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

  • Xiangjin Kang
  • Wenyin He
  • Yuling Huang
  • Qian Yu
  • Yaoyong Chen
  • Xingcheng Gao
  • Xiaofang Sun
  • Yong Fan
Technological Innovations

DOI: 10.1007/s10815-016-0710-8

Cite this article as:
Kang, X., He, W., Huang, Y. et al. J Assist Reprod Genet (2016) 33: 581. doi:10.1007/s10815-016-0710-8

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.

Keywords

CRISPR/Cas9 Genetic modification CCR5 Human 3PN embryos 

Supplementary material

10815_2016_710_MOESM1_ESM.pdf (177 kb)
Table S1Oligonucleotides used for making in vitro transcription template, CCR5 genotyping and as HDR-mediated repair template. (PDF 177 kb)
10815_2016_710_MOESM2_ESM.pdf (27 kb)
Table S2Off-target analysis for CRISPR-Cas9–mediated targeting in human 3PN zygotes. (PDF 27 kb)
10815_2016_710_Fig3_ESM.gif (192 kb)
Fig S1

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

10815_2016_710_MOESM3_ESM.tif (449 kb)
High Resolution Image (TIF 449 kb)

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Xiangjin Kang
    • 1
  • Wenyin He
    • 1
  • Yuling Huang
    • 1
  • Qian Yu
    • 1
  • Yaoyong Chen
    • 1
  • Xingcheng Gao
    • 1
  • Xiaofang Sun
    • 1
  • Yong Fan
    • 1
  1. 1.Key Laboratory for Major Obstetric Diseases of Guangdong Province, Key Laboratory of Reproduction and Genetics of Guangdong Higher Education InstitutesThe Third Affiliated Hospital of Guangzhou Medical UniversityGuangzhouChina