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

J Assist Reprod Genet. 2016 May;33(5):581-588. doi: 10.1007/s10815-016-0710-8. Epub 2016 Apr 6.


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: CCR5; CRISPR/Cas9; Genetic modification; Human 3PN embryos.

MeSH terms

  • CRISPR-Cas Systems*
  • DNA End-Joining Repair
  • Disease Resistance / genetics
  • Embryo, Mammalian
  • Embryonic Development
  • Fertilization
  • Gene Editing / ethics
  • Gene Editing / methods*
  • Genetic Diseases, Inborn / genetics*
  • Genetic Diseases, Inborn / prevention & control
  • Humans
  • Receptors, CCR5 / genetics*
  • Sequence Analysis, DNA


  • CCR5 protein, human
  • Receptors, CCR5