Introducing precise genetic modifications into human 3PN embryos by CRISPR/Cas-mediated genome editing

An Erratum to this article is available

Abstract

Purpose

As a powerful technology for genome engineering, the CRISPR/Cas system has been successfully applied to modify the genomes of various species. The purpose of this study was to evaluate the technology and establish principles for the introduction of precise genetic modifications in early human embryos.

Methods

3PN zygotes were injected with Cas9 messenger RNA (mRNA) (100 ng/μl) and guide RNA (gRNA) (50 ng/μl). For oligo-injections, donor oligo-1 (99 bp) or oligo-2 (99 bp) (100 ng/μl) or dsDonor (1 kb) was mixed with Cas9 mRNA (100 ng/μl) and gRNA (50 ng/μl) and injected into the embryos.

Results

By co-injecting Cas9 mRNA, gRNAs, and donor DNA, we successfully introduced the naturally occurring CCR5Δ32 allele into early human 3PN embryos. In the embryos containing the engineered CCR5Δ32 allele, however, the other alleles at the same locus could not be fully controlled because they either remained wild type or contained indel mutations.

Conclusions

This work has implications for the development of therapeutic treatments of genetic disorders, and it demonstrates that significant technical issues remain to be addressed. We advocate preventing any application of genome editing on the human germline until after a rigorous and thorough evaluation and discussion are undertaken by the global research and ethics communities.

This is a preview of subscription content, log in to check access.

Fig. 1
Fig. 2

References

  1. 1.

    Lander ES, Linton LM, Birren B, Nusbaum C, Zody MC, Baldwin J, et al. Initial sequencing and analysis of the human genome. Nature. 2001;409(6822):860–921.

    CAS  Article  PubMed  Google Scholar 

  2. 2.

    Venter JC, Adams MD, Myers EW, Li PW, Mural RJ, Sutton GG, et al. The sequence of the human genome. Science. 2001;291(5507):1304–51.

    CAS  Article  PubMed  Google Scholar 

  3. 3.

    Urnov FD, Rebar EJ, Holmes MC, Zhang HS, Gregory PD. Genome editing with engineered zinc finger nucleases. Nat Rev Genet. 2010;11(9):636–46.

    CAS  Article  PubMed  Google Scholar 

  4. 4.

    Bogdanove AJ, Voytas DF. TAL effectors: customizable proteins for DNA targeting. Science. 2011;333(6051):1843–6.

    CAS  Article  PubMed  Google Scholar 

  5. 5.

    Hsu PD, Lander ES, Zhang F. Development and applications of CRISPR-Cas9 for genome engineering. Cell. 2014;157(6):1262–78.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  6. 6.

    Wang H, Yang H, Shivalila CS, Dawlaty MM, Cheng AW, Zhang F, et al. One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell. 2013;153(4):910–8.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  7. 7.

    Yang H, Wang H, Shivalila CS, Cheng AW, Shi L, Jaenisch R. One-step generation of mice carrying reporter and conditional alleles by CRISPR/Cas-mediated genome engineering. Cell. 2013;154(6):1370–9.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  8. 8.

    Wu Y, Liang D, Wang Y, Bai M, Tang W, Bao S, et al. Correction of a genetic disease in mouse via use of CRISPR-Cas9. Cell Stem Cell. 2013;13(6):659–62.

    CAS  Article  PubMed  Google Scholar 

  9. 9.

    Long C, McAnally JR, Shelton JM, Mireault AA, Bassel-Duby R, Olson EN. Prevention of muscular dystrophy in mice by CRISPR/Cas9-mediated editing of germline DNA. Science. 2014;345(6201):1184–8.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  10. 10.

    Niu Y, Shen B, Cui Y, Chen Y, Wang J, Wang L, et al. Generation of gene-modified cynomolgus monkey via Cas9/RNA-mediated gene targeting in one-cell embryos. Cell. 2014;156(4):836–43.

    CAS  Article  PubMed  Google Scholar 

  11. 11.

    Liu H, Chen Y, Niu Y, Zhang K, Kang Y, Ge W, et al. TALEN-mediated gene mutagenesis in rhesus and cynomolgus monkeys. Cell Stem Cell. 2014;14:323–8.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  12. 12.

    Liang P, Xu Y, Zhang X, Ding C, Huang R, Zhang Z, et al. CRISPR/Cas9-mediated gene editing in human tripronuclear zygotes. Protein Cell. 2015;6(5):363–72.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  13. 13.

    Liu P, Chen S, Li X, Qin L, Huang K, Wang L et al. Low immunogenicity of neural progenitor cells differentiated from induced pluripotent stem cells derived from less immunogenic somatic cells. PLoS One. 8(7):e69617.

  14. 14.

    Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N et al. Multiplex genome engineering using CRISPR/Cas systems. Science. 339(6121):819–23.

  15. 15.

    Kola I, Trounson A, Dawson G, Rogers P. Tripronuclear human oocytes: altered cleavage patterns and subsequent karyotypic analysis of embryos. Biol Reprod. 1987;37(2):395–401.

    CAS  Article  PubMed  Google Scholar 

  16. 16.

    Feenan K, Herbert M. Can “abnormally” fertilized zygotes give rise to viable embryos? Hum Fertil. 2006;9(3):157–69.

    Article  Google Scholar 

  17. 17.

    Balakier H. Tripronuclear human zygotes: the first cell cycle and subsequent development. Hum Reprod. 1993;8(11):1892–7.

    CAS  Article  PubMed  Google Scholar 

  18. 18.

    Martinson JJ, Chapman NH, Rees DC, Liu YT, Clegg JB. Global distribution of the CCR5 gene 32-basepair deletion. Nat Genet. 1997;16(1):100–3.

    CAS  Article  PubMed  Google Scholar 

  19. 19.

    Marmor M, Sheppard HW, Donnell D, Bozeman S, Celum C, Buchbinder S, et al. Homozygous and heterozygous CCR5-Delta32 genotypes are associated with resistance to HIV infection. J Acquir Immune Defic Syndr. 2001;27(5):472–81.

    CAS  Article  PubMed  Google Scholar 

  20. 20.

    Samson M, Libert F, Doranz BJ, Rucker J, Liesnard C, Farber CM, et al. Resistance to HIV-1 infection in Caucasian individuals bearing mutant alleles of the CCR-5 chemokine receptor gene. Nature. 1996;382(6593):722–5.

    CAS  Article  PubMed  Google Scholar 

  21. 21.

    Holt N, Wang J, Kim K, Friedman G, Wang X, Taupin V, et al. Human hematopoietic stem/progenitor cells modified by zinc-finger nucleases targeted to CCR5 control HIV-1 in vivo. Nat Biotechnol. 2010;28(8):839–47.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  22. 22.

    Li L, Krymskaya L, Wang J, Henley J, Rao A, Cao LF, et al. Genomic editing of the HIV-1 coreceptor CCR5 in adult hematopoietic stem and progenitor cells using zinc finger nucleases. Mol Ther : J Am Soc Gene Ther. 2013;21(6):1259–69.

    CAS  Article  Google Scholar 

  23. 23.

    Didigu CA, Wilen CB, Wang J, Duong J, Secreto AJ, Danet-Desnoyers GA, et al. Simultaneous zinc-finger nuclease editing of the HIV coreceptors ccr5 and cxcr4 protects CD4+ T cells from HIV-1 infection. Blood. 2014;123(1):61–9.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  24. 24.

    Yao Y, Nashun B, Zhou T, Qin L, Qin L, Zhao S, et al. Generation of CD34+ cells from CCR5-disrupted human embryonic and induced pluripotent stem cells. Hum Gene Ther. 2012;23(2):238–42.

    CAS  Article  PubMed  Google Scholar 

  25. 25.

    Ye L, Wang J, Beyer AI, Teque F, Cradick TJ, Qi Z, et al. Seamless modification of wild-type induced pluripotent stem cells to the natural CCR5Delta32 mutation confers resistance to HIV infection. Proc Natl Acad Sci U S A. 2014;111(26):9591–6.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  26. 26.

    Ramalingam S, London V, Kandavelou K, Cebotaru L, Guggino W, Civin C, et al. Generation and genetic engineering of human induced pluripotent stem cells using designed zinc finger nucleases. Stem Cells Dev. 2013;22(4):595–610.

    CAS  Article  PubMed  Google Scholar 

  27. 27.

    Tesson L, Usal C, Menoret S, Leung E, Niles BJ, Remy S, et al. Knockout rats generated by embryo microinjection of TALENs. Nat Biotechnol. 2011;29(8):695–6.

    CAS  Article  PubMed  Google Scholar 

  28. 28.

    Hsu PD, Scott DA, Weinstein JA, Ran FA, Konermann S, Agarwala V, et al. DNA targeting specificity of RNA-guided Cas9 nucleases. Nat Biotechnol. 2013;31:827–32.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  29. 29.

    Hong SG, Winkler T, Wu C, Guo V, Pittaluga S, Nicolae A et al. Path to the clinic: assessment of iPSC-based cell therapies in vivo in a nonhuman primate model. Cell Rep. 7(4):1298–309.

  30. 30.

    Frock RL, Hu J, Meyers RM, Ho YJ, Kii E, Alt FW. Genome-wide detection of DNA double-stranded breaks induced by engineered nucleases. Nat Biotechnol. 2015;33(2):179–86.

    CAS  Article  PubMed  Google Scholar 

  31. 31.

    Tsai SQ, Zheng Z, Nguyen NT, Liebers M, Topkar VV, Thapar V, et al. GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases. Nat Biotechnol. 2015;33(2):187–97.

    CAS  Article  PubMed  Google Scholar 

  32. 32.

    Kim D, Bae S, Park J, Kim E, Kim S, Yu HR, et al. Digenome-seq: genome-wide profiling of CRISPR-Cas9 off-target effects in human cells. Nat Methods. 2015;12(3):237–43.

    CAS  Article  PubMed  Google Scholar 

  33. 33.

    Ran FA, Hsu PD, Lin CY, Gootenberg JS, Konermann S, Trevino AE, et al. Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. Cell. 2013;154(6):1380–9.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  34. 34.

    Tsai SQ, Wyvekens N, Khayter C, Foden JA, Thapar V, Reyon D, et al. Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing. Nat Biotechnol. 2014;32(6):569–76.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  35. 35.

    Fu Y, Sander JD, Reyon D, Cascio VM, Joung JK. Improving CRISPR-Cas nuclease specificity using truncated guide RNAs. Nat Biotechnol. 2014;32(3):279–84.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  36. 36.

    Doench JG, Fusi N, Sullender M, Hegde M, Vaimberg EW, Donovan KF, et al. Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. Nat Biotechnol. 2016;34(2):184–91.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  37. 37.

    Yu C, Liu Y, Ma T, Liu K, Xu S, Zhang Y, et al. Small molecules enhance CRISPR genome editing in pluripotent stem cells. Cell Stem Cell. 2015;16(2):142–7.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  38. 38.

    Wu Y, Zhou H, Fan X, Zhang Y, Zhang M, Wang Y, et al. Correction of a genetic disease by CRISPR-Cas9-mediated gene editing in mouse spermatogonial stem cells. Cell Res. 2015;25(1):67–79.

    CAS  Article  PubMed  Google Scholar 

  39. 39.

    Baltimore D, Berg P, Botchan M, Carroll D, Charo RA, Church G, et al. Biotechnology: a prudent path forward for genomic engineering and germline gene modification. Science. 2015;348(6230):36–8.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgments

We thank Dr. Haoyi Wang from Institute of Zoology in Chinese Academy of Sciences for helpful advice. This work was supported in part by the National Natural Science Foundation of China (81370766, 81570101, 81370670), Guangdong Province Higher Education Funding (2013KJCX0149, Yq2013135), and Guangdong Province Science and Technology Project (2014A02011029, 2015B020227002). Y. F. was supported by the State 863 project 2015AA020307.

Authors’ contributions

Y.F. designed the study. Y.F., X.K., and Y.H. recruited the patients and signed the informed consent. Y.F. performed embryo injection. X.K. and Y.H. collected embryos. W.H. and Q.Y. performed the WGA and PCR genotyping and off-target prediction. Y.C., X.G., and X.S. contributed to off-target sites genotyping. Y.F. analyzed the data and wrote the paper.

Author information

Affiliations

Authors

Corresponding author

Correspondence to Yong Fan.

Ethics declarations

This study was approved by the ethics committee of the Third Affiliated Hospital of Guangzhou Medical University (LLSC2014018). The methods used in the present study closely followed the guidelines legislated and posted by the Ministry of Health of the People’s Republic of China. The patients involved in this study knew about and understood the usage of polyspermic zygotes and voluntarily donated them after providing informed consent.

Conflict of interest

The authors declare that they have no conflict of interest.

Additional information

Capsule As a powerful technology for genome engineering, the CRISPR/Cas system has been successfully applied to modify the genomes of various species.

Xiangjin Kang, Wenyin He and Yuling Huang contributed equally to this work.

An erratum to this article is available at http://dx.doi.org/10.1007/s10815-017-0946-y.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Fig S1
figure3

The sequences of the CCR5 gene in human 3PN embryos carrying CRISPR/Cas9-induced gene modifications (GIF 192 kb)

Table S1

Oligonucleotides used for making in vitro transcription template, CCR5 genotyping and as HDR-mediated repair template. (PDF 177 kb)

Table S2

Off-target analysis for CRISPR-Cas9–mediated targeting in human 3PN zygotes. (PDF 27 kb)

High Resolution Image (TIF 449 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Kang, X., He, W., Huang, Y. et al. Introducing precise genetic modifications into human 3PN embryos by CRISPR/Cas-mediated genome editing. J Assist Reprod Genet 33, 581–588 (2016). https://doi.org/10.1007/s10815-016-0710-8

Download citation

Keywords

  • CRISPR/Cas9
  • Genetic modification
  • CCR5
  • Human 3PN embryos