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

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

Fig. 1

Gene editing at CCR5 locus in human 3PN embryos. a Schematic of the targeted CCR5 locus. The 32 bp sequence deleted in natural occurring CCR5Δ32 allele is capitalized and marked by black box. The two gRNAs are bold and underlined, and their PAM regions are labeled in green. b Pictures showing the injection of a human 3PN zygote (left panel) and the development to 8–16-cell stage in vitro (right panel). Scale bar = 100 μm. c Schematic of all the alleles identified in each mutant embryos. The gRNAs injected in each embryo are labeled at the left side, and the mutations are labeled in red. d Sequence chromatography of the CCR5Δ32 allele from the targeted human 3PN embryo. The black line indicates the position of 32 bp deletion

Fig. 2

All alleles identified in human 3PN embryos containing CCR5Δ32 allele. Schematic of all the alleles identified in each mutant embryo. The 32 bp sequence deleted in natural occurring CCR5Δ32 allele is capitalized. The gRNAs and donor DNA injected in each embryo are labeled at the left side, and the mutations are labeled in red