F0 mice showed nonsense mutations with an efficiency of 44–57% and allelic frequencies of up to 100%, demonstrating an efficient method to generate mice with targeted point mutations.

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Figure 1: Dystrophin-deficient mutant mice generated by cytidine-deaminase-mediated base editing.
Figure 2: Generation of an albinism mouse model by cytidine-deaminase-mediated base editing.

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References

  1. Komor, A.C., Kim, Y.B., Packer, M.S., Zuris, J.A. & Liu, D.R. Nature 533, 420–424 (2016).

    Article  CAS  Google Scholar 

  2. Conticello, S.G. Genome Biol. 9, 229 (2008).

    Article  Google Scholar 

  3. Li, J., Sun, Y., Du, J., Zhao, Y. & Xia, L. Mol. Plant http://dx.doi.org/10.1016/j.molp.2016.12.001 (2016).

  4. Lu, Y. & Zhu, J.-k. Mol. Plant http://dx.doi.org/10.1016/j.molp.2016.11.013

  5. Ma, Y. et al. Nat. Methods 13, 1029–1035 (2016).

    Article  CAS  Google Scholar 

  6. Nishida, K. et al. Science http://dx.doi.org/10.1126/science.aaf8729

  7. Cho, S.W. et al. Genome Res. 24, 132–141 (2014).

    Article  CAS  Google Scholar 

  8. Ran, F.A. et al. Cell 154, 1380–1389 (2013).

    Article  CAS  Google Scholar 

  9. Mali, P. et al. Nat. Biotechnol. 31, 833–838 (2013).

    Article  CAS  Google Scholar 

  10. Hur, J.K. et al. Nat. Biotechnol. 34, 807–808 (2016).

    Article  CAS  Google Scholar 

  11. Sung, Y.H. et al. Nat. Biotechnol. 31, 23–24 (2013).

    Article  CAS  Google Scholar 

  12. Sung, Y.H. et al. Genome Res. 24, 125–131 (2014).

    Article  CAS  Google Scholar 

  13. Bae, S., Park, J. & Kim, J.S. Bioinformatics 30, 1473–1475 (2014).

    Article  CAS  Google Scholar 

  14. Kim, D. et al. Nat. Methods 12, 237–243, 1, 243 (2015).

    Article  CAS  Google Scholar 

  15. Cho, S.W., Kim, S., Kim, J.M. & Kim, J.S. Nat. Biotechnol. 31, 230–232 (2013).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by the Institute for Basic Science (IBS-R021-D1 to J.-S.K.).

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Author notes

  1. Kyoungmi Kim and Seuk-Min Ryu: These authors contributed equally to this work.

Authors and Affiliations

  1. Center for Genome Engineering, Institute for Basic Science, Seoul, Republic of Korea

    Kyoungmi Kim, Seuk-Min Ryu, Sang-Tae Kim, Gayoung Baek, Kayeong Lim, Eugene Chung, Sunghyun Kim & Jin-Soo Kim

  2. Department of Chemistry, Seoul National University, Seoul, Republic of Korea

    Seuk-Min Ryu, Daesik Kim, Kayeong Lim, Eugene Chung, Sunghyun Kim & Jin-Soo Kim

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  1. Kyoungmi Kim
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Contributions

K.K., S.-M.R., and J.-S.K. designed the research. K.K., S.-M.R., S.-T.K., G.B., D.K., K.L., E.C. and S.K. performed the experiments. J.-S.K. supervised the research. All authors discussed the results and commented on the manuscript.

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Correspondence to Jin-Soo Kim.

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J.-S.K. is a founder of and shareholder in ToolGen.

Integrated supplementary information

Supplementary Figure 1 Targeted mutagenesis in mouse embryos by microinjection of deaminse-nCas9 mRNA and sgRNAs.

(a) and (b) Alignments of mutant sequences from blastocysts that developed after microinjection of BE3-encoding mRNA and sgRNA into zygotes. The target sequence is underlined. The PAM site and substitutions are shown in blue and red, respectively. The column on the right indicates frequencies of mutant alleles. Wt, wild-type.

Supplementary Figure 2 Sanger sequencing chromatograms of wild type and Dmd mutant mice.

Sanger sequencing chromatograms of DNA from wild-type and D108 mutant mice. The red arrow indicates the substituted nucleotide. The relevant codon identities at the target site are shown under the DNA sequence.

Supplementary Figure 3 Germline transmission of the mutant Dmd mouse.

(a) Targeted deep sequencing analysis using gDNA of Wt (Wild type) and D108 testis. (b) Alignments of mutant sequences from Wt (Wild type) and D108 testis. The PAM site and substitutions are shown in blue and red, respectively. The column on the right indicates frequencies of mutant alleles. (C) Germline transmission of the mutant allele in pups (D201 to D204 and D207 to D212) obtained from the Dmd mutant mouse (D102). Deep sequencing was used to confirm the genotype. The PAM site and substitutions are shown in blue and red, respectively. The column on the right indicates frequencies of mutant alleles.

Supplementary Figure 4 Targeted mutagenesis in mouse embryos by electroporation of rAPOBEC1-nCas9 (D10A)-UGI RNPs.

(a) and (b) Alignments of mutant sequences from blastocysts that developed after electroporation of the BE3 RNP into zygotes. The target sequence is underlined. The PAM site and substitutions are shown in blue and red, respectively. The column on the right indicates frequencies of mutant alleles. Wt, wild-type.

Supplementary Figure 5 No off-target mutations were detectably induced at potential off-target sites in Dmd mutant mice.

Targeted deep sequencing was used to measure base editing efficiencies at potential off-target sites in Dmd mutant mice (n=3). Mismatched nucleotides and PAM sequences are shown in red and in blue, respectively.

Supplementary Figure 6 No off-target mutations were detectably induced at potential off-target sites in Tyr mutant mice.

Targeted deep sequencing was used to measure base editing efficiencies at potential off-target sites in Tyr mutant mice (n=2). Mismatched nucleotides and PAM sequences are shown in red and in blue, respectively.

Supplementary Figure 7 Whole genome sequencing of Dmd mutant (D108) and wild-type (Wt) mice.

(a) Summary of whole genome sequencing analysis. A Dmd mutant mouse (D108) and a wild type mouse (Wt) were separately sequenced using Illumina HiSeq X10. Total variants (SNPs + small indels) were identified using ISACC16. After filtering out naturally-occurring variants in the SNP database (dbSNP) and excluding SNPs also found in the wild-type genome, we obtained 8,722 SNPs in the D108 genome. We further excluded base substitutions other than C to T or A or G conversions. We next compared the DNA sequences at the remaining SNP sites with the on-target sequence. Among 319,663 or 296,518 proto-spacer adjacent motif (PAM)-containing sites that differ from the on-target site by up to 7 or 5 mismatches, respectively, with 0 or up to 2 bulges, respectively, just a single site was identified as a potential off-target site. (b) The off-target candidate site was invalidated using targeted deep sequencing of genomic DNA isolated from various organs (heart, kidney, lung, muscle, tail, and ear). Mismatched nucleotides and PAM sequences are shown in red and in blue, respectively.

16. Raczy, C. et al. Bioinformatics. 29, 2041–2043 (2013).

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Supplementary figures 1–7, Supplementary Tables 1–3 (PDF 2635 kb)

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Kim, K., Ryu, SM., Kim, ST. et al. Highly efficient RNA-guided base editing in mouse embryos. Nat Biotechnol 35, 435–437 (2017). https://doi.org/10.1038/nbt.3816

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