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Molecular Genetics and Genomics

, Volume 292, Issue 3, pp 525–533 | Cite as

CRISPR/Cas9-mediated gene editing in human zygotes using Cas9 protein

Original Article

Abstract

Previous works using human tripronuclear zygotes suggested that the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system could be a tool in correcting disease-causing mutations. However, whether this system was applicable in normal human (dual pronuclear, 2PN) zygotes was unclear. Here we demonstrate that CRISPR/Cas9 is also effective as a gene-editing tool in human 2PN zygotes. By injection of Cas9 protein complexed with the appropriate sgRNAs and homology donors into one-cell human embryos, we demonstrated efficient homologous recombination-mediated correction of point mutations in HBB and G6PD. However, our results also reveal limitations of this correction procedure and highlight the need for further research.

Keywords

CRISPR/Cas9 Homology-directed repair (HDR) Cas9 protein Gene modification Human zygotes 

Notes

Acknowledgements

We thank Dr. Yuanfeng Li of Beijing Proteome Research Center and Dr. Zhongsheng Sun of Beijing Institutes of Life Science, Chinese Academy of Sciences for help with genome sequencing and analysis. We thank Dr. Xingxu Huang of the School of Life Science and Technology, Shanghai Tech University for helpful advice.

Compliance with ethical standards

Funding

This study was funded by a Grant from the Beijing Municipal Committee on Science and Technology (#Z141100000214015), by an international collaboration Grant from the Chinese Minister of Science and Technology (#2013DFB30210), by The National Basic Research Program (973 Program, No. 2013CB910300), by a Grant from the Science and Technology Program of Guangzhou, China (#2014KZDXM047), by The Innovation of Science and Technology Commission of Guangzhou, China (No. 201604020075), by The Department of Science and Technology of Guangdong, China (No. 2016A020218012), and a Grant from the China Postdoctoral Science Foundation (#2013M532214).

Conflict of interest

All authors declare that they have no conflict of interest.

Ethical approval

This study was approved by the ethics committee of the Third Affiliated Hospital of Guangzhou Medical University, numbered [2015] No. 068. 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 tripronuclear zygotes, immature oocytes and leftover sperm, and voluntarily donated them after providing informed consent.

Informed consent

Informed consent was obtained from all individual participants included in the study.

Supplementary material

438_2017_1299_MOESM1_ESM.docx (223 kb)
Supplementary material 1 (DOCX 222 KB)

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Copyright information

© Springer-Verlag Berlin Heidelberg 2017

Authors and Affiliations

  1. 1.State Key Laboratory of Proteomics, Beijing Proteome Research CenterBeijing Institute of Radiation MedicineBeijingChina
  2. 2.National Center for Protein Sciences BeijingBeijingChina
  3. 3.Center for Reproductive Medicine, The Third Affiliated HospitalGuangzhou Medical UniversityGuangzhouChina
  4. 4.National Center for International Research of Biological Targeting Diagnosis and Therapy, Collaborative Innovation Center for Targeting Tumor Diagnosis and TherapyGuangxi Medical UniversityGuangxiChina
  5. 5.Houston Fertility InstituteHoustonUSA
  6. 6.Department of CardiologyBayi Hospital Affiliated Nanjing University of Chineses MedicineNanjingChina

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