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06.04.2016 | Technological Innovations | Ausgabe 5/2016

Journal of Assisted Reproduction and Genetics 5/2016

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

Zeitschrift:
Journal of Assisted Reproduction and Genetics > Ausgabe 5/2016
Autoren:
Xiangjin Kang, Wenyin He, Yuling Huang, Qian Yu, Yaoyong Chen, Xingcheng Gao, Xiaofang Sun, Yong Fan
Wichtige Hinweise

Electronic supplementary material

The online version of this article (doi:10.​1007/​s10815-016-0710-8) contains supplementary material, which is available to authorized users.
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.

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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