Introducing precise genetic modifications into human 3PN embryos by CRISPR/Cas-mediated genome editing
- 15k Downloads
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.
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.
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.
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.
KeywordsCRISPR/Cas9 Genetic modification CCR5 Human 3PN embryos
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.
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.
Compliance with ethical standards
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.
- 13.Liu P, Chen S, Li X, Qin L, Huang K, Wang L et al. Low immunogenicity of neural progenitor cells differentiated from induced pluripotent stem cells derived from less immunogenic somatic cells. PLoS One. 8(7):e69617.Google Scholar
- 14.Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N et al. Multiplex genome engineering using CRISPR/Cas systems. Science. 339(6121):819–23.Google Scholar
- 29.Hong SG, Winkler T, Wu C, Guo V, Pittaluga S, Nicolae A et al. Path to the clinic: assessment of iPSC-based cell therapies in vivo in a nonhuman primate model. Cell Rep. 7(4):1298–309.Google Scholar