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

  • Technological Innovations
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Journal of Assisted Reproduction and Genetics Aims and scope Submit manuscript

An Erratum to this article was published on 15 May 2017

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.

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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

Author notes

    Authors and Affiliations

    1. Key Laboratory for Major Obstetric Diseases of Guangdong Province, Key Laboratory of Reproduction and Genetics of Guangdong Higher Education Institutes, The Third Affiliated Hospital of Guangzhou Medical University, Guangzhou, 510150, China

      Xiangjin Kang, Wenyin He, Yuling Huang, Qian Yu, Yaoyong Chen, Xingcheng Gao, Xiaofang Sun & Yong Fan

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    1. Xiangjin Kang
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    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.

    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)

    Fig S1

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

    High Resolution Image (TIF 449 kb)

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    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

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    • DOI: https://doi.org/10.1007/s10815-016-0710-8

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